AU2021103139A4

AU2021103139A4 - Differential detection kit for common SARS-CoV-2 variants in COVID-19 patients

Info

Publication number: AU2021103139A4
Application number: AU2021103139A
Authority: AU
Inventors: Sorayya Ghasemi; Leila Mousavizadeh; Marcel Mozafari; Milint Neleptchenko Wintrasiri
Original assignee: Mousavizadeh Leila Dr; Ghasemi Sorayya Dr; Neleptchenko Wintrasiri Milint Mrs
Current assignee: Mousavizadeh Leila Dr; Ghasemi Sorayya Dr; Neleptchenko Wintrasiri Milint Mrs
Priority date: 2021-06-06
Filing date: 2021-06-06
Publication date: 2021-08-26
Anticipated expiration: 2029-06-06

Abstract

The invention relates to a differential detection kit for common SARS-CoV-2 variants in COVID-19 patients, the kit comprising a set of probes and primers specific for spike gene mutations, as well as to the nucleocapsid gene and the RNA-dependent RNA polymerase gene. Detection using real time PCR methods are disclosed using probes with different colors for different mutations.

Description

Title of the invention:

Differential detection kit for common SARS-CoV-2 variants

in COVID-19 patients

Inventors:

Marcel Reza Mozafari 1, Sorayya Ghasemi 2, Milint Neleptchenko Wintrasiri 3

, and Leila Mousavizadeh4

Affiliations of Inventors:

1. Australasian Nanoscience and Nanotechnology Initiative (ANNI), Monash University

LPO, Clayton, Victoria 3168, Australia. dr.m.r.mozafari@gmail.com

2. Cellular and Molecular Research Center, Basic Health Sciences Institute, Shahrekord

University of Medical Sciences, Shahrekord, Iran. sorayya.ghasemi@gmail.com

3. Supreme NanoBiotics Co. Ltd. and Supreme Pharmatech Co. Ltd., 399/90-95 Moo 13

Kingkaew Rd. Soi 25/1, T. Rachateva, A. Bangplee, Samutprakan 10540, Thailand.

milint@supremepharmatech.org

4. Department of Virus-host Interaction, Heinrich-Pette-Institut (HPI), Martinistrasse 52,

20251 Hamburg, Germany. lmosavi2007@yahoo.com

DESCRIPTION

[0001] The embodiments herein relate to a diagnostic kit for identifying virus

variants in COVID-19 patients.

[0002] The present invention more particularly relates to new types of probe

and primer designs for accurate identification of SARS-CoV-2 variants.

[0003] The mixture of primer-probe in this kit is designed based on the six

target gene, which targets the specific conserved sequence encoding the RdRp

(RNA-dependent RNA polymerase) region and the nucleocapsid protein N region

as well as detection of different variants based on specific mutations (N501Y,

L452R, D614G, P681H), which have been separated into two segments, such as

two vials.

BACKGROUND OF THE INVENTION

Technicalfield

[0004] Coronavirus is a parasitic organism containing single-stranded RNA,

of positive polarity, with the size of approximately 30 kilobases that replicates in

the cytoplasm of the host cells; being Human cells or animal cells. The 5' end of

the genome of Coronavirus has a capped structure and the 3' end contains a polyA

tail. This virus is enveloped and comprises, at its surface, peplomeric (spike)

structures known as spicules.

[0005] The N protein or nucleocapsid protein (45-50 kDa) that is the most

conserved among the coronavirus structural proteins is necessary for encapsulating

the genomic RNA and also for directing its incorporation into the virion. It is

known that this protein is also involved in the replication of the RNA.

[0006] When the host cell is infected, the open reading frame (ORF) situated

at the 5' of the viral genome is translated into a polyprotein that is cleaved by the

viral proteases, and consequently releases several nonstructural proteins such as the

RNA-dependent RNA polymerase and the ATPase helicase (Hel). These two

proteins are involved in the replication of the viral genome and in the generation of

transcripts that are employed in the synthesis of the viral proteins. The mechanisms by which these subgenomic mRNAs are produced are not completely elucidated.

Nevertheless, recent evidences indicate that the sequences for regulation of

transcription at the 5' end of each gene represent signals which regulate the

discontinuous transcription of the subgenomic mRNAs (1).

[0007] The proteins of the viral membrane (S, E and M proteins) are inserted

into the intermediate compartment, whereas the replicated RNA (+ strand) is

assembled with the N (nucleocapsid) protein. In a later stage, this protein-RNA

complex combines with the M protein contained in the membranes of the

endoplasmic reticulum of the host cell. The viral particles form when the

nucleocapsid complex buds into the endoplasmic reticulum. The virus then

migrates across the Golgi complex and eventually leaves the cell (e.g., by

exocytosis). The site of attachment of the virus to the host cell is at the level of the

spike protein (S protein).

[0008] Amplification of RNA-dependent RNA polymerase (RdRp), N

protein, internal control, and two mutant variants (N501Y, L452R) can be

qualitatively monitored by the increasing fluorescence signal detected by a Real

time PCR instrument. However, in the second vial reaction buffer is able to detect

two other mutant variants (D614G, P681H). The PCR detection system includes an

internal control primer-probe. The cycle threshold (CT) results / values of the

internal control provide the accuracy of sampling and extraction process. This is in order to avoid false-negative results.

DESCRIPTION OF THE RELATED ART

[0009] Since 2002, we have faced several outbreaks of coronavirus strains in

the world. SARS-CoV-2 affected around 800 people in 2002 which led to the death

of 10 percent of the infected individuals. On the other hand, MERS-CoV affected

around 2300 people in 2012 with a 35 percent death rate.

[0010] The third outbreak of this family of viruses is the pandemic of SARS

CoV-2 (i.e., the cause of COVID-19 disease) in 2019 and 2020, with 9.678.494

confirmed cases and a 2.72 percent mortality rate. Lack of safety-related

experience from confrontation with this virus has led to the susceptibility and

sensitivity of people to this infection (2).

[0011] SARS-CoV-2 is a positive-strand RNA virus with four main structural

proteins including spike, envelope, membrane, and nucleoprotein. Spike protein (S

protein) plays a key role in the attachment of the virus to the receptor of the host

cell (ACE2). S protein consists of S subunit (i.e., receptor-binding subunit) and

S2 subunit (i.e., membrane fusion subunit). S1 subunit contains a receptor-binding

domain (RBD) which is responsible for the attachment of the virus to the cellular

receptor (3).

[0012] The mutation of coronavirus varies from moderate to intense. The

most common recombinant mutation of coronavirus occurs in the S protein

expressing gene located at the RBP section and leads to the change of amino acid

sequence of the part which is required for the attachment to the host cell.

[0013] Recently, in the early stages of the COVID-19 pandemic, 149

mutations were detected in 103 gene sequences which have led to the identification

of the two mainstream mutations called L and S. The number of mutations is

increasing with the progression of the pandemic. More detailed reviews on the

characteristics of these strains can help assess the potential risks, development of

the treatment methods, and preventive strategies.

[0014] Still, it is not clear that these mutations contribute to the deterioration

of the disease or intensity of the virus transmission, as it has been shown that these

mutations can occur in genomic parts that do not affect the characteristics of the

virus. Although, some evidence indicates that the D614G strain can enhance the

infectivity of COVID-19.

[0015] Recently, B.1.1.7 (a new mutated strain of SARS-CoV-2 emerged in

England) indicated a 70 percent higher transmission rate in comparison to the wild

type. This mutation has occurred at the N501Y locus which translates to the

surface of the spike protein. Although this mutation has not affected the

vaccination, it appears that it has enhanced the transmission rate and it is less likely to be detected by the Real time-PCR technique.

[0016] N501Y mutation is one of the main six sequences of the RBD and it

was revealed by prior studies that this mutation can enhance the ability of the

attachment to ACE2 in human and mouse models. N501Y is associated with the

increase of infectivity and virulence in the mouse model.

[0017] In addition to this mutation in coronavirus, there is another mutation

called P681H which is one of the main four sequences of the viral entry and causes

a furin cleavage site between S Iand S2 in the S protein. This region was not

observed in other coronaviruses, and it facilitates virus entry to the epithelial cells

of the respiratory system and virus transmission in the animal model.

[0018] Two other strains which are created by mutations of the spike protein

are K417N and E484K, which cause viral transformation and can affect the

vaccines based on the primary type of the virus. However, recent studies revealed

no indication of a decrease in the antibody levels after the administration of the

Pfizer vaccine.

[0019] One study on the current mutations including N501Y, A570D,

D614G, P681H, T7161, Dl1l8H, and S982A revealed that they share the same

forward primer which contains ACC TCA AGG TAT TGG GAA CCT sequence

(3). Among these mutations, N501Y is the most frequent with 0.02% frequency.

Therefore, the designed reverse primer contains the CCA ACC CAC TTA TGG

TGT TGG sequence, and it has been tested.

[0020] A new SARS-CoV2 mutant called L452R with a mutation in spike

sequence is rapidly spreading in Denmark and the United States since 2020, and

2021, respectively. The analysis of the receptor-binding domain (RBD) indicates

that some of the mutations can enhance the attachment of S protein to ACE2 and

subsequently, the infectivity of the SARS-CoV-2 would be increased.

[0021] Studies have shown that L452R mutation is one of the monoclonal

antibody-resistant mutations and is of great importance in vaccination and

treatment of the patients. Henceforth, in terms of resistance to antibodies,

infectivity, and transmission rate, this mutation is important for public health and

especially the vaccination of the population.

[0022] It is revealed that two different SARS-CoV-2 strains can combine

their genomes and produce a third mutant hybrid. This recombination was first

observed in the California strain of the virus and indicates the probability of the

initiation of a new phase of the pandemic. This hybrid was the result of the

recombination of a highly contagious strain called B.1.1.7 from the U.K. with the

B.1.429 strain originated from California which can cause a new outbreak in Los

Angeles or even other parts of the world. This characteristic is due to the resistance

of this strain to some of the antibodies.

[0023] Since the new variants of COVID-19 are rapidly spreading through the countries and according to the reports of the ministry of health of different countries, several cases have been diagnosed with these new variants; therefore, the next outbreak of coronavirus is imminent.

OBJECTIVES OF THE EMBODIMENTS

[0024] The first objective is to provide the primers and probes which are

designed based on the conserved sequence of the novel coronavirus (SARS-CoV

2), and have a high detection rate of the target gene fragment.

[0025] The main object of the embodiments herein is to provide an accurate

differential detection kit for different variants of SARS-CoV-2 in COVID-19

patients.

[0026] The purpose of the embodiments is to provide a kit to detect the

presence and type of virus variant.

[0027] Accurate determination of the mutant region of various variants of the

SARS-CoV2.

[0028] The specific purpose of these embodiments is to design and prepare

specific probes for mutant regions in the new variants of the SARS-CoV-2.

[0029] Another object of the embodiments herein is to provide suitable

primers for amplification of the mutant regions, in different variants of the virus genome, simultaneously.

[0030] Next object of the embodiments herein is to provide a multiplex

reaction to perform a Real-time PCR.

[0031] Another object of the embodiments herein is to provide usable primers

and probes set to make the kit. The important point that should be considered is

this kit should possess no cross-reactions among positive samples of Coronavirus

(NL63, HKU1, 229E, OC43), Influenza A virus, Influenza B virus, Respiratory

syncytial virus, Adenovirus, Parainfluenza virus, Klebsiella pneumoniae,

Streptococcus pneumoniae, Haemophilus influenza, Pseudomonas aeruginosa,

Legionella pneumophila, Pertussis, Staphylococcus aureus, Mycoplasma

pneumoniae, and Chlamydia pneumoniae.

SUMMARY

[0032] This invention relates to the preparation and design of a diagnostic kit

for accurate detection of various variants as well as the wild type of SARS-CoV-2.

[0033] This kit is designed to identify the current common strains of the

virus.

[0034] According to this design, the steps will be as follows:

1- Accurate identification of mutated regions in the virus genome;

2- Designing primers and probes for replication and detection of mutated regions;

3- Simultaneous use of these sequences in a Real-time PCR run;

4- Setting the temperature to perform simultaneously reactions related to the

genes of conserved regions such as RdRp, N, spike, and mutated regions

in spike such as N501Y, L452R, D614G, and P681H.

[0035] During the operation steps, depending on the conditions of the tests,

changes and adjustments will probably be made in the work steps.

DETAILED DESCRIPTION OF THE EMBODIMENTS

[0036] Reagent Preparation (to be performed in the Reagent Processing

Area):

The component must be taken out and thawed at room temperature until

equilibrated. The Lyophilized Enzyme Mix must be resuspended in 400 pL

Enzyme Mix Buffer. Add 500 pL RNase-free water and mix by gently pipetting up

and down. Avoid generating air bubbles. To prevent wastage of the lyophilized

powder (i.e., to avoid leaving material in the tube), wash the wall of the tube by

pipetting. The tube will then be placed aside for 30 min.

[0037] Reaction Mix Preparation:

The recommended sample volume used in the reaction is 5 pL or 10 pL. This

mixture has to be prepared in two separate tubes designated, for instance, as vial 1 and vial 2. Aliquot of 15 pL (or 10 pL, depending on the sample volume) of the above reaction will be mixed into the PCR plate of the chosen PCR platform in two wells (first vial in first well and second vial in second well for each sample).

[0038] Aliquot into wells according to the number of samples to be tested

include one well for the positive control and one well for the negative control.

Transfer the reaction mix to the Sample Processing Area.

[0039] Sample Adding (to be performed in the Sample Processing Area):

For the 5 pL sample: Add 5 pL of the following into the appropriate wells

according to plate setup: Sample(s), positive control, and negative control. For 10

pL sample: Add 10 pL of the following into the appropriate wells according to

plate setup: Samples consist of diluted positive control, and negative control. After

adding the samples, cover the lid immediately. Spin down briefly using a

centrifuge to remove air bubbles. Transfer the mixture to the amplification area.

[0040] PCR Amplification (to be performed in the amplification and analysis

area):

Place the tubes on the sample holder in the instrument. Set up the test panel

according to the positions of positive control, negative control, and RNA samples.

Select the detection channels as follows:

a) Select FAM (RdRp gene) and HEX (N gene), and cy5 (spike gene) channels to

detect SARS-CoV-2 RNA by using the first vial.

b) Select ROX (RNase P) channel to detect the internal control.

c) Select VIC (D614G), CY5 (L452R), FAM (N501Y), JOE (P681H) channels to

detect one or more mutants of spike gene by using the second vial.

[0041] Program setting:

1. Reverse Transcription: 50°C, 15 min, 1 cycle;

2. cDNA Initial Denaturation: 95°C, 3 min, 1 cycle;

3. Denaturation: 95°C, 15 sec, 45-50 cycles;

4. Annealing, Extension and Fluorescence measurement: 55°C, 10 sec;

5. Cooling: 25°C, 10 sec, 1 cycle.

[0042] Result Interpretation:

After the reaction is completed, the results are automatically saved and the

amplification curves of the detected target RNA and the internal control are

analyzed separately. According to the analysis, the amplification plot will adjust

the start value, end value, and threshold value of the baseline (operators can adjust

the values according to the actual situation. Start value can be set within 3~15, end

value can be set within 5~20; operators can adjust the amplification curve of

negative control to make it linear or below the threshold line). Click "Analyze" to

perform the analysis and the parameters should meet the requirements mentioned

above.

[0043] Quality Control:

Lastly, record the qualitative results in the Plate window. COVID-19 PCR

Negative Control: None of the FAM, HEX, and Internal Control (ROX) channels

have a Ct value or Ct > 40. COVID-19 PCR Positive Control: FAM, HEX and

Internal Control (ROX) channels Ct < 35. The above requirements must be met at

the same time in the same experiment. Otherwise, this experiment is invalid and

needs to be repeated. According to the study of the reference value, the Ct

reference value for the target gene detected by this kit is 40, and the Ct reference

value of internal control is 40.

Claims

CLAIMS [0044] The main claim of the embodiment herein is that in addition to detecting a positive / negative result for COVID-19 disease, the design of specific probes and primers of spike gene in positive patients, make a Real-time PCR-based method to rapidly identify and diagnose mutant variants in patients, carriers, and people who are suspected of being infected with mutated variants of SARS-CoV-2 with high accuracy. [0045] The next claim of the embodiment is that method is based on the use of Real-time PCR kits to diagnose SARS-CoV-2 infections. The basis for the result in this test is the proliferation detection containing specific mutation regions. [0046] Another claim of this embodiment is that in this method, probes with different colors are used for different mutations. The proliferation of the target areas is a sign of mutation(s) and the non-proliferation indicates no mutation of the related gene or genomic region. [0047] The next claim of the embodiment is that the reactions can be performed in more than one tube or vessel if the probe can be designed for different areas. In order to validate the specificity of the primer and probe set specific for the mutant region of interest, a positive control is necessary. References 1- Marra, M. A., Jones, S. J., Astell, C. R., Holt, R. A., Brooks-Wilson, A., Butterfield, Y. S., ... & Roper, R. L. (2003). The genome sequence of the SARS-associated coronavirus. Science, 300(5624), 1399-1404. 2- AminJafari, A., & Ghasemi, S. (2020). The possible of immunotherapy for COVID-19: A systematic review. InternationalImmunopharmacology, 83, 106455. 3- Mousavizadeh, L., & Ghasemi, S. (2021). Genotype and phenotype of COVID-19: Their roles in pathogenesis. Journal of Microbiology, Immunology and Infection, 54(2), pp. 1 59-163. 4- Lopez-Rincon, A., Perez-Romero, C., Tonda, A., Mendoza-Maldonado, L., Claassen, E., Garssen, J., & Kraneveld, A. D. (2021). Design of Specific Primer Sets for the Detection of B. 1.1. 7, B. 1.351, P. 1, B. 1.617. 2 and B.

1.1. 519 Variants of SARS-CoV-2 using Artificial Intelligence. bioRxiv.