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WO2023041564A1 - Composition and method for detecting sars-cov-2 - Google Patents

Composition and method for detecting sars-cov-2 Download PDF

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Publication number
WO2023041564A1
WO2023041564A1 PCT/EP2022/075495 EP2022075495W WO2023041564A1 WO 2023041564 A1 WO2023041564 A1 WO 2023041564A1 EP 2022075495 W EP2022075495 W EP 2022075495W WO 2023041564 A1 WO2023041564 A1 WO 2023041564A1
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Prior art keywords
seq
sequence
cov
sars
polynucleotide
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PCT/EP2022/075495
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French (fr)
Inventor
Gemma STOKES
Andrew Dawson
Thomas Jefferson
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Primer Design Limited
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Publication of WO2023041564A1 publication Critical patent/WO2023041564A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C12Q1/701—Specific hybridization probes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/16—Primer sets for multiplex assays

Definitions

  • the invention relates to novel primers, probes, kits and methods for determining the presence or absence of SARS-CoV-2.
  • Coronaviruses are members of the subfamily Coronavirinae in the family Coronaviridae and the order Nidovirales.
  • the virus belongs to genera Betacoronavirus and has close similarities to severe acute respiratory syndrome-related coronaviruses (SARS-CoV).
  • SARS-CoV severe acute respiratory syndrome-related coronaviruses
  • the virus uses ACE2 as the entry receptor-like SARS-CoV. These similarities had lead the Coronavirus Study Group of the International Committee on Taxonomy of Viruses to term the virus SARS-CoV-2. Since the virus is novel, new methods are required to test for the virus and distinguish it from other similar coronaviruses.
  • the sequence of SARS-Cov-2 has been published under accession no. MN908947
  • the inventors have surprisingly identified new primers, probes, kits and methods for determining the presence or absence of SARS-CoV-2 in a variety of samples.
  • the first set of new primers and probes target the ORFlab gene in SARS-CoV-2.
  • the ORFlab gene encodes the ORFla/ORFlab poly-proteins.
  • the second set of new primers and probes target the S gene in SARS-CoV-2.
  • the S gene encodes the spike glycoprotein which facilitates entry of the virus into cells.
  • the third set of new primers and probes target the M gene in SARS-CoV-2.
  • the M gene encodes the membrane (M) glycoprotein.
  • the three sets of primers and probes are present together in the kits of the invention and are used together in the triplex methods of the invention.
  • the invention provides: three pairs of primers for determining the presence or absence of SARS-CoV-2 in a sample, wherein (a) in the first pair the forward primer comprises a polynucleotide having the sequence shown in SEQ ID NO: 1 or a variant thereof having at least about 80% homology to SEQ ID NO: 1 based on sequence identity over its entire length and the reverse primer comprises a polynucleotide having the sequence shown in SEQ ID NO: 2 or a variant thereof having at least about 75% homology to SEQ ID NO: 2 based on sequence identity over its entire length; (b) in the second pair the forward primer comprises a polynucleotide having the sequence shown in SEQ ID NO: 6 or a variant thereof having at least about 75% homology to SEQ ID NO: 6 based on sequence identity over its entire length and the reverse primer comprises a polynucleotide having the sequence shown in SEQ ID NO: 7 or a variant thereof having at least about 75% homology to SEQ ID NO: 7 based on sequence
  • Figure 1 shows the amplification plot for ORFlab (FAM).
  • Figure 2 shows the amplification plot for M-gene (ROX).
  • FIG. 3 shows the amplification plot for S-gene (ATTO).
  • Figure 4 shows the amplification plot for L.lactis IEC (HEX).
  • SEQ ID NO: 1 shows the forward ORFlab gene primer used in Example.
  • SEQ ID NO: 2 shows the reverse ORFlab gene primer used in Example.
  • SEQ ID NO: 3 shows the ORFlab gene probe used in Example.
  • SEQ ID NO: 4 shows the ORFlab gene cDNA amplified in the Example.
  • SEQ ID NO: 5 shows the reverse complement of SEQ ID NO: 3.
  • SEQ ID NO: 6 shows the forward S gene primer used in the Example.
  • SEQ ID NO: 7 shows the reverse S gene primer used in the Example.
  • SEQ ID NO: 8 shows the S gene probe used in the Example. The probe specifically hybridises to part of the complementary strand of the cDNA amplicon sequence shown in SEQ ID NO: 9.
  • SEQ ID NO: 9 shows the S gene cDNA amplified in the Example.
  • SEQ ID NO: 10 shows the reverse complement of SEQ ID NO: 8.
  • SEQ ID NO: 13 shows the M gene probe used in the Example.
  • SEQ ID NO: 14 shows the M gene cDNA amplified in the Example.
  • SEQ ID NO: 15 shows the reverse complement of SEQ ID NO: 13.
  • SEQ ID NO: 16 shows the Lactococcus lactis (L. lactis) forward primer.
  • SEQ ID NO: 17 shows the L. lactis reverse primer.
  • SEQ ID NO: 18 shows the L. lactis probe 1.
  • SEQ ID NO: 19 shows the L. lactis probe 2.
  • SARS-CoV-2 is interchangeable with 2019-nCoV or COVID-19.
  • determining is interchangeable with detecting.
  • the new primers, probes, kits and methods of the invention may be for detecting the presence or absence of SARS-CoV-2 in a sample or patient. Also, in all instances, the new primers, probes, kits and methods of the invention may be for determining/detecting/identifying whether or not a sample contains or comprises SARS-CoV-2 or whether or not a patient is infected with SARS-CoV-2.
  • the inventors have surprisingly identified new primers, probes, kits and methods for determining the presence or absence of SARS-CoV-2 in a variety of samples.
  • the compositions and methods have several advantages.
  • the new primers, probes, kits and methods are capable of detecting the presence of SARS-CoV-2 with a high degree of sensitivity. As shown in the Example, the method is capable of detecting only 1 copy of the SARS-CoV-2 RNA genome per ul of sample.
  • the new primers, probes, kits and methods do not cross react with other human viruses, such as other human coronaviruses.
  • the new primers, probes, kits and methods do not cross react with SARS-CoV. Positive results from the new primers, probes, kits and methods can therefore be trusted as being indicative of the presence of SARS-CoV-2.
  • the new primers, probes, kits and methods therefore do not give false positives.
  • the new primers, probes, kits and methods are capable of measuring the SARS-CoV-2 titre in sample and distinguishing between a low SARS-CoV-2 titre and a high SARS-CoV-2 titre. This is particularly helpful in determining the level of infection in patients and allowing physicians to tailor their treatments to those levels.
  • the present invention provides three pairs of primers for determining the presence or absence of SARS-CoV-2 in a sample.
  • the forward primer comprises a polynucleotide having the sequence shown in SEQ ID NO: 1 or a variant thereof having at least about 80% homology to SEQ ID NO: 1 based on sequence identity over its entire length.
  • the reverse primer comprises a polynucleotide having the sequence shown in SEQ ID NO: 2 or a variant thereof having at least about 75% homology to SEQ ID NO: 2 based on sequence identity over its entire length.
  • the forward primer comprises a polynucleotide having the sequence shown in SEQ ID NO: 6 or a variant thereof having at least about 75% homology to SEQ ID NO: 6 based on sequence identity over its entire length.
  • the reverse primer comprises a polynucleotide having the sequence shown in SEQ ID NO: 7 or a variant thereof having at least about 75% homology to SEQ ID NO: 7 based on sequence identity over its entire length.
  • the forward primer comprises a polynucleotide having the sequence shown in SEQ ID NO: 11 or a variant thereof having at least about 75% homology to SEQ ID NO: 11 based on sequence identity over its entire length.
  • the reverse primer comprises a polynucleotide having the sequence shown in SEQ ID NO: 12 or a variant thereof having at least about 75% homology to SEQ ID NO: 12 based on sequence identity over its entire length.
  • a polynucleotide such as a nucleic acid, is a polymer comprising two or more nucleotides.
  • the nucleotides can be naturally occurring or artificial.
  • a nucleotide typically contains a nucleobase, a sugar and at least one linking group, such as a phosphate, 2'0-methyl, 2' methoxy-ethyl, phosphoramidate, methylphosphonate or phosphorothioate group.
  • the nucleobase is typically heterocyclic.
  • Nucleobases include, but are not limited to, purines and pyrimidines and more specifically adenine (A), guanine (G), thymine (T), uracil (U) and cytosine (C).
  • the sugar is typically a pentose sugar.
  • Nucleotide sugars include, but are not limited to, ribose and deoxyribose.
  • nucleosides include, but are not limited to, adenosine, guanosine, 5-methyluridine, uridine, cytidine, deoxyadenosine, deoxyguanosine, thymidine, deoxyuridine and deoxycytidine.
  • the nucleosides are most preferably adenosine, guanosine, uridine and cytidine
  • the nucleotides are typically ribonucleotides or deoxyribonucleotides.
  • the nucleotides are preferably deoxyribonucleotides.
  • the nucleotides typically contain a monophosphate, diphosphate or triphosphate. Phosphates may be attached on the 5' or 3' side of a nucleotide.
  • Nucleotides include, but are not limited to, adenosine monophosphate (AMP), adenosine diphosphate (ADP), adenosine triphosphate (ATP), guanosine monophosphate (GMP), guanosine diphosphate (GDP), guanosine triphosphate (GTP), thymidine monophosphate (TMP), thymidine diphosphate (TDP), thymidine triphosphate (TTP), uridine monophosphate (UMP), uridine diphosphate (UDP), uridine triphosphate (UTP), cytidine monophosphate (CMP), cytidine diphosphate (CDP), cytidine triphosphate (CTP), 5-methylcytidine monophosphate, 5-methylcytidine diphosphate, 5-methylcytidine triphosphate, 5- hydroxymethylcytidine monophosphate, 5-hydroxymethylcytidine diphosphate, 5- hydroxymethylcytidine diphosphate, 5-
  • the nucleotides are preferably selected from AMP, UMP, GMP, CMP, dAMP, dTMP, dGMP or dCMP.
  • the nucleotides are preferably selected from dAMP, dTMP, dGMP or dCMP.
  • nucleotides may contain additional modifications.
  • suitable modified nucleotides include, but are not limited to, 2'amino pyrimidines (such as 2'-amino cytidine and 2'-amino uridine), 2'-hyrdroxyl purines (such as , 2'-fluoro pyrimidines (such as 2'- fluorocytidine and 2'fluoro uridine), hydroxyl pyrimidines (such as 5'-o-P-borano uridine), 2'-O-methyl nucleotides (such as 2'-O-methyl adenosine, 2'-O-methyl guanosine, 2'-O- methyl cytidine and 2'-O-methyl uridine), 4'-thio pyrimidines (such as 4'-thio uridine and 4'- thio cytidine) and nucleotides have modifications of the nucleobase (such as 5-pent
  • One or more nucleotides in the polynucleotide may be modified, for instance with a label or a tag.
  • the label may be any suitable label which allows the polynucleotide to be detected. Suitable labels include, but are not limited to, fluorescent molecules, radioisotopes, e.g. 125 I, 35 S, enzymes, antibodies, antigens, other polynucleotides and ligands such as biotin.
  • the nucleotides in the polynucleotide may be attached to each other in any manner.
  • the nucleotides may be linked by phosphate, 2'0-methyl, 2' methoxy-ethyl, phosphoramidate, methylphosphonate or phosphorothioate linkages.
  • the nucleotides are typically attached by their sugar and phosphate groups as in nucleic acids.
  • the nucleotides may be connected via their nucleobases as in pyrimidine dimers.
  • the polynucleotide can be a nucleic acid, such as deoxyribonucleic acid (DNA) or a ribonucleic acid (RIMA).
  • the polynucleotide may be any synthetic nucleic acid known in the art, such as peptide nucleic acid (PNA), glycerol nucleic acid (GNA), threose nucleic acid (TNA), locked nucleic acid (LNA), morpholino nucleic acid or other synthetic polymers with nucleotide side chains.
  • PNA peptide nucleic acid
  • GMA glycerol nucleic acid
  • TAA threose nucleic acid
  • LNA locked nucleic acid
  • morpholino nucleic acid or other synthetic polymers with nucleotide side chains.
  • the polynucleotide may comprise any of the nucleotides discussed above, including the modified nucleotides.
  • the polynucleotide may be DNA or RNA.
  • a primer polynucleotide that is RNA has the sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2 if all of the instances of dAMP, dTMP, dGMP and dCMP are replaced with AMP, UMP, GMP and CMP respectively.
  • the DNA nucleotides are replaced with their corresponding RNA nucleotides and T is replaced with U.
  • the polynucleotide is preferably DNA.
  • the polynucleotide may be single stranded or double stranded.
  • the primer polynucleotide is preferably single stranded. Any of the polynucleotides discussed herein, such as the primers, probes, cDNA amplicons and target cDNA sequences, may be isolated, substantially isolated, purified or substantially purified. The polynucleotide is isolated or purified if it is completely free of any other components, such as buffer, other polynucleotides, virus material or cells.
  • the polynucleotide is substantially isolated or substantially purified if it is only mixed with carriers or diluents, such as buffers or excipients, which will not interfere with its intended use, such as in a RT-PCR assay.
  • carriers or diluents such as buffers or excipients, which will not interfere with its intended use, such as in a RT-PCR assay.
  • the primer polynucleotides are not naturally occurring.
  • the primer of the invention may comprise a variant sequence based on SEQ ID NO: 1, 2, 6, 7, 11 or 12.
  • a variant sequence is a polynucleotide that has a nucleotide sequence which varies from that of SEQ ID NO: 1, 2, 6, 7, 11 or 12 and which retains its ability to specifically hybridise to the target sequence of SEQ ID NO: 1, 2, 6, 7, 11 or 12 .
  • a variant sequence is a polynucleotide that has a nucleotide sequence which varies from that of SEQ ID NO: 1, 2, 6, 7, 11 or 12 and which retains its ability to specifically hybridise to a sequence that is complementary to SEQ ID NO: 1, 2, 6, 7, 11 or 12 .
  • a variant sequence is a polynucleotide that has a nucleotide sequence which varies from that of SEQ ID NO: 1, 2, 6, 7, 11 or 12 and which retains its ability to amplify the complementary DNA (cDNA) amplicon amplified from SARS-CoV-2 cDNA by SEQ ID NOs: 1, 2, 6, 7, 11 or 12 (/.e. SEQ ID NO: 4, 9 or 14).
  • cDNA complementary DNA
  • amplified refers to the process of making multiple copies of the polynucleotide, such as cDNA, from a single polynucleotide or fewer polynucleotides.
  • Conditions that permit the hybridisation are well-known in the art (for example, Sambrook et al., 2001, Molecular Cloning: a laboratory manual, 3rd edition, Cold Spring Harbour Laboratory Press; and Current Protocols in Molecular Biology, Chapter 2, Ausubel et al., Eds., Greene Publishing and Wiley-lnterscience, New York (1995)).
  • Hybridisation can be carried out under low stringency conditions, for example in the presence of a buffered solution of 30 to 35% formamide, 1 M NaCI and 1 % SDS (sodium dodecyl sulfate) at 37 °C followed by a 20 wash in from IX (0.1650 M Na+) to 2X (0.33 M Na+) SSC (standard sodium citrate) at 50 °C.
  • Hybridisation can be carried out under moderate stringency conditions, for example in the presence of a buffer solution of 40 to 45% formamide, 1 M NaCI, and 1 % SDS at 37 °C, followed by a wash in from 0.5X (0.0825 M Na+) to IX (0.1650 M Na+) SSC at 55 °C.
  • Hybridisation can be carried out under high stringency conditions, for example in the presence of a buffered solution of 50% formamide, 1 M NaCI, 1% SDS at 37 °C, followed by a wash in 0.1X (0.0165 M Na+) SSC at 60 °C.
  • a variant “specifically hybridises” if it hybridises to its partner with a melting temperature (Tm) that is at least 2 °C, such as at least 3 °C, at least 4 °C, at least 5 °C, at least 6 °C, at least 7 °C, at least 8 °C, at least 9 °C or at least 10 °C, greater than its Tm for other polynucleotides.
  • Tm melting temperature
  • the variant hybridises to its partner with a Tm that is at least 2 °C, such as at least 3 °C, at least 4 °C, at least 5 °C, at least 6 °C, at least 7 °C, at least 8 °C, at least 9 °C, at least 10 °C, at least 20 °C, at least 30 °C or at least 40 °C, greater than its Tm for other polynucleotides.
  • the variant typically hybridises to its target sequence with a Tm of at least 90 °C, such as at least 92 °C or at least 95 °C.
  • Tm can be measured experimentally using known techniques, including the use of DNA microarrays, or can be calculated using publicly available Tm calculators, such as those available over the internet.
  • the variant sequence may comprise any of the nucleotides discussed above, including the modified nucleotides.
  • the variant sequence is typically the same length as SEQ ID NO: 1, 2, 6, 7, 11 or 12, but may be longer or shorter.
  • a variant of SEQ ID NO: 1 is preferably at least 20 nucleotides in length, such as 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides in length.
  • a variant of SEQ ID NO: 2 is preferably at least 19 nucleotides in length, such as 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 or 29 nucleotides in length.
  • a variant of SEQ ID NO: 6 or 7 is preferably at least 17 nucleotides in length, such as 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 or 27 nucleotides in length.
  • a variant of SEQ ID NO: 11 is preferably at least 15 nucleotides in length, such as 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides in length.
  • a variant of SEQ ID NO: 12 is preferably at least 16 nucleotides in length, such as 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or 26 nucleotides in length.
  • a variant sequence is at least about 80% or at least about 83.3% homologous to that sequence based on nucleotide identity. Over the entire length of the sequence of SEQ ID NO: 1, a variant sequence has at least about 80% or at least about 83.3% identity to that sequence. Homology based on sequence identity and identity are interchangeable herein. This allows for variation, deletion, addition or a combination thereof of five nucleotides within the sequence of SEQ ID NO: 1.
  • a variant sequence is preferably at least about 84% or at least about 86.2% homologous to that sequence based on nucleotide identity. Over the entire length of the sequence of SEQ ID NO: 1, a variant sequence preferably has at least about 84% or at least about 86.2% identity to that sequence. This allows for variation, deletion, addition or a combination thereof of four nucleotides within the sequence of SEQ ID NO: 1. Over the entire length of the sequence of SEQ ID NO: 1, a variant sequence is preferably at least about 88% or at least about 89.2% homologous to that sequence based on nucleotide identity.
  • a variant sequence preferably has at least about 88% or at least about 89.2% identity to that sequence. This allows for variation, deletion, addition or a combination thereof of three nucleotides within the sequence of SEQ ID NO: 1.
  • a variant sequence is preferably at least about 92% or at least about 92.5% homologous to that sequence based on nucleotide identity. Over the entire length of the sequence of SEQ ID NO: 1, a variant sequence preferably has at least about 92% or at least about 92.5% identity to that sequence. This allows for variation, deletion, addition or a combination thereof of two nucleotides within the sequence of SEQ ID NO: 1.
  • a variant sequence is preferably at least about 96% or at least about 96.1% homologous to that sequence based on nucleotide identity. Over the entire length of the sequence of SEQ ID NO: 1, a variant sequence preferably has at least about 96% or at least about 96.1% identity to that sequence. This allows for variation, deletion, addition or a combination thereof of one nucleotide within the sequence of SEQ ID NO: 1.
  • a variant sequence is preferably at least about 79%, at least about 79.16%, at least about 79.17% or at least about 82.7% homologous to that sequence based on nucleotide identity.
  • a variant sequence preferably has at least about 79%, at least about 79.16%, at least about 79.17% or at least about 82.7% identity to that sequence. This allows for variation, deletion, addition or a combination thereof of five nucleotides within the sequence of SEQ ID NO: 2.
  • a variant sequence is preferably at least about 80%, at least about 83.3% or at least about 85.7% homologous to that sequence based on nucleotide identity. Over the entire length of the sequence of SEQ ID NO: 2, a variant sequence preferably has at least about 80%, at least about 83.3% or at least about 85.7% identity to that sequence. This allows for variation, deletion, addition or a combination thereof of four nucleotides within the sequence of SEQ ID NO: 2.
  • a variant sequence is preferably at least about 85%, at least about 87.5% or at least about 88.8% homologous to that sequence based on nucleotide identity. Over the entire length of the sequence of SEQ ID NO: 2, a variant sequence preferably has at least about 85%, at least about 87.5% or at least about 88.8% identity to that sequence. This allows for variation, deletion, addition or a combination thereof of three nucleotides within the sequence of SEQ ID NO: 2.
  • a variant sequence is preferably at least about 90%, at least about 91.66%, at least about 91.67% or at least about 92.3% homologous to that sequence based on nucleotide identity. Over the entire length of the sequence of SEQ ID NO: 2, a variant sequence preferably has at least about 90%, at least about 91.66%, at least about 91.67% or at least about 92.3% identity to that sequence. This allows for variation, deletion, addition or a combination thereof of two nucleotides within the sequence of SEQ ID NO: 2.
  • a variant sequence is preferably at least about 95%, at least about 95.8% or at least about 96% homologous to that sequence based on nucleotide identity. Over the entire length of the sequence of SEQ ID NO: 2, a variant sequence preferably has at least about 95%, at least about 95.8% or at least about 96% identity to that sequence. This allows for variation, deletion, addition or a combination thereof of one nucleotide within the sequence of SEQ ID NO: 2.
  • a variant sequence is preferably at least about 77%, at least about 77.27%, at least about 81% or at least about 81.48% homologous to that sequence based on nucleotide identity. Over the entire length of the sequence of SEQ ID NO: 6 or 7 , a variant sequence preferably has at least about 77%, at least about 77.27%, at least about 81% or at least about 81.48% identity to that sequence. This allows for variation, deletion, addition or a combination thereof of five nucleotides within the sequence of SEQ ID NO: 6 or 7.
  • a variant sequence is preferably at least about 81%, at least about 81.18%, at least about 84% or at least about 84.61% homologous to that sequence based on nucleotide identity. Over the entire length of the sequence of SEQ ID NO: 6 or 7, a variant sequence preferably has at least about 81%, at least about 81.18%, at least about 84% or at least about 84.61% identity to that sequence. This allows for variation, deletion, addition or a combination thereof of four nucleotides within the sequence of SEQ ID NO: 6 or 7.
  • a variant sequence is preferably at least about 86%, at least about 86.36, at least about 95% or at least about 95.6% homologous to that sequence based on nucleotide identity. Over the entire length of the sequence of SEQ ID NO: 6 or 7, a variant sequence preferably has at least about 86%, at least about 86.36, at least about 95% or at least about 95.6% identity to that sequence. This allows for variation, deletion, addition or a combination thereof of three nucleotides within the sequence of SEQ ID NO: 6 or 7.
  • a variant sequence is preferably at least about 90%, at least about 90.90%, at least about 91% or at least about 91.67% homologous to that sequence based on nucleotide identity. Over the entire length of the sequence of SEQ ID NO: 6 or 7, a variant sequence preferably has at least about 90%, at least about 90.90%, at least about 91% or at least about 91.67% identity to that sequence. This allows for variation, deletion, addition or a combination thereof of two nucleotides within the sequence of SEQ ID NO: 6 or 7.
  • a variant sequence is preferably at least about 95%, at least about 95.45% or at least about 95.65% homologous to that sequence based on nucleotide identity. Over the entire length of the sequence of SEQ ID NO: 6 or 7, a variant sequence preferably has at least about 95%, at least about 95.45% or at least about 95.65% identity to that sequence. This allows for variation, deletion, addition or a combination thereof of one nucleotide within the sequence of SEQ ID NO: 6 or 7.
  • a variant sequence is at least about 75% or at least about 80% homologous to that sequence based on nucleotide identity. Over the entire length of the sequence of SEQ ID NO: 11, a variant sequence has at least about 75% or at least about 80% identity to that sequence. Homology based on sequence identity and identity are interchangeable herein. This allows for variation, deletion, addition or a combination thereof of five nucleotides within the sequence of SEQ ID NO: 11.
  • a variant sequence is preferably at least about 80%, at least about 83% or at least about 83.33% homologous to that sequence based on nucleotide identity. Over the entire length of the sequence of SEQ ID NO: 11, a variant sequence preferably has at least about 80%, at least about 83% or at least about 83.33% identity to that sequence. This allows for variation, deletion, addition or a combination thereof of four nucleotides within the sequence of SEQ ID NO: 11.
  • a variant sequence is preferably at least about 85%, at least about 86% or at least about 86.95% homologous to that sequence based on nucleotide identity. Over the entire length of the sequence of SEQ ID NO: 11, a variant sequence preferably has at least about 85%, at least about 86% or at least about 86.95% identity to that sequence. This allows for variation, deletion, addition or a combination thereof of three nucleotides within the sequence of SEQ ID NO: 11.
  • a variant sequence is preferably at least about 90% or at least about 90.90% homologous to that sequence based on nucleotide identity. Over the entire length of the sequence of SEQ ID NO: 11, a variant sequence preferably has at least about 90% or at least about 90.90% identity to that sequence. This allows for variation, deletion, addition or a combination thereof of two nucleotides within the sequence of SEQ ID NO: 11.
  • a variant sequence is preferably at least about 95% or at least about 95.23% homologous to that sequence based on nucleotide identity. Over the entire length of the sequence of SEQ ID NO: 11, a variant sequence preferably has at least about 95% or at least about 95.23% identity to that sequence. This allows for variation, deletion, addition or a combination thereof of one nucleotide within the sequence of SEQ ID NO: 11.
  • a variant sequence is preferably at least about 76%, at least about 79.19%, at least about 80% or at least about 80.76% homologous to that sequence based on nucleotide identity. Over the entire length of the sequence of SEQ ID NO: 12, a variant sequence preferably has at least about 76%, at least about 79.19%, at least about 80% or at least about 80.76% identity to that sequence. This allows for variation, deletion, addition or a combination thereof of five nucleotides within the sequence of SEQ ID NO: 12.
  • a variant sequence is preferably at least about 80%, at least about 80.95% or at least about 84% homologous to that sequence based on nucleotide identity. Over the entire length of the sequence of SEQ ID NO: 12, a variant sequence preferably has at least about 80%, at least about 80.95% or at least about 84% identity to that sequence. This allows for variation, deletion, addition or a combination thereof of four nucleotides within the sequence of SEQ ID NO: 12.
  • a variant sequence is preferably at least about 85%, at least about 85.71%, at least about 87% or at least about 87.5% homologous to that sequence based on nucleotide identity. Over the entire length of the sequence of SEQ ID NO: 12, a variant sequence preferably has at least about 85%, at least about 85.71%, at least about 87% or at least about 87.5% identity to that sequence. This allows for variation, deletion, addition or a combination thereof of three nucleotides within the sequence of SEQ ID NO: 12.
  • a variant sequence is preferably at least about 90%, at least about 90.47%, at least about 91% or at least about 91.30% homologous to that sequence based on nucleotide identity. Over the entire length of the sequence of SEQ ID NO: 12, a variant sequence preferably has at least about 90%, at least about 90.47%, at least about 91% or at least about 91.30% identity to that sequence.
  • a variant sequence is preferably at least about 95%, at least about 95.23% or at least about 95.45% homologous to that sequence based on nucleotide identity.
  • a variant sequence preferably has at least about 95%, at least about 95.23% or at least about 95.45% identity to that sequence. This allows for variation, deletion, addition or a combination thereof of one nucleotide within the sequence of SEQ ID NO: 12.
  • homology based on sequence identity or sequence identity is measured over the entire length of the nonvariant/ reference sequence (e.g. SEQ ID NO: 1 or 2). This may also be referred to as global homology based on sequence identity or global sequence identity.
  • the PILEUP and BLAST algorithms can also be used to calculate identity, homology or line up sequences (typically on their default settings), for example as described in Altschul S.F. (1993) J Mol Evol 36:290-300; Altschul, S, F et al (1990) J Mol Biol 215:403-10.
  • HSPs high scoring sequence pair
  • Extensions for the word hits in each direction are halted when: the cumulative alignment score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached.
  • the BLAST algorithm parameters W, T and X determine the sensitivity and speed of the alignment.
  • the BLAST algorithm performs a statistical analysis of the similarity between two sequences; see e.g., Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5787.
  • One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance.
  • P(N) the smallest sum probability
  • a sequence is considered similar to another sequence if the smallest sum probability in comparison of the first sequence to the second sequence is less than about 1, preferably less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001.
  • a variant is typically identical to SEQ ID NO: 1 over at least about 20, at least about 21, at least about 22, at least about 23, at least about 24 or at least about 25 consecutive nucleotides.
  • a variant is typically identical to SEQ ID NO: 2 over at least about 19, at least about 20, at least about 21, at least about 22, at least about 23 or at least about 24 consecutive nucleotides.
  • a variant is typically identical to SEQ ID NO: 6 or 7 over at least about 17, at least about 18, at least about 19, at least about 20, at least about 21 or at least about 22 consecutive nucleotides.
  • a variant is typically identical to SEQ ID NO: 11 over at least about 15, at least about 16, at least about 17, at least about 18, at least about 19 or at least about 20 consecutive nucleotides.
  • a variant is typically identical to SEQ ID NO: 12 over at least about 16, at least about 17, at least about 18, at least about 19, at least about 20 or at least about 21 consecutive nucleotides.
  • the first forward primer (ORFlab gene) is preferably at least 20 nucleotides in length, such as 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides in length.
  • the first reverse primer (ORFlab gene) is preferably at least 19 nucleotides in length, such as 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 or 29 nucleotides in length.
  • the second forward and reverse primers (S gene) are preferably at least 17 nucleotides in length, such as 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 or 27 nucleotides in length.
  • the third forward primer (M gene) is preferably at least 15 nucleotides in length, such as 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides in length.
  • the third reverse primers (M gene) is preferably at least 16 nucleotides in length, such as 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or 26 nucleotides in length.
  • the forward primer preferably comprises or consists of the exact sequence shown in SEQ ID NO: 1.
  • the reverse primer preferably comprises or consists of the exact sequence shown in SEQ ID NO: 2.
  • the forward primer preferably is the exact sequence shown in SEQ ID NO: 1.
  • the reverse primer preferably is the exact sequence shown in SEQ ID NO: 2.
  • the forward primer preferably is DNA and comprises/consist of the exact sequence shown in SEQ ID NO: 1.
  • the reverse primer preferably is DNA and comprises/consists of the exact sequence shown in SEQ ID NO: 2.
  • the first pair of primers may comprise any combination of primers discussed above.
  • the first pair of primers may comprise a forward primer that comprises/consist of the exact sequence shown in SEQ ID NO: 1 and a reverse primer that comprises/consists of a variant of SEQ ID NO: 2 as discussed above.
  • the first pair of primers may comprise a forward primer that comprises/consists of a variant of SEQ ID NO: 1 as discussed above and a reverse primer that comprises/consists of the exact sequence of SEQ ID NO: 2.
  • the first pair of primers may comprise a forward primer that comprises/consists a variant of SEQ ID NO: 1 as discussed above and a reverse primer that comprises/consists of a variant of SEQ ID NO: 2 as discussed above.
  • the first pair of primers more preferably comprises a forward primer which comprises or consists of the exact sequence shown in SEQ ID NO: 1 and a reverse primer which comprises or consists of the exact sequence shown in SEQ ID NO: 2.
  • the first pair of primers more preferably comprises a forward primer that is the exact sequence shown in SEQ ID NO: 1 and a reverse primer that is the exact sequence shown in SEQ ID NO: 2.
  • the first pair of primers most preferably comprises a forward primer that is DNA and comprises/ or consists of the exact sequence shown in SEQ ID NO: 1 and a reverse primer that is preferably DNA and comprises/ or consists of the exact sequence shown in SEQ ID NO: 2.
  • the first primer preferably comprises or consists of the exact sequence shown in SEQ ID NO: 6.
  • the reverse primer preferably comprises or consists of the exact sequence shown in SEQ ID NO: 7.
  • the forward primer preferably is the exact sequence shown in SEQ ID NO: 6.
  • the reverse primer preferably is the exact sequence shown in SEQ ID NO: 7.
  • the forward primer preferably is DNA and comprises/consist of the exact sequence shown in SEQ ID NO: 6.
  • the reverse primer preferably is DNA and comprises/consists of the exact sequence shown in SEQ ID NO: 7.
  • the second pair of primers may comprise any combination of primers discussed above.
  • the second pair of primers may comprise a forward primer that comprises/consist of the exact sequence shown in SEQ ID NO: 6 and a reverse primer that comprises/consists of a variant of SEQ ID NO: 7 as discussed above.
  • the second pair of primers may comprise a forward primer that comprises/consists of a variant of SEQ ID NO: 6 as discussed above and a reverse primer that comprises/consists of the exact sequence of SEQ ID NO: 7.
  • the second pair of primers may comprise a forward primer that comprises/consists a variant of SEQ ID NO: 6 as discussed above and a reverse primer that comprises/consists of a variant of SEQ ID NO: 7 as discussed above.
  • the second pair of primers more preferably comprises a forward primer which comprises or consists of the exact sequence shown in SEQ ID NO: 6 and a reverse primer which comprises or consists of the exact sequence shown in SEQ ID NO: 7.
  • the second pair of primers more preferably comprises a forward primer that is the exact sequence shown in SEQ ID NO: 6 and a reverse primer that is the exact sequence shown in SEQ ID NO: 7.
  • the second pair of primers most preferably comprises a forward primer that is DNA and comprises/ or consists of the exact sequence shown in SEQ ID NO: 6 and a reverse primer that is preferably DNA and comprises/ or consists of the exact sequence shown in SEQ ID NO: 7.
  • the first primer preferably comprises or consists of the exact sequence shown in SEQ ID NO: 11.
  • the reverse primer preferably comprises or consists of the exact sequence shown in SEQ ID NO: 12.
  • the forward primer preferably is the exact sequence shown in SEQ ID NO: 11.
  • the reverse primer preferably is the exact sequence shown in SEQ ID NO: 12.
  • the forward primer preferably is DNA and comprises/consist of the exact sequence shown in SEQ ID NO: 11.
  • the reverse primer preferably is DNA and comprises/consists of the exact sequence shown in SEQ ID NO: 12.
  • the third pair of primers may comprise any combination of primers discussed above.
  • the third pair of primers may comprise a forward primer that comprises/consist of the exact sequence shown in SEQ ID NO: 11 and a reverse primer that comprises/consists of a variant of SEQ ID NO: 12 as discussed above.
  • the third pair of primers may comprise a forward primer that comprises/consists of a variant of SEQ ID NO: 11 as discussed above and a reverse primer that comprises/consists of the exact sequence of SEQ ID NO: 12.
  • the third pair of primers may comprise a forward primer that comprises/consists a variant of SEQ ID NO: 11 as discussed above and a reverse primer that comprises/consists of a variant of SEQ ID NO: 12 as discussed above.
  • the third pair of primers more preferably comprises a forward primer which comprises or consists of the exact sequence shown in SEQ ID NO: 11 and a reverse primer which comprises or consists of the exact sequence shown in SEQ ID NO: 12.
  • the third pair of primers more preferably comprises a forward primer that is the exact sequence shown in SEQ ID NO: 11 and a reverse primer that is the exact sequence shown in SEQ ID NO: 12.
  • the third pair of primers most preferably comprises a forward primer that is DNA and comprises/ or consists of the exact sequence shown in SEQ ID NO: 11 and a reverse primer that is preferably DNA and comprises/ or consists of the exact sequence shown in SEQ ID NO: 12.
  • Primers having any of the sequences above may be manufactured using standard techniques. Custom primers having specific sequences are commercially available from various suppliers (such as Biolegio). cDNA amplicons of the invention
  • the invention also provides three complementary DNA (cDNA) amplicons amplified from SARS-CoV-2 cDNA by the three pairs of primers of the invention.
  • the invention also provides first, second and third cDNA amplicons amplified from SARS- CoV-2 cDNA.
  • the first cDNA amplicon comprises or consists of a sequence (a) to which the sequence shown in SEQ ID NO: 3 specifically hybridises and/or (b) which is complementary to the sequence shown in SEQ ID NO: 3. Specific hybridisation is defined above with reference to the primers of the invention.
  • the first cDNA amplicon is preferably amplified from SARS-CoV-2 cDNA and comprises or consists of a sequence which is complementary to the sequence shown in SEQ ID NO: 3.
  • the second cDNA amplicon comprises or consists of a sequence (a) to which the sequence shown in SEQ ID NO: 8 specifically hybridises and/or (b) which is complementary to the sequence shown in SEQ ID NO: 8. Specific hybridisation is defined above with reference to the primers of the invention.
  • the second cDNA amplicon is preferably amplified from SARS- CoV-2 cDNA and comprises or consists of a sequence which is complementary to the sequence shown in SEQ ID NO: 8.
  • the third cDNA amplicon comprises or consists of a sequence (a) to which the sequence shown in SEQ ID NO: 13 specifically hybridises and/or (b) which is complementary to the sequence shown in SEQ ID NO: 13. Specific hybridisation is defined above with reference to the primers of the invention.
  • the second cDNA amplicon is preferably amplified from SARS- CoV-2 cDNA and comprises or consists of a sequence which is complementary to the sequence shown in SEQ ID NO: 13.
  • SARS-CoV-2 cDNA is cDNA produced by the reverse transcription of SARS-CoV-2 RNA.
  • the sequence of SARS-CoV-2 cDNA is published and methods for reverse transcribing it are known in the art and discussed in the Example.
  • the pairs of primers of the invention may be used to amplify the cDNA amplicons of the invention using polymerase chain reaction (PCR). Methods for conducting PCR using primers are known in the art and discussed below and in the Example.
  • a cDNA amplicon is amplified from SARS-CoV-2 cDNA if it comprises a portion/part of the SARS- CoV-2 cDNA. In other words, it comprises a portion/part of the sequence of the SARS-CoV- 2 cDNA.
  • amplified refers to the process of making multiple copies of the cDNA from a single polynucleotide or fewer polynucleotides.
  • An amplicon is the cDNA that results from amplification.
  • primers such as the primers of the invention, may be used to amplify a part of the SARS-CoV-2 cDNA resulting in the shorter cDNA amplicons of the invention.
  • These amplicons typically contain the cDNA target sequence being used in the context of the invention.
  • the cDNA amplicon is typically a polynucleotide comprising nucleotides selected from dAMP, dTMP, dGMP or dCMP as discussed above.
  • the cDNA may be double stranded or single stranded.
  • the cDNA is preferably single stranded.
  • the first cDNA amplicon may be any length, such as from about 20 to about 150 nucleotides in the length, such as from about 25 to about 100, from about 30 to about 95 or from about 40 to about 90 nucleotides in length.
  • the first cDNA amplicon is preferably 82 nucleotides in length.
  • the second cDNA amplicon may be any length, such as from about 20 to about 200 nucleotides in the length, such as from about 25 to about 180, from about 30 to about 150 or from about 40 to about 120 nucleotides in length.
  • the second cDNA amplicon is preferably 119 nucleotides in length.
  • the third cDNA amplicon may be any length, such as from about 20 to about 150 nucleotides in the length, such as from about 25 to about 100, from about 30 to about 95 or from about 40 to about 90 nucleotides in length.
  • the third cDNA amplicon is preferably 89 nucleotides in length.
  • the first cDNA amplicon preferably comprises or consists of the sequence shown in SEQ ID NO: 4.
  • the first cDNA amplicon preferably comprises or consists of the sequence shown in SEQ ID NO: 5.
  • the second cDNA amplicon preferably comprises or consists of the sequence shown in SEQ ID NO: 9.
  • the second cDNA amplicon preferably comprises or consists of the sequence shown in SEQ ID NO: 10.
  • the third cDNA amplicon preferably comprises or consists of the sequence shown in SEQ ID NO: 14.
  • the third cDNA amplicon preferably comprises or consists of the sequence shown in SEQ ID NO: 15.
  • the cDNA amplicons may be isolated, substantially isolated, purified or substantially purified as discussed above.
  • the cDNA amplicons are not naturally occurring.
  • the invention also provides three polynucleotide probes for determining the presence or absence of SARS-CoV-2 in a sample.
  • the three polynucleotide probes specifically hybridise to the three cDNA amplicons of the invention.
  • Each probe specifically hybridises to one of the cDNA amplicons of the invention.
  • Each polynucleotide probe comprises or consists of a sequence which specifically hybridises to the corresponding cDNA amplicon of the invention.
  • Each polynucleotide probe preferably comprises or consists of a sequence which specifically hybridises to a target sequence on the corresponding cDNA amplicon of the invention.
  • the target sequence may be any length, such as from 17 to 32 nucleotides in length, such as 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or 32 nucleotides in length.
  • Each probe polynucleotide may be any of the polynucleotides discussed above.
  • Each polynucleotide probe is preferably DNA.
  • Each polynucleotide probe is preferably single stranded DNA.
  • Each polynucleotide probe may be any length, such as from 17 to 32 nucleotides in length, such as 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or 32 nucleotides in length.
  • Each polynucleotide probe is preferably the same length as the target sequence it detects.
  • Each target sequence is preferably specific for SARS- CoV-2.
  • Each target sequence is preferably found/present in SARS-Cov-2 cDNA but is not found in the RNA of any other virus or human virus.
  • Each target sequence is preferably found/present in SARS-Cov-2 cDNA but is not found in the RNA of any other virus or human virus or in any human RNA.
  • Each target sequence is preferably found/present in a cDNA amplicon of the invention.
  • the ORFlab gene target sequence is preferably part of SEQ ID NO: 4.
  • the ORFlab gene target sequence is preferably the sequence shown in SEQ ID NO: 5.
  • the S gene target sequence is preferably part of SEQ ID NO: 9.
  • the S gene target sequence is preferably the sequence shown in SEQ ID NO: 10.
  • the M gene target sequence is preferably part of SEQ ID NO: 14.
  • the M gene target sequence is preferably the sequence shown in SEQ ID NO: 15.
  • the three polynucleotide probes comprise or consist of specific sequences of the invention.
  • the first (ORFlab gene targeting) polynucleotide probe comprises or consists of the sequence shown in SEQ ID NO: 3 or a variant thereof having at least about 80% homology to SEQ ID NO: 3 based on sequence identity over its entire length.
  • the second (S gene targeting) polynucleotide probe comprises or consists of the sequence shown in SEQ ID NO: 8 or a variant thereof having at least about 80% homology to SEQ ID NO: 8 based on sequence identity over its entire length.
  • the third (M gene targeting) polynucleotide probe comprises or consists of the sequence shown in SEQ ID NO: 13 or a variant thereof having at least about 80% homology to SEQ ID NO: 13 based on sequence identity over its entire length.
  • Polynucleotides are defined above with reference to the primers of the invention.
  • Each probe polynucleotide may be any of the polynucleotides discussed above.
  • Each polynucleotide probe is preferably DNA.
  • Each polynucleotide probe is preferably single stranded DNA.
  • Each polynucleotide probe may be any length.
  • the first polynucleotide probe is preferably from 20 to 30 nucleotides in length, such as 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides in length.
  • the second polynucleotide probe is preferably from 22 to 32 nucleotides in length, such as 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 or 32 nucleotides in length.
  • the third polynucleotide probe is preferably from 20 to 30 nucleotides in length, such as 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides in length.
  • a variant sequence is a polynucleotide that has a nucleotide sequence which varies from that of SEQ ID NO: 3, 8 or 13 and which retains its ability to specifically hybridise to the target sequence of SEQ ID NO: 5, 10 or 15. Specific hybridisation is discussed above. The variant sequence must not recognise or hybridise to any cDNA sequence from any other virus.
  • the variant sequence may comprise any of the nucleotides discussed above, including the modified nucleotides.
  • the variant sequence is typically the same length as SEQ ID NO: 3, 8 or 13, but may be longer or shorter.
  • a variant of SEQ ID NO: 3 is preferably at least 20 nucleotides in length, such as 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides in length.
  • a variant of SEQ ID NO: 8 is preferably at least 22 nucleotides in length, such as 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 or 32 nucleotides in length.
  • a variant of SEQ ID NO: 13 is preferably at least 20 nucleotides in length, such as 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides in length.
  • a variant sequence is at least about 80%, at least about 83% or at least about 83.33% homologous to that sequence based on nucleotide identity. Over the entire length of the sequence of SEQ ID NO: 3 or 13, a variant sequence has at least about 80%, at least about 83% or at least about 83.33% identity to that sequence. Homology based on sequence identity and identity are interchangeable herein. This allows for variation, deletion, addition or a combination thereof of five nucleotides within the sequence of SEQ ID NO: 3 or 13.
  • a variant sequence is preferably at least about 84%, at least 86% or at least about 86.20% homologous to that sequence based on nucleotide identity. Over the entire length of the sequence of SEQ ID NO: 3 or 13, a variant sequence preferably has at least about 84%, at least 86% or at least about 86.20% identity to that sequence. This allows for variation, deletion, addition or a combination thereof of four nucleotides within the sequence of SEQ ID NO: 3 or 13.
  • a variant sequence is preferably at least about 88%, at least 89% or at least about 89.28% homologous to that sequence based on nucleotide identity. Over the entire length of the sequence of SEQ ID NO: 3 or 13, a variant sequence preferably has at least about 88%, at least 89% or at least about 89.28% identity to that sequence. This allows for variation, deletion, addition or a combination thereof of three nucleotides within the sequence of SEQ ID NO: 3 or 13.
  • a variant sequence is preferably at least about 92% or at least about 92.59% homologous to that sequence based on nucleotide identity. Over the entire length of the sequence of SEQ ID NO: 3 or 13, a variant sequence preferably has at least about 92% or at least about 92.59% identity to that sequence. This allows for variation, deletion, addition or a combination thereof of two nucleotides within the sequence of SEQ ID NO: 3 or 13. Over the entire length of the sequence of SEQ ID NO: 3 or 13, a variant sequence is preferably at least about 96% or at least about 96.15% homologous to that sequence based on nucleotide identity.
  • a variant sequence preferably has at least about 96% or at least about 96.15% identity to that sequence. This allows for variation, deletion, addition or a combination thereof of one nucleotide within the sequence of SEQ ID NO: 3 or 13.
  • a variant sequence is at least about 80%, at least about 81.48%, at least about 84% or at least about 84.37% homologous to that sequence based on nucleotide identity. Over the entire length of the sequence of SEQ ID NO: 8, a variant sequence has at least about 80%, at least about 81.48%, at least about 84% or at least about 84.37% identity to that sequence. Homology based on sequence identity and identity are interchangeable herein. This allows for variation, deletion, addition or a combination thereof of five nucleotides within the sequence of SEQ ID NO: 8.
  • a variant sequence is preferably at least about 85%, at least about 85.18%, at least about 87% or at least about 87.09% homologous to that sequence based on nucleotide identity. Over the entire length of the sequence of SEQ ID NO: 8, a variant sequence preferably has at least about 85%, at least about 85.18%, at least about 87% or at least about 87.09% identity to that sequence. This allows for variation, deletion, addition or a combination thereof of four nucleotides within the sequence of SEQ ID NO: 8.
  • a variant sequence is preferably at least about 88%, at least about 88.88% or at least about 90% homologous to that sequence based on nucleotide identity. Over the entire length of the sequence of SEQ ID NO: 8, a variant sequence preferably has at least about 88%, at least about 88.88% or at least about 90% identity to that sequence. This allows for variation, deletion, addition or a combination thereof of three nucleotides within the sequence of SEQ ID NO: 8.
  • a variant sequence is preferably at least about 92%, at least about 92.59%, at least about 93% or at least about 93.10% homologous to that sequence based on nucleotide identity. Over the entire length of the sequence of SEQ ID NO: 8, a variant sequence preferably has at least about 92%, at least about 92.59%, at least about 93% or at least about 93.10% identity to that sequence. This allows for variation, deletion, addition or a combination thereof of two nucleotides within the sequence of SEQ ID NO: 8.
  • a variant sequence is preferably at least about 96%, at least about 96.29% or at least about 96.42% homologous to that sequence based on nucleotide identity. Over the entire length of the sequence of SEQ ID NO: 8, a variant sequence preferably has at least about 96%, at least about 96.29% or at least about 96.42% identity to that sequence. This allows for variation, deletion, addition or a combination thereof of one nucleotide within the sequence of SEQ ID NO: 8.
  • the first polynucleotide probe preferably comprises or consists of the sequence shown in SEQ ID NO: 3.
  • the second polynucleotide probe preferably comprises or consists of the sequence shown in SEQ ID NO: 8.
  • the third polynucleotide probe preferably comprises or consists of the sequence shown in SEQ ID NO: 13.
  • Each polynucleotide probe may be isolated, substantially isolated, purified or substantially purified as discussed above. Each polynucleotide probe is not naturally occurring.
  • Each polynucleotide probe is preferably DNA probe, a TaqMan probe, a molecular beacon or a scorpion probe.
  • DNA probes hybridise to the, typically complementary, target sequence and then can be detected.
  • TaqMan probes are known in the art and are polynucleotides that have a fluorescent dye attached to the 5' end and a quencher to the 3' end. The polymerase used in PCR cleaves hybridised probes freeing the fluorescent dye from quenching such it can be detected.
  • Molecular beacon probes are known in the art and are similar to TaqMan probes except (rather than using cleavage to separate the dye from the quencher) hybridisation to the target sequence separates the dye from the quencher.
  • Scorpion probes are known the art and are similar to molecular beacons except the 3' end also contains a sequence that is complementary to the extension product of the primer on the 5' end which opens the probe on hybridisation and allows the dye to be detected.
  • Each polynucleotide probe is preferably a TaqMan probe.
  • the polynucleotide probes of the invention are preferably detectably-labelled.
  • Suitable detectable labels are known in the art.
  • the detectable labels are preferably fluorescent molecules or dyes, such as fluorescein derivatives.
  • Suitable fluorescent molecules or dyes include, but are not limited to, 6-carboxyfluorescein (FAM), 6-Carboxyl-X-Rhodamine (ROX), 2'-chloro-7'phenyl-l,4-dichloro-6-carboxy-fluorescein (VIC®), Hexachloro- Fluorescein (HEX), tetrachlorofluorescein (TET) and ATTO 647N (ATTO647N in the Cy5 spectral range (ATTO-TEC GmbH, Germany)).
  • a suitable quencher for use with these dyes in TaqMan, molecular beacon or scorpion probes is tetramethylrhodamine (TAMRA).
  • the detectable label is most preferably 6-carboxyfluorescein (FAM), ATTO 647N or 6- Carboxyl-X-Rhodamine (ROX).
  • FAM 6-carboxyfluorescein
  • ROX 6- Carboxyl-X-Rhodamine
  • the first probe is preferably labelled with FAM.
  • the second probe is preferably labelled with ATTO 647N.
  • the third probe is preferably labelled with ROX.
  • the first probe is preferably labelled with FAM
  • the second probe is preferably labelled with ATT and the third probe is preferably labelled with ROX.
  • Polynucleotide probes are also available from commercial sources (such as Biolegio). Target cDNA sequence
  • the invention also provides three target cDNA polynucleotides.
  • the first (derived from the ORFlab gene) comprises or consists of a sequence (a) to which the sequence shown in SEQ ID NO: 3 specifically hybridises and/or (b) which is complementary to the sequence shown in SEQ ID NO: 3.
  • the second (derived from the S gene) comprises or consists of a sequence (a) to which the sequence shown in SEQ ID NO: 8 specifically hybridises and/or (b) which is complementary to the sequence shown in SEQ ID NO: 8.
  • the third (derived from the M gene) comprises or consists of a sequence (a) to which the sequence shown in SEQ ID NO: 13 specifically hybridises and/or (b) which is complementary to the sequence shown in SEQ ID NO: 13.
  • the target cDNA may be any length.
  • the first is typically 25 nucleotides in length but may be any of the lengths discussed above for the first probe.
  • the second is typically 27 nucleotides in length but may be any of the lengths discussed above for the second probe.
  • the third is typically 25 nucleotides in length but may be any of the lengths discussed above for the third probe.
  • the first target cDNA polynucleotide preferably comprises or consists of the sequence shown in SEQ ID NO: 5.
  • the second target cDNA polynucleotide preferably comprises or consists of the sequence shown in SEQ ID NO: 10.
  • the third target cDNA polynucleotide preferably comprises or consists of the sequence shown in SEQ ID NO: 15.
  • Each target cDNA polynucleotide may be double stranded or single stranded.
  • Each target cDNA polynucleotide is preferably single stranded.
  • Each target cDNA polynucleotide may be isolated, substantially isolated, purified or substantially purified as discussed above. Each target cDNA polynucleotide is not naturally occurring.
  • the invention also provides a kit for determining the presence or absence of SARS-CoV-2 in a sample.
  • the kit comprises the three pairs of primers of the invention.
  • the kit also comprises the three polynucleotide probes of the invention. Any of the primers and probes discussed above may be used.
  • the primers and probes may be isolated, substantially isolated, purified or substantially purified as discussed above.
  • the kit preferably further comprises three positive control polynucleotides comprising or consisting of three cDNA amplicons of the invention and/or three target cDNA polynucleotides of the invention. These may form the basis of a positive control for RT-PCR. Any of the cDNA amplicons and cDNA target sequences discussed above may be used.
  • the kit preferably comprises a polynucleotide comprising or consisting of the sequence shown in SEQ ID NO: 4 or 5, a polynucleotide comprising or consisting of the sequence shown in SEQ ID NO: 9 or 10 and a polynucleotide comprising or consisting of the sequence shown in SEQ ID NO: 14 or 15.
  • the cDNA polynucleotides may be isolated, substantially isolated, purified or substantially purified as discussed above.
  • the kit preferably contains a negative control, such as DNAase-free and RNAase-free water.
  • the kit of the invention preferably further comprises a RNA Internal Extraction Control (IEC).
  • the kit more preferably comprises a set of RNA IEC primers and probe.
  • the RNA IEC primers and probe are preferably DNA. DNA primers and probes are discussed above.
  • the set of RNA IEC primers and probe preferably comprises:
  • RNA IEC L.lactis Forward primer SEQ ID NO: 16
  • RNA IEC L.lactis Reverse primer SEQ ID NO: 17
  • RNA IEC L.lactis Probe 1 SEQ ID NO: 18
  • RNA IEC L.lactis Probe 2 (SEQ ID NO: 19).
  • the kit most preferably comprises the following components in the specified concentrations.
  • the various components may be resuspended to generate the concentrations discussed below with reference to the methods of the invention.
  • the kit may additionally comprise one or more other reagents or instruments which enable any of the embodiments of the methods below to be carried out.
  • reagents or instruments include, but are not limited to, one or more of the following: suitable buffer(s) (aqueous solutions), means to obtain a sample from a patient (such as a vessel or an instrument comprising a needle or a swab) or tubes in which quantitative reactions can be done.
  • suitable buffer(s) aqueous solutions
  • means to obtain a sample from a patient such as a vessel or an instrument comprising a needle or a swab
  • the kit may, optionally, comprise instructions to enable the kit to be used in the methods of the invention or details regarding which patients may be tested.
  • the kit may further comprise any of the reagents required to conduct RT-PCR discussed below, including a reverse transcriptase and/or heat-stable DNA polymerase.
  • the invention provides various methods of determining the presence or absence of SARS- CoV-2 in a sample.
  • the methods are triplex methods targeting three genes in SARS-CoV-2, namely the ORFlab gene, the S gene and the M gene.
  • RT-PCR reverse-transcription polymerase chain reaction
  • Methods for conducting RT-PCR are well known in the art and any suitable conditions may be used.
  • Reverse transcription involves the use of the enzyme reverse transcriptase to convert viral RNA into cDNA.
  • PCR involves using the three pairs of primers to amplify three specific portions of the cDNA, namely the cDNA amplicons, which typically contains the target cDNAs of interest.
  • reverse transcriptases are commercially available (e.g. Superscript® II reverse transcriptase (Invitrogen) and Affinity script (Agilent)). Methods typically construct cDNA from random cDNA hexamers included in the reaction mixture.
  • PCR involves amplifying a cDNA amplicon using a heat-stable DNA polymerase and three pairs of forward and reverse polynucleotide primers. Because the newly synthesized cDNA strands can subsequently serve as additional templates for the same primer sequences, successive rounds of primer annealing, strand elongation, and dissociation produce rapid and highly specific amplification of the desired sequence.
  • Many polymerase chain methods are known to those of skill in the art and may be used in the methods of the invention.
  • cDNA can be subjected to 20 to 40 cycles of amplification in a thermocycler as follows: 95°C for 30 sec, 52° to 60°C for 1 min, and 72°C for 1 min, with a final extension step of 72°C for 5 min.
  • cDNA can be subjected to 20 to 40 polymerase chain reaction cycles in a thermocycler at a denaturing temperature of 95°C for 30 sec, followed by varying annealing temperatures ranging from 54°C to 58°C for 1 min, an extension step at 70°C for 1 min, with a final extension step at 70°C for 5 min.
  • Heat stable DNA polymerases are commercially available (such as GoTaq G2 (Promega)).
  • the RT-PCR assay may be conducted using the conditions set out in the Example.
  • the reverse transcriptase is preferably Affinity script (Agilent).
  • the DNA polymerase is preferably GoTaq G2 (Promega). Both enzymes are preferably present in OneStep Lyophilised Master Mix.
  • the reaction conditions are preferably (1) one cycle of reverse transcription for 10 minutes at 55°C, (2) one cycle of initial denaturation and Taq activation for 2 minutes at 95°C and (3) 45 cycles of denaturation for 10 seconds at 95°C and annealing and extension for 60 seconds at 60°C.
  • the reverse transcriptase is preferably Luna® Reverse Transcriptase (NEB).
  • the DNA polymerase is preferably GoTaq G2 (Promega). Both enzymes are preferably present in an Onestep Lyophilised Master Mix. The composition of Onestep Lyophilised Master Mix can be found in the Example.
  • the reaction mix is preferably lOul OneStep Lyophilised Master Mix resuspended in 525ul Oasig resuspension buffer, lul primer/probe mix of each of the three sets of probes and primers, and from 5ul to 500ul sample, preferably 5ul sample.
  • the primers and probes of the invention are preferably used in the following concentrations:
  • the reaction mix is preferably lOul OneStep Lyophilised Master Mix resuspended in 525ul Oasig resuspension buffer, lul primer/probe mix of each of the three sets of probes and primers, lul diluted RIMA IEC, lul of IEC probes and primers (SEQ ID NOs: 16 to 19), and from 5ul to 500ul sample, preferably 5ul sample.
  • the primers and probes of the invention and the RNA IEC primers and probes are preferably used in the following concentrations:
  • the reaction mix is most preferably lOpi of resuspended OneStep Lyophislised Master Mix, 2pl of resuspended primers & probes (including all three sets of probes and primers and IEC RIMA primers and probes) and 8pl of sample/DNase/RNase free water.
  • the reaction conditions are preferably (1) one cycle of reverse transcription for 10 minutes at 55°C, (2) one cycle of initial denaturation and Taq activation for 2 minutes at 95°C and (3) 45 cycles of denaturation for 10 seconds at 95°C and annealing and extension for 60 seconds at 60°C.
  • the RT-PCR method is preferably real time RT-PCR. This method is known in the art.
  • the method comprises using three pairs of primers of the invention and detecting the cDNA amplicons amplified by the primers if present.
  • the method may comprise using all three pairs of primers of the invention and detecting the cDNA amplicons amplified by the primers if present.
  • using "all three" primers of the invention means using a pair of primers of the invention targeting the ORFlab gene, a pair of primers of the invention targeting the S gene and a pair of primers of the invention targeting the M gene. Any method may be used to detect the cDNA amplicons.
  • the method may use SYBR Green to detect the cDNA amplicons.
  • SYBR Green binds to the cDNA, it emits light and the intensity of the fluorescence increases as the cDNA amplicons accumulate.
  • the method preferably uses three polynucleotide probes of the invention which specifically hybridise to the cDNA amplicons.
  • the probes may be any of those discussed above.
  • the use of the probes allows the specific cDNA amplicons to be identified if present.
  • the probes are preferably a TaqMan probes. Different targets can be detected (and different Cq values can be measured) by using different detectable labels on the three polynucleotide probes (so each label is specific for a particular probe/cDNA amplicon).
  • the method comprises using three pairs of primers that amplify three cDNA amplicons of the invention.
  • the first cDNA amplicon preferably comprises (or consists of) a sequence (a) to which the sequence shown in SEQ ID NO: 3 specifically hybridises and/or (b) which is complementary to the sequence shown in SEQ ID NO: 3, if present.
  • the first cDNA amplicon may be any of those discussed above.
  • the first cDNA amplicon preferably comprises or consists of SEQ ID NO: 4.
  • the first cDNA amplicon preferably comprises or consists of SEQ ID NO: 5.
  • the second cDNA amplicon preferably comprises (or consists of) a sequence (a) to which the sequence shown in SEQ ID NO: 8 specifically hybridises and/or (b) which is complementary to the sequence shown in SEQ ID NO: 8, if present.
  • the cDNA amplicon may be any of those discussed above.
  • the second cDNA amplicon preferably comprises or consists of SEQ ID NO: 9.
  • the second cDNA amplicon preferably comprises or consists of SEQ ID NO: 10.
  • the third cDNA amplicon preferably comprises (or consists of) a sequence (a) to which the sequence shown in SEQ ID NO: 13 specifically hybridises and/or (b) which is complementary to the sequence shown in SEQ ID NO: 13, if present.
  • the cDNA amplicon may be any of those discussed above.
  • the third cDNA amplicon preferably comprises or consists of SEQ ID NO: 14.
  • the method also comprises using three polynucleotide probes of the invention.
  • the first polynucleotide probe preferably comprises the sequence shown in SEQ ID NO: 3 or a variant thereof as defined above to detect the ORFlab gene cDNA amplicon if present.
  • the first polynucleotide probe may be any of those discussed above.
  • the second polynucleotide probe preferably comprises the sequence shown in SEQ ID NO: 8 or a variant thereof as defined above to detect the S gene cDNA amplicon, if present.
  • the second polynucleotide probe may be any of those discussed above.
  • the third polynucleotide probe preferably comprises the sequence shown in SEQ ID NO: 13 or a variant thereof as defined above to detect the M gene cDNA amplicon, if present.
  • the third polynucleotide probe may be any of those discussed above.
  • the three probes are preferably TaqMan probes.
  • the three polynucleotide probes specifically hybridise to three cDNA amplicons if present.
  • the method comprises using a kit of the invention.
  • the kit comprises three pairs of primers of the invention and three polynucleotide probes of the invention. This allows the three cDNA amplicons of the invention to be amplified and detected, if present.
  • the kit may comprise any of the primers and probes discussed above.
  • the first pair of primers preferably comprises a forward primer which comprises or consists of the sequence shown in SEQ ID NO: 1 and a reverse primer which comprises of consists of the sequence shown in SEQ ID NO: 2.
  • the second pair of primers preferably comprises a forward primer which comprises or consists of the sequence shown in SEQ ID NO: 6 and a reverse primer which comprises of consists of the sequence shown in SEQ ID NO: 7.
  • the third pair of primers preferably comprises a forward primer which comprises or consists of the sequence shown in SEQ ID NO: 11 and a reverse primer which comprises of consists of the sequence shown in SEQ ID NO: 12.
  • the first polynucleotide probe preferably comprises or consists of the sequence shown in SEQ ID NO: 3.
  • the second polynucleotide probe preferably comprises or consists of the sequence shown in SEQ ID NO: 8.
  • the third polynucleotide probe preferably comprises or consists of the sequence shown in SEQ ID NO: 13.
  • detection of all three cDNA amplicons indicates that the sample contains SARS-CoV-2 (/.e. indicates the presence of SARS-CoV-2 in the sample).
  • a lack of detection of all three cDNA amplicons indicates that the sample does not contain SARS-CoV-2 (/.e. indicates the absence of SARS-CoV-2 from the sample).
  • Detection of multiple copies of all three cDNA amplicons indicates that the sample contains SARS-CoV-2 (/.e. indicates the presence of SARS-CoV-2 in the sample).
  • all three cDNA amplicons refers to the three cDNA amplicons of the invention (one derived from the ORFlab gene, one from the S gene and one from the M gene).
  • detection of one or two of the three cDNA amplicons indicates that the method should be repeated to confirm the presence or the absence of SARS-CoV-2 in the sample.
  • Detection of multiple copies of one or two of the three cDNA amplicons indicates that the method should be repeated to confirm the presence or the absence of SARS-CoV-2 in the sample.
  • the method preferably comprises (a) reverse transcribing any RNA in the sample, (b) contacting any cDNA produced in (a) with the three pairs of primers under conditions which allow the primers to amplify their cDNA amplicons and (c) determining the presence or absence of the cDNA amplicons.
  • Step (c) preferably uses three polynucleotide probes as described above.
  • the RT-PCR assay is preferably a one-step RT-PCR assay conducted in one tube or vessel. Such one-step reactions are routine in the art.
  • the method preferably does not comprise extracting RNA from the sample before conducting the RT-PCR assay.
  • the methods may comprise extracting any RNA from the sample before conducting the RT-PCR assay.
  • Suitable kits for doing this are commercial available (such as QIAamp® virus RNA mini kit (Qiagen)).
  • Magnetic bead kits for RNA extraction are also well known in the art, such as Automated extraction system GenoXtract® from HAIN Lifescience GmbH (Brucker) using GXT DNA/RNA Extraction kit (Catalogue no: 12.01.02, 96 samples).
  • the sample may be any sample.
  • the invention is typically carried out on a sample that is known to contain or suspected to contain SARS-CoV-2. Alternatively, the invention may be carried out on any sample whose SARS-CoV-2 status is unknown to confirm the presence or absence of SARS-CoV-2.
  • the sample may be a biological sample.
  • the sample may be obtained from or extracted from any organism or microorganism.
  • the organism or microorganism may be archaeal, prokaryotic or eukaryotic and typically belongs to one of the five kingdoms: plantae, animalia, fungi, monera and protista.
  • the sample may be obtained from or extracted from any virus.
  • the sample is human in origin, but alternatively it may be from another mammal animal such as from commercially farmed animals such as horses, cattle, sheep, fish, chickens or pigs or may alternatively be pets such as cats or dogs.
  • the sample may be of plant origin, such as a sample obtained from a commercial crop, such as a cereal, legume, fruit or vegetable, for example wheat, barley, oats, canola, maize, soya, rice, rhubarb, bananas, apples, tomatoes, potatoes, grapes, tobacco, beans, lentils, sugar cane, cocoa, cotton.
  • the sample may be derived from human or animal food.
  • the sample is preferably a fluid sample.
  • the sample typically comprises a body fluid of the patient.
  • the sample may be urine, lymph, saliva, mucus, amniotic fluid, blood, plasma or serum.
  • the sample is preferably a nasopharyngeal sample, a saliva sample or a blood sample.
  • the sample may be a non-biological sample. Any non-biological sample can be tested.
  • the non-biological sample is preferably a fluid sample.
  • Example of non-biological samples include, but are not limited to, surgical fluids, water such as drinking water, sea water or river water, reagents for laboratory tests and wet swabs of surfaces or materials.
  • the sample is typically processed prior to being used in the invention, for example by centrifugation or by passage through a membrane that filters out unwanted molecules or cells, such as red blood cells.
  • the sample may be tested immediately upon being taken.
  • the sample may also be typically stored prior to assay, preferably below -70°C.
  • the invention also provides methods for measuring the SARS-CoV-2 titre in a sample.
  • the method comprises conducting any of the methods discussed above. Any of the methods for determining the presence or absence of SARS-CoV-2 in a sample as discussed above may be used.
  • the quantitative method uses real time RT-PCR. If SARS-CoV-2 is present, the method comprises evaluating the cycle quantification (Cq) value and thereby measuring the SARS-CoV-2 titre.
  • the method preferably comprises evaluating the Cq value against standard Cq values generated from standard dilution curves.
  • the threshold line is the level of detection or the point at which a reaction reaches a fluorescent intensity above background levels.
  • the threshold level is preferably 10% of the end point fluorescence. Autocalling may be used to set the threshold level.
  • the Cq is the PCR cycle number at which the sample reaction curve intersects the threshold line. This value indicates how many cycles it took to detect a real signal from the sample.
  • Real-Time PCR machines typically calculate the Cq value for each of sample.
  • the Cq value for the triplex assay changes depending on the amount of SARS-CoV-2 present in the sample. These standard curves allow the amount of SARS-CoV-2 to be measured in sample. In other words, the Cq is predictive of the amount of the SARS-CoV-2 in the sample.
  • the following discussion concerning preferred Cq values applies to all three targets (ORFlab, S gene and M gene).
  • a Cq value of from about 20.00 to about 27.99, preferably from about 21.94 to about 23.00 indicates the sample contains about 2xl0 3 virions/pl
  • a Cq value of from about 28.00 to about 31.99, preferably from about 28.64 to about 29.33 indicates the sample contains about 20 virions/pl
  • a Cq value of from about 32.00 to about 32.99, preferably from about 32.55 to about 32.90 indicates the sample contains about 2 virions/pl
  • a Cq value of above 33.00, or above preferably 33.07 or above indicates the sample contains 2 virions/pl.
  • the virions are SARS-CoV-2 virions. All of the Cq values above are preferably mean Cq values derived from multiple experiments. The Cq values are typically expressed to two significant figures.
  • the sample refers to the (undiluted) sample that is added to (and diluted by) the RT-PCR assay mixture. The concentrations do not relate to the final concentration of the virions in the diluted RT-PCR assay mixture. If the sample, for instance from a patient, is diluted or concentrated before it is used in the RT-PCR assay, the dilution factor or concentration factor needs to be taken into account when calculating the amount of SARS-CoV-2 present in the sample.
  • the invention also provides a method for differentiating between high and low SARS-CoV-2 titres in a sample.
  • the method comprises conducting any of the methods discussed above. Any of the methods for determining the presence or absence of SARS-CoV-2 in a sample as discussed above may be used.
  • the method uses real time RT-PCR. If SARS-CoV-2 is present, the method comprises evaluating the cycle quantification (Cq) value and thereby determining whether the sample has a low or high SARS-CoV-2 titre. Cq measurement and evaluation is discussed above.
  • the Cq value for the triplex assay changes depending on the amount of SARS-CoV-2 in a sample.
  • These standard curves allow the amount of SARS-CoV- 2 to be measured in a sample.
  • the Cq is predictive of the amount of the SARS-CoV-2 in the sample.
  • a low SARS-CoV-2 titre is about 20 virions/pl or lower and a high SARS-CoV-2 titre is about 2xl0 3 virions/pl or higher.
  • a Cq value of from about 28.00 or 28.64 or above indicates the sample has a low SARS-CoV-2 titre and a Cq value of about 27.99 or about 23.00 or lower indicates the sample has a high SARS-CoV-2 titre.
  • a low SARS-CoV-2 titre is about 2 virions/pl or lower and a high SARS-CoV-2 titre is about 2xl0 3 virions/pl or higher.
  • a Cq value of about 32.00 or about 32.55 or above indicates the sample has a low SARS-CoV-2 titre and a Cq value of about 27.99 or about 23.00 or lower indicates the sample has a high SARS-CoV-2 titre.
  • a low SARS-CoV-2 titre is about 1 virions/pl or lower and a high SARS-CoV-2 titre is about 2xl0 3 virions/pl or higher.
  • a Cq value of about 33.00 or about 32.07 or above indicates the sample has a low SARS-CoV-2 titre and a Cq value of about 27.99 or about 23.00 or lower indicates the sample has a high SARS-CoV-2 titre.
  • the virions are SARS-CoV-2 virions. All of the Cq values above are preferably mean Cq values derived from multiple experiments. The Cq values are typically expressed to two significant figures.
  • the invention also provides a method of determining whether or not a patient is infected with SARS-CoV-2.
  • the invention therefore relates to the diagnosis of SARS-CoV-2 infection.
  • the diagnostic method of the invention may be carried out in conjunction with other assays or genetic tests.
  • the method comprises conducting a method of the invention for determining the presence or absence of SARS-CoV-2 on a sample from the patient. Any of the methods discussed above may be used.
  • the presence of SARS-CoV-2 in the sample indicates the presence of SARS-CoV-2 in the patient.
  • the presence of SARS-CoV-2 in the sample indicates the patient is infected with SARS-CoV-2.
  • the absence of SARS-CoV-2 from the sample typically indicates the absence of SARS-CoV-2 in the patient.
  • the absence of SARS-CoV-2 from the sample typically indicates the patient is not infected with SARS- CoV-2.
  • the absence of SARS-CoV-2 from the sample may indicate that the particular sample from the patient does not contain SARS-CoV-2 and does not necessarily mean the patient is not infected.
  • the diagnostic method preferably uses a nasopharyngeal sample or a saliva sample. The absence of SARS-CoV-2 from these samples does typically indicate the patient is not infected with SARS-CoV-2.
  • the invention also provides a method of measuring the titre of SARS-CoV-2 in a patient.
  • the invention also provides a method for differentiating between high and low SARS-CoV-2 titres in a patient. These methods comprise conducting the quantitative method of the invention for measuring the titre of SARS-CoV-2 or for differentiating between high and low SARS-CoV-2 titres on a sample from the patient. Low SARS-CoV-2 titres, high SARS-CoV-2 titres and methods for measuring them are discussed above. Any of these may be used on a sample from the patient.
  • the quantitative method preferably uses a nasopharyngeal sample or a saliva sample.
  • the diagnostic and quantitative methods preferably comprise taking a sample from the patient before conducting the RT-PCR assay.
  • the patient displays the symptoms of SARS-CoV-2, i.e. the patient is known or expected to be infected with SARS-CoV-2.
  • the patient may be asymptomatic, i.e. the patient's SARS-CoV-2 status is unknown or the patient is expected not to be infected with SARS-CoV-2.
  • the patient may be susceptible to, or at risk from, infection with SARS-CoV- 2.
  • the patient is may have underlying health conditions which make infection with SARS- CoV-2 particularly serious.
  • the patient is generally a human patient.
  • the patient may be a fetus, a newborn, an infant, a juvenile or an adult.
  • the invention provides a method of treating SARS-CoV-2 in a patient identified as being infected with SARS-CoV-2 using a method of the invention.
  • the method comprises administering to the patient a therapeutically effective amount of an anti-SARS-CoV-2 treatment.
  • the invention also provides a method of treating SARS-CoV-2 in a patient.
  • the method comprises (a) identifying the patient as being infected with SARS-CoV-2 using a method of the invention and (b) administering to the patient a therapeutically effective amount of an anti-SARS-CoV-2 treatment.
  • the invention also provides a substance or composition for use in a method of treating SARS-CoV-2 in a patient identified as being infected with SARS-CoV-2 using a method of the invention.
  • the invention also provides a substance or composition for use in a method of treating SARS-CoV-2 in a patient, wherein the method comprises (a) identifying the patient as being infected with SARS-CoV-2 using a method of the invention and (b) administering the substance of composition to the patient.
  • the substance or composition is preferably an anti-SARS-CoV-2 substance or composition.
  • the invention also provides a method of treating SARS-CoV-2 in a patient identified as having a high SARS-CoV-2 titre using a method of the invention.
  • the method comprises administering to the patient a therapeutically effective amount of an anti-SARS-CoV-2 treatment.
  • the invention also provides a method of treating SARS-CoV-2 in a patient comprising (a) identifying the patient as having a high SARS-CoV-2 titre using a method of the invention and (b) administering to the patient a therapeutically effective amount of an anti-SARS-CoV-2 treatment.
  • a high SARS-CoV-2 titre is about 2xl0 3 virions/pl or higher.
  • a Cq value of 27.99 or 23.00 or lower indicates the sample has a high SARS-CoV-2 titre.
  • the invention also provides a substance or composition for use in a method of treating SARS-CoV-2 in a patient identified as being having a high SARS-CoV-2 titre using a method of the invention.
  • the invention also provides a substance or composition for use in a method of treating SARS-CoV-2 in a patient, wherein the method comprises (a) identifying the patient as being having a high SARS-CoV-2 titre using a method of the invention and (b) administering the substance or composition to the patient.
  • the substance or composition is preferably an anti-SARS-CoV-2 substance or composition
  • any treatment, substance or composition may be used in the invention.
  • Suitable anti- SARS-CoV-2 treatments, substances and compositions are well known.
  • the anti-SARS-CoV- 2 treatment, substance or composition may inhibit viral entry into cells by inhibiting ACE2 receptors, such as ACE2 receptor antibodies or arbidol, or inhibiting TMPRSS2, such as camosat mesylate.
  • the anti-SARS-CoV-2 treatment, substance or composition may inhibit 3-chymotrypsin-like protease, such as lopinavir or darunavir.
  • the anti-SARS-CoV-2 treatment, substance or composition may inhibit viral replication by inhibiting viral RNA- dependent RNA polymerase (RdRP), such as ribavirin, remdesivir or favipiravir.
  • RdRP viral RNA- dependent RNA polymerase
  • the anti- SARS-CoV-2 treatment, substance or composition may be an anti-SARS-CoV-2 small interfering RNA (siRNA) designed to inhibit entry of the virus into cells and/or inhibit viral replication by targeting SARS-CoV-2 genes involved in these processes.
  • siRNA small interfering RNA
  • the anti-SARS-CoV-2 treatment, substance or composition may be an anti-IL-6 therapy or antibody, such as tocilizumab or sarilumab.
  • the anti-SARS-CoV-2 treatment, substance or composition may be any anti-inflammatory and inhibitor of immune responses.
  • the invention also provides a method of treating SARS-CoV-2 in a patient identified as having a high SARS-CoV-2 titre using a method of the invention.
  • the method comprises administering to the patient a therapeutically effective amount of an anti-IL-6 therapy or antibody.
  • the invention also provides a method of treating SARS-CoV-2 in a patient comprising (a) identifying the patient as having a high SARS-CoV-2 titre using a method of the invention and (b) administering to the patient a therapeutically effective amount of an anti-IL-6 therapy or antibody.
  • High SARS-CoV-2 titres and methods of measuring them are discussed above.
  • the invention also provides an anti-IL-6 therapy or antibody for use in a method of treating SARS-CoV-2 in a patient identified as being having a high SARS-CoV-2 titre using a method of the invention.
  • the invention also provides an anti-IL-6 therapy or antibody for use in a method of treating SARS-CoV-2 in a patient, wherein the method comprises (a) identifying the patient as being having a high SARS-CoV-2 titre using a method of the invention and (b) administering the an anti-IL-6 therapy or antibody to the patient.
  • the invention concerns administering to the patient a therapeutically effective mount of the treatment, substance or composition to the patient.
  • a therapeutically effective amount is an amount which ameliorates one or more symptoms of the SARS-CoV-2 infection.
  • a therapeutically effective amount is preferably a number which abolishes one or symptoms of the SARS-CoV-2 infection.
  • a therapeutically effective amount may cure or abolish the SARS-CoV-2 infection. Suitable amounts are discussed in more detail below.
  • composition of the invention may be administered to any suitable patient. Suitable patients are discussed above with reference to the diagnostic embodiments of the invention.
  • the invention may be used in combination with other means of, and substances for, treating the disease or disorder or providing pain relief.
  • the treatment, substance or composition may be used in combination with existing treatments for the SARS-CoV-2 infection including intensive care treatment and the use of ventilators.
  • the treatment, substance or composition of the invention may be formulated using any suitable method. Formulation with standard pharmaceutically acceptable carriers and/or excipients may be carried out using routine methods in the pharmaceutical art. The exact nature of a formulation will depend upon several factors including the composition to be administered and the desired route of administration. Suitable types of formulation are fully described in Remington's Pharmaceutical Sciences, 19 th Edition, Mack Publishing Company, Eastern Pennsylvania, USA.
  • the treatment, substance or composition may be administered by any route. Suitable routes include, but are not limited to, enteral or parenteral routes such as via buccal, anal, pulmonary, intravenous, intra-arterial, intramuscular, intraperitoneal, intraarticular, topical and other appropriate routes. If the lungs are being treated, the treatment, substance or composition is preferably administered by inhalation.
  • compositions may be prepared together with a physiologically acceptable carrier or diluent.
  • the treatment, substance or composition may be mixed with an excipient which is pharmaceutically acceptable and compatible with the active ingredient.
  • excipients are, for example, water, saline, dextrose, glycerol, of the like and combinations thereof.
  • Liquid dispersions for oral administration may be syrups, emulsions or suspensions.
  • the syrups may contain as carriers, for example, saccharose or saccharose with glycerine and/or mannitol and/or sorbitol.
  • Suspensions and emulsions may contain as carrier, for example a natural gum, agar, sodium alginate, pectin, methylcellulose, carboxymethylcellulose, or polyvinyl alcohol.
  • the suspensions or solutions for intramuscular injections may contain, together with the active substance, a pharmaceutically acceptable carrier, e.g. sterile water, olive oil, ethyl oleate, glycols, e.g. propylene glycol, and if desired, a suitable amount of lidocaine hydrochloride.
  • Solutions for intravenous administration or infusion may contain as carrier, for example, sterile water or preferably they may be in the form of sterile, aqueous, isotonic saline solutions.
  • binders and carriers may include, for example, polyalkylene glycols or triglycerides; such suppositories may be formed from mixtures containing the active ingredient in the range of 0.5% to 10%, preferably 1% to 2%.
  • Oral formulations include such normally employed excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like. These compositions typically take the form of solutions or suspensions and contain 10% to 95% of active ingredient, preferably 25% to 70%. Where the pharmaceutical composition is lyophilised, the lyophilised material may be reconstituted prior to administration, e.g. a suspension. Reconstitution is preferably effected in buffer.
  • compositions of the invention may contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, and/or adjuvants which enhance effectiveness.
  • the composition preferably comprises human serum albumin.
  • Plasma-Lyte A® is a sterile, nonpyrogenic isotonic solution for intravenous administration.
  • Each 100 mL contains 526 mg of Sodium Chloride, USP (NaCI); 502 mg of Sodium Gluconate (C6HllNaO7); 368 mg of Sodium Acetate Trihydrate, USP (C2H3NaO2*3H2O); 37 mg of Potassium Chloride, USP (KOI); and 30 mg of Magnesium Chloride, USP (MgCI2*6H2O). It contains no antimicrobial agents.
  • the pH is adjusted with sodium hydroxide. The pH is 7.4 (6.5 to 8.0).
  • the most preferred pharmaceutically acceptable carrier or diluent is a pharmaceutically acceptable transfection reagent.
  • a pharmaceutical acceptable transfection reagent is suitable for administration to patients.
  • the pharmaceutical acceptable transfection reagent may be liposomes, preferably cationic liposomes, polymers, preferably cationic polymers, and dendrimers.
  • the pharmaceutical acceptable transfection reagent is more preferably a pharmaceutically acceptable PEI transfection reagent, such as a linear PEI transfection reagent.
  • the most preferred pharmaceutical acceptable transfection reagent is GMP in v/ o-jetPEI®.
  • composition is administered in a manner compatible with the dosage formulation and in such amount will be therapeutically effective.
  • quantity to be administered depends on the subject to be treated, capacity of the patient's immune system and the degree of treatment desired. Precise amounts required to be administered may depend on the judgement of the practitioner and may be peculiar to each patient.
  • any suitable dose of the treatment, substance or composition may be administered to a patient.
  • the dose may be determined according to various parameters, especially according to the substance used; the age, weight and condition of the patient to be treated; the route of administration; and the required regimen. Again, a physician will be able to determine the required route of administration and dosage for any particular patient.
  • a typical daily dose is from about 0.01 to 50 mg per kg of body weight, according to the activity of the specific substance, the age, weight and conditions of the subject to be treated and the frequency and route of administration.
  • daily dosage levels are from 5 mg to 2 g.
  • from about 0.01 to about 50mg per kg of patient of sRNA may administered, such as from about 0.05 to about 40, from about 0.1 to about 30, from about 0.5 to about 20, from about 1 to about 10 or from about 2 to about 5 mg per kg.
  • At least about 0.01 mg per kg of patient may administered, such as at least about 0.05, at least about 0.1, at least about 0.5, at least about 1, at least about 2, at least about 5, at least about 10, at least about 20, at least at least about 30 or at least about 40 mg per kg.
  • doses may be provided as a single dose or may be provided as multiple doses, for example taken at regular intervals, for example 2, 3 or 4 doses administered daily.
  • regular intervals include, but are not limited to, every day, every week, every fortnight or every month.
  • the objective of this report is to select optimum primers and probe concentration for the COVID-19 3G kit.
  • the COVID-19 3G kit has the following targets:
  • SARS-CoV-2 whole genome RIMA was diluted down to 2 x 10 3 copies/
  • Table 3 Tested primers and probe concentrations for each target Each combination was tested with OneStep Lyophilised Master Mix following recipe in Table:
  • Table 8 Cq values, average Cq values, Cq values standard deviation, standard curve efficiency and R2 for primers and probe concentrations in Table 7
  • the COVID-19 3G has the same setup and sample-to-reaction ratio as the COVID-19 CE kit and the COVID-19 2G kit, which is shown below:
  • primers and probes will be resuspended with the double of total number of reactions volume (e.g., if the total number of reactions is 110 per tube, then the resuspension volume should be 220pl).
  • Table 9 shows manufacturing and PCR reaction concentrations: Table 9: Manufacture and PCR reaction primers and probes concentrations

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Abstract

The invention relates to novel primers, probes, kits and methods for determining the presence or absence of SARS-CoV-2.

Description

COMPOSITION AND METHOD FOR DETECTING SARS-COV-2
TECHNICAL FIELD
The invention relates to novel primers, probes, kits and methods for determining the presence or absence of SARS-CoV-2.
BACKGROUND
Coronaviruses are members of the subfamily Coronavirinae in the family Coronaviridae and the order Nidovirales. A novel coronavirus, 2019-nCoV or COVID-19, capable of infecting humans was identified in Wuhan, China and then led to a worldwide pandemic. Based on its phylogenetic relationships and genomic structures, the virus belongs to genera Betacoronavirus and has close similarities to severe acute respiratory syndrome-related coronaviruses (SARS-CoV). The virus uses ACE2 as the entry receptor-like SARS-CoV. These similarities had lead the Coronavirus Study Group of the International Committee on Taxonomy of Viruses to term the virus SARS-CoV-2. Since the virus is novel, new methods are required to test for the virus and distinguish it from other similar coronaviruses. The sequence of SARS-Cov-2 has been published under accession no. MN908947
(https ://www. ncbi.nlm.nih.gov/nuccore/MN908947.3) ■ Sequence information from around the world nevertheless indicates that numerous variants of the virus exist.
SUMMARY OF THE INVENTION
The inventors have surprisingly identified new primers, probes, kits and methods for determining the presence or absence of SARS-CoV-2 in a variety of samples. The first set of new primers and probes target the ORFlab gene in SARS-CoV-2. The ORFlab gene encodes the ORFla/ORFlab poly-proteins. The second set of new primers and probes target the S gene in SARS-CoV-2. The S gene encodes the spike glycoprotein which facilitates entry of the virus into cells. The third set of new primers and probes target the M gene in SARS-CoV-2. The M gene encodes the membrane (M) glycoprotein. The three sets of primers and probes are present together in the kits of the invention and are used together in the triplex methods of the invention.
The invention provides: three pairs of primers for determining the presence or absence of SARS-CoV-2 in a sample, wherein (a) in the first pair the forward primer comprises a polynucleotide having the sequence shown in SEQ ID NO: 1 or a variant thereof having at least about 80% homology to SEQ ID NO: 1 based on sequence identity over its entire length and the reverse primer comprises a polynucleotide having the sequence shown in SEQ ID NO: 2 or a variant thereof having at least about 75% homology to SEQ ID NO: 2 based on sequence identity over its entire length; (b) in the second pair the forward primer comprises a polynucleotide having the sequence shown in SEQ ID NO: 6 or a variant thereof having at least about 75% homology to SEQ ID NO: 6 based on sequence identity over its entire length and the reverse primer comprises a polynucleotide having the sequence shown in SEQ ID NO: 7 or a variant thereof having at least about 75% homology to SEQ ID NO: 7 based on sequence identity over its entire length; and (c) in the third pair the forward primer comprises a polynucleotide having the sequence shown in SEQ ID NO: 11 or a variant thereof having at least about 75% homology to SEQ ID NO: 11 based on sequence identity over its entire length and the reverse primer comprises a polynucleotide having the sequence shown in SEQ ID NO: 12 or a variant thereof having at least about 75% homology to SEQ ID NO: 12 based on sequence identity over its entire length; three complementary DNA (cDNA) amplicons amplified from SARS-CoV-2 cDNA by three pairs of primers of the invention; three polynucleotide probes which specifically hybridise to three cDNA amplicons of the invention; three polynucleotide probes for determining the presence or absence of SARS-CoV-2 in a sample, wherein (a) the first polynucleotide probe comprises the sequence shown in SEQ ID NO: 3 or a variant thereof having at least about 80% homology to SEQ ID NO: 3 based on sequence identity over its entire length, (b) the second polynucleotide probe comprises the sequence shown in SEQ ID NO: 8 or a variant thereof having at least about 80% homology to SEQ ID NO: 8 based on sequence identity over its entire length and (c) the third polynucleotide probe comprises the sequence shown in SEQ ID NO: 13 or a variant thereof having at least about 80% homology to SEQ ID NO: 13 based on sequence identity over its entire length; three cDNA amplicons amplified from SARS-CoV-2 cDNA wherein (a) the first cDNA amplicon comprises a sequence to which the sequence shown in SEQ ID NO: 3 specifically hybridises and/or which is complementary to the sequence shown in SEQ ID NO: 3, (b) the second cDNA amplicon comprises a sequence to which the sequence shown in SEQ ID NO: 8 specifically hybridises and/or which is complementary to the sequence shown in SEQ ID NO: 8 and (c) the third cDNA amplicon comprises a sequence to which the sequence shown in SEQ ID NO: 13 specifically hybridises and/or which is complementary to the sequence shown in SEQ ID NO: 13; three target cDNA polynucleotides, wherein (a) the first target cDNA polynucleotide comprises a sequence to which the sequence shown in SEQ ID NO: 3 specifically hybridises and/or which is complementary to the sequence shown in SEQ ID NO: 3, (b) the second target cDNA polynucleotide comprises a sequence to which the sequence shown in SEQ ID NO: 8 specifically hybridises and/or which is complementary to the sequence shown in SEQ ID NO: 8 and (c) the third target cDNA polynucleotide comprises a sequence to which the sequence shown in SEQ ID NO: 13 specifically hybridises and/or which is complementary to the sequence shown in SEQ ID NO: 13; a kit for determining the presence or absence of SARS-CoV-2 in a sample, comprising (a) three pairs of primers of the invention and (b) three polynucleotide probes of the invention; a method of determining the presence or absence of SARS-CoV-2 in a sample, wherein the method comprises conducting a reverse-transcription polymerase chain reaction (RT-PCR) assay on the sample using three pairs of primers of the invention and detecting the cDNA amplicons amplified by the primers, if present, and thereby determining the presence or absence of SARS-CoV-2 in the sample; a method of determining the presence or absence of SARS-CoV-2 in a sample, wherein the method comprises conducting a reverse-transcription polymerase chain reaction (RT-PCR) assay on the sample using three pairs of primers that amplifies three cDNA amplicons of the invention and three polynucleotide probes of the invention and thereby determining the presence or absence of SARS-CoV-2 in the sample; a method of determining the presence or absence of SARS-CoV-2 in a sample, wherein the method comprises conducting a reverse-transcription polymerase chain reaction (RT-PCR) assay on the sample using a kit of the invention and thereby determining the presence or absence of SARS-CoV-2 in the sample; a method for measuring the SARS-CoV-2 titre in a sample, wherein the method comprises conducting a real time RT-PCR method of the invention and, if SARS-CoV-2 is present, evaluating the cycle quantification (Cq) value and thereby measuring the SARS- CoV-2 titre; a method of determining whether or not a patient is infected with SARS-CoV-2, wherein the method comprises conducting a method of the invention on a sample from the patient and thereby determining whether or not a patient is infected with SARS-CoV-2; a method of measuring the titre of SARS-CoV-2 in a patient, wherein the method comprises conducting a method of the invention on a sample from the patient and thereby measuring the viral titre in the patient; a method of treating SARS-CoV-2 in a patient identified as being infected with SARS- CoV-2 using a method of the invention, comprising administering to the patient a therapeutically effective amount of an anti-SARS-CoV-2 treatment and thereby treating the SARS-CoV-2 infection in the patient; a substance or composition for use in a method of treating SARS-CoV-2 in a patient identified as being infected with SARS-CoV-2 using a method of the invention; a method of treating SARS-CoV-2 in a patient identified as having a high SARS-CoV- 2 titre using a method of the invention, comprising administering to the patient a therapeutically effective amount of an anti-SARS-CoV-2 treatment and thereby treating the SARS-CoV-2 infection in the patient; a substance or composition for use in a method of treating SARS-CoV-2 in a patient identified as being having a high SARS-CoV-2 titre using a method of the invention.
DESCRIPTION OF THE FIGURES
Figure 1 shows the amplification plot for ORFlab (FAM).
Figure 2 shows the amplification plot for M-gene (ROX).
Figure 3 shows the amplification plot for S-gene (ATTO).
Figure 4 shows the amplification plot for L.lactis IEC (HEX).
DESCRIPTION OF THE SEQUENCE LISTING
SEQ ID NO: 1 shows the forward ORFlab gene primer used in Example.
SEQ ID NO: 2 shows the reverse ORFlab gene primer used in Example.
SEQ ID NO: 3 shows the ORFlab gene probe used in Example.
SEQ ID NO: 4 shows the ORFlab gene cDNA amplified in the Example.
SEQ ID NO: 5 shows the reverse complement of SEQ ID NO: 3.
SEQ ID NO: 6 shows the forward S gene primer used in the Example.
SEQ ID NO: 7 shows the reverse S gene primer used in the Example.
SEQ ID NO: 8 shows the S gene probe used in the Example. The probe specifically hybridises to part of the complementary strand of the cDNA amplicon sequence shown in SEQ ID NO: 9.
SEQ ID NO: 9 shows the S gene cDNA amplified in the Example.
SEQ ID NO: 10 shows the reverse complement of SEQ ID NO: 8. SEQ ID NO: 11 shows the forward M gene primer used in the Example (Y=C or T).
SEQ ID NO: 12 shows the reverse M gene primer used in the Example (M = A or C).
SEQ ID NO: 13 shows the M gene probe used in the Example.
SEQ ID NO: 14 shows the M gene cDNA amplified in the Example.
SEQ ID NO: 15 shows the reverse complement of SEQ ID NO: 13.
SEQ ID NO: 16 shows the Lactococcus lactis (L. lactis) forward primer.
SEQ ID NO: 17 shows the L. lactis reverse primer.
SEQ ID NO: 18 shows the L. lactis probe 1.
SEQ ID NO: 19 shows the L. lactis probe 2.
DETAILED DESCRIPTION
It is to be understood that different applications of the disclosed products and methods may be tailored to the specific needs in the art. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments of the invention only and is not intended to be limiting.
In addition, as used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to "a primer" includes two or more primers, reference to "a probe" includes two or more probes, reference to "a sample" includes two or more such samples, reference to "a patient" includes two or more such patients, and the like.
All publications, patents and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety.
Terminology
In all instances herein, SARS-CoV-2 is interchangeable with 2019-nCoV or COVID-19.
In all instances herein, determining is interchangeable with detecting. The new primers, probes, kits and methods of the invention may be for detecting the presence or absence of SARS-CoV-2 in a sample or patient. Also, in all instances, the new primers, probes, kits and methods of the invention may be for determining/detecting/identifying whether or not a sample contains or comprises SARS-CoV-2 or whether or not a patient is infected with SARS-CoV-2.
Advantages of the invention The inventors have surprisingly identified new primers, probes, kits and methods for determining the presence or absence of SARS-CoV-2 in a variety of samples. The compositions and methods have several advantages.
First, the new primers, probes, kits and methods are capable of detecting the presence of SARS-CoV-2 with a high degree of sensitivity. As shown in the Example, the method is capable of detecting only 1 copy of the SARS-CoV-2 RNA genome per ul of sample.
Second, the new primers, probes, kits and methods do not cross react with other human viruses, such as other human coronaviruses. The new primers, probes, kits and methods do not cross react with SARS-CoV. Positive results from the new primers, probes, kits and methods can therefore be trusted as being indicative of the presence of SARS-CoV-2. The new primers, probes, kits and methods therefore do not give false positives.
Third, the new primers, probes, kits and methods are capable of measuring the SARS-CoV-2 titre in sample and distinguishing between a low SARS-CoV-2 titre and a high SARS-CoV-2 titre. This is particularly helpful in determining the level of infection in patients and allowing physicians to tailor their treatments to those levels.
Primers of the invention
The present invention provides three pairs of primers for determining the presence or absence of SARS-CoV-2 in a sample.
In the first pair targeting the ORFlab gene, the forward primer comprises a polynucleotide having the sequence shown in SEQ ID NO: 1 or a variant thereof having at least about 80% homology to SEQ ID NO: 1 based on sequence identity over its entire length. The reverse primer comprises a polynucleotide having the sequence shown in SEQ ID NO: 2 or a variant thereof having at least about 75% homology to SEQ ID NO: 2 based on sequence identity over its entire length.
In the second pair targeting the S gene, the forward primer comprises a polynucleotide having the sequence shown in SEQ ID NO: 6 or a variant thereof having at least about 75% homology to SEQ ID NO: 6 based on sequence identity over its entire length. The reverse primer comprises a polynucleotide having the sequence shown in SEQ ID NO: 7 or a variant thereof having at least about 75% homology to SEQ ID NO: 7 based on sequence identity over its entire length.
In the third pair targeting the M gene, the forward primer comprises a polynucleotide having the sequence shown in SEQ ID NO: 11 or a variant thereof having at least about 75% homology to SEQ ID NO: 11 based on sequence identity over its entire length. The reverse primer comprises a polynucleotide having the sequence shown in SEQ ID NO: 12 or a variant thereof having at least about 75% homology to SEQ ID NO: 12 based on sequence identity over its entire length.
A polynucleotide, such as a nucleic acid, is a polymer comprising two or more nucleotides. The nucleotides can be naturally occurring or artificial.
A nucleotide typically contains a nucleobase, a sugar and at least one linking group, such as a phosphate, 2'0-methyl, 2' methoxy-ethyl, phosphoramidate, methylphosphonate or phosphorothioate group. The nucleobase is typically heterocyclic. Nucleobases include, but are not limited to, purines and pyrimidines and more specifically adenine (A), guanine (G), thymine (T), uracil (U) and cytosine (C). The sugar is typically a pentose sugar. Nucleotide sugars include, but are not limited to, ribose and deoxyribose. The sugar and the nucleobase together form a nucleoside. Preferred nucleosides include, but are not limited to, adenosine, guanosine, 5-methyluridine, uridine, cytidine, deoxyadenosine, deoxyguanosine, thymidine, deoxyuridine and deoxycytidine. The nucleosides are most preferably adenosine, guanosine, uridine and cytidine
The nucleotides are typically ribonucleotides or deoxyribonucleotides. The nucleotides are preferably deoxyribonucleotides. The nucleotides typically contain a monophosphate, diphosphate or triphosphate. Phosphates may be attached on the 5' or 3' side of a nucleotide.
Nucleotides include, but are not limited to, adenosine monophosphate (AMP), adenosine diphosphate (ADP), adenosine triphosphate (ATP), guanosine monophosphate (GMP), guanosine diphosphate (GDP), guanosine triphosphate (GTP), thymidine monophosphate (TMP), thymidine diphosphate (TDP), thymidine triphosphate (TTP), uridine monophosphate (UMP), uridine diphosphate (UDP), uridine triphosphate (UTP), cytidine monophosphate (CMP), cytidine diphosphate (CDP), cytidine triphosphate (CTP), 5-methylcytidine monophosphate, 5-methylcytidine diphosphate, 5-methylcytidine triphosphate, 5- hydroxymethylcytidine monophosphate, 5-hydroxymethylcytidine diphosphate, 5- hydroxy methylcytidine triphosphate, cyclic adenosine monophosphate (cAMP), cyclic guanosine monophosphate (cGMP), deoxyadenosine monophosphate (dAMP), deoxyadenosine diphosphate (dADP), deoxyadenosine triphosphate (dATP), deoxyguanosine monophosphate (dGMP), deoxyguanosine diphosphate (dGDP), deoxyguanosine triphosphate (dGTP), deoxythymidine monophosphate (dTMP), deoxythymidine diphosphate (dTDP), deoxythymidine triphosphate (dTTP), deoxyuridine monophosphate (dUMP), deoxyuridine diphosphate (dUDP), deoxyuridine triphosphate (dUTP), deoxycytidine monophosphate (dCMP), deoxycytidine diphosphate (dCDP) and deoxycytidine triphosphate (dCTP), 5-methyl-2'-deoxycytidine monophosphate, 5-methyl-2'-deoxycytidine diphosphate, 5-methyl-2'-deoxycytidine triphosphate, 5-hydroxymethyl-2'-deoxycytidine monophosphate, 5-hydroxymethyl-2'-deoxycytidine diphosphate and 5-hydroxymethyl-2'- deoxycytidine triphosphate. The nucleotides are preferably selected from AMP, UMP, GMP, CMP, dAMP, dTMP, dGMP or dCMP. The nucleotides are preferably selected from dAMP, dTMP, dGMP or dCMP.
The nucleotides may contain additional modifications. In particular, suitable modified nucleotides include, but are not limited to, 2'amino pyrimidines (such as 2'-amino cytidine and 2'-amino uridine), 2'-hyrdroxyl purines (such as , 2'-fluoro pyrimidines (such as 2'- fluorocytidine and 2'fluoro uridine), hydroxyl pyrimidines (such as 5'-o-P-borano uridine), 2'-O-methyl nucleotides (such as 2'-O-methyl adenosine, 2'-O-methyl guanosine, 2'-O- methyl cytidine and 2'-O-methyl uridine), 4'-thio pyrimidines (such as 4'-thio uridine and 4'- thio cytidine) and nucleotides have modifications of the nucleobase (such as 5-pentynyl-2'- deoxy uridine, 5-(3-aminopropyl)-uridine and l,6-diaminohexyl-N-5-carbamoylmethyl uridine).
One or more nucleotides in the polynucleotide may be modified, for instance with a label or a tag. The label may be any suitable label which allows the polynucleotide to be detected. Suitable labels include, but are not limited to, fluorescent molecules, radioisotopes, e.g. 125I, 35S, enzymes, antibodies, antigens, other polynucleotides and ligands such as biotin.
The nucleotides in the polynucleotide may be attached to each other in any manner. The nucleotides may be linked by phosphate, 2'0-methyl, 2' methoxy-ethyl, phosphoramidate, methylphosphonate or phosphorothioate linkages. The nucleotides are typically attached by their sugar and phosphate groups as in nucleic acids. The nucleotides may be connected via their nucleobases as in pyrimidine dimers.
The polynucleotide can be a nucleic acid, such as deoxyribonucleic acid (DNA) or a ribonucleic acid (RIMA). The polynucleotide may be any synthetic nucleic acid known in the art, such as peptide nucleic acid (PNA), glycerol nucleic acid (GNA), threose nucleic acid (TNA), locked nucleic acid (LNA), morpholino nucleic acid or other synthetic polymers with nucleotide side chains. The polynucleotide may comprise any of the nucleotides discussed above, including the modified nucleotides.
The polynucleotide may be DNA or RNA. In the context of the invention, a primer polynucleotide that is RNA has the sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2 if all of the instances of dAMP, dTMP, dGMP and dCMP are replaced with AMP, UMP, GMP and CMP respectively. In other words, the DNA nucleotides are replaced with their corresponding RNA nucleotides and T is replaced with U.
The polynucleotide is preferably DNA.
The polynucleotide may be single stranded or double stranded. The primer polynucleotide is preferably single stranded. Any of the polynucleotides discussed herein, such as the primers, probes, cDNA amplicons and target cDNA sequences, may be isolated, substantially isolated, purified or substantially purified. The polynucleotide is isolated or purified if it is completely free of any other components, such as buffer, other polynucleotides, virus material or cells. The polynucleotide is substantially isolated or substantially purified if it is only mixed with carriers or diluents, such as buffers or excipients, which will not interfere with its intended use, such as in a RT-PCR assay. The primer polynucleotides are not naturally occurring.
The primer of the invention may comprise a variant sequence based on SEQ ID NO: 1, 2, 6, 7, 11 or 12. A variant sequence is a polynucleotide that has a nucleotide sequence which varies from that of SEQ ID NO: 1, 2, 6, 7, 11 or 12 and which retains its ability to specifically hybridise to the target sequence of SEQ ID NO: 1, 2, 6, 7, 11 or 12 . A variant sequence is a polynucleotide that has a nucleotide sequence which varies from that of SEQ ID NO: 1, 2, 6, 7, 11 or 12 and which retains its ability to specifically hybridise to a sequence that is complementary to SEQ ID NO: 1, 2, 6, 7, 11 or 12 . A variant sequence is a polynucleotide that has a nucleotide sequence which varies from that of SEQ ID NO: 1, 2, 6, 7, 11 or 12 and which retains its ability to amplify the complementary DNA (cDNA) amplicon amplified from SARS-CoV-2 cDNA by SEQ ID NOs: 1, 2, 6, 7, 11 or 12 (/.e. SEQ ID NO: 4, 9 or 14). The term "amplified" refers to the process of making multiple copies of the polynucleotide, such as cDNA, from a single polynucleotide or fewer polynucleotides.
A variant "specifically hybridises" to its target sequence when it hybridises with preferential or high affinity to the partner but does not substantially hybridise, does not hybridise or hybridises with only low affinity to other polynucleotides, especially other sequences in SARS-CoV-2. Conditions that permit the hybridisation are well-known in the art (for example, Sambrook et al., 2001, Molecular Cloning: a laboratory manual, 3rd edition, Cold Spring Harbour Laboratory Press; and Current Protocols in Molecular Biology, Chapter 2, Ausubel et al., Eds., Greene Publishing and Wiley-lnterscience, New York (1995)). Hybridisation can be carried out under low stringency conditions, for example in the presence of a buffered solution of 30 to 35% formamide, 1 M NaCI and 1 % SDS (sodium dodecyl sulfate) at 37 °C followed by a 20 wash in from IX (0.1650 M Na+) to 2X (0.33 M Na+) SSC (standard sodium citrate) at 50 °C. Hybridisation can be carried out under moderate stringency conditions, for example in the presence of a buffer solution of 40 to 45% formamide, 1 M NaCI, and 1 % SDS at 37 °C, followed by a wash in from 0.5X (0.0825 M Na+) to IX (0.1650 M Na+) SSC at 55 °C. Hybridisation can be carried out under high stringency conditions, for example in the presence of a buffered solution of 50% formamide, 1 M NaCI, 1% SDS at 37 °C, followed by a wash in 0.1X (0.0165 M Na+) SSC at 60 °C. A variant "specifically hybridises" if it hybridises to its partner with a melting temperature (Tm) that is at least 2 °C, such as at least 3 °C, at least 4 °C, at least 5 °C, at least 6 °C, at least 7 °C, at least 8 °C, at least 9 °C or at least 10 °C, greater than its Tm for other polynucleotides. More preferably, the variant hybridises to its partner with a Tm that is at least 2 °C, such as at least 3 °C, at least 4 °C, at least 5 °C, at least 6 °C, at least 7 °C, at least 8 °C, at least 9 °C, at least 10 °C, at least 20 °C, at least 30 °C or at least 40 °C, greater than its Tm for other polynucleotides. Preferably, the variant hybridises to its partner with a Tm that is at least 2 °C, such as at least 3 °C, at least 4 °C, at least 5 °C, at least 6 °C, at least 7 °C, at least 8 °C, at least 9 °C, at least 10 °C, at least 20 °C, at least 30 °C or at least 40 °C, greater than its Tm for a polynucleotide which differs from its partner by one or more nucleotides, such as by 1, 2, 3, 4 or 5 or more nucleotides. The variant typically hybridises to its target sequence with a Tm of at least 90 °C, such as at least 92 °C or at least 95 °C. Tm can be measured experimentally using known techniques, including the use of DNA microarrays, or can be calculated using publicly available Tm calculators, such as those available over the internet.
The variant sequence may comprise any of the nucleotides discussed above, including the modified nucleotides. The variant sequence is typically the same length as SEQ ID NO: 1, 2, 6, 7, 11 or 12, but may be longer or shorter. A variant of SEQ ID NO: 1 is preferably at least 20 nucleotides in length, such as 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides in length. A variant of SEQ ID NO: 2 is preferably at least 19 nucleotides in length, such as 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 or 29 nucleotides in length. A variant of SEQ ID NO: 6 or 7 is preferably at least 17 nucleotides in length, such as 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 or 27 nucleotides in length. A variant of SEQ ID NO: 11 is preferably at least 15 nucleotides in length, such as 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides in length. A variant of SEQ ID NO: 12 is preferably at least 16 nucleotides in length, such as 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or 26 nucleotides in length.
Over the entire length of the sequence of SEQ ID NO: 1, a variant sequence is at least about 80% or at least about 83.3% homologous to that sequence based on nucleotide identity. Over the entire length of the sequence of SEQ ID NO: 1, a variant sequence has at least about 80% or at least about 83.3% identity to that sequence. Homology based on sequence identity and identity are interchangeable herein. This allows for variation, deletion, addition or a combination thereof of five nucleotides within the sequence of SEQ ID NO: 1.
Over the entire length of the sequence of SEQ ID NO: 1, a variant sequence is preferably at least about 84% or at least about 86.2% homologous to that sequence based on nucleotide identity. Over the entire length of the sequence of SEQ ID NO: 1, a variant sequence preferably has at least about 84% or at least about 86.2% identity to that sequence. This allows for variation, deletion, addition or a combination thereof of four nucleotides within the sequence of SEQ ID NO: 1. Over the entire length of the sequence of SEQ ID NO: 1, a variant sequence is preferably at least about 88% or at least about 89.2% homologous to that sequence based on nucleotide identity. Over the entire length of the sequence of SEQ ID NO: 1, a variant sequence preferably has at least about 88% or at least about 89.2% identity to that sequence. This allows for variation, deletion, addition or a combination thereof of three nucleotides within the sequence of SEQ ID NO: 1.
Over the entire length of the sequence of SEQ ID NO: 1, a variant sequence is preferably at least about 92% or at least about 92.5% homologous to that sequence based on nucleotide identity. Over the entire length of the sequence of SEQ ID NO: 1, a variant sequence preferably has at least about 92% or at least about 92.5% identity to that sequence. This allows for variation, deletion, addition or a combination thereof of two nucleotides within the sequence of SEQ ID NO: 1.
Over the entire length of the sequence of SEQ ID NO: 1, a variant sequence is preferably at least about 96% or at least about 96.1% homologous to that sequence based on nucleotide identity. Over the entire length of the sequence of SEQ ID NO: 1, a variant sequence preferably has at least about 96% or at least about 96.1% identity to that sequence. This allows for variation, deletion, addition or a combination thereof of one nucleotide within the sequence of SEQ ID NO: 1.
Over the entire length of the sequence of SEQ ID NO: 2, a variant sequence is preferably at least about 79%, at least about 79.16%, at least about 79.17% or at least about 82.7% homologous to that sequence based on nucleotide identity. Over the entire length of the sequence of SEQ ID NO: 2, a variant sequence preferably has at least about 79%, at least about 79.16%, at least about 79.17% or at least about 82.7% identity to that sequence. This allows for variation, deletion, addition or a combination thereof of five nucleotides within the sequence of SEQ ID NO: 2.
Over the entire length of the sequence of SEQ ID NO: 2, a variant sequence is preferably at least about 80%, at least about 83.3% or at least about 85.7% homologous to that sequence based on nucleotide identity. Over the entire length of the sequence of SEQ ID NO: 2, a variant sequence preferably has at least about 80%, at least about 83.3% or at least about 85.7% identity to that sequence. This allows for variation, deletion, addition or a combination thereof of four nucleotides within the sequence of SEQ ID NO: 2.
Over the entire length of the sequence of SEQ ID NO: 2, a variant sequence is preferably at least about 85%, at least about 87.5% or at least about 88.8% homologous to that sequence based on nucleotide identity. Over the entire length of the sequence of SEQ ID NO: 2, a variant sequence preferably has at least about 85%, at least about 87.5% or at least about 88.8% identity to that sequence. This allows for variation, deletion, addition or a combination thereof of three nucleotides within the sequence of SEQ ID NO: 2.
Over the entire length of the sequence of SEQ ID NO: 2, a variant sequence is preferably at least about 90%, at least about 91.66%, at least about 91.67% or at least about 92.3% homologous to that sequence based on nucleotide identity. Over the entire length of the sequence of SEQ ID NO: 2, a variant sequence preferably has at least about 90%, at least about 91.66%, at least about 91.67% or at least about 92.3% identity to that sequence. This allows for variation, deletion, addition or a combination thereof of two nucleotides within the sequence of SEQ ID NO: 2.
Over the entire length of the sequence of SEQ ID NO: 2, a variant sequence is preferably at least about 95%, at least about 95.8% or at least about 96% homologous to that sequence based on nucleotide identity. Over the entire length of the sequence of SEQ ID NO: 2, a variant sequence preferably has at least about 95%, at least about 95.8% or at least about 96% identity to that sequence. This allows for variation, deletion, addition or a combination thereof of one nucleotide within the sequence of SEQ ID NO: 2.
Over the entire length of the sequence of SEQ ID NO: 6 or 7, a variant sequence is preferably at least about 77%, at least about 77.27%, at least about 81% or at least about 81.48% homologous to that sequence based on nucleotide identity. Over the entire length of the sequence of SEQ ID NO: 6 or 7 , a variant sequence preferably has at least about 77%, at least about 77.27%, at least about 81% or at least about 81.48% identity to that sequence. This allows for variation, deletion, addition or a combination thereof of five nucleotides within the sequence of SEQ ID NO: 6 or 7.
Over the entire length of the sequence of SEQ ID NO: 6 or 7, a variant sequence is preferably at least about 81%, at least about 81.18%, at least about 84% or at least about 84.61% homologous to that sequence based on nucleotide identity. Over the entire length of the sequence of SEQ ID NO: 6 or 7, a variant sequence preferably has at least about 81%, at least about 81.18%, at least about 84% or at least about 84.61% identity to that sequence. This allows for variation, deletion, addition or a combination thereof of four nucleotides within the sequence of SEQ ID NO: 6 or 7.
Over the entire length of the sequence of SEQ ID NO: 6 or 7, a variant sequence is preferably at least about 86%, at least about 86.36, at least about 95% or at least about 95.6% homologous to that sequence based on nucleotide identity. Over the entire length of the sequence of SEQ ID NO: 6 or 7, a variant sequence preferably has at least about 86%, at least about 86.36, at least about 95% or at least about 95.6% identity to that sequence. This allows for variation, deletion, addition or a combination thereof of three nucleotides within the sequence of SEQ ID NO: 6 or 7. Over the entire length of the sequence of SEQ ID NO: 6 or 7, a variant sequence is preferably at least about 90%, at least about 90.90%, at least about 91% or at least about 91.67% homologous to that sequence based on nucleotide identity. Over the entire length of the sequence of SEQ ID NO: 6 or 7, a variant sequence preferably has at least about 90%, at least about 90.90%, at least about 91% or at least about 91.67% identity to that sequence. This allows for variation, deletion, addition or a combination thereof of two nucleotides within the sequence of SEQ ID NO: 6 or 7.
Over the entire length of the sequence of SEQ ID NO: 6 or 7, a variant sequence is preferably at least about 95%, at least about 95.45% or at least about 95.65% homologous to that sequence based on nucleotide identity. Over the entire length of the sequence of SEQ ID NO: 6 or 7, a variant sequence preferably has at least about 95%, at least about 95.45% or at least about 95.65% identity to that sequence. This allows for variation, deletion, addition or a combination thereof of one nucleotide within the sequence of SEQ ID NO: 6 or 7.
Over the entire length of the sequence of SEQ ID NO: 11, a variant sequence is at least about 75% or at least about 80% homologous to that sequence based on nucleotide identity. Over the entire length of the sequence of SEQ ID NO: 11, a variant sequence has at least about 75% or at least about 80% identity to that sequence. Homology based on sequence identity and identity are interchangeable herein. This allows for variation, deletion, addition or a combination thereof of five nucleotides within the sequence of SEQ ID NO: 11.
Over the entire length of the sequence of SEQ ID NO: 11, a variant sequence is preferably at least about 80%, at least about 83% or at least about 83.33% homologous to that sequence based on nucleotide identity. Over the entire length of the sequence of SEQ ID NO: 11, a variant sequence preferably has at least about 80%, at least about 83% or at least about 83.33% identity to that sequence. This allows for variation, deletion, addition or a combination thereof of four nucleotides within the sequence of SEQ ID NO: 11.
Over the entire length of the sequence of SEQ ID NO: 11, a variant sequence is preferably at least about 85%, at least about 86% or at least about 86.95% homologous to that sequence based on nucleotide identity. Over the entire length of the sequence of SEQ ID NO: 11, a variant sequence preferably has at least about 85%, at least about 86% or at least about 86.95% identity to that sequence. This allows for variation, deletion, addition or a combination thereof of three nucleotides within the sequence of SEQ ID NO: 11.
Over the entire length of the sequence of SEQ ID NO: 11, a variant sequence is preferably at least about 90% or at least about 90.90% homologous to that sequence based on nucleotide identity. Over the entire length of the sequence of SEQ ID NO: 11, a variant sequence preferably has at least about 90% or at least about 90.90% identity to that sequence. This allows for variation, deletion, addition or a combination thereof of two nucleotides within the sequence of SEQ ID NO: 11.
Over the entire length of the sequence of SEQ ID NO: 11, a variant sequence is preferably at least about 95% or at least about 95.23% homologous to that sequence based on nucleotide identity. Over the entire length of the sequence of SEQ ID NO: 11, a variant sequence preferably has at least about 95% or at least about 95.23% identity to that sequence. This allows for variation, deletion, addition or a combination thereof of one nucleotide within the sequence of SEQ ID NO: 11.
Over the entire length of the sequence of SEQ ID NO: 12, a variant sequence is preferably at least about 76%, at least about 79.19%, at least about 80% or at least about 80.76% homologous to that sequence based on nucleotide identity. Over the entire length of the sequence of SEQ ID NO: 12, a variant sequence preferably has at least about 76%, at least about 79.19%, at least about 80% or at least about 80.76% identity to that sequence. This allows for variation, deletion, addition or a combination thereof of five nucleotides within the sequence of SEQ ID NO: 12.
Over the entire length of the sequence of SEQ ID NO: 12, a variant sequence is preferably at least about 80%, at least about 80.95% or at least about 84% homologous to that sequence based on nucleotide identity. Over the entire length of the sequence of SEQ ID NO: 12, a variant sequence preferably has at least about 80%, at least about 80.95% or at least about 84% identity to that sequence. This allows for variation, deletion, addition or a combination thereof of four nucleotides within the sequence of SEQ ID NO: 12.
Over the entire length of the sequence of SEQ ID NO: 12, a variant sequence is preferably at least about 85%, at least about 85.71%, at least about 87% or at least about 87.5% homologous to that sequence based on nucleotide identity. Over the entire length of the sequence of SEQ ID NO: 12, a variant sequence preferably has at least about 85%, at least about 85.71%, at least about 87% or at least about 87.5% identity to that sequence. This allows for variation, deletion, addition or a combination thereof of three nucleotides within the sequence of SEQ ID NO: 12.
Over the entire length of the sequence of SEQ ID NO: 12, a variant sequence is preferably at least about 90%, at least about 90.47%, at least about 91% or at least about 91.30% homologous to that sequence based on nucleotide identity. Over the entire length of the sequence of SEQ ID NO: 12, a variant sequence preferably has at least about 90%, at least about 90.47%, at least about 91% or at least about 91.30% identity to that sequence.
This allows for variation, deletion, addition or a combination thereof of two nucleotides within the sequence of SEQ ID NO: 12. Over the entire length of the sequence of SEQ ID NO: 12, a variant sequence is preferably at least about 95%, at least about 95.23% or at least about 95.45% homologous to that sequence based on nucleotide identity. Over the entire length of the sequence of SEQ ID NO: 12, a variant sequence preferably has at least about 95%, at least about 95.23% or at least about 95.45% identity to that sequence. This allows for variation, deletion, addition or a combination thereof of one nucleotide within the sequence of SEQ ID NO: 12.
In all instances herein (including for probe polynucleotides below), homology based on sequence identity or sequence identity is measured over the entire length of the nonvariant/ reference sequence (e.g. SEQ ID NO: 1 or 2). This may also be referred to as global homology based on sequence identity or global sequence identity.
Methods of measuring homology based on sequence identity or identity are known in the art. For example the UWGCG Package provides the BESTFIT program which can be used to calculate homology or identity (e.g. used on its default settings) (Devereux et al (1984) Nucleic Acids Research 12, p387-395).
The PILEUP and BLAST algorithms can also be used to calculate identity, homology or line up sequences (typically on their default settings), for example as described in Altschul S.F. (1993) J Mol Evol 36:290-300; Altschul, S, F et al (1990) J Mol Biol 215:403-10.
Software for performing BLAST analysis is publicly available through the National Centre for Biotechnology Information (http://www.ncbi.nlm.nih.gov/). This algorithm involves first identifying high scoring sequence pair (HSPs) by identifying short words of length W in the query sequence that either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighbourhood word score threshold (Altschul et al, supra). These initial neighbourhood word hits act as seeds for initiating searches to find HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Extensions for the word hits in each direction are halted when: the cumulative alignment score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T and X determine the sensitivity and speed of the alignment. The BLAST program uses as defaults a word length (W) of 11, the BLOSUM62 scoring matrix (see Henikoff and Henikoff (1992) Proc. Natl. Acad. Sci. USA 89: 10915-10919) alignments (B) of 50, expectation (E) of 10, M = 5, N=4, and a comparison of both strands.
The BLAST algorithm performs a statistical analysis of the similarity between two sequences; see e.g., Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5787. One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a sequence is considered similar to another sequence if the smallest sum probability in comparison of the first sequence to the second sequence is less than about 1, preferably less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001.
A variant is typically identical to SEQ ID NO: 1 over at least about 20, at least about 21, at least about 22, at least about 23, at least about 24 or at least about 25 consecutive nucleotides. A variant is typically identical to SEQ ID NO: 2 over at least about 19, at least about 20, at least about 21, at least about 22, at least about 23 or at least about 24 consecutive nucleotides. A variant is typically identical to SEQ ID NO: 6 or 7 over at least about 17, at least about 18, at least about 19, at least about 20, at least about 21 or at least about 22 consecutive nucleotides. A variant is typically identical to SEQ ID NO: 11 over at least about 15, at least about 16, at least about 17, at least about 18, at least about 19 or at least about 20 consecutive nucleotides. A variant is typically identical to SEQ ID NO: 12 over at least about 16, at least about 17, at least about 18, at least about 19, at least about 20 or at least about 21 consecutive nucleotides.
Each polynucleotide in the primer may be any length. The first forward primer (ORFlab gene) is preferably at least 20 nucleotides in length, such as 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides in length. The first reverse primer (ORFlab gene) is preferably at least 19 nucleotides in length, such as 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 or 29 nucleotides in length. The second forward and reverse primers (S gene) are preferably at least 17 nucleotides in length, such as 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 or 27 nucleotides in length. The third forward primer (M gene) is preferably at least 15 nucleotides in length, such as 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides in length. The third reverse primers (M gene) is preferably at least 16 nucleotides in length, such as 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or 26 nucleotides in length.
In the first pair of primers targeting the ORFlab gene, the forward primer preferably comprises or consists of the exact sequence shown in SEQ ID NO: 1. The reverse primer preferably comprises or consists of the exact sequence shown in SEQ ID NO: 2. The forward primer preferably is the exact sequence shown in SEQ ID NO: 1. The reverse primer preferably is the exact sequence shown in SEQ ID NO: 2. The forward primer preferably is DNA and comprises/consist of the exact sequence shown in SEQ ID NO: 1. The reverse primer preferably is DNA and comprises/consists of the exact sequence shown in SEQ ID NO: 2.
The first pair of primers may comprise any combination of primers discussed above. The first pair of primers may comprise a forward primer that comprises/consist of the exact sequence shown in SEQ ID NO: 1 and a reverse primer that comprises/consists of a variant of SEQ ID NO: 2 as discussed above. The first pair of primers may comprise a forward primer that comprises/consists of a variant of SEQ ID NO: 1 as discussed above and a reverse primer that comprises/consists of the exact sequence of SEQ ID NO: 2. The first pair of primers may comprise a forward primer that comprises/consists a variant of SEQ ID NO: 1 as discussed above and a reverse primer that comprises/consists of a variant of SEQ ID NO: 2 as discussed above.
The first pair of primers more preferably comprises a forward primer which comprises or consists of the exact sequence shown in SEQ ID NO: 1 and a reverse primer which comprises or consists of the exact sequence shown in SEQ ID NO: 2. The first pair of primers more preferably comprises a forward primer that is the exact sequence shown in SEQ ID NO: 1 and a reverse primer that is the exact sequence shown in SEQ ID NO: 2.
The first pair of primers most preferably comprises a forward primer that is DNA and comprises/ or consists of the exact sequence shown in SEQ ID NO: 1 and a reverse primer that is preferably DNA and comprises/ or consists of the exact sequence shown in SEQ ID NO: 2. In the second pair of primers targeting the S gene, the first primer preferably comprises or consists of the exact sequence shown in SEQ ID NO: 6. The reverse primer preferably comprises or consists of the exact sequence shown in SEQ ID NO: 7.
The forward primer preferably is the exact sequence shown in SEQ ID NO: 6. The reverse primer preferably is the exact sequence shown in SEQ ID NO: 7.
The forward primer preferably is DNA and comprises/consist of the exact sequence shown in SEQ ID NO: 6. The reverse primer preferably is DNA and comprises/consists of the exact sequence shown in SEQ ID NO: 7.
The second pair of primers may comprise any combination of primers discussed above. The second pair of primers may comprise a forward primer that comprises/consist of the exact sequence shown in SEQ ID NO: 6 and a reverse primer that comprises/consists of a variant of SEQ ID NO: 7 as discussed above. The second pair of primers may comprise a forward primer that comprises/consists of a variant of SEQ ID NO: 6 as discussed above and a reverse primer that comprises/consists of the exact sequence of SEQ ID NO: 7. The second pair of primers may comprise a forward primer that comprises/consists a variant of SEQ ID NO: 6 as discussed above and a reverse primer that comprises/consists of a variant of SEQ ID NO: 7 as discussed above.
The second pair of primers more preferably comprises a forward primer which comprises or consists of the exact sequence shown in SEQ ID NO: 6 and a reverse primer which comprises or consists of the exact sequence shown in SEQ ID NO: 7. The second pair of primers more preferably comprises a forward primer that is the exact sequence shown in SEQ ID NO: 6 and a reverse primer that is the exact sequence shown in SEQ ID NO: 7. The second pair of primers most preferably comprises a forward primer that is DNA and comprises/ or consists of the exact sequence shown in SEQ ID NO: 6 and a reverse primer that is preferably DNA and comprises/ or consists of the exact sequence shown in SEQ ID NO: 7.
In the third pair of primers targeting the M gene, the first primer preferably comprises or consists of the exact sequence shown in SEQ ID NO: 11. The reverse primer preferably comprises or consists of the exact sequence shown in SEQ ID NO: 12.
The forward primer preferably is the exact sequence shown in SEQ ID NO: 11. The reverse primer preferably is the exact sequence shown in SEQ ID NO: 12.
The forward primer preferably is DNA and comprises/consist of the exact sequence shown in SEQ ID NO: 11. The reverse primer preferably is DNA and comprises/consists of the exact sequence shown in SEQ ID NO: 12.
The third pair of primers may comprise any combination of primers discussed above. The third pair of primers may comprise a forward primer that comprises/consist of the exact sequence shown in SEQ ID NO: 11 and a reverse primer that comprises/consists of a variant of SEQ ID NO: 12 as discussed above. The third pair of primers may comprise a forward primer that comprises/consists of a variant of SEQ ID NO: 11 as discussed above and a reverse primer that comprises/consists of the exact sequence of SEQ ID NO: 12. The third pair of primers may comprise a forward primer that comprises/consists a variant of SEQ ID NO: 11 as discussed above and a reverse primer that comprises/consists of a variant of SEQ ID NO: 12 as discussed above.
The third pair of primers more preferably comprises a forward primer which comprises or consists of the exact sequence shown in SEQ ID NO: 11 and a reverse primer which comprises or consists of the exact sequence shown in SEQ ID NO: 12. The third pair of primers more preferably comprises a forward primer that is the exact sequence shown in SEQ ID NO: 11 and a reverse primer that is the exact sequence shown in SEQ ID NO: 12.
The third pair of primers most preferably comprises a forward primer that is DNA and comprises/ or consists of the exact sequence shown in SEQ ID NO: 11 and a reverse primer that is preferably DNA and comprises/ or consists of the exact sequence shown in SEQ ID NO: 12.
Primers having any of the sequences above may be manufactured using standard techniques. Custom primers having specific sequences are commercially available from various suppliers (such as Biolegio). cDNA amplicons of the invention The invention also provides three complementary DNA (cDNA) amplicons amplified from SARS-CoV-2 cDNA by the three pairs of primers of the invention.
The invention also provides first, second and third cDNA amplicons amplified from SARS- CoV-2 cDNA. The first cDNA amplicon comprises or consists of a sequence (a) to which the sequence shown in SEQ ID NO: 3 specifically hybridises and/or (b) which is complementary to the sequence shown in SEQ ID NO: 3. Specific hybridisation is defined above with reference to the primers of the invention. The first cDNA amplicon is preferably amplified from SARS-CoV-2 cDNA and comprises or consists of a sequence which is complementary to the sequence shown in SEQ ID NO: 3.
The second cDNA amplicon comprises or consists of a sequence (a) to which the sequence shown in SEQ ID NO: 8 specifically hybridises and/or (b) which is complementary to the sequence shown in SEQ ID NO: 8. Specific hybridisation is defined above with reference to the primers of the invention. The second cDNA amplicon is preferably amplified from SARS- CoV-2 cDNA and comprises or consists of a sequence which is complementary to the sequence shown in SEQ ID NO: 8.
The third cDNA amplicon comprises or consists of a sequence (a) to which the sequence shown in SEQ ID NO: 13 specifically hybridises and/or (b) which is complementary to the sequence shown in SEQ ID NO: 13. Specific hybridisation is defined above with reference to the primers of the invention. The second cDNA amplicon is preferably amplified from SARS- CoV-2 cDNA and comprises or consists of a sequence which is complementary to the sequence shown in SEQ ID NO: 13.
SARS-CoV-2 cDNA is cDNA produced by the reverse transcription of SARS-CoV-2 RNA. The sequence of SARS-CoV-2 cDNA is published and methods for reverse transcribing it are known in the art and discussed in the Example. Once the SARS-CoV-2 cDNA has been reversed transcribed, the pairs of primers of the invention may be used to amplify the cDNA amplicons of the invention using polymerase chain reaction (PCR). Methods for conducting PCR using primers are known in the art and discussed below and in the Example. A cDNA amplicon is amplified from SARS-CoV-2 cDNA if it comprises a portion/part of the SARS- CoV-2 cDNA. In other words, it comprises a portion/part of the sequence of the SARS-CoV- 2 cDNA.
The term "amplified" refers to the process of making multiple copies of the cDNA from a single polynucleotide or fewer polynucleotides. An amplicon is the cDNA that results from amplification. In the context of the invention, primers, such as the primers of the invention, may be used to amplify a part of the SARS-CoV-2 cDNA resulting in the shorter cDNA amplicons of the invention. These amplicons typically contain the cDNA target sequence being used in the context of the invention. The cDNA amplicon is typically a polynucleotide comprising nucleotides selected from dAMP, dTMP, dGMP or dCMP as discussed above. The cDNA may be double stranded or single stranded. The cDNA is preferably single stranded.
The first cDNA amplicon may be any length, such as from about 20 to about 150 nucleotides in the length, such as from about 25 to about 100, from about 30 to about 95 or from about 40 to about 90 nucleotides in length. The first cDNA amplicon is preferably 82 nucleotides in length. The second cDNA amplicon may be any length, such as from about 20 to about 200 nucleotides in the length, such as from about 25 to about 180, from about 30 to about 150 or from about 40 to about 120 nucleotides in length. The second cDNA amplicon is preferably 119 nucleotides in length. The third cDNA amplicon may be any length, such as from about 20 to about 150 nucleotides in the length, such as from about 25 to about 100, from about 30 to about 95 or from about 40 to about 90 nucleotides in length. The third cDNA amplicon is preferably 89 nucleotides in length.
The first cDNA amplicon preferably comprises or consists of the sequence shown in SEQ ID NO: 4. The first cDNA amplicon preferably comprises or consists of the sequence shown in SEQ ID NO: 5.
The second cDNA amplicon preferably comprises or consists of the sequence shown in SEQ ID NO: 9. The second cDNA amplicon preferably comprises or consists of the sequence shown in SEQ ID NO: 10.
The third cDNA amplicon preferably comprises or consists of the sequence shown in SEQ ID NO: 14. The third cDNA amplicon preferably comprises or consists of the sequence shown in SEQ ID NO: 15.
The cDNA amplicons may be isolated, substantially isolated, purified or substantially purified as discussed above. The cDNA amplicons are not naturally occurring.
Polynucleotide probes of the invention
The invention also provides three polynucleotide probes for determining the presence or absence of SARS-CoV-2 in a sample.
In one embodiment, the three polynucleotide probes specifically hybridise to the three cDNA amplicons of the invention. Each probe specifically hybridises to one of the cDNA amplicons of the invention. Each polynucleotide probe comprises or consists of a sequence which specifically hybridises to the corresponding cDNA amplicon of the invention. Each polynucleotide probe preferably comprises or consists of a sequence which specifically hybridises to a target sequence on the corresponding cDNA amplicon of the invention. The target sequence may be any length, such as from 17 to 32 nucleotides in length, such as 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or 32 nucleotides in length. Polynucleotides and specific hybridisation are defined above with reference to the primers of the invention. Each probe polynucleotide may be any of the polynucleotides discussed above. Each polynucleotide probe is preferably DNA. Each polynucleotide probe is preferably single stranded DNA. Each polynucleotide probe may be any length, such as from 17 to 32 nucleotides in length, such as 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or 32 nucleotides in length. Each polynucleotide probe is preferably the same length as the target sequence it detects. Each target sequence is preferably specific for SARS- CoV-2. Each target sequence is preferably found/present in SARS-Cov-2 cDNA but is not found in the RNA of any other virus or human virus. Each target sequence is preferably found/present in SARS-Cov-2 cDNA but is not found in the RNA of any other virus or human virus or in any human RNA. Each target sequence is preferably found/present in a cDNA amplicon of the invention. The ORFlab gene target sequence is preferably part of SEQ ID NO: 4. The ORFlab gene target sequence is preferably the sequence shown in SEQ ID NO: 5. The S gene target sequence is preferably part of SEQ ID NO: 9. The S gene target sequence is preferably the sequence shown in SEQ ID NO: 10. The M gene target sequence is preferably part of SEQ ID NO: 14. The M gene target sequence is preferably the sequence shown in SEQ ID NO: 15.
In another embodiment, the three polynucleotide probes comprise or consist of specific sequences of the invention. The first (ORFlab gene targeting) polynucleotide probe comprises or consists of the sequence shown in SEQ ID NO: 3 or a variant thereof having at least about 80% homology to SEQ ID NO: 3 based on sequence identity over its entire length. The second (S gene targeting) polynucleotide probe comprises or consists of the sequence shown in SEQ ID NO: 8 or a variant thereof having at least about 80% homology to SEQ ID NO: 8 based on sequence identity over its entire length. The third (M gene targeting) polynucleotide probe comprises or consists of the sequence shown in SEQ ID NO: 13 or a variant thereof having at least about 80% homology to SEQ ID NO: 13 based on sequence identity over its entire length. Polynucleotides are defined above with reference to the primers of the invention. Each probe polynucleotide may be any of the polynucleotides discussed above. Each polynucleotide probe is preferably DNA. Each polynucleotide probe is preferably single stranded DNA. Each polynucleotide probe may be any length. The first polynucleotide probe is preferably from 20 to 30 nucleotides in length, such as 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides in length. The second polynucleotide probe is preferably from 22 to 32 nucleotides in length, such as 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 or 32 nucleotides in length. The third polynucleotide probe is preferably from 20 to 30 nucleotides in length, such as 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides in length.
A variant sequence is a polynucleotide that has a nucleotide sequence which varies from that of SEQ ID NO: 3, 8 or 13 and which retains its ability to specifically hybridise to the target sequence of SEQ ID NO: 5, 10 or 15. Specific hybridisation is discussed above. The variant sequence must not recognise or hybridise to any cDNA sequence from any other virus.
The variant sequence may comprise any of the nucleotides discussed above, including the modified nucleotides. The variant sequence is typically the same length as SEQ ID NO: 3, 8 or 13, but may be longer or shorter. A variant of SEQ ID NO: 3 is preferably at least 20 nucleotides in length, such as 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides in length. A variant of SEQ ID NO: 8 is preferably at least 22 nucleotides in length, such as 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 or 32 nucleotides in length. A variant of SEQ ID NO: 13 is preferably at least 20 nucleotides in length, such as 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides in length.
Over the entire length of the sequence of SEQ ID NO: 3 or 13, a variant sequence is at least about 80%, at least about 83% or at least about 83.33% homologous to that sequence based on nucleotide identity. Over the entire length of the sequence of SEQ ID NO: 3 or 13, a variant sequence has at least about 80%, at least about 83% or at least about 83.33% identity to that sequence. Homology based on sequence identity and identity are interchangeable herein. This allows for variation, deletion, addition or a combination thereof of five nucleotides within the sequence of SEQ ID NO: 3 or 13.
Over the entire length of the sequence of SEQ ID NO: 3 or 13, a variant sequence is preferably at least about 84%, at least 86% or at least about 86.20% homologous to that sequence based on nucleotide identity. Over the entire length of the sequence of SEQ ID NO: 3 or 13, a variant sequence preferably has at least about 84%, at least 86% or at least about 86.20% identity to that sequence. This allows for variation, deletion, addition or a combination thereof of four nucleotides within the sequence of SEQ ID NO: 3 or 13.
Over the entire length of the sequence of SEQ ID NO: 3 or 13, a variant sequence is preferably at least about 88%, at least 89% or at least about 89.28% homologous to that sequence based on nucleotide identity. Over the entire length of the sequence of SEQ ID NO: 3 or 13, a variant sequence preferably has at least about 88%, at least 89% or at least about 89.28% identity to that sequence. This allows for variation, deletion, addition or a combination thereof of three nucleotides within the sequence of SEQ ID NO: 3 or 13.
Over the entire length of the sequence of SEQ ID NO: 3 or 13, a variant sequence is preferably at least about 92% or at least about 92.59% homologous to that sequence based on nucleotide identity. Over the entire length of the sequence of SEQ ID NO: 3 or 13, a variant sequence preferably has at least about 92% or at least about 92.59% identity to that sequence. This allows for variation, deletion, addition or a combination thereof of two nucleotides within the sequence of SEQ ID NO: 3 or 13. Over the entire length of the sequence of SEQ ID NO: 3 or 13, a variant sequence is preferably at least about 96% or at least about 96.15% homologous to that sequence based on nucleotide identity. Over the entire length of the sequence of SEQ ID NO: 3 or 13, a variant sequence preferably has at least about 96% or at least about 96.15% identity to that sequence. This allows for variation, deletion, addition or a combination thereof of one nucleotide within the sequence of SEQ ID NO: 3 or 13.
Over the entire length of the sequence of SEQ ID NO: 8, a variant sequence is at least about 80%, at least about 81.48%, at least about 84% or at least about 84.37% homologous to that sequence based on nucleotide identity. Over the entire length of the sequence of SEQ ID NO: 8, a variant sequence has at least about 80%, at least about 81.48%, at least about 84% or at least about 84.37% identity to that sequence. Homology based on sequence identity and identity are interchangeable herein. This allows for variation, deletion, addition or a combination thereof of five nucleotides within the sequence of SEQ ID NO: 8.
Over the entire length of the sequence of SEQ ID NO: 8, a variant sequence is preferably at least about 85%, at least about 85.18%, at least about 87% or at least about 87.09% homologous to that sequence based on nucleotide identity. Over the entire length of the sequence of SEQ ID NO: 8, a variant sequence preferably has at least about 85%, at least about 85.18%, at least about 87% or at least about 87.09% identity to that sequence. This allows for variation, deletion, addition or a combination thereof of four nucleotides within the sequence of SEQ ID NO: 8.
Over the entire length of the sequence of SEQ ID NO: 8, a variant sequence is preferably at least about 88%, at least about 88.88% or at least about 90% homologous to that sequence based on nucleotide identity. Over the entire length of the sequence of SEQ ID NO: 8, a variant sequence preferably has at least about 88%, at least about 88.88% or at least about 90% identity to that sequence. This allows for variation, deletion, addition or a combination thereof of three nucleotides within the sequence of SEQ ID NO: 8.
Over the entire length of the sequence of SEQ ID NO: 8, a variant sequence is preferably at least about 92%, at least about 92.59%, at least about 93% or at least about 93.10% homologous to that sequence based on nucleotide identity. Over the entire length of the sequence of SEQ ID NO: 8, a variant sequence preferably has at least about 92%, at least about 92.59%, at least about 93% or at least about 93.10% identity to that sequence. This allows for variation, deletion, addition or a combination thereof of two nucleotides within the sequence of SEQ ID NO: 8.
Over the entire length of the sequence of SEQ ID NO: 8, a variant sequence is preferably at least about 96%, at least about 96.29% or at least about 96.42% homologous to that sequence based on nucleotide identity. Over the entire length of the sequence of SEQ ID NO: 8, a variant sequence preferably has at least about 96%, at least about 96.29% or at least about 96.42% identity to that sequence. This allows for variation, deletion, addition or a combination thereof of one nucleotide within the sequence of SEQ ID NO: 8.
The first polynucleotide probe preferably comprises or consists of the sequence shown in SEQ ID NO: 3. The second polynucleotide probe preferably comprises or consists of the sequence shown in SEQ ID NO: 8. The third polynucleotide probe preferably comprises or consists of the sequence shown in SEQ ID NO: 13.
Each polynucleotide probe may be isolated, substantially isolated, purified or substantially purified as discussed above. Each polynucleotide probe is not naturally occurring.
Each polynucleotide probe is preferably DNA probe, a TaqMan probe, a molecular beacon or a scorpion probe. DNA probes hybridise to the, typically complementary, target sequence and then can be detected. TaqMan probes are known in the art and are polynucleotides that have a fluorescent dye attached to the 5' end and a quencher to the 3' end. The polymerase used in PCR cleaves hybridised probes freeing the fluorescent dye from quenching such it can be detected. Molecular beacon probes are known in the art and are similar to TaqMan probes except (rather than using cleavage to separate the dye from the quencher) hybridisation to the target sequence separates the dye from the quencher. Scorpion probes are known the art and are similar to molecular beacons except the 3' end also contains a sequence that is complementary to the extension product of the primer on the 5' end which opens the probe on hybridisation and allows the dye to be detected. Each polynucleotide probe is preferably a TaqMan probe.
The polynucleotide probes of the invention are preferably detectably-labelled. Suitable detectable labels are known in the art. The detectable labels are preferably fluorescent molecules or dyes, such as fluorescein derivatives. Suitable fluorescent molecules or dyes include, but are not limited to, 6-carboxyfluorescein (FAM), 6-Carboxyl-X-Rhodamine (ROX), 2'-chloro-7'phenyl-l,4-dichloro-6-carboxy-fluorescein (VIC®), Hexachloro- Fluorescein (HEX), tetrachlorofluorescein (TET) and ATTO 647N (ATTO647N in the Cy5 spectral range (ATTO-TEC GmbH, Germany)). A suitable quencher for use with these dyes in TaqMan, molecular beacon or scorpion probes is tetramethylrhodamine (TAMRA).
The detectable label is most preferably 6-carboxyfluorescein (FAM), ATTO 647N or 6- Carboxyl-X-Rhodamine (ROX). The first probe is preferably labelled with FAM. The second probe is preferably labelled with ATTO 647N. The third probe is preferably labelled with ROX. The first probe is preferably labelled with FAM, the second probe is preferably labelled with ATT and the third probe is preferably labelled with ROX.
Polynucleotide probes are also available from commercial sources (such as Biolegio). Target cDNA sequence
The invention also provides three target cDNA polynucleotides. The first (derived from the ORFlab gene) comprises or consists of a sequence (a) to which the sequence shown in SEQ ID NO: 3 specifically hybridises and/or (b) which is complementary to the sequence shown in SEQ ID NO: 3. The second (derived from the S gene) comprises or consists of a sequence (a) to which the sequence shown in SEQ ID NO: 8 specifically hybridises and/or (b) which is complementary to the sequence shown in SEQ ID NO: 8. The third (derived from the M gene) comprises or consists of a sequence (a) to which the sequence shown in SEQ ID NO: 13 specifically hybridises and/or (b) which is complementary to the sequence shown in SEQ ID NO: 13. Specific hybridisation is discussed above. The target cDNA may be any length. The first is typically 25 nucleotides in length but may be any of the lengths discussed above for the first probe. The second is typically 27 nucleotides in length but may be any of the lengths discussed above for the second probe. The third is typically 25 nucleotides in length but may be any of the lengths discussed above for the third probe.
The first target cDNA polynucleotide preferably comprises or consists of the sequence shown in SEQ ID NO: 5. The second target cDNA polynucleotide preferably comprises or consists of the sequence shown in SEQ ID NO: 10. The third target cDNA polynucleotide preferably comprises or consists of the sequence shown in SEQ ID NO: 15. Each target cDNA polynucleotide may be double stranded or single stranded. Each target cDNA polynucleotide is preferably single stranded. Each target cDNA polynucleotide may be isolated, substantially isolated, purified or substantially purified as discussed above. Each target cDNA polynucleotide is not naturally occurring.
Kits of the invention
The invention also provides a kit for determining the presence or absence of SARS-CoV-2 in a sample.
The kit comprises the three pairs of primers of the invention. The kit also comprises the three polynucleotide probes of the invention. Any of the primers and probes discussed above may be used.
The primers and probes may be isolated, substantially isolated, purified or substantially purified as discussed above.
The kit preferably further comprises three positive control polynucleotides comprising or consisting of three cDNA amplicons of the invention and/or three target cDNA polynucleotides of the invention. These may form the basis of a positive control for RT-PCR. Any of the cDNA amplicons and cDNA target sequences discussed above may be used. The kit preferably comprises a polynucleotide comprising or consisting of the sequence shown in SEQ ID NO: 4 or 5, a polynucleotide comprising or consisting of the sequence shown in SEQ ID NO: 9 or 10 and a polynucleotide comprising or consisting of the sequence shown in SEQ ID NO: 14 or 15. The cDNA polynucleotides may be isolated, substantially isolated, purified or substantially purified as discussed above. The kit preferably contains a negative control, such as DNAase-free and RNAase-free water.
The kit of the invention preferably further comprises a RNA Internal Extraction Control (IEC). The kit more preferably comprises a set of RNA IEC primers and probe. The RNA IEC primers and probe are preferably DNA. DNA primers and probes are discussed above. The set of RNA IEC primers and probe preferably comprises:
RNA IEC L.lactis Forward primer (SEQ ID NO: 16);
RNA IEC L.lactis Reverse primer (SEQ ID NO: 17); RNA IEC L.lactis Probe 1 (SEQ ID NO: 18); and
RNA IEC L.lactis Probe 2 (SEQ ID NO: 19).
The kit most preferably comprises the following components in the specified concentrations. The various components may be resuspended to generate the concentrations discussed below with reference to the methods of the invention.
Figure imgf000027_0001
Figure imgf000028_0001
The kit may additionally comprise one or more other reagents or instruments which enable any of the embodiments of the methods below to be carried out. Such reagents or instruments include, but are not limited to, one or more of the following: suitable buffer(s) (aqueous solutions), means to obtain a sample from a patient (such as a vessel or an instrument comprising a needle or a swab) or tubes in which quantitative reactions can be done. The kit may, optionally, comprise instructions to enable the kit to be used in the methods of the invention or details regarding which patients may be tested. The kit may further comprise any of the reagents required to conduct RT-PCR discussed below, including a reverse transcriptase and/or heat-stable DNA polymerase.
Method of determining the presence or absence of SARS-CoV-2 in a sample
The invention provides various methods of determining the presence or absence of SARS- CoV-2 in a sample. The methods are triplex methods targeting three genes in SARS-CoV-2, namely the ORFlab gene, the S gene and the M gene.
All of the methods comprise conducting a reverse-transcription polymerase chain reaction (RT-PCR) assay on the sample. Methods for conducting RT-PCR are well known in the art and any suitable conditions may be used. Reverse transcription involves the use of the enzyme reverse transcriptase to convert viral RNA into cDNA. In the context of the invention, PCR involves using the three pairs of primers to amplify three specific portions of the cDNA, namely the cDNA amplicons, which typically contains the target cDNAs of interest.
Methods for reverse transcription are well known in the art and reverse transcriptases are commercially available (e.g. Superscript® II reverse transcriptase (Invitrogen) and Affinity script (Agilent)). Methods typically construct cDNA from random cDNA hexamers included in the reaction mixture.
PCR involves amplifying a cDNA amplicon using a heat-stable DNA polymerase and three pairs of forward and reverse polynucleotide primers. Because the newly synthesized cDNA strands can subsequently serve as additional templates for the same primer sequences, successive rounds of primer annealing, strand elongation, and dissociation produce rapid and highly specific amplification of the desired sequence. Many polymerase chain methods are known to those of skill in the art and may be used in the methods of the invention. For example, cDNA can be subjected to 20 to 40 cycles of amplification in a thermocycler as follows: 95°C for 30 sec, 52° to 60°C for 1 min, and 72°C for 1 min, with a final extension step of 72°C for 5 min. For another example, cDNA can be subjected to 20 to 40 polymerase chain reaction cycles in a thermocycler at a denaturing temperature of 95°C for 30 sec, followed by varying annealing temperatures ranging from 54°C to 58°C for 1 min, an extension step at 70°C for 1 min, with a final extension step at 70°C for 5 min. Heat stable DNA polymerases are commercially available (such as GoTaq G2 (Promega)).
The RT-PCR assay may be conducted using the conditions set out in the Example. The reverse transcriptase is preferably Affinity script (Agilent). The DNA polymerase is preferably GoTaq G2 (Promega). Both enzymes are preferably present in OneStep Lyophilised Master Mix. The reaction conditions are preferably (1) one cycle of reverse transcription for 10 minutes at 55°C, (2) one cycle of initial denaturation and Taq activation for 2 minutes at 95°C and (3) 45 cycles of denaturation for 10 seconds at 95°C and annealing and extension for 60 seconds at 60°C.
The reverse transcriptase is preferably Luna® Reverse Transcriptase (NEB). The DNA polymerase is preferably GoTaq G2 (Promega). Both enzymes are preferably present in an Onestep Lyophilised Master Mix. The composition of Onestep Lyophilised Master Mix can be found in the Example.
The reaction mix is preferably lOul OneStep Lyophilised Master Mix resuspended in 525ul Oasig resuspension buffer, lul primer/probe mix of each of the three sets of probes and primers, and from 5ul to 500ul sample, preferably 5ul sample. The primers and probes of the invention are preferably used in the following concentrations:
Figure imgf000029_0001
Figure imgf000030_0001
The reaction mix is preferably lOul OneStep Lyophilised Master Mix resuspended in 525ul Oasig resuspension buffer, lul primer/probe mix of each of the three sets of probes and primers, lul diluted RIMA IEC, lul of IEC probes and primers (SEQ ID NOs: 16 to 19), and from 5ul to 500ul sample, preferably 5ul sample. The primers and probes of the invention and the RNA IEC primers and probes are preferably used in the following concentrations:
Figure imgf000030_0002
Figure imgf000031_0001
The reaction mix is most preferably lOpi of resuspended OneStep Lyophislised Master Mix, 2pl of resuspended primers & probes (including all three sets of probes and primers and IEC RIMA primers and probes) and 8pl of sample/DNase/RNase free water. The reaction conditions are preferably (1) one cycle of reverse transcription for 10 minutes at 55°C, (2) one cycle of initial denaturation and Taq activation for 2 minutes at 95°C and (3) 45 cycles of denaturation for 10 seconds at 95°C and annealing and extension for 60 seconds at 60°C.
The RT-PCR method is preferably real time RT-PCR. This method is known in the art. In one embodiment, the method comprises using three pairs of primers of the invention and detecting the cDNA amplicons amplified by the primers if present. The method may comprise using all three pairs of primers of the invention and detecting the cDNA amplicons amplified by the primers if present. In the context of the invention, using "all three" primers of the invention means using a pair of primers of the invention targeting the ORFlab gene, a pair of primers of the invention targeting the S gene and a pair of primers of the invention targeting the M gene. Any method may be used to detect the cDNA amplicons. For instance, the method may use SYBR Green to detect the cDNA amplicons. When the SYBR Green binds to the cDNA, it emits light and the intensity of the fluorescence increases as the cDNA amplicons accumulate. This technique is easy to use since designing of probes is not necessary given lack of specificity of its binding. The method preferably uses three polynucleotide probes of the invention which specifically hybridise to the cDNA amplicons. The probes may be any of those discussed above. The use of the probes allows the specific cDNA amplicons to be identified if present. The probes are preferably a TaqMan probes. Different targets can be detected (and different Cq values can be measured) by using different detectable labels on the three polynucleotide probes (so each label is specific for a particular probe/cDNA amplicon).
In another embodiment, the method comprises using three pairs of primers that amplify three cDNA amplicons of the invention. The first cDNA amplicon preferably comprises (or consists of) a sequence (a) to which the sequence shown in SEQ ID NO: 3 specifically hybridises and/or (b) which is complementary to the sequence shown in SEQ ID NO: 3, if present. The first cDNA amplicon may be any of those discussed above. The first cDNA amplicon preferably comprises or consists of SEQ ID NO: 4. The first cDNA amplicon preferably comprises or consists of SEQ ID NO: 5. The second cDNA amplicon preferably comprises (or consists of) a sequence (a) to which the sequence shown in SEQ ID NO: 8 specifically hybridises and/or (b) which is complementary to the sequence shown in SEQ ID NO: 8, if present. The cDNA amplicon may be any of those discussed above. The second cDNA amplicon preferably comprises or consists of SEQ ID NO: 9. The second cDNA amplicon preferably comprises or consists of SEQ ID NO: 10. The third cDNA amplicon preferably comprises (or consists of) a sequence (a) to which the sequence shown in SEQ ID NO: 13 specifically hybridises and/or (b) which is complementary to the sequence shown in SEQ ID NO: 13, if present. The cDNA amplicon may be any of those discussed above. The third cDNA amplicon preferably comprises or consists of SEQ ID NO: 14. The method also comprises using three polynucleotide probes of the invention. The first polynucleotide probe preferably comprises the sequence shown in SEQ ID NO: 3 or a variant thereof as defined above to detect the ORFlab gene cDNA amplicon if present. The first polynucleotide probe may be any of those discussed above. The second polynucleotide probe preferably comprises the sequence shown in SEQ ID NO: 8 or a variant thereof as defined above to detect the S gene cDNA amplicon, if present. The second polynucleotide probe may be any of those discussed above. The third polynucleotide probe preferably comprises the sequence shown in SEQ ID NO: 13 or a variant thereof as defined above to detect the M gene cDNA amplicon, if present. The third polynucleotide probe may be any of those discussed above.
The three probes are preferably TaqMan probes. The three polynucleotide probes specifically hybridise to three cDNA amplicons if present.
In another embodiment, the method comprises using a kit of the invention. The kit comprises three pairs of primers of the invention and three polynucleotide probes of the invention. This allows the three cDNA amplicons of the invention to be amplified and detected, if present. The kit may comprise any of the primers and probes discussed above. The first pair of primers preferably comprises a forward primer which comprises or consists of the sequence shown in SEQ ID NO: 1 and a reverse primer which comprises of consists of the sequence shown in SEQ ID NO: 2. The second pair of primers preferably comprises a forward primer which comprises or consists of the sequence shown in SEQ ID NO: 6 and a reverse primer which comprises of consists of the sequence shown in SEQ ID NO: 7. The third pair of primers preferably comprises a forward primer which comprises or consists of the sequence shown in SEQ ID NO: 11 and a reverse primer which comprises of consists of the sequence shown in SEQ ID NO: 12. The first polynucleotide probe preferably comprises or consists of the sequence shown in SEQ ID NO: 3. The second polynucleotide probe preferably comprises or consists of the sequence shown in SEQ ID NO: 8. The third polynucleotide probe preferably comprises or consists of the sequence shown in SEQ ID NO: 13.
In all embodiments, detection of all three cDNA amplicons (/.e. the presence of all three cDNA amplicons) indicates that the sample contains SARS-CoV-2 (/.e. indicates the presence of SARS-CoV-2 in the sample). A lack of detection of all three cDNA amplicons (/.e. the absence of all three cDNA amplicons) indicates that the sample does not contain SARS-CoV-2 (/.e. indicates the absence of SARS-CoV-2 from the sample). Detection of multiple copies of all three cDNA amplicons (/.e. the presence of multiple copies of all three cDNA amplicons) indicates that the sample contains SARS-CoV-2 (/.e. indicates the presence of SARS-CoV-2 in the sample). A lack of detection of multiple copies of all three cDNA amplicons (/.e. the absence of multiple copies of all three cDNA amplicons) indicates that the sample does not contain SARS-CoV-2 (/.e. indicates the absence of SARS-CoV-2 from the sample). In these contexts, "all three cDNA amplicons" refers to the three cDNA amplicons of the invention (one derived from the ORFlab gene, one from the S gene and one from the M gene).
In all embodiments, detection of one or two of the three cDNA amplicons (/.e. the presence of one or two of the cDNA amplicons) but not the other cDNA amplicon(s) (/.e. the absence of the other cDNA amplicon(s)) indicates that the method should be repeated to confirm the presence or the absence of SARS-CoV-2 in the sample. Detection of multiple copies of one or two of the three cDNA amplicons (/.e. the presence of multiple copies of one or two of the cDNA amplicons) but not the other(s) (/.e. the absence of multiple copies of the other cDNA amplicon(s)) indicates that the method should be repeated to confirm the presence or the absence of SARS-CoV-2 in the sample.
In all embodiments, the method preferably comprises (a) reverse transcribing any RNA in the sample, (b) contacting any cDNA produced in (a) with the three pairs of primers under conditions which allow the primers to amplify their cDNA amplicons and (c) determining the presence or absence of the cDNA amplicons. Step (c) preferably uses three polynucleotide probes as described above.
The RT-PCR assay is preferably a one-step RT-PCR assay conducted in one tube or vessel. Such one-step reactions are routine in the art.
In all the embodiments, the method preferably does not comprise extracting RNA from the sample before conducting the RT-PCR assay. In some instances, the methods may comprise extracting any RNA from the sample before conducting the RT-PCR assay. Suitable kits for doing this are commercial available (such as QIAamp® virus RNA mini kit (Qiagen)). Magnetic bead kits for RNA extraction are also well known in the art, such as Automated extraction system GenoXtract® from HAIN Lifescience GmbH (Brucker) using GXT DNA/RNA Extraction kit (Catalogue no: 12.01.02, 96 samples).
The sample may be any sample. The invention is typically carried out on a sample that is known to contain or suspected to contain SARS-CoV-2. Alternatively, the invention may be carried out on any sample whose SARS-CoV-2 status is unknown to confirm the presence or absence of SARS-CoV-2. The sample may be a biological sample. The sample may be obtained from or extracted from any organism or microorganism. The organism or microorganism may be archaeal, prokaryotic or eukaryotic and typically belongs to one of the five kingdoms: plantae, animalia, fungi, monera and protista. The sample may be obtained from or extracted from any virus. Typically, the sample is human in origin, but alternatively it may be from another mammal animal such as from commercially farmed animals such as horses, cattle, sheep, fish, chickens or pigs or may alternatively be pets such as cats or dogs. Alternatively, the sample may be of plant origin, such as a sample obtained from a commercial crop, such as a cereal, legume, fruit or vegetable, for example wheat, barley, oats, canola, maize, soya, rice, rhubarb, bananas, apples, tomatoes, potatoes, grapes, tobacco, beans, lentils, sugar cane, cocoa, cotton. The sample may be derived from human or animal food.
The sample is preferably a fluid sample. The sample typically comprises a body fluid of the patient. The sample may be urine, lymph, saliva, mucus, amniotic fluid, blood, plasma or serum. The sample is preferably a nasopharyngeal sample, a saliva sample or a blood sample.
The sample may be a non-biological sample. Any non-biological sample can be tested. The non-biological sample is preferably a fluid sample. Example of non-biological samples include, but are not limited to, surgical fluids, water such as drinking water, sea water or river water, reagents for laboratory tests and wet swabs of surfaces or materials.
The sample is typically processed prior to being used in the invention, for example by centrifugation or by passage through a membrane that filters out unwanted molecules or cells, such as red blood cells. The sample may be tested immediately upon being taken. The sample may also be typically stored prior to assay, preferably below -70°C.
Methods of measuring the SARS-CoV-2 titre
The invention also provides methods for measuring the SARS-CoV-2 titre in a sample. The method comprises conducting any of the methods discussed above. Any of the methods for determining the presence or absence of SARS-CoV-2 in a sample as discussed above may be used. The quantitative method uses real time RT-PCR. If SARS-CoV-2 is present, the method comprises evaluating the cycle quantification (Cq) value and thereby measuring the SARS-CoV-2 titre. The method preferably comprises evaluating the Cq value against standard Cq values generated from standard dilution curves.
In real time RT-PCR, the threshold line is the level of detection or the point at which a reaction reaches a fluorescent intensity above background levels. The threshold level is preferably 10% of the end point fluorescence. Autocalling may be used to set the threshold level.
The Cq is the PCR cycle number at which the sample reaction curve intersects the threshold line. This value indicates how many cycles it took to detect a real signal from the sample. Real-Time PCR machines typically calculate the Cq value for each of sample.
As is clear from the Example, the Cq value for the triplex assay changes depending on the amount of SARS-CoV-2 present in the sample. These standard curves allow the amount of SARS-CoV-2 to be measured in sample. In other words, the Cq is predictive of the amount of the SARS-CoV-2 in the sample. The following discussion concerning preferred Cq values applies to all three targets (ORFlab, S gene and M gene).
In a preferred method, a Cq value of from about 20.00 to about 27.99, preferably from about 21.94 to about 23.00, indicates the sample contains about 2xl03 virions/pl, a Cq value of from about 28.00 to about 31.99, preferably from about 28.64 to about 29.33, indicates the sample contains about 20 virions/pl, a Cq value of from about 32.00 to about 32.99, preferably from about 32.55 to about 32.90, indicates the sample contains about 2 virions/pl, and a Cq value of above 33.00, or above preferably 33.07 or above, indicates the sample contains 2 virions/pl.
In all cases, the virions are SARS-CoV-2 virions. All of the Cq values above are preferably mean Cq values derived from multiple experiments. The Cq values are typically expressed to two significant figures. In all concentration cases in this paragraph, the sample refers to the (undiluted) sample that is added to (and diluted by) the RT-PCR assay mixture. The concentrations do not relate to the final concentration of the virions in the diluted RT-PCR assay mixture. If the sample, for instance from a patient, is diluted or concentrated before it is used in the RT-PCR assay, the dilution factor or concentration factor needs to be taken into account when calculating the amount of SARS-CoV-2 present in the sample.
The invention also provides a method for differentiating between high and low SARS-CoV-2 titres in a sample. The method comprises conducting any of the methods discussed above. Any of the methods for determining the presence or absence of SARS-CoV-2 in a sample as discussed above may be used. The method uses real time RT-PCR. If SARS-CoV-2 is present, the method comprises evaluating the cycle quantification (Cq) value and thereby determining whether the sample has a low or high SARS-CoV-2 titre. Cq measurement and evaluation is discussed above.
As is clear from the Example, the Cq value for the triplex assay changes depending on the amount of SARS-CoV-2 in a sample. These standard curves allow the amount of SARS-CoV- 2 to be measured in a sample. In other words, the Cq is predictive of the amount of the SARS-CoV-2 in the sample.
In a preferred method, a low SARS-CoV-2 titre is about 20 virions/pl or lower and a high SARS-CoV-2 titre is about 2xl03 virions/pl or higher. In this method, a Cq value of from about 28.00 or 28.64 or above indicates the sample has a low SARS-CoV-2 titre and a Cq value of about 27.99 or about 23.00 or lower indicates the sample has a high SARS-CoV-2 titre.
In a preferred method, a low SARS-CoV-2 titre is about 2 virions/pl or lower and a high SARS-CoV-2 titre is about 2xl03 virions/pl or higher. In this method, a Cq value of about 32.00 or about 32.55 or above indicates the sample has a low SARS-CoV-2 titre and a Cq value of about 27.99 or about 23.00 or lower indicates the sample has a high SARS-CoV-2 titre.
In a preferred method, a low SARS-CoV-2 titre is about 1 virions/pl or lower and a high SARS-CoV-2 titre is about 2xl03 virions/pl or higher. In this method, a Cq value of about 33.00 or about 32.07 or above indicates the sample has a low SARS-CoV-2 titre and a Cq value of about 27.99 or about 23.00 or lower indicates the sample has a high SARS-CoV-2 titre.
In all cases, the virions are SARS-CoV-2 virions. All of the Cq values above are preferably mean Cq values derived from multiple experiments. The Cq values are typically expressed to two significant figures.
Method of diagnosis of the invention
The invention also provides a method of determining whether or not a patient is infected with SARS-CoV-2. The invention therefore relates to the diagnosis of SARS-CoV-2 infection. The diagnostic method of the invention may be carried out in conjunction with other assays or genetic tests. The method comprises conducting a method of the invention for determining the presence or absence of SARS-CoV-2 on a sample from the patient. Any of the methods discussed above may be used. The presence of SARS-CoV-2 in the sample indicates the presence of SARS-CoV-2 in the patient. The presence of SARS-CoV-2 in the sample indicates the patient is infected with SARS-CoV-2. The absence of SARS-CoV-2 from the sample typically indicates the absence of SARS-CoV-2 in the patient. The absence of SARS-CoV-2 from the sample typically indicates the patient is not infected with SARS- CoV-2. The absence of SARS-CoV-2 from the sample may indicate that the particular sample from the patient does not contain SARS-CoV-2 and does not necessarily mean the patient is not infected. The diagnostic method preferably uses a nasopharyngeal sample or a saliva sample. The absence of SARS-CoV-2 from these samples does typically indicate the patient is not infected with SARS-CoV-2.
The invention also provides a method of measuring the titre of SARS-CoV-2 in a patient. The invention also provides a method for differentiating between high and low SARS-CoV-2 titres in a patient. These methods comprise conducting the quantitative method of the invention for measuring the titre of SARS-CoV-2 or for differentiating between high and low SARS-CoV-2 titres on a sample from the patient. Low SARS-CoV-2 titres, high SARS-CoV-2 titres and methods for measuring them are discussed above. Any of these may be used on a sample from the patient.
The quantitative method preferably uses a nasopharyngeal sample or a saliva sample.
The diagnostic and quantitative methods preferably comprise taking a sample from the patient before conducting the RT-PCR assay.
Typically, the patient displays the symptoms of SARS-CoV-2, i.e. the patient is known or expected to be infected with SARS-CoV-2. The patient may be asymptomatic, i.e. the patient's SARS-CoV-2 status is unknown or the patient is expected not to be infected with SARS-CoV-2. The patient may be susceptible to, or at risk from, infection with SARS-CoV- 2. The patient is may have underlying health conditions which make infection with SARS- CoV-2 particularly serious.
The patient is generally a human patient. The patient may be a fetus, a newborn, an infant, a juvenile or an adult.
Therapeutic methods of the invention
The invention provides a method of treating SARS-CoV-2 in a patient identified as being infected with SARS-CoV-2 using a method of the invention. The method comprises administering to the patient a therapeutically effective amount of an anti-SARS-CoV-2 treatment. The invention also provides a method of treating SARS-CoV-2 in a patient. The method comprises (a) identifying the patient as being infected with SARS-CoV-2 using a method of the invention and (b) administering to the patient a therapeutically effective amount of an anti-SARS-CoV-2 treatment.
The invention also provides a substance or composition for use in a method of treating SARS-CoV-2 in a patient identified as being infected with SARS-CoV-2 using a method of the invention. The invention also provides a substance or composition for use in a method of treating SARS-CoV-2 in a patient, wherein the method comprises (a) identifying the patient as being infected with SARS-CoV-2 using a method of the invention and (b) administering the substance of composition to the patient. The substance or composition is preferably an anti-SARS-CoV-2 substance or composition.
The invention also provides a method of treating SARS-CoV-2 in a patient identified as having a high SARS-CoV-2 titre using a method of the invention. The method comprises administering to the patient a therapeutically effective amount of an anti-SARS-CoV-2 treatment. The invention also provides a method of treating SARS-CoV-2 in a patient comprising (a) identifying the patient as having a high SARS-CoV-2 titre using a method of the invention and (b) administering to the patient a therapeutically effective amount of an anti-SARS-CoV-2 treatment.
In a preferred method, a high SARS-CoV-2 titre is about 2xl03 virions/pl or higher. In this method, a Cq value of 27.99 or 23.00 or lower indicates the sample has a high SARS-CoV-2 titre.
The invention also provides a substance or composition for use in a method of treating SARS-CoV-2 in a patient identified as being having a high SARS-CoV-2 titre using a method of the invention. The invention also provides a substance or composition for use in a method of treating SARS-CoV-2 in a patient, wherein the method comprises (a) identifying the patient as being having a high SARS-CoV-2 titre using a method of the invention and (b) administering the substance or composition to the patient. The substance or composition is preferably an anti-SARS-CoV-2 substance or composition
Any treatment, substance or composition may be used in the invention. Suitable anti- SARS-CoV-2 treatments, substances and compositions are well known. The anti-SARS-CoV- 2 treatment, substance or composition may inhibit viral entry into cells by inhibiting ACE2 receptors, such as ACE2 receptor antibodies or arbidol, or inhibiting TMPRSS2, such as camosat mesylate. The anti-SARS-CoV-2 treatment, substance or composition may inhibit 3-chymotrypsin-like protease, such as lopinavir or darunavir. The anti-SARS-CoV-2 treatment, substance or composition may inhibit viral replication by inhibiting viral RNA- dependent RNA polymerase (RdRP), such as ribavirin, remdesivir or favipiravir. The anti- SARS-CoV-2 treatment, substance or composition may be an anti-SARS-CoV-2 small interfering RNA (siRNA) designed to inhibit entry of the virus into cells and/or inhibit viral replication by targeting SARS-CoV-2 genes involved in these processes. Some patients infected with SARS-Cov-2, especially patients with a high SARS-Cov-2 titre, suffer serious immune responses, including cytokine storms. IL-6 has been shown to be partly responsible for such cytokine storms. The anti-SARS-CoV-2 treatment, substance or composition may be an anti-IL-6 therapy or antibody, such as tocilizumab or sarilumab. The anti-SARS-CoV-2 treatment, substance or composition may be any anti-inflammatory and inhibitor of immune responses.
The invention also provides a method of treating SARS-CoV-2 in a patient identified as having a high SARS-CoV-2 titre using a method of the invention. The method comprises administering to the patient a therapeutically effective amount of an anti-IL-6 therapy or antibody. The invention also provides a method of treating SARS-CoV-2 in a patient comprising (a) identifying the patient as having a high SARS-CoV-2 titre using a method of the invention and (b) administering to the patient a therapeutically effective amount of an anti-IL-6 therapy or antibody. High SARS-CoV-2 titres and methods of measuring them are discussed above.
The invention also provides an anti-IL-6 therapy or antibody for use in a method of treating SARS-CoV-2 in a patient identified as being having a high SARS-CoV-2 titre using a method of the invention. The invention also provides an anti-IL-6 therapy or antibody for use in a method of treating SARS-CoV-2 in a patient, wherein the method comprises (a) identifying the patient as being having a high SARS-CoV-2 titre using a method of the invention and (b) administering the an anti-IL-6 therapy or antibody to the patient.
The invention concerns administering to the patient a therapeutically effective mount of the treatment, substance or composition to the patient. A therapeutically effective amount is an amount which ameliorates one or more symptoms of the SARS-CoV-2 infection. A therapeutically effective amount is preferably a number which abolishes one or symptoms of the SARS-CoV-2 infection. A therapeutically effective amount may cure or abolish the SARS-CoV-2 infection. Suitable amounts are discussed in more detail below.
The composition of the invention may be administered to any suitable patient. Suitable patients are discussed above with reference to the diagnostic embodiments of the invention.
The invention may be used in combination with other means of, and substances for, treating the disease or disorder or providing pain relief. In some cases, the treatment, substance or composition may be used in combination with existing treatments for the SARS-CoV-2 infection including intensive care treatment and the use of ventilators. The treatment, substance or composition of the invention may be formulated using any suitable method. Formulation with standard pharmaceutically acceptable carriers and/or excipients may be carried out using routine methods in the pharmaceutical art. The exact nature of a formulation will depend upon several factors including the composition to be administered and the desired route of administration. Suitable types of formulation are fully described in Remington's Pharmaceutical Sciences, 19th Edition, Mack Publishing Company, Eastern Pennsylvania, USA.
The treatment, substance or composition may be administered by any route. Suitable routes include, but are not limited to, enteral or parenteral routes such as via buccal, anal, pulmonary, intravenous, intra-arterial, intramuscular, intraperitoneal, intraarticular, topical and other appropriate routes. If the lungs are being treated, the treatment, substance or composition is preferably administered by inhalation.
Pharmaceutical compositions may be prepared together with a physiologically acceptable carrier or diluent. The treatment, substance or composition may be mixed with an excipient which is pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients are, for example, water, saline, dextrose, glycerol, of the like and combinations thereof.
Liquid dispersions for oral administration may be syrups, emulsions or suspensions. The syrups may contain as carriers, for example, saccharose or saccharose with glycerine and/or mannitol and/or sorbitol.
Suspensions and emulsions may contain as carrier, for example a natural gum, agar, sodium alginate, pectin, methylcellulose, carboxymethylcellulose, or polyvinyl alcohol. The suspensions or solutions for intramuscular injections may contain, together with the active substance, a pharmaceutically acceptable carrier, e.g. sterile water, olive oil, ethyl oleate, glycols, e.g. propylene glycol, and if desired, a suitable amount of lidocaine hydrochloride.
Solutions for intravenous administration or infusion may contain as carrier, for example, sterile water or preferably they may be in the form of sterile, aqueous, isotonic saline solutions.
For suppositories, traditional binders and carriers may include, for example, polyalkylene glycols or triglycerides; such suppositories may be formed from mixtures containing the active ingredient in the range of 0.5% to 10%, preferably 1% to 2%.
Oral formulations include such normally employed excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like. These compositions typically take the form of solutions or suspensions and contain 10% to 95% of active ingredient, preferably 25% to 70%. Where the pharmaceutical composition is lyophilised, the lyophilised material may be reconstituted prior to administration, e.g. a suspension. Reconstitution is preferably effected in buffer.
In addition, if desired, the pharmaceutical compositions of the invention may contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, and/or adjuvants which enhance effectiveness. The composition preferably comprises human serum albumin.
One suitable carrier or diluents is Plasma-Lyte A®. This is a sterile, nonpyrogenic isotonic solution for intravenous administration. Each 100 mL contains 526 mg of Sodium Chloride, USP (NaCI); 502 mg of Sodium Gluconate (C6HllNaO7); 368 mg of Sodium Acetate Trihydrate, USP (C2H3NaO2*3H2O); 37 mg of Potassium Chloride, USP (KOI); and 30 mg of Magnesium Chloride, USP (MgCI2*6H2O). It contains no antimicrobial agents. The pH is adjusted with sodium hydroxide. The pH is 7.4 (6.5 to 8.0).
If the treatment, substance or composition is a siRNA, the most preferred pharmaceutically acceptable carrier or diluent is a pharmaceutically acceptable transfection reagent. A pharmaceutical acceptable transfection reagent is suitable for administration to patients. The pharmaceutical acceptable transfection reagent may be liposomes, preferably cationic liposomes, polymers, preferably cationic polymers, and dendrimers. The pharmaceutical acceptable transfection reagent is more preferably a pharmaceutically acceptable PEI transfection reagent, such as a linear PEI transfection reagent. The most preferred pharmaceutical acceptable transfection reagent is GMP in v/ o-jetPEI®.
The composition is administered in a manner compatible with the dosage formulation and in such amount will be therapeutically effective. The quantity to be administered depends on the subject to be treated, capacity of the patient's immune system and the degree of treatment desired. Precise amounts required to be administered may depend on the judgement of the practitioner and may be peculiar to each patient.
Any suitable dose of the treatment, substance or composition may be administered to a patient. The dose may be determined according to various parameters, especially according to the substance used; the age, weight and condition of the patient to be treated; the route of administration; and the required regimen. Again, a physician will be able to determine the required route of administration and dosage for any particular patient. A typical daily dose is from about 0.01 to 50 mg per kg of body weight, according to the activity of the specific substance, the age, weight and conditions of the subject to be treated and the frequency and route of administration. Preferably, daily dosage levels are from 5 mg to 2 g. For example, from about 0.01 to about 50mg per kg of patient of sRNA may administered, such as from about 0.05 to about 40, from about 0.1 to about 30, from about 0.5 to about 20, from about 1 to about 10 or from about 2 to about 5 mg per kg. At least about 0.01 mg per kg of patient may administered, such as at least about 0.05, at least about 0.1, at least about 0.5, at least about 1, at least about 2, at least about 5, at least about 10, at least about 20, at least at least about 30 or at least about 40 mg per kg.
These doses may be provided as a single dose or may be provided as multiple doses, for example taken at regular intervals, for example 2, 3 or 4 doses administered daily. Other suitable regular intervals include, but are not limited to, every day, every week, every fortnight or every month.
The following Example illustrates the invention.
EXAMPLE
Objective
The objective of this report is to select optimum primers and probe concentration for the COVID-19 3G kit. The COVID-19 3G kit has the following targets:
• ORFlab (FAM-probe)
• S gene (ATTO-probe)
• M-gene (ROX-probe)
• L.lactis Internal Extraction Control (HEX-probe)
Materials
Reagents and PCR instruments serial numbers are listed in Tables 1 and 2:
Table 1: Reagents batch numbers
Figure imgf000042_0001
Figure imgf000043_0001
Table 2: PCR Instruments serial numbers
Figure imgf000043_0002
Figure imgf000044_0001
Method
SARS-CoV-2 whole genome RIMA was diluted down to 2 x 103 copies/|jl and 1 copy/pl and was tested with different combinations of COVID-19 CE (ORFlab), M-gene (SARS_M_V2) and S gene (S-gene_V3) primer and probe concentrations multiplexed with L.lactis Internal Extraction Control (IEC) primers and probes. Tested primers and probes concentrations (final PCR concentrations) are shown in table below:
Table 3: Tested primers and probe concentrations for each target
Figure imgf000044_0002
Each combination was tested with OneStep Lyophilised Master Mix following recipe in Table:
Table 4: Reaction mix components
Figure imgf000045_0001
Each combination was tested with a SARS-CoV-2 whole genome RIMA standard curve (tested dilutions are shown in Table 5):
Table 5: Tested SARS-CoV-2 whole genome RNA dilutions
Figure imgf000045_0002
Table 6: Onestep Lyophilised Master Mix
Figure imgf000045_0003
Figure imgf000046_0001
Results
The following concentrations produced optimal results (see Table 7):
Table 7: Optimum primers and probe concentrations for each target
Figure imgf000046_0002
Results are shown in Table 8: Table 8: Cq values, average Cq values, Cq values standard deviation, standard curve efficiency and R2 for primers and probe concentrations in Table 7
Figure imgf000047_0001
46
SUBSTITUTE SHEET (RULE 26) The results are shown in Figures 1 to 4.
Conclusion
The COVID-19 3G has the same setup and sample-to-reaction ratio as the COVID-19 CE kit and the COVID-19 2G kit, which is shown below:
• lOpI of resuspended OneStep Lyophilised Master Mix
• 2pl of resuspended primers & probes (including IEC RNA primers & probes)
• 8pl of sample/DNase/RNase free water/Positive Control Template (PCT)
To maintain the manufacturing process consistent with the COVID-19 CE and the COVID-19 2G CE kits, primers and probes will be resuspended with the double of total number of reactions volume (e.g., if the total number of reactions is 110 per tube, then the resuspension volume should be 220pl).
Manufacturing concentrations are listed below:
• COVID-19 CE (ORFlab) Forward primer: 2 pmol/pl
• COVID-19 CE (ORFlab) Reverse primer: 2 pmol/pl
• COVID-19 CE (ORFlab) Probe (FAM): 2 pmol/pl
• SARS_M_V2 Forward primer: 6 pmol/pl
• SARS_M_V2 Reverse primer: 6 pmol/pl
• SARS_M_V2 Probe (ROX): 3 pmol/pl
• S gene_V3 Forward primer: 2.50 pmol/pl
• S gene_V3 Reverse primer: 2.50 pmol/pl
• S gene_V3 Probe (ATTO): 2 pmol/pl
• L.lactis Forward primer: 0.75 pmol/pl
• L.lactis Reverse primer: 0.75 pmol/pl
• L.lactis Probe 1 (HEX): 1 pmol/pl
• L.lactis Probe 2 (HEX): 1 pmol/pl
Table 9 shows manufacturing and PCR reaction concentrations: Table 9: Manufacture and PCR reaction primers and probes concentrations
Figure imgf000049_0001
Figure imgf000050_0001
SEQUENCE LISTING
SEQ ID NO : 1
G C GAAGAAG C T AT AAGAC AT GT AC G
SEQ ID NO : 2
GTACCAACAGCTTCTCTAGTAGCA
SEQ ID NO : 3
C C T C GACAT C GAAG C C AAT C CAT G C
SEQ ID NO : 4
GCGAAGAAGCTATAAGACATGTACGTGCATGGATTGGCTTCGATGTCGAGGGGTGTCATGCTACTAG AGAAGCTGTTGGTAC
SEQ ID NO : 5 (underlined section in SEQ ID NO : 4)
GCATGGATTGGCTTCGATGTCGAGG
SEQ ID NO : 6
GT GGT CAACCAAAAT GCACAAG
SEQ ID NO : 7
TCAGCCTCAACTTTGTCAAGAC
SEQ ID NO : 8
ACACGCTTGTTAAACAACTTAGCTCCA
SEQ ID NO : 9 GT GGT CAACCAAAAT GCACAAGCTTTAAACACGCTTGTTAAACAACTTAGCTCCAATTT T GGT GCAA TTTCAAGTGTTTTAAATGATATCCTTTCACGTCTTGACAAAGTTGAGGCTGA
SEQ ID NO : 10 (reverse complement of the underlined section in SEQ ID NO : 9)
TGGAGCTAAGTTGTTTAACAAGCGTGT
SEQ ID NO : 11
GTGTAGCAGGTGAYTCAGGT
SEQ ID NO : 12
GTCACTGCTACTGGAATGGTM
SEQ ID NO : 13 CCAATCCTGTAGCGACTGTATGCAG
SEQ ID NO : 14
GT GTAGCAGGT GACT CAGGT TT T GCTGCATACAGTCGCTACAGGATTGGCAACTATAAATTAAACAC
AGAC CAT T C C AG TAG C AGT GAC
SEQ ID NO : 15 (underlined section in SEQ ID NO : 14)
CTGCATACAGTCGCTACAGGATTGG
SEQ ID NO : 16
GAGGGGACGGAACTTTTGAC
SEQ ID NO : 17
TGCTTGTGACCACGAGAGT
SEQ ID NO : 18
TTCTTTCCACGGGTTGCCATATTGTAATG
SEQ ID NO : 19
T T CAT GOAT GAT T CCAAAGGCAT TACAATAT G

Claims

CLAIMS Three pairs of primers for determining the presence or absence of SARS-CoV-2 in a sample, wherein (a) in the first pair the forward primer comprises a polynucleotide having the sequence shown in SEQ ID NO: 1 or a variant thereof having at least about 80% homology to SEQ ID NO: 1 based on sequence identity over its entire length and the reverse primer comprises a polynucleotide having the sequence shown in SEQ ID NO: 2 or a variant thereof having at least about 75% homology to SEQ ID NO: 2 based on sequence identity over its entire length; (b) in the second pair the forward primer comprises a polynucleotide having the sequence shown in SEQ ID NO: 6 or a variant thereof having at least about 75% homology to SEQ ID NO: 6 based on sequence identity over its entire length and the reverse primer comprises a polynucleotide having the sequence shown in SEQ ID NO: 7 or a variant thereof having at least about 75% homology to SEQ ID NO: 7 based on sequence identity over its entire length; and (c) in the third pair the forward primer comprises a polynucleotide having the sequence shown in SEQ ID NO: 11 or a variant thereof having at least about 75% homology to SEQ ID NO: 11 based on sequence identity over its entire length and the reverse primer comprises a polynucleotide having the sequence shown in SEQ ID NO: 12 or a variant thereof having at least about 75% homology to SEQ ID NO: 2 based on sequence identity over its entire length. Three pairs of primers according to claim 1, wherein in the first pair the forward primer comprises a polynucleotide having the sequence shown in SEQ ID NO: 1 and/or the reverse primer comprises a polynucleotide having the sequence shown in SEQ ID NO: 2. Three pairs of primers according to claim 1 or 2, wherein in the second pair the forward primer comprises a polynucleotide having the sequence shown in SEQ ID NO: 6 and/or the reverse primer comprises a polynucleotide having the sequence shown in SEQ ID NO: 7. Three pairs of primers according to any one of claims 1 to 3, wherein in the third pair the forward primer comprises a polynucleotide having the sequence shown in SEQ ID NO: 11 and/or the reverse primer comprises a polynucleotide having the sequence shown in SEQ ID NO: 12. Three complementary DNA (cDNA) amplicons amplified from SARS-CoV-2 cDNA by three pairs of primers according to any one of claims 1 to 4. Three polynucleotide probes which specifically hybridise to three cDNA amplicons according to claim 5. Three polynucleotide probes for determining the presence or absence of SARS-CoV-2 in a sample, wherein (a) the first polynucleotide probe comprises the sequence shown in SEQ 52 ID NO: 3 or a variant thereof having at least about 80% homology to SEQ ID NO: 3 based on sequence identity over its entire length, (b) the second polynucleotide probe comprises the sequence shown in SEQ ID NO: 8 or a variant thereof having at least about 80% homology to SEQ ID NO: 8 based on sequence identity over its entire length and (c) the third polynucleotide probe comprises the sequence shown in SEQ ID NO: 13 or a variant thereof having at least about 80% homology to SEQ ID NO: 13 based on sequence identity over its entire length. Three polynucleotide probes according to claim 7, wherein the first polynucleotide probe comprises the sequence shown in SEQ ID NO: 3. Three polynucleotide probes according to claim 7 or 8, wherein the second polynucleotide probe comprises the sequence shown in SEQ ID NO: 8. . Three polynucleotide probes according to any one of claims 7 to 9, wherein the third polynucleotide probe comprises the sequence shown in SEQ ID NO: 13. . Three polynucleotide probes according to any one of claims 8 or 10, wherein the two probes are DNA probes, a TaqMan probes, molecular beacons or a scorpion probes. . Three polynucleotide probes according to any one of claims 8 to 10, wherein the two polynucleotide probes are detectably-labelled. . Three polynucleotide probes according to claim 12, wherein the detectable label is a fluorescent molecule or dye. . Three cDNA amplicons amplified from SARS-CoV-2 cDNA wherein (a) the first cDNA amplicon comprises a sequence to which the sequence shown in SEQ ID NO: 3 specifically hybridises and/or which is complementary to the sequence shown in SEQ ID NO: 3, (b) the second cDNA amplicon comprises a sequence to which the sequence shown in SEQ ID NO: 8 specifically hybridises and/or which is complementary to the sequence shown in SEQ ID NO: 8 and (c) the third cDNA amplicon comprises a sequence to which the sequence shown in SEQ ID NO: 13 specifically hybridises and/or which is complementary to the sequence shown in SEQ ID NO: 13. . Three target cDNA polynucleotides, wherein (a) the first target cDNA polynucleotide comprises a sequence to which the sequence shown in SEQ ID NO: 3 specifically hybridises and/or which is complementary to the sequence shown in SEQ ID NO: 3, (b) the second target cDNA polynucleotide comprises a sequence to which the sequence shown in SEQ ID NO: 8 specifically hybridises and/or which is complementary to the sequence shown in SEQ ID NO: 8 and (c) the third target cDNA polynucleotide comprises
53 a sequence to which the sequence shown in SEQ ID NO: 13 specifically hybridises and/or which is complementary to the sequence shown in SEQ ID NO: 13. . A kit for determining the presence or absence of SARS-CoV-2 in a sample, comprising (a) three pairs of primers according to any one of claims 1 to 4 and (b) three polynucleotide probes according to any one of claims 6 to 13. . A kit according to claim 16, wherein the kit further comprises (a) three positive control polynucleotides comprising three cDNA amplicons according to claim 14 and/or three target cDNA polynucleotides according to claim 15 and/or (b) a negative control. . A method of determining the presence or absence of SARS-CoV-2 in a sample, wherein the method comprises conducting a reverse-transcription polymerase chain reaction (RT- PCR) assay on the sample using three pairs of primers according to any one of claims 1 to 4 and detecting the cDNA amplicons amplified by the primers, if present, and thereby determining the presence or absence of SARS-CoV-2 in the sample. . A method of determining the presence or absence of SARS-CoV-2 in a sample, wherein the method comprises conducting a reverse-transcription polymerase chain reaction (RT- PCR) assay on the sample using three pairs of primers that amplifies three cDNA amplicons according to claim 5 or 14 and three polynucleotide probes according to any one of claims 6 to 13 and thereby determining the presence or absence of SARS-CoV-2 in the sample. . A method of determining the presence or absence of SARS-CoV-2 in a sample, wherein the method comprises conducting a reverse-transcription polymerase chain reaction (RT- PCR) assay on the sample using a kit according to claim 16 or 17 and thereby determining the presence or absence of SARS-CoV-2 in the sample. . A method according to any one of claims 16 to 18, wherein the method comprises (a) reverse transcribing any RNA in the sample, (b) contacting any cDNA produced in (a) with the three pairs of primers under conditions which allow the primers to amplify their cDNA amplicons and (c) determining the presence or absence of the cDNA amplicons. . A method according to any one of claims 18 to 21, wherein the RT-PCR assay is a one- step RT-PCR assay conducted in one tube or vessel. . A method according to any one of claims 18 to 22, wherein the method comprises extracting any RNA from the sample before conducting the RT-PCR assay. . A method according to any one of claims 18 to 23, wherein the sample is a human sample.
. A method according to any one of claims 18 to 24, wherein the sample is a nasopharyngeal sample, a saliva sample or a blood sample. . A method for measuring the SARS-CoV-2 titre in a sample, wherein the method comprises conducting a real time RT-PCR method according to any one of claims 18 to 25 and, if SARS-CoV-2 is present, evaluating the cycle quantification (Cq) value and thereby measuring the SARS-CoV-2 titre. . A method of determining whether or not a patient is infected with SARS-CoV-2, wherein the method comprises conducting a method according to any one of claims 18 to 25 on a sample from the patient and thereby determining whether or not a patient is infected with SARS-CoV-2. . A method of measuring the titre of SARS-CoV-2 in a patient, wherein the method comprises conducting a method according to claim 26 on a sample from the patient and thereby measuring the viral titre in the patient. . A method according to claim 27 or 28, wherein the method comprises taking a sample from the patient before conducting the RT-PCR assay. . A method of treating SARS-CoV-2 in a patient identified as being infected with SARS- CoV-2 using a method according to claim 27, comprising administering to the patient a therapeutically effective amount of an anti-SARS-CoV-2 treatment and thereby treating the SARS-CoV-2 infection in the patient. . A substance or composition for use in a method of treating SARS-CoV-2 in a patient identified as being infected with SARS-CoV-2 using a method according to claim 27. . A method of treating SARS-CoV-2 in a patient identified as having a high SARS-CoV-2 titre using a method according to claim 28, comprising administering to the patient a therapeutically effective amount of an anti-SARS-CoV-2 treatment and thereby treating the SARS-CoV-2 infection in the patient. . A substance or composition for use in a method of treating SARS-CoV-2 in a patient identified as being having a high SARS-CoV-2 titre using a method according to claim 28.
55
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