KR102695295B1 - Novel guide RNA capable of specific detection of norovirus GII.3 based on CRISPR-Cas and uses thereof - Google Patents

Abstract

The present invention relates to a novel guide RNA capable of specific detection of norovirus GII.3 based on CRISPR-Cas and a method for detecting norovirus GII.3 and diagnosing infection thereof using the same. Using the guide RNA provided by the present invention, norovirus GII.3 in a specimen can be rapidly detected with very high sensitivity and specificity.

Description

{Novel guide RNA capable of specific detection of norovirus GII.3 based on CRISPR-Cas and uses thereof}

The present invention relates to a novel guide RNA capable of specific detection of norovirus GII.3 based on CRISPR-Cas, and a method for detecting norovirus GII.3 and diagnosing infection thereof using the same.

Norovirus is known worldwide as a cause of nonbacterial acute gastroenteritis, and is a type of enteric virus that causes infectious gastroenteritis in humans when contaminated food or water is consumed. Norovirus is a virus belonging to the family Calicivirdae and has a single-stranded RNA genome of 7.5 to 7.5 kb in size, consisting of three open reading frames (ORFs). Norovirus was first discovered in 1968 from the diarrhea of a patient with mass food poisoning at an elementary school in Norwalk, Ohio, and was called (Norwalk Viruses) at the time. After that, viruses similar to Norwalk virus were continuously discovered in similar food poisoning patients, and they were named after the places where they were discovered, such as Hawaii virus, Montgomery virus, and Taunton virus. In 1972, its shape was revealed under an electron microscope, and because this virus was small and spherical among viruses, it was also called small round-structured virus (SRSV). After that, small spherical viruses similar to Norwalk virus were successively discovered from patients with nonbacterial acute gastroenteritis, so it was temporarily called Norwalk-like virus. In August 2002, the International Committee on Taxonomy of Viruses (ICTV) unified the name as norovirus. Currently, among the five types of norovirus classified by genotype (GI, GII, GIII, GIV, GV), the genotypes that cause human infection are the GI, GII, and GIV groups. Of these, genotype GII.4 is involved in most norovirus food poisoning worldwide, and in Korea, GII.3 has been reported to have a high detection rate following GII.4.

If you are infected with norovirus, the main symptoms are nausea, vomiting, diarrhea, and abdominal pain. In most cases, the symptoms are mild and recover naturally after 1-2 days. The incubation period is 24-48 hours, and the virus in the stool or vomit of an infected person contaminates food or water, or the virus enters the mouth when you eat, drink, or come into contact with contaminated objects or food that the infected person has touched. It is easily transmitted to the point that you can easily get infected with just a small amount of the virus, and the contagiousness is most severe when symptoms appear and lasts from 3 days to up to 2 weeks after recovery (Korea Centers for Disease Control and Prevention Health Information).

Although commercial enzyme immunoassays (EIA) diagnostic kits for detecting norovirus antigens have been developed, the enzyme immunoassay analysis method has a disadvantage in that its sensitivity varies depending on the norovirus strain. Therefore, although it can be used as an auxiliary means for identifying the cause of an outbreak caused by norovirus, it has been reported to have technical limitations for use as a standard protocol for diagnosis. In general, reverse transcription polymerase chain reaction (RT-PCR) and real-time RT-PCR methods, which directly amplify and detect viral genes from the patient's stool, are widely used for detection and diagnosis. However, since it is difficult to detect with high sensitivity with a single primer pair, there is a disadvantage in that the cost of producing primers or probes is high.

Recently, CRISPR-Cas-based DNA detection platforms, including SHERLOCK (Cas13), DETECTR (Cas12a), and Cas14-DETECTR, with high detection sensitivity and specificity have been developed, but no related technology has been developed yet that can detect norovirus GII.3 with very high specificity.

Therefore, to improve the accuracy and sensitivity of the accurate detection of norovirus GII.3 and the diagnosis of its infection, it is necessary to develop a novel guide RNA and a method using the same for use in a CRISPR-Cas-based gene detection method.

Accordingly, the inventors of the present invention developed a novel guide RNA capable of specifically detecting norovirus GII.3 together with a CRISPR-Cas protein and evaluated its effect, thereby completing the present invention.

Accordingly, the purpose of the present invention is to provide a norovirus GII.3 specific guide RNA comprising the base sequence of sequence number 1.

Another object of the present invention is to provide a composition and kit for detecting norovirus GII.3 comprising the guide RNA.

Another object of the present invention is to provide a method for detecting norovirus GII.3 comprising the following steps:

(a) A step of amplifying nucleic acid obtained from a sample.

(b) a step of adding the guide RNA, endonuclease and reporter gene to the amplification product and reacting them; and

(c) a step of detecting a signal of the reporter gene.

Another object of the present invention is to provide a composition for diagnosing food poisoning comprising the guide RNA.

However, the above-described tasks of the present invention are exemplary and do not limit the scope of the present invention. In addition, other objects and advantages of the present invention will become more apparent from the detailed description of the invention, claims, and drawings below.

In order to achieve the above-mentioned object of the present invention, the present invention provides a norovirus GII.3 specific guide RNA comprising the base sequence of sequence number 1.

In order to achieve another object of the present invention, the present invention provides a composition and kit for detecting norovirus GII.3 comprising the guide RNA.

In order to achieve another object of the present invention, the present invention provides a method for detecting norovirus GII.3, comprising the following steps:

(a) A step of amplifying nucleic acid obtained from a sample.

(b) a step of adding the guide RNA, endonuclease and reporter gene to the amplification product and reacting them; and

(c) a step of detecting a signal of the reporter gene.

In order to achieve another object of the present invention, the present invention provides a composition for diagnosing food poisoning comprising the guide RNA.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. The following references provide one of the art having common definitions of various terms used in the specification of the present invention; Singleton et al., DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOTY (2nd ed. 1994); THE CAMBRIDGE DICTIONARY OF SCIENCE AND TECHNOLOGY (Walkered., 1988); and Hale & Marham, THE HARPER COLLINS DICTIONARY OF BIOLOGY.

The present invention is described in detail below.

The present invention provides a norovirus GII.3 specific guide RNA comprising a base sequence of SEQ ID NO: 1.

In the present invention, the guide RNA may be crRNA or gRNA. crRNA is called CRISPR RNA. In addition, gRNA refers to guide RNA. crRNA and gRNA may be single strand RNA. In addition, crRNA may bind to tracrRNA to operate a CRISPR-associated protein, and crRNA may be used in a form bound to tracrRNA. At this time, crRNA may have a sequence complementary to a gene sequence specifically present in norovirus GII.3. In addition, gRNA may also bind to a gene sequence specifically present in norovirus GII.3 to cause the CRISPR-associated protein to exhibit activity. The crRNA or gRNA may be RNA composed of 15 to 40 nucleic acids. In one specific example, the crRNA or gRNA may be composed of 24 nucleic acids. Additionally, the crRNA or gRNA may include additional sequences (e.g., tracrRNA, scoutRNA) at the 3' end to activate a CRISPR-associated protein such as Cas12, Cas13 or Cas14. The guide RNA may include a polynucleotide complementary to a nucleotide sequence of 2 to 24 nucleotides (e.g., about 20 nt) (hereinafter referred to as 'nt') in the 5' or 3' direction of the PAM in the target nucleic acid. The length of the above guide RNA may be 10 nt to 100 nt, 10 nt to 90 nt, 10 nt to 80 nt, 10 nt to 70 nt, 10 nt to 60 nt, 10 nt to 50 nt, 15 nt to 50 nt, 20 nt to 50 nt, 25 nt to 50 nt, 30 nt to 50 nt, 35 nt to 50 nt, 40 nt to 50 nt, or 45 nt to 50 nt. In one embodiment, the gRNA may be characterized by including a base sequence of SEQ ID NO: 1.

The present invention also provides a composition for detecting norovirus GII.3 comprising the guide RNA.

The composition according to the present invention may further include additional components for specifically detecting norovirus GII.3 using the guide RNA, specifically, an endonuclease and a reporter gene, and may further include a pair of primers capable of amplifying the norovirus GII.3 gene and including a sequence complementary to the same.

The term "primer" in the present invention means a short nucleic acid sequence having a short free 3' hydroxyl group, which can form base pairs with a complementary template of a norovirus nucleic acid, and which functions as a starting point for template strand copying. The composition according to the present invention may include any primer pair of any sequence as long as it contains a sequence complementary to a gene of norovirus GII.3 and can amplify it, and preferably, the primer pair may be one that can specifically amplify the VP1 capsid region of the ORF2 gene of norovirus GII.3.

In one embodiment of the present invention, the primer pair may include a forward primer comprising a base sequence selected from the group consisting of SEQ ID NOs: 2 to 4 and a reverse primer comprising a base sequence of SEQ ID NO: 5, preferably may include a forward primer comprising a base sequence of SEQ ID NO: 3 and a reverse primer comprising a base sequence of SEQ ID NO: 5, and most preferably may include a forward primer comprising a base sequence of SEQ ID NO: 3 and a reverse primer comprising a base sequence of SEQ ID NO: 5. The forward primer selected from the group consisting of SEQ ID NOs: 2 to 4 and the reverse primer of SEQ ID NO: 5 specifically amplify the VP1 capsid region of the ORF2 gene of norovirus GII.3, and norovirus GII.3 can be detected by analysis of the amplification product.

The above amplification can be performed by any method used in the art to amplify an amplification target (gene, etc.), and for example, can be performed by recombinase polymerase amplification (RPA), but is not limited thereto.

The above amplification product may be, for example, an amplicon, but is not limited thereto.

In this specification, the term "Recombinase Polymerase Amplification (RPA)" refers to a technology that can confirm DNA and RNA amplification, and unlike the existing PCR, it is a technology that can quickly and accurately amplify a target sequence by using a DNA binding protein and recombinase. RPA is similar to the existing PCR method, but since amplification of a specific gene is possible at a constant temperature without temperature change, the reaction time can be shortened, no special equipment is required, so the test cost is low, and it has excellent transportability so that on-site testing is possible, and it has the advantage of. Recombinase Polymerase Amplification (RPA) is a method that causes dissociation of DNA double strands using bacteriophage T4 recombinase and at the same time amplifies specific DNA using DNA polymerase and specific primers. This can amplify DNA of a specific base sequence using a target template and a pair of primers (oligonucleotides) like in the case of PCR, but unlike in the case of PCR, it has the advantage of being able to cause an amplification reaction under isothermal conditions in a certain temperature range (37℃ - 42℃). Currently, RPA has been developed to be able to produce an amplification product very quickly, and this is widely recognized along with the advantage of isothermal conditions, and RPA is being applied in various ways to detect specific pathogens through specific gene amplification.

It is recommended that the length of the RPA amplification product does not exceed 500 nt (nucleotides), and the amplification product length is generally between 100 and 250 nt. Unlike PCR, the primer length for RPA is recommended to be at least 30 nucleotides, and generally 32 to 35 nt is mainly used. In addition, since it is not sensitive to the ratio of GC (GC%) and Tm (melting temperature) value in the sequence like PCR, the RPA primer should converge to GC% (20 to 70%) and Tm (50 to 100℃) (Analyst, 2019, 144, 31).

In the present invention, the endonuclease may be a CRISPR-associated protein. The term CRISPR-associated protein used in the present invention refers to an enzyme that can recognize and cleave a nucleic acid such as DNA or RNA when it has a double strand or single strand (dsDNA/RNA and ssDNA/RNA). Specifically, they can recognize and cleave a double-stranded or single-stranded nucleic acid bound to crRNA or gRNA.

In the present invention, one specific example of the endonuclease may be one that recognizes that gRNA has bound to a target site and thus has an endonuclease function activated. In addition, as the endonuclease function is activated, it may have an exonuclease activity capable of non-specifically cleaving double-stranded and/or single-stranded DNA and/or RNA. In addition, once a CRISPR-associated protein such as Cas12, Cas13 or Cas14 is activated, non-specific exonuclease activity may be exhibited. It may exhibit an activity of cleaving a target nucleic acid to which a guide RNA is bound (cis-cleavage) or may non-specifically cleave any external nucleic acid, specifically, a reporter nucleic acid (DNA and/or RNA) (trans-cleavage).

Therefore, in a composition of one embodiment of the present invention, crRNA or gRNA is bound to a specific target site of norovirus GII. 3, and a reporter is cleaved by a non-specific nuclease activated thereby, so that norovirus GII. 3 can be detected by emission of a fluorescent substance bound to the reporter.

Specifically, one specific example of the CRISPR-associated protein may be Cas12, Cas13 or Cas14, and specifically any one selected from the group consisting of Cas12a, Cas12b, Cas12c, Cas12d, Cas12e, Cas12g, Cas12h, Cas12i, Cas13a, Cas13b, Cas13c, Cas13d and Cas14.

The above Cas12 and Cas14 can cleave ssDNA of any sequence or dsDNA of the target nucleic acid by exhibiting endonuclease activity and non-specific exonuclease activity in the presence or absence of a PAM sequence in the target nucleic acid after binding to a guide RNA complementary to the target nucleic acid.

The above Cas13 can cleave ssRNA of any sequence or ssRNA of the target nucleic acid after binding to a guide RNA complementary to the target nucleic acid.

Since the TTTA PAM sequence exists in the gene region of norovirus GII.3 to which the guide RNA according to the present invention complementarily binds, the present invention can be applied in combination with a CRISPR-associated protein that requires a PAM sequence of (T)TTV, TTN, or TN to exhibit endonuclease or exonuclease activity, or does not require a PAM sequence.

In the present invention, the reporter gene refers to any sequence of DNA or RNA that is cleaved by the exonuclease activity of a CRISPR-associated protein, and it is preferable that the reporter gene is labeled with a detectable label. The detectable label can be bound to the end or the inside of the reporter gene. The detectable label can be a chemical label, an enzymatic label, a radioactive label, a fluorescent label, a luminescent label, a chemiluminescent label, a FRET (fluorescence resonance energy transfer) label, a metal label, or a combination thereof. The fluorescent label can be selected from the group consisting of FAM, TET, JOE, YAKYE, HEX, CY3, ATTO550, TAM, ROX, TxRed, CY35, LC610, LC640, ATTO647N, CY5, VIC, evergreen dye, and ATTO680, but is not limited thereto.

In one aspect of the present invention, the composition may be characterized in that it is used in a method for detecting norovirus GII.3 by an isothermal amplification reaction and a subsequent CRISPR gene scissors reaction.

Specifically, if a target region exists in an amplified product amplified by a primer pair capable of amplifying the norovirus GII.3 gene, preferably a primer pair capable of specifically amplifying the VP1 capsid region of the ORF2 gene of norovirus GII.3, the guide RNA complementarily binds to the target region and an endonuclease, preferably a CRISPR-associated protein, is activated to cut the sequence of the reporter gene. As a result, norovirus GII.3 can be detected by a label bound to the reporter gene.

The present invention also provides a kit for detecting norovirus GII.3 comprising the composition.

The above kit may be a kit containing the essential elements necessary for performing detection of norovirus GII.3, and may include, in addition to the composition, a test tube or other appropriate container, a reaction buffer (having various pH and magnesium concentrations), deoxynucleotides (dNTPs), an enzyme such as Taq polymerase, DNase, RNAse inhibitor, DEPC water, and sterile water.

The present invention also provides a method for detecting norovirus GII.3, comprising the following steps:

(a) A step of amplifying nucleic acid obtained from a sample.

(b) a step of adding a guide RNA, an endonuclease and a reporter gene according to claim 1 to the amplification product and reacting them; and

(c) a step of detecting a signal of the reporter gene.

In the present invention, the “specimen” refers to a material or organism used for testing, inspection, analysis, etc., and its type is not particularly limited, but may be a food or biological sample suspected of having norovirus GII.3.

In the present invention, the biological sample may include, but is not limited to, tissue, cells, whole blood, serum, plasma, cerebrospinal fluid, colostrum, synovial fluid, saliva, feces, cell culture supernatant, and the like.

Nucleic acid can be obtained from a sample using a conventionally known nucleic acid obtaining method. If the starting material is gDNA, the isolation of gDNA can be performed according to a conventional method known in the art, and if the starting material is mRNA, it can be synthesized into cDNA using reverse transcriptase. For example, the RNA isolation is not particularly limited as long as it is a method for isolating RNA from a sample. For example, a commercially available RNA isolation kit such as Qiagen's RNeasy mini kit can be used.

In the present invention, the “amplification” may be one selected from the group consisting of PCR, RT (reverse transcription)-PCR, isothermal amplification (RPA) and RT (reverse transcription)-RPA, preferably RPA or RT-RPA, and most preferably RT-RPA.

The above “isothermal amplification” refers to a method of amplifying a target nucleic acid sequence (template) under a constant reaction temperature condition, and may be any one of recombinase polymerase amplification (RPA), loop-mediated isothermal amplification (LAMP), helicase-dependent amplification (HDA), rolling circle amplification (RCA), multiple displacement amplification (MDA), strand displacement amplification (SDA), and nucleic acid sequence-based amplification (NASBA), and preferably recombinase polymerase amplification (RPA).

Detection of the above amplification product can be performed via, but is not limited to, capillary electrophoresis, DNA chip, gel electrophoresis, radiometric measurement, fluorescence measurement or phosphorescence measurement.

As one of the methods for detecting the amplification product, for example, capillary electrophoresis can be performed. Capillary electrophoresis can be performed, for example, using ABi Sequencer. In addition, gel electrophoresis can be performed, and gel electrophoresis can use agarose gel electrophoresis or acrylamide gel electrophoresis depending on the size of the amplification product. In addition, a fluorescence measurement method is performed by labeling the 5'-end of a primer with Cy-5 or Cy-3, and when PCR is performed, the target sequence is labeled with a detectable fluorescent labeling substance, and the fluorescence thus labeled can be measured using a fluorescence meter. In addition, a radioactivity measurement method is performed by adding a radioactive isotope such as 32P or 35S to the PCR reaction solution during PCR to label the amplification product, and then measuring the radioactivity using a radioactivity measuring device such as a Geiger counter or a liquid scintillation counter. In addition, RPA amplification products can be obtained and separated by methods such as agarose gel electrophoresis, and the presence of DNA having a length corresponding to DNA polymerized by the primer set used, specifically, a length of 211 bp, can be confirmed to detect norovirus GII.3.

In one aspect of the present invention, when the endonuclease is Cas13, a step of transcribing the amplification product of step (a) may be additionally performed.

Specific examples of detecting norovirus GII.3 using the method of the present invention are as follows:

(1) The gene of norovirus GII.3 present in the specimen is amplified using a primer pair. If the gene is DNA, the gene is amplified by PCR, RPA, etc., and if the gene is RNA, the gene is amplified by RT (reverse transcription)-PCR, RT-RPA, etc.

(2) In the case of Cas13, the amplification product is further transcribed (e.g., T7 transcription) and converted into RNA, while Cas12 and Cas14 use the amplification product of (1) as is.

(3) The amplification product of (1) is mixed with guide RNA, Cas12 or Cas14, and reporter DNA, or the amplification product transcript of (2) is mixed with guide RNA, Cas13, and reporter RNA and reacted.

(4) Detects a signal generated when the above reporter is cut.

The present invention also provides a composition for diagnosing food poisoning.

The term “diagnosis” as used herein includes determining whether a subject currently has a particular disease or condition, or determining the prognosis of a subject having a particular disease or condition.

Since the composition including the guide RNA provided by the present invention can specifically detect norovirus GII.3, by using the composition, norovirus GII.3 in a biological sample to be diagnosed (e.g., feces, blood, etc.) can be detected quickly and accurately, and diseases caused by norovirus GII.3 infection, specifically, food poisoning, can be diagnosed very quickly and accurately.

Using the guide RNA provided by the present invention, norovirus GII.3 in a sample can be rapidly detected with very high sensitivity and specificity.

Figure 1 is a diagram showing the location and sequence of the gene targeted by gRNA for detection of norovirus GII.3, and the PAM sequence.
Figure 2 is a diagram showing the process and results of constructing a primer pair combination capable of amplifying the gene of norovirus GII.3 with high sensitivity.
Figure 3 shows the experimental conditions for amplifying the gene of norovirus GII.3 using a primer pair and detecting norovirus GII.3 using the amplified product using gRNA, CRISPR-Cas12a (Cpf1), and a reporter gene.
Figure 4 shows the results of confirming the detection sensitivity by amplifying the gene of norovirus GII.3 using a primer pair and detecting norovirus GII.3 using the amplified product, gRNA, CRISPR-Cas12a (Cpf1), and a reporter gene.
Figure 5 shows the results of confirming the detection specificity by detecting various viruses using gRNA according to the present invention (MBC111-R: norovirus GII.3, NCCP76067: norovirus GII.4).

Hereinafter, preferred examples are presented to help understand the present invention. However, the following examples are provided only to help understand the present invention more easily, and the content of the present invention is not limited thereby.

Example 1: Selection of gRNA for detection of norovirus GII.3

The core of diagnostic research using CRISPR gene scissors technology is the applied Cas protein and gRNA. In this study, cpf1 (Cas12a), which is active on dsDNA, was selected, and the protein is known to require T-rich PAM (TTTV) in the target.

The gRNA specific to GII.3 was selected by aligning the VP1 capsid region of the Norovirus GII ORF2 gene with each ref. genome (GII.3: MK764020) of the GII.3 genotype and each standard pathogen base sequence (Fig. 1).

Example 2: RPA primer design

For the selected gRNA target and optimal RPA primer design, three combinations of forward 3 and reverse 1 regions were selected and RPA primer validation targeting Norovirus GII.3 (MBC111-R) was performed, and the primer pair with the best reaction combination was used in the following experiments (Fig. 2).

Example 3: Detection experiment of norovirus GII.3 using gRNA, primer pair, and CRISPR-Cas12a

After amplification of the norovirus GII.3 gene using the final selected primer pair, the gRNA and CRISPR-Cas12a application reactions were performed, and the detailed reaction conditions are shown in Fig. 3. The mixture dispensed by applying the protocol was measured every minute using an isothermal fluorescence detector at 37°C, and the results are shown in Fig. 4.

As shown in Figure 4, it was confirmed to exhibit very high detection sensitivity at the 1 copy level.

Example 4: Evaluation of detection specificity

Detection experiments for various food-related viruses were conducted using the same method as in Example 3 above. As a result, as shown in Fig. 5, no viruses other than norovirus were detected, and furthermore, norovirus GII.4 was not detected either, confirming that it exhibited very high specificity.

The RNA provided by the present invention can rapidly detect norovirus GII.3 in a sample with very high sensitivity and specificity, and thus has very high potential for industrial use.

Attach electronic file of sequence list

Claims (11)

Norovirus GII.3 specific guide RNA comprising the nucleotide sequence of sequence number 1;
A forward primer comprising the base sequence of sequence number 2 or 3;
A reverse primer comprising the base sequence of sequence number 5;
A composition for detecting norovirus GII.3 comprising a CRISPR-associated protein.
delete delete delete A composition according to claim 1, wherein the CRISPR-associated protein is selected from the group consisting of Cas12, Cas13, and Cas14.
A composition according to claim 1, characterized in that the composition further comprises a reporter gene.
A composition according to claim 6, characterized in that the reporter gene is labeled with a detectable label.
A kit for detecting norovirus GII.3 comprising the composition of claim 1.
A method for detecting norovirus GII.3 comprising the following steps:
(a) A step of amplifying nucleic acid obtained from a sample.
(b) a step of adding a composition according to claim 1 and a reporter gene to the amplification product and reacting them; and
(c) a step of detecting a signal of the reporter gene.
A detection method according to claim 9, characterized in that the sample is a food or biological sample.
A composition for diagnosing food poisoning comprising the composition of claim 1.