CN115125330A

CN115125330A - Detection kit for detecting different variants of novel coronavirus and application thereof

Info

Publication number: CN115125330A
Application number: CN202210163663.XA
Authority: CN
Inventors: 唐时幸; 林洪青; 梁源浩; 王海鹰
Original assignee: Southern Medical University
Current assignee: Southern Medical University
Priority date: 2022-02-22
Filing date: 2022-02-22
Publication date: 2022-09-30

Abstract

The invention discloses a detection kit for detecting different variants of a novel coronavirus and application thereof. The detection kit comprises an RAA primer group and crRNA. The detection kit can rapidly identify the current SARS-CoV-2 main variant strains (including Alpha, Beta, Delta and Omicron), has low requirement on reaction conditions, and is suitable for detection by relatively cheap instant detection (POCT) equipment in basic laboratories; the detection result analysis method can realize diversification, and the direct visual identification can be realized by naked eyes under blue light; short time consumption, high speed compared with PCR technology, obvious detection signal within 1-1.5h, high detection sensitivity up to 1.0 x 104 copy/microliter, and very high application prospect.

Description

Detection kit for detecting different variants of novel coronavirus and application thereof

Technical Field

The invention relates to the field of virus detection, in particular to a detection kit for detecting different variants of a novel coronavirus and application thereof.

Background

Novel coronaviruses (SARS-CoV-2) have evolved a variety of variants and subtypes, with variants of interest including Alpha, Beta, Delta, and Omicron, among others. The existing novel coronavirus variant is mainly characterized in that key mutation of spike protein is adopted, so that the pathogenicity and the transmission efficiency of the virus are changed, the protection rate of the vaccine is influenced, and the control of the epidemic situation of the virus is important.

In the related art, although the variant and subtype of SARS-CoV-2 can be found and diagnosed by virus genome sequence analysis, an effective means for realizing field detection is lacked, and a large number of samples cannot be detected at high throughput, which brings certain difficulty to the high efficiency of epidemic prevention and control. At present, the identification of SARS-CoV-2 virus variant mainly depends on gene sequencing, but no matter the first generation sequencing technology, or metagenome sequencing based on the second generation gene sequencing and third generation nanopore high-throughput sequencing, the cDNA library is constructed by utilizing PCR amplification technology, the method has high detection cost, needs professional instruments and has certain requirements on the technical level of operators. After sequencing, the sequencing data needs to be cleaned and analyzed by using professional bioinformatics software to obtain the virus gene sequence and related variation conditions. Although the homology of different strains can be found out through analyzing the virus gene sequence, the source of the strains can be identified, the evolution process of the strains can be analyzed, and the mutation condition of the strains can be tracked, the virus gene sequencing has high requirements on laboratory configuration, long time consumption and high cost, has extremely high requirements on the technical and professional knowledge of operators, and is limited in the rapid identification application of SARS-CoV-2 variant strains.

Therefore, the development of a rapid, sensitive, convenient and low-cost SARS-CoV-2 variant strain identification method and a detection kit have important significance for preventing and controlling epidemic situation and early detecting mutant strains.

Disclosure of Invention

The present invention is directed to solving at least one of the problems of the prior art described above. Therefore, the invention provides a detection kit for detecting different variants of the novel coronavirus and application thereof, the detection kit can rapidly identify the current SARS-CoV-2 main variants (including Alpha, Beta, Delta and Omicron), has low requirement on reaction conditions, and is suitable for relatively cheap point-of-care testing (POCT) equipment for basic laboratories to detect; the detection result analysis method can realize diversification, and the direct visual identification can be realized by naked eyes under blue light; the time consumption is short, the speed is high compared with that of the PCR technology, and the method can obviously detect signals within 1-1.5h and has extremely high application prospect.

In a first aspect of the invention, there is provided a set of RAA primer sets targeting at least one mutation site of K417N, T478K, E484K, N501Y and D614G in a novel coronavirus variant.

According to a first aspect of the invention, in some embodiments of the invention, the novel coronavirus comprises a wild-type and a mutant novel coronavirus, wherein the mutant comprises Alpha, Beta, Delta, Omicron.

In some embodiments of the invention, the K417N, T478K, E484K, N501Y and D614G mutation sites are from the sequence 21563-25384bp of SARS-CoV-2-S protein fragment, in particular, the GenBank accession number MN908947 strain.

According to the first aspect of the invention, in some embodiments of the invention, the 5' end of the mutation site further comprises a PAM sequence.

Since activation and cleavage activity of Cas12a protein requires the presence of a T-rich PAM sequence (5 ' -TTTN-3 ', where N is a/G/C) at the 5 ' end of the target sequence, and the 5 ' ends of the three mutation sites K417N, T478K, and E484K all lack PAM sequences and cannot be identified using Cas12a protein mediation, the inventors introduced PAM sequences to the 5 ' ends of the three mutation sites. And the suitable PAM sequences are arranged near the N501Y and D614G sites, so that the PAM sequences do not need to be specially introduced.

According to the first aspect of the present invention, in some embodiments of the present invention, the nucleotide sequence of the RAA primer set is shown in SEQ ID NO. 1-11.

In some preferred embodiments of the present invention, the upstream primer of the RAA primer set is any one of SEQ ID NOs 1-2, 4-8 and 10, and the downstream primer of the RAA primer set is any one of SEQ ID NOs 3, 9 and 11.

In some more preferred embodiments of the present invention, the upstream and downstream primers corresponding to each mutation site are: for K417N, the upstream primer was RAA-417F-1(SEQ ID NO:1) and the downstream primer was RAA-R1(SEQ ID NO: 3); for T478K, the upstream primer was RAA-478F-1(SEQ ID NO:4) and the downstream primer was RAA-R2(SEQ ID NO: 9); for E484K, the upstream primer was RAA-484F-2(SEQ ID NO:7) and the downstream primer was RAA-R2; for N501Y, the upstream primer is RAA-F2(SEQ ID NO:10), and the downstream primer is RAA-R3(SEQ ID NO: 11); for D614G, the upstream primer was RAA-F and the downstream primer was RAA-R3.

In a second aspect of the present invention, there is provided a set of crrnas comprising a stem-loop structure sequence at the 5 'end and a target gene complementary sequence at the 3' end, wherein the target gene is a novel coronavirus S protein-encoding gene comprising any one of sites K417N, T478K, E484K, N501Y and D614G.

According to a second aspect of the invention, in some embodiments of the invention, the nucleotide sequence of the crRNA is as shown in SEQ ID NO 12-30.

In some embodiments of the invention, the 5' end of crRNA-417N, crRNA-478K, crRNA-501N, crRNA-614D introduces a single base mutation.

In some embodiments of the invention, the crRNA may be obtained by synthesis or by in vitro transcription of T7.

In some preferred embodiments of the present invention, the crRNA is obtained by T7 in vitro transcription, which is prepared by: synthesizing single-stranded DNA oligonucleotide and a complementary strand thereof, wherein the single-stranded DNA oligonucleotide comprises three parts of a T7 promoter, a conservative scaffold sequence and a target gene complementary sequence, then denaturing the single-stranded DNA oligonucleotide and the complementary strand thereof at 95 ℃ for 10min, naturally annealing to room temperature, complementarily combining double strands, transcribing and purifying to obtain the DNA.

In a third aspect of the present invention, there is provided a novel coronavirus detection reagent, wherein the detection reagent comprises the RAA primer set according to the first aspect of the present invention and the crRNA according to the second aspect of the present invention.

According to a third aspect of the invention, in some embodiments of the invention, the paired combination of the RAA primer set and the crRNA is as set forth in table 4 of the specification.

In some preferred embodiments of the invention, the RAA primer and the crRNA in combination are:

aiming at K417N, crRNA is crRNA-417N-3, an upstream primer is RAA-417F-1, and a downstream primer is RAA-R1;

aiming at T478K, crRNA is crRNA-478K-5, an upstream primer is RAA-478F-1, and a downstream primer is RAA-R2;

aiming at E484K, crRNA is crRNA-484K-2, an upstream primer is RAA-484F-2, and a downstream primer is RAA-R2;

aiming at N501Y, the crRNA is crRNA-501N-3, the upstream primer is RAA-F2, and the downstream primer is RAA-R3;

for D614G, the crRNA is crRNA-614D-2, the upstream primer is RAA-F2, and the downstream primer is RAA-R3.

According to a third aspect of the invention, in some embodiments of the invention, the detection reagent further comprises a single-stranded DNA probe and a Cas12a protein.

In some embodiments of the invention, the nucleotide sequence of the single-stranded DNA probe is: 5 '-TTATT-3'.

In some preferred embodiments of the present invention, the single-stranded DNA probe is labeled with a reporter group, and the reporter group is labeled on both ends of the single-stranded DNA probe.

In some preferred embodiments of the invention, the reporter group is specifically: the 5 'end is modified with a fluorescence reporter group FAM, and the 3' end is modified with a fluorescence quenching group BHQ 1.

In some embodiments of the invention, the Cas12a protein may be selected from any conventional Cas12a protein in the art, including but not limited to assas 12a and LbCas12 a. Wherein, the source of the Cas12a protein does not affect the action effect thereof in the invention, and can comprise a Cas12a protein which is commercially purchased or synthesized in self.

In some embodiments of the invention, the Cas12a protein is AsCas12a, and the preparation method of the AsCas12a is: a plasmid (Addge #114073) containing the henAsCas12a (Acylaminococcus sp.) sequence was purchased, transformed into DE3 competent cells, expressed in large quantities and purified to obtain the AsCas12a protein.

In a fourth aspect of the present invention, there is provided a use of the RAA primer set according to the first aspect of the present invention and/or the crRNA according to the second aspect of the present invention in the preparation of a novel coronavirus detection kit.

According to a fourth aspect of the present invention, in some embodiments of the present invention, the functions of the novel coronavirus detection kit include, but are not limited to, at least one of the following (1) to (3):

(1) detecting whether the unknown sample contains the novel coronavirus virus;

(2) determining the typing condition of the variant strains of the novel coronavirus in an unknown sample;

(3) differentiating or screening specific novel coronavirus variant strains.

The novel coronavirus detection kit disclosed by the invention is based on 5 characteristic mutations (K417N, T478K, E484K, N501Y and D614G) of spike protein of SARS-CoV-2 variant strains, integrates RAA and CRISPR/Cas12a reactions through 4 specific groups of RAA primer groups and 5 pieces of allele specific crRNA, completes amplification reaction and enzyme digestion detection in the same closed system, and finally integrates the detection results of the 5 characteristic mutations to realize the typing of different SARS-CoV-2 variant strains. Through the mediation of Cas12a, specific crRNA and a universal probe, the detection kit can realize the rapid identification of different SARS-CoV-2 variant strains within 1-1.5h at the temperature of 37-39 ℃. Compared with viral gene sequencing, the CRISPR-Cas12a does not depend on expensive instruments and professional technicians, greatly shortens the detection time, and has the advantages of high specificity, high sensitivity, rapidness, high efficiency, simplicity and convenience in operation, easily-readable results and the like. In the prior art, the RPA nucleic acid amplification and the CRISPR/Cas12a are required to be separately used, so that the operation complexity is increased, and the uncovering operation is easy to form aerosol, which causes pollution to a nucleic acid laboratory and is not beneficial to clinical application.

According to a fourth aspect of the invention, in some embodiments of the invention, the method of using the test kit is: adding 1 tube filled with freeze-dried RAA reaction enzyme powder into an upstream primer and a downstream primer of an RAA primer group, a magnesium acetate solution, an RAA hydration buffer solution and a nucleic acid sample to be detected, which are all described in the first aspect of the invention, uniformly mixing, adding crRNA, Cas12a protein, a single-stranded DNA probe and a CRISPR buffer solution which are all described in the second aspect of the invention, placing the mixture in an environment with a constant temperature of 37-39 ℃ for reaction for 35-40 min, and judging a detection result.

In some preferred embodiments of the present invention, the interpretation of the detection result may be performed in any one of the following manners a to B:

A. judging according to the real-time fluorescent signal, removing the background signal, and then judging the result, wherein the result is negative: the fluorescence value is less than 2 times of the fluorescence value of the negative control; positive: the fluorescence value is greater than or equal to 2 times the fluorescence value of the negative control.

The specific calculation formula is as follows:

wherein, F: fluorescence value at 40-60 min; b: fluorescence value at 0 min; PC: experimental group or positive control; NC: and (5) negative control. FC > 2 is positive, FC <2 is negative.

B. According to the judgment of the result of direct visual observation under blue light, the person emitting green fluorescence is positive, and the person emitting no fluorescence is negative.

In some preferred embodiments of the present invention, the detection system for each different mutation site is configured to:

(1) K417N characteristic mutation site detection system configuration: taking 1 tube filled with freeze-dried RAA reaction enzyme powder, adding 45 mu L of RAA hydration buffer solution, 2 mu L of each RAA primer group (upstream primer RAA-417F-1 and downstream primer RAA-R1), 2.5 mu L of magnesium acetate solution and 2 mu L of nucleic acid sample to be detected into each tube, mixing uniformly, and adding 0.4 mu L of crRNA-417N-3, 0.2 mu L of AsCas12a, 1 mu L of single-stranded DNA probe and 3 mu L of NEBbuffer2.1 into a tube cover of the reaction tube after mixing to obtain the nucleic acid sample.

(2) T478K characteristic mutation site detection system configuration: taking 1 tube filled with freeze-dried RAA reaction enzyme powder, adding 45 mu L of RAA hydration buffer solution, 2 mu L of each RAA primer group (upstream primer RAA-478F-1 and downstream primer RAA-R2), 2.5 mu L of magnesium acetate solution and 2 mu L of nucleic acid sample to be detected into each tube, mixing uniformly, and adding 0.4 mu L of crRNA-478K-5, 0.2 mu L of AsCas12a, 1 mu L of single-stranded DNA probe and 3 mu L of NEBbuffer2.1 into a tube cover of the reaction tube after mixing to obtain the nucleic acid sample.

(3) E484K characteristic mutation site detection system configuration: taking 1 tube filled with freeze-dried RAA reaction enzyme powder, adding 45 mu L of RAA hydration buffer solution, 2 mu L of each RAA primer group (upstream primer RAA-484F-2 and downstream primer RAA-R2), 2.5 mu L of magnesium acetate solution and 2 mu L of nucleic acid sample to be detected into each tube, mixing uniformly, and adding 0.4 mu L of crRNA-484K-2, 0.2 mu L of AsCas12a, 1 mu L of single-stranded DNA probe and 3 mu L of NEBbuffer2.1 into a tube cover of the reaction tube after mixing to obtain the nucleic acid sample.

(4) N501Y characteristic mutation site detection system configuration: taking 1 tube filled with freeze-dried RAA reaction enzyme powder, adding 45 mu L of RAA hydration buffer solution into each tube, 2 mu L of each RAA primer group (upstream primer RAA-F2 and downstream primer RAA-R3), 2.5 mu L of magnesium acetate solution, 2 mu L of nucleic acid sample to be detected, mixing uniformly, and adding 0.4 mu L of crRNA-501N-3, 0.2 mu L of AsCas12a, 1 mu L of single-stranded DNA probe and 3 mu L of NEBbuffer2.1 into a tube cover of the reaction tube after mixing to obtain the product.

(5) D614G characteristic mutation site detection system configuration: taking 1 tube filled with freeze-dried RAA reaction enzyme powder, adding 45 mu L of RAA hydration buffer solution, 2 mu L of each RAA primer group (upstream primer RAA-F2 and downstream primer RAA-R3), 2.5 mu L of magnesium acetate solution, 2 mu L of nucleic acid sample to be detected, mixing uniformly, and adding 0.4 mu L of crRNA-614D-2, 0.2 mu L of AsCas12a, 1 mu L of single-stranded DNA probe and 3 mu L of NEBbuffer2.1 into a tube cover of the reaction tube after mixing to obtain the product.

Wherein, according to the detection condition of each site, the virus sample is judged to belong to which variant strain, and the judgment is specifically as follows:

wherein, + indicates positive and-indicates negative.

According to the fourth aspect of the present invention, in some embodiments of the present invention, the detection kit further comprises a positive quality control and a negative quality control

The nucleic acid to be detected containing the mutation sites of K417N, T478K, E484K, N501Y and D614G is selected as a SARS-CoV-2-S protein fragment (namely 417N, 478K, 484K positive, 501N, 614D negative), in particular 21563-25384bp of the sequence of the strain with GenBank accession number MN 908947. And constructing a wild-type DNA plasmid (negative control) and a mutant-type DNA plasmid (positive control) based on the fragment, wherein the nucleotide sequences of the wild-type DNA plasmid and the mutant-type DNA plasmid are SEQ ID NO. 31 and SEQ ID NO. 32, respectively. Of course, other negative and positive controls can be reasonably selected by one skilled in the art according to actual use requirements.

The invention has the beneficial effects that:

1. the RAA primer group and the crRNA have good combination effect, can accurately target a specific mutation site, and cannot generate hybridization reaction with other viruses or similar genes to cause false positive.

2. The detection kit disclosed by the invention can realize one-tube reaction of RAA and CRISPR/Cas12a, greatly reduces the operation complexity of detection and professional requirements of personnel, and effectively improves the rapid and efficient detection of the novel coronavirus.

3. The detection kit can specifically distinguish the main SARS-CoV-2 variant strains (including Alpha, Beta, Delta and Omicron), has low requirement on reaction conditions, can be carried out at the temperature of about 37-39 ℃, does not need fine temperature control conditions and complex temperature change like PCR amplification, is suitable for relatively cheap point-of-care testing (POCT) equipment for detection in basic laboratories, and meets the requirement of general field detection.

4. The detection kit has short detection time, high speed compared with PCR technology, high sensitivity and high sensitivity of 1.0 multiplied by 10 for different new coronavirus strains, and can obviously detect signals within 1 to 1.5 hours ⁴ Copies/. mu.L.

Drawings

FIG. 1 is a diagram showing the results of screening crRNA and RAA primers in the present invention.

Fig. 2 is a graph showing the effect of different RAA reaction volumes and CRISPR/Cas12a reaction volumes on the detection effect.

Fig. 3 is a graph showing the effect of different Cas12a proteins on the detection effect.

FIG. 4 is a graph showing the effect of different RAA amplification reaction temperatures on the detection effect.

FIG. 5 is a graph showing the effect of different RAA amplification reaction times on the detection effect.

FIG. 6 is a graph showing the results of detecting the relative fluorescence intensity values of K417N, T478K, E484K, N501Y and D614G mutation sites in the RAA-Cas12a new coronavirus detection reaction system of the present invention, wherein n.s. represents P > 0.05; represents P < 0.05; represents P < 0.01; represents P < 0.001; represents P < 0.0001.

FIG. 7 is a blue light fluorescence image of RAA-Cas12a new coronavirus detection reaction system detecting K417N, T478K, E484K, N501Y and D614G mutation sites in the invention.

FIG. 8 is a diagram showing the specific detection result of the RAA-Cas12a new coronavirus detection reaction system in the present invention.

FIG. 9 is a relative fluorescence intensity heat map of 54 SARS-CoV-2 positive clinical samples detected by the RAA-Cas12a new coronavirus detection reaction system of the present invention.

FIG. 10 is a blue light fluorescence image of a part of SARS-CoV-2 positive clinical samples detected by RAA-Cas12a new coronavirus detection reaction system in the invention.

Detailed Description

In order to make the objects, technical solutions and technical effects of the present invention more clear, the present invention will be described in further detail with reference to specific embodiments. It should be understood that the detailed description and specific examples, while indicating the preferred embodiment of the invention, are intended for purposes of illustration only and are not intended to limit the scope of the invention.

The experimental materials and reagents used are, unless otherwise specified, all consumables and reagents which are conventionally available from commercial sources.

Design of RAA primers and crRNA

The genome of each variant strain of the novel coronavirus SARS-CoV-2 is downloaded through NCBI for complete gene sequence analysis, and 5 variant sites of genes for coding spike protein (S protein) are obtained by screening, including K417N, T478K, E484K, N501Y and D614G.

Wherein, the corresponding relationship between 5 mutation sites of coding spike protein (S protein) gene and each variant of the novel coronavirus SARS-CoV-2 is shown in Table 1.

TABLE 1

Wherein, + indicates positive and-indicates negative.

(1) Designing RAA primers:

designing a recombinase-mediated isothermal nucleic acid amplification (RAA) primer based on the mutation sites, wherein a T-rich PAM sequence (5 ' -TTTN-3 ', wherein N is A/G/C) is required at the 5 ' end of a target sequence for activation and cleavage activity of the Cas12a protein, and the 5 ' ends of the three mutation sites of K417N, T478K and E484K lack the PAM sequence and cannot be identified by Cas12a protein mediation, so that the PAM sequence is introduced into the 5 ' ends of the three mutation sites. And the suitable PAM sequences are arranged near the N501Y and D614G sites, so that the PAM sequences do not need to be specially introduced. Specific RAA primer nucleotide sequences are shown in table 2.

TABLE 2

After the primers were designed, they were committed to be synthesized by Guangzhou Rui Boxing Ke Biotechnology GmbH.

(2) Design of crRNA:

designing crRNA based on the variation sites, wherein the sequence of the crRNA consists of two parts: a conserved stem-loop structure sequence at the 5 'end (scaffold part), and a complementary sequence of the target gene sequence at the 3' end. Wherein, the 5' end of the crRNA-417N, crRNA-478K, crRNA-501N, crRNA-614D is introduced with single base mutation. The designed crRNA is shown in Table 3.

TABLE 3

The designed crRNA is synthesized by biological companies, or can be obtained by means of in vitro transcription of T7.

In the present example, the crRNA was obtained by in vitro transcription of T7. The crRNA of each nucleic acid detection target obtained by the in vitro transcription of T7 in the embodiment is specifically obtained by the following steps: a single-stranded DNA oligonucleotide comprising three portions of a T7 promoter, a conserved scaffold sequence and a target gene complementary sequence and a complementary strand thereof were synthesized by Rui Boxing, Guangzhou, and then the single-stranded DNA oligonucleotide and the complementary strand thereof were denatured at 95 ℃ for 10 minutes and naturally annealed to room temperature to complementarily bind double strands, followed by purification using an in vitro transcription Kit (HiScribe T7 HighyieldRNASynthesis Kit, available from New England Biolabs), followed by RNA purification Kit (MiRNeasy Serum/Plasma Kit, available from Qiagen), and subpackaging after measuring crRNA concentration using a NanoDrop 2000 spectrophotometer, and storage at-80 ℃.

Negative control and positive control

The nucleic acid to be detected containing the mutation sites of K417N, T478K, E484K, N501Y and D614G is selected as a SARS-CoV-2-S protein fragment (namely 417N, 478K, 484K positive, 501N, 614D negative), in particular 21563-25384bp of the sequence of the strain with GenBank accession number MN 908947. And constructing a wild-type DNA plasmid (negative control) and a mutant-type DNA plasmid (positive control) based on the fragments, wherein the nucleotide sequences of the wild-type DNA plasmid and the mutant-type DNA plasmid are shown below.

The nucleotide sequence of the wild type DNA plasmid is shown in SEQ ID NO. 31.

The nucleotide sequence of the mutant DNA plasmid is shown as SEQ ID NO. 32.

The sizes of the wild type DNA plasmid and the mutant type DNA plasmid are 3822bp, and the DNA plasmid is synthesized by the Competition Biotechnology engineering (Shanghai) corporation. And (3) measuring the concentration of the synthesized plasmid by using an ultraviolet spectrophotometer, calculating the copy number, and diluting to obtain a working standard substance. Wherein the standard substance 1-5 is diluted to 1.0 × 10 ² ～1.0×10 ⁶ Copy/. mu.L wild type DNA plasmid diluent, standard 6-10 for gradient dilution to 1.0 × 10 ² ～1.0×10 ⁶ Copies/. mu.L of dilutions of mutant DNA plasmids.

RAA primers and screening for crRNA

In this embodiment, after amplifying the target to be detected by using the RAA method, the inventors selected the RAA amplification product to add into the CRISPR/Cas12a system for detection, and selected the most specific and sensitive crRNA for each target and the RAA primer corresponding to the crRNA for amplifying each target according to the ratio of the fluorescence intensity values of the wild-type DNA plasmid and the mutant-type DNA plasmid.

The specific crRNA and RAA primer pairing test in this example is shown in table 4.

TABLE 4

The specific detection method comprises the following steps:

(1) RAA amplification:

adding a hydration buffer solution, an upstream primer, a downstream primer and a magnesium acetate solution into a tube filled with a freeze-dried RAA reaction enzyme according to a system shown in Table 5, adding a detection sample (in the embodiment, a positive control, a negative control or a blank control is respectively adopted, the blank control is non-ribozyme water and is purchased from Esciurel biological company), uniformly mixing, and reacting in a water bath kettle at 39 ℃ for 40min to obtain an amplification product.

Wherein, the hydration buffer solution, the magnesium acetate solution and the freeze-dried RAA reaction enzyme powder tube are all from an RAA basic nucleic acid amplification kit and purchased from Jiangsu Qitian instrument company Limited. The freeze-dried RAA reaction enzyme powder tube comprises components required by constant-temperature reaction, such as recombinase, single-chain binding protein, polymerase, recombinase coenzyme, exonuclease, trehalose, dNTPs, ATP, CK, creatine phosphate disodium and the like.

TABLE 5

Components	Amount of the composition
		Hydration buffer	45μL
(10. mu.M) upstream primer	2μL
		(10. mu.M) downstream primer	2μL
(280mM) magnesium acetate solution	2.5μL
		Lyophilized RAA reaction enzyme powder	1 pipe
Test samples (Positive control, negative control or blank control)	2μL

(2) CRISPR-Cas12a reaction:

according to the mixture ratio shown in Table 5, NEBbuffer2.1 (purchased from New England Biolabs, the components of which comprise 50mM NaCl, 10mM Tris-HCl, 10mM MgCl2, 100 mu g/ml recombinant albumin, pH 7.9), a single-stranded DNA probe, AsCas12a protein and ribozyme-free water are mixed uniformly, then crRNA is added, the mixture is placed in a fluorescence signal detector for real-time signal detection after being mixed fully, the reaction time is 60min, fluorescence signals are collected every 1min, and the judgment of the detection result is carried out. After the reaction is finished, the result is observed by naked eyes through a blue light excitation device, and the detection result is interpreted.

Wherein, the single-stranded DNA probe is a universal probe with 5 variation sites (K417N, T478K, E484K, N501Y and D614G), and the nucleotide sequence of the universal probe is as follows: 5 '-TTATT-3'. The single-stranded DNA probe is marked with a reporter group, specifically a 5 'end modified fluorescence reporter group FAM and a 3' end modified fluorescence quenching group BHQ 1.

TABLE 6

Components	Dosage of
		CRISPR buffer (NEBbufferr2.1)	3μL
(10. mu.M) Single-stranded DNA Probe	1μL
		(20. mu.M) Cas12a protein	0.2μL
Ribozyme-free water	24μL
		Step (a)1) RAA amplification product in (1)	2μL
(100μM)crRNA	0.4μL

In the screening experiment of crRNA-417N, crRNA-478K, crRNA-484K in this example, the RAA amplification reaction in step (1) was performed using Standard No. 3 as a positive sample (mutant DNA plasmid, concentration 1.0X 10 ⁴ Copy/. mu.L), and a sample (wild-type DNA plasmid, concentration 1.0X 10) negative to Standard 10 ⁶ Copy/. mu.L). In the screening experiment of crRNA-501N, crRNA-614D, the RAA amplification reaction in step (1) was performed using standard 5 as a positive sample (mutant DNA plasmid, concentration 1.0X 10 ⁶ Copies/. mu.L), negative sample (wild type DNA plasmid, concentration 1.0X 10) against standard 8 ⁴ Copy/. mu.L).

In this example, taking the K417N site as an example, the specific operation steps of the CRISPR-Cas12a reaction are: according to the mixture ratio shown in table 6, nebbufferr 2.1, single-stranded DNA probe, AsCas12a protein and ribozyme-free water were mixed uniformly to prepare 12 reaction units, which were divided into 3 groups of 4 reaction units, wherein 2 μ L of the positive control RAA amplification product in step (1) was added to

group

1, 2 μ L of the negative control RAA amplification product in step (1) was added to

group

2, 2 μ L of the blank control RAA amplification product in step (1) was added to group 3, and 0.4 μ L of crRNA-417N-1, crRNA-417N-2, crRNA-417N-3, and crRNA-417N-4 was added to each reaction unit to determine which crRNA was the most effective.

(3) Judging the detection result:

and calculating the ratio of the fluorescence intensity values of the crRNA to the mutant type DNA plasmid and the wild type DNA plasmid, and screening the crRNA according to the ratio to obtain the crRNA with the best effect of distinguishing the 5 mutant sites in the mutant strain and the wild strain.

The results are shown in FIG. 1.

After practical experiment screening of the RAA primer and crRNA combination, the RAA primer and crRNA combination which can accurately and sensitively identify 5 characteristic mutations of SARS-CoV-2 is found to be:

for T478K, the crRNA is crRNA-478K-5, the upstream primer is RAA-478F-1, and the downstream primer is RAA-R2;

Optimization of reaction systems

In order to combine the RAA and the CRISPR/Cas12a to perform reaction in the same closed system, thereby simplifying the operation and avoiding aerosol contamination, the present embodiment takes K417N as an example, and verifies the influence of different RAA reaction volumes, different CRISPR/Cas12a reaction volumes, different Cas12a enzymes, different RAA amplification reaction temperatures and times on the detection effect in the mixed system.

In this example, the test sample was selected as Standard 3 (mutant DNA plasmid, concentration 1.0X 10) ⁴ Copies/. mu.L) and standard 10 (wild type DNA plasmid, concentration 1.0X 10 ⁶ Copy/. mu.L), a blank control (no ribozyme water) was set.

(1) Effect of different RAA reaction volumes, CRISPR/Cas12a reaction volumes on assay effect:

the experimental groups were set up as per table 7.

TABLE 7

The components are uniformly mixed, placed in a constant temperature environment of 39 ℃ for reaction for 40min, then centrifuged, placed in a fluorescence signal detector for real-time signal detection, reacted for 60min, and fluorescence signals are collected every 1min for interpretation of detection results. After the reaction is finished, the result is observed by naked eyes through a blue light excitation device, and the detection result is interpreted. And (3) calculating the ratio of the fluorescence intensity values of the single base mutation of K417N in the mutant form and the wild type DNA plasmid in each system, and screening the reaction system combinations according to the ratio to obtain the RAA and CRISPR/Cas12a reaction volumes (N is 3) with the best effect of distinguishing the single base mutation.

The results are shown in FIG. 2.

Under the integrated condition, when the RAA system is 50 mu L, CRISPR/Cas12a system is 5 mu L, the effect of distinguishing the mutant strain and the wild-type plasmid K417N single-base mutation is the best when the RAA and the CRISPR/Cas12a are combined to react in the same closed system.

(2) Effect of different Cas12a proteins on assay effect:

the experimental groups were set up as per table 8.

TABLE 8

Wherein, AsCas12a (0.2 μ L, 20 μ M) and LbCas12a (1 μ L, 40 μ M) are self-made purified Cas12a proteins, and the specific obtaining mode is as follows: the LbCas12a (Lachnospiraceae) gene is obtained from pcDNA3.1-hLbCpf1 plasmid (Addge #69988), and cloned into expression vector pET-28a (+) to obtain recombinant plasmid pET-28a (+) -hLbCpf1, which is transformed into DE3 competent cell for mass expression and purification to obtain LbCas12a protein, which is stored at-80 ℃. A plasmid (Addge #114073) containing the henAsCas12a (Acylaminococcus sp.) sequence was purchased, transformed into DE3 competent cells, expressed in large quantities and purified to obtain AsCas12a protein, which was stored at-80 ℃ after split charging. LbCas12a (0.1. mu.L, 100. mu.M) was purchased from NewEngland Biolabs. LbCas12a (1. mu.L, 10. mu.M) was purchased from Bio-lifesci.

The components are uniformly mixed, placed in a constant temperature environment of 39 ℃ for reaction for 40min, then centrifuged, placed in a fluorescence signal detector for real-time signal detection, reacted for 60min, and fluorescence signals are collected every 1min for interpretation of detection results. And after the reaction is finished, carrying out interpretation on the detection result by using blue light excitation equipment and observing the result by naked eyes. And (3) calculating the ratio of the fluorescence intensity values of the single base mutation of K417N in the mutant form and the wild type DNA plasmid in each system, and screening the Cas12a enzyme according to the ratio to obtain the Cas12a protein (N-3) with the best single base mutation distinguishing effect.

The results are shown in FIG. 3.

It can be found that the fluorescence intensity ratio of assas 12a (0.2 μ L, 20 μ M) is the largest, but the fluorescence intensity ratio of LbCas12a obtained by other methods is not different from the largest, so that the protein can be used as Cas12a protein.

(3) Effect of different RAA amplification reaction temperatures on detection effect:

the experimental groups were set up as per table 9.

TABLE 9

Test sample	Standard substance 3(2 mu L)	Standard substance 10(2 mu L)	Ribozyme-free water (2 μ L)
				Upstream and downstream primers of RAA primer set	2 μ L each (total 4 μ L)	2 μ L each (total 4 μ L)	2 μ L each (total 4 μ L)
Magnesium acetate solution	2.5μL	2.5μL	2.5μL
				RAA hydration buffer	45μL	45μL	45μL
Freeze-dried RAA reaction enzyme powder	1 tube	1 tube	1 tube
				crRNA	0.4μL	0.4μL	0.4μL
Cas12a protein	0.2μLAsCas12a20μM	0.2μLAsCas12a20μM	0.2μLAsCas12a20μM
				Single-stranded DNA probe	1μL	1μL	1μL
NEBbuffer2.1	3μL	3μL	3μL

After the components are uniformly mixed, 3 parts of each group are respectively placed in the environment with the constant temperature of 37 ℃, 39 ℃ and 42 ℃ for reaction for 40min, then centrifugation is carried out, the reaction is carried out in a fluorescence signal detector for real-time signal detection, the reaction time is 60min, a fluorescence signal is collected every 1min, and the judgment of the detection result is carried out. After the reaction is finished, the result is observed by naked eyes through a blue light excitation device, and the detection result is interpreted. The ratio of the fluorescence intensity values of the single base mutation of K417N in the mutation type and the wild type DNA plasmid in each system is calculated, and the RAA amplification reaction temperature is screened according to the ratio, so that the RAA amplification reaction temperature with the best discrimination effect on the single base mutation is obtained (N is 3).

The results are shown in FIG. 4.

It was found that the fluorescence intensity ratio was the largest at 39 ℃ and thus 39 ℃ was selected as the RAA amplification reaction temperature.

(4) Effect of different RAA amplification reaction times on detection effect:

the experimental groups were set up as per table 9.

After the components are uniformly mixed, 4 parts of each group are respectively placed in an environment with the constant temperature of 39 ℃ to react for 25min, 30min, 35min and 40min, then centrifugation is carried out, the mixture is placed in a fluorescence signal detector to carry out real-time signal detection, the reaction time is 60min, a fluorescence signal is collected every 1min, and the interpretation of the detection result is carried out. After the reaction is finished, the result is observed by naked eyes through a blue light excitation device, and the detection result is interpreted. And (3) calculating the ratio of the fluorescence intensity values of the single base mutation of K417N in the mutant form and the wild type DNA plasmid in each system, and screening the RAA amplification reaction time according to the ratio to obtain the RAA amplification reaction time with the best effect of distinguishing the single base mutation (N-3).

The results are shown in FIG. 5.

It was found that the ratio of fluorescence intensity was the largest at 40min, and therefore 40min was selected as the RAA amplification reaction time.

In conclusion, when RAA system of 50 mu L, CRISPR/Cas12a system reacts at 39 ℃ for 40min at 5 mu L, RAA, the effect of differentiating mutant and wild-type plasmid K417N single-base mutation is best when RAA and CRISPR/Cas12a are combined to react in the same closed system. In addition, both the AsCas12a protein and LbCas12a proteins from different sources can react, and the ratio of fluorescence intensity values has no obvious statistical difference, so that the Cas12a protein in the method can be obtained after expression and purification, and can also be directly purchased as a commercial Cas12a protein.

Detection effect of RAA-Cas12a new coronavirus detection reaction system

(1) Detection sensitivity:

a RAA-Cas12a novel coronavirus detection reaction system was constructed according to Table 10.

TABLE 10

The detection sample is recombinant plasmid containing S gene fragment of SARS-CoV-2 with different copy concentration, wherein in sensitivity experiment of K417N, T478K and E484K loci, the target nucleic acid components to be detected are respectively 1-5 (concentration 1.0X 10) ² Copy/. mu.L, 1.0X 10 ³ Copy/. mu.L, 1.0X 10 ⁴ Copy/. mu.L, 1.0X 10 ⁵ Copy/. mu.L, 1.0X 10 ⁶ Copies/. mu.L of mutant plasmid) and standard 10 (concentration 1.0X 10) ⁶ Copies/. mu.L of wild-type plasmid, negative control). Water without ribozymes served as a blank control. In the sensitivity experiments of the sites of N501Y and D614G, the nucleic acid component of the target to be detected is 6-10 of a standard substance, 5 of the standard substance is used as a negative control, and water without ribozyme is used as a blank control.

The detection method is the same as the above embodiment.

Wherein, the interpretation of the detection result can be carried out by any one of the following methods A to B:

The specific calculation formula is as follows:

The results are shown in FIGS. 6 to 7.

It was found that the detection sensitivity of the characteristic mutation sites of K417N, T478K, E484K, N501Y and D614G was 1.0X 10, respectively ⁴ The copy/. mu.L, therefore, it can be determined that the sensitivity of the RAA-Cas12a new coronavirus detection reaction system in the above example to different new coronavirus strains is 1.0X 10 ⁴ Copies/. mu.L.

(2) Detection specificity:

the specificity test is carried out by taking 19 clinical samples infected with common respiratory pathogens as detection objects, taking a standard 5 as a negative control, taking a standard 10 as a positive control and taking ribozyme-free water as a blank control and adopting the RAA-Cas12a new coronavirus detection reaction system in the table 10.

Among them, 19 common pathogens infecting the respiratory tract are: 3 cases of human coronavirus 229E (HCoV-229E), 1 case of human coronavirus OC43(HCoV-OC43), 3 cases of human coronavirus HKU1(HCoV-HKU1), 1 case of respiratory syncytial virus type A (RSV-A) and 1 case of respiratory syncytial virus type B (RSV-B), 1 case of parainfluenza virus type 1 (HPIV-1) and 1 case of parainfluenza virus type 4 (HPIV-4), 3 cases of rhinovirus (HRV), 1 case of adenovirus (AdV), 3 cases of human Beard virus (HBoV), 1 case of human pneumonia virus (HMPV).

The detection system, detection method and detection result judgment standard are the same as those of the above embodiment.

The results are shown in FIG. 8.

It can be found that only the detection result of the positive control (SARS-CoV-2-S fragment plasmid containing target mutation site) is positive, and the other nucleic acid results are negative, thus proving the high specificity of the method.

(3) The practical detection effect of the RAA-Cas12a new coronavirus detection reaction system is as follows:

in order to prove the practical feasibility of the RAA-Cas12a new coronavirus detection reaction system, 54 clinical samples are selected in the embodiment, and the RAA-Cas12a new coronavirus detection reaction system and the Sanger sequencing method are used for parallel detection to verify the consistency of the method disclosed by the invention and Sanger sequencing. Among them, 54 clinical samples were obtained from Guangdong provincial disease control and prevention center, and cDNA samples subjected to reverse transcription after nucleic acid extraction from throat swabs of patients with COVID-19 was confirmed were subjected to Sanger sequencing, and according to the sequence typing results, 4 cases of Wild type (Wild type), 16 cases of Aplha variants, 14 cases of Beta variants, 15 cases of Delta variants, and 5 cases of Omicron variants were included.

The detection system, detection method and detection result judgment criteria are the same as those of the above embodiment.

The results are shown in FIGS. 9 to 10.

It can be found that the RAA-Cas12a new coronavirus detection reaction system can correctly type 50 parts of SARS-CoV-2 positive clinical samples, the consistency with the Sanger sequencing result is 92.59%, the detection result positive predictive value of various variants is 100.0%, and the negative predictive value is 92.9-100.0% (see the following Table 11). The 4 samples that were not correctly typed were 1 Alpha variant and 3 Delta variants, of which 1 Delta variant (2021A-XG08905) was a sublay of Delta variants (AY.1), and the sequencing results indicated that it had an additional K417N mutation compared to the Delta variant, which could not be correctly typed using the above-described principle of RAA-Cas12a New crown Virus detection reaction system. In addition, N501Y mutation was not detected in 2 Delta variants (2021A-XG09089 and 2021A-XG08715) and an additional 484K mutation was detected in 1 Alpha variant (2021A-XG04560), all of which failed to correctly type.

TABLE 11

In conclusion, the RAA-Cas12a new coronavirus detection reaction system has high accuracy in detecting and identifying different variants of SARS-CoV-2 in practical application, the consistency with a Sanger sequencing result is 92.59% (50/54), and compared with gene sequencing, the RAA-Cas12a new coronavirus detection reaction system has the characteristics of short time consumption, low cost, visualization, no need of large instruments, and suitability for primary laboratories.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

SEQUENCE LISTING

<110> southern medical university

<120> detection kit for detecting different variants of novel coronavirus and application thereof

<130>

<160> 32

<170> PatentIn version 3.5

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ggcctgatag atttcagttg aaatatctct ct 32

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tatttcaact gaaatctatc aggccgtttg ca 32

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agatatttca actgaaatct ttcaggccgg tag 33

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tctatcaggc cggtagcaca ctttgtaatg gtg 33

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tcaggccggt agcacacctt ttaatggtgt t 31

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ttgagagaga tatttcaact gaaatctatc ag 32

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ttgttagact cagtaagaac acctgtgcct gt 32

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taggaagtct aatctcaaac cttttgagag ag 32

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agtaagttga tctgcatgaa tagcaacagg ga 32

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uaauuucuac uaaguguaga aaauauugcu gauuauaauu 40

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uaauuucuac uaaguguaga acauauugcu gauuauaauu 40

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uaauuucuac uaaguguaga aaauagugcu gauuauaauu 40

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uaauuucuac uaaguguaga gcaaaccuug uaaugguguu 40

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uaauuucuac uaaguguaga aggccgguag caaaccuugu 40

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uaauuucuac uaaguguaga cgauggccgg acuaucuaaa 40

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uaauuucuac uaaguguaga ccaaaccuug uaaugguguu 40

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uaauuucuac uaaguguaga gcaaagcuug uaaugguguu 40

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uaauuucuac uaaguguaga ggaaaccuug uaaugguguu 40

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uaauuucuac uaaguguaga uaaugguguu aaagguuuua 40

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uaauuucuac uaaguguaga aaugguguua aagguuuuaa 40

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uaauuucuac uaaguguaga aacaccauua caaggugugc u 41

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uaauuucuac uaaguguaga caacccacua augguguugg 40

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uaauuucuac uaaguguaga caacccacua aagguguugg 40

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uaauuucuac uaaguguaga caacccagua augguguugg 40

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uaauuucuac uaaguguaga caacccacaa augguguugg 40

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uaauuucuac uaaguguaga ucaggauguu aacugcacag 40

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uaauuucuac uaaguguaga ucaggacguu aacugcacag 40

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<212> DNA

<213> Artificial sequence

<400> 31

atgtttgttt tttttgtttt attgccacta gtctctagtc agtgtgttaa tcttacaacc 60

agaactcaat taccccctgc atacactaat tctttcacac gtggtgttta ttaccctgac 120

aaagttttca gatcctcagt tttacattca actcaggact tgttcttacc tttcttttcc 180

aatgttactt ggttccatgc tatacatgtc tctgggacca atggtactaa gaggtttgct 240

aaccctgtcc taccatttaa tgatggtgtt tattttgctt ccactgagaa gtctaacata 300

ataagaggct ggatttttgg tactacttta gattcgaaga cccagtccct acttattgtt 360

aataacgcta ctaatgttgt tattaaagtc tgtgaatttc aattttgtaa tgatccattt 420

ttgggtgttt attaccacaa aaacaacaaa agttggatgg aaagtgagtt cagagtttat 480

tctagtgcga ataattgcac ttttgaatat gtctctcagc cttttcttat ggaccttgaa 540

ggaaaacagg gtaatttcaa aaatcttagg gaatttgtgt ttaagaatat tgatggttat 600

tttaaaatat attctaagca cacgcctatt aatttagtgc gtggtctccc tcagggtttt 660

tcggctttag aaccattggt agatttgcca ataggtatta acatcactag gtttcaaact 720

ttacttgctt tacatataag ttatttgact cctggtgatt cttcttcagg ttggacagct 780

ggtgctgcag cttattatgt gggttatctt caacctagga cttttctatt aaaatataat 840

gaaaatggaa ccattacaga tgctgtagac tgtgcacttg accctctctc agaaacaaag 900

tgtacgttga aatccttcac tgtagaaaaa ggaatctatc aaacttctaa ctttagagtc 960

caaccaacag aatctattgt tagatttcct aatattacaa acttgtgccc ttttggtgaa 1020

gtttttaacg ccaccagatt tgcatctgtt tatgcttgga acaggaagag aatcagcaac 1080

tgtgttgctg attattctgt cctatataat tccgcatcat tttccacttt taagtgttat 1140

ggagtgtctc ctactaaatt aaatgatctc tgctttacta atgtctatgc agattcattt 1200

gtaattagag gtgatgaagt cagacaaatc gctccagggc aaactggaaa cattgctgat 1260

tataattata aattaccaga tgattttaca ggctgcgtta tagcttggaa ttctaacaat 1320

cttgattcta aggttggtgg taattataat tacctgttta gattgtttag gaagtctaat 1380

ctcaaacctt ttgagagaga tatttcaact gaaatctatc aggccggtag cacaccttgt 1440

aatggtgtta aaggttttaa ttgttacttt cctttacaat catatggttt ccaacccact 1500

tatggtgttg gttaccaacc atacagagta gtagtacttt cttttgaact tctacatgca 1560

ccagcaactg tttgtggacc taaaaagtct actaatttgg ttaaaaacaa atgtgtcaat 1620

ttcaacttca atggtttaac aggcacaggt gttcttactg agtctaacaa aaagtttctg 1680

cctttccaac aatttggcag agacattgat gacactactg atgctgtccg tgatccacag 1740

acacttgaga ttcttgacat tacaccatgt tcttttggtg gtgtcagtgt tataacacca 1800

ggaacaaata cttctaacca ggttgctgtt ctttatcagg gtgttaactg cacagaagtc 1860

cctgttgcta ttcatgcaga tcaacttact cctacttggc gtgtttattc tacaggttct 1920

aatgtttttc aaacacgtgc aggctgttta ataggggctg aacatgtcaa caactcatat 1980

gagtgtgaca tacccattgg tgcaggtata tgcgctagtt atcagactca gactaattct 2040

catcggcggg cacgtagtgt agctagtcaa tccatcattg cctacactat gtcacttggt 2100

gtagaaaatt cagttgctta ctctaataac tctattgcca tacccataaa ttttactatt 2160

agtgttacca cagaaattct accagtgtct atgaccaaga catcagtaga ttgtacaatg 2220

tacatttgtg gtgattcaac tgaatgcagc aatcttttgt tgcaatatgg cagtttttgt 2280

acacaattaa accgtgcttt aactggaata gctgttgaac aagacaaaaa cacccaagaa 2340

gtttttgcac aagtcaaaca aatttacaaa acaccaccaa ttaaagattt tggtggtttt 2400

aatttttcac aaatattacc agatccatca aaaccaagca agaggtcatt tattgaagat 2460

ctacttttca acaaagtgac acttgcagat gctggcttca tcaaacaata tggtgattgc 2520

cttggtgata ttgctgctag agacctcatt tgtgcacaaa agtttaacgg ccttactgtt 2580

ttgccacctt tgctcacaga tgaaatgatt gctcaataca cttctgcact gttagcgggt 2640

acaatcactt ctggttggac ctttggtgca ggtgctgcat tacaaatacc atttgctatg 2700

caaatggctt ataggtttaa tggtattgga gttacacaga atgttctcta tgagaaccaa 2760

aaattgattg ccaaccaatt taatagtgct attggcaaaa ttcaagactc actttcttcc 2820

acagcaagtg cacttggaaa acttcaagat gtggtcaacc aaaatgcaca agctttaaac 2880

acgcttgtta aacaacttag ctccaatttt ggtgcaattt caagtgtttt aaatgatatc 2940

cttgcacgtc ttgacaaagt tgaggctgaa gtgcaaattg ataggttgat cacaggcaga 3000

cttcaaagtt tgcagacata tgtgactcaa caattaatta gagctgcaga aatcagagct 3060

tctgctaatc ttgctgctac taaaatgtca gagtgtgtac ttggacaatc aaaaagagtt 3120

gatttttgtg gaaagggcta tcatcttatg tccttccctc agtcagcacc tcatggtgta 3180

gtcttcttgc atgtgactta tgtccctgca caagaaaaga acttcacaac tgctcctgcc 3240

atttgtcatg atggaaaagc acactttcct cgtgaaggtg tctttgtttc aaatggcaca 3300

cactggtttg taacacaaag gaatttttat gaaccacaaa tcattactac acacaacaca 3360

tttgtgtctg gtaactgtga tgttgtaata ggaattgtca acaacacagt ttatgatcct 3420

ttgcaacctg aattagactc attcaaggag gagttagata aatattttaa gaatcataca 3480

tcaccagatg ttgatttagg tgacatctct ggcattaatg cttcagttgt aaacattcaa 3540

aaagaaattg accgcctcaa tgaggttgcc aagaatttaa atgaatctct catcgatctc 3600

caagaacttg gaaagtatga gcagtatata aaatggccat ggtacatttg gctaggtttt 3660

atagctggct tgattgccat agtaatggtg acaattatgc tttgctgtat gaccagttgc 3720

tgtagttgtc tcaagggctg ttgttcttgt ggatcctgct gcaaatttga tgaagacgac 3780

tctgagctag tgctcaaagg agtcaaatta cattacacat aa 3822

<210> 32

<211> 3822

<212> DNA

<213> Artificial sequence

<400> 32

atgtttgttt tttttgtttt attgccacta gtctctagtc agtgtgttaa tcttacaacc 60

agaactcaat taccccctgc atacactaat tctttcacac gtggtgttta ttaccctgac 120

aaagttttca gatcctcagt tttacattca actcaggact tgttcttacc tttcttttcc 180

aatgttactt ggttccatgc tatacatgtc tctgggacca atggtactaa gaggtttgct 240

aaccctgtcc taccatttaa tgatggtgtt tattttgctt ccactgagaa gtctaacata 300

ataagaggct ggatttttgg tactacttta gattcgaaga cccagtccct acttattgtt 360

aataacgcta ctaatgttgt tattaaagtc tgtgaatttc aattttgtaa tgatccattt 420

ttgggtgttt attaccacaa aaacaacaaa agttggatgg aaagtgagtt cagagtttat 480

tctagtgcga ataattgcac ttttgaatat gtctctcagc cttttcttat ggaccttgaa 540

ggaaaacagg gtaatttcaa aaatcttagg gaatttgtgt ttaagaatat tgatggttat 600

tttaaaatat attctaagca cacgcctatt aatttagtgc gtggtctccc tcagggtttt 660

tcggctttag aaccattggt agatttgcca ataggtatta acatcactag gtttcaaact 720

ttacttgctt tacatataag ttatttgact cctggtgatt cttcttcagg ttggacagct 780

ggtgctgcag cttattatgt gggttatctt caacctagga cttttctatt aaaatataat 840

gaaaatggaa ccattacaga tgctgtagac tgtgcacttg accctctctc agaaacaaag 900

tgtacgttga aatccttcac tgtagaaaaa ggaatctatc aaacttctaa ctttagagtc 960

caaccaacag aatctattgt tagatttcct aatattacaa acttgtgccc ttttggtgaa 1020

gtttttaacg ccaccagatt tgcatctgtt tatgcttgga acaggaagag aatcagcaac 1080

tgtgttgctg attattctgt cctatataat tccgcatcat tttccacttt taagtgttat 1140

ggagtgtctc ctactaaatt aaatgatctc tgctttacta atgtctatgc agattcattt 1200

gtaattagag gtgatgaagt cagacaaatc gctccagggc aaactggaaa tattgctgat 1260

tataattata aattaccaga tgattttaca ggctgcgtta tagcttggaa ttctaacaat 1320

cttgattcta aggttggtgg taattataat taccggttta gattgtttag gaagtctaat 1380

ctcaaacctt ttgagagaga tatttcaact gaaatctatc aggccggtag caaaccttgt 1440

aatggtgttc aaggttttaa ttgttacttt cctttacaat catatggttt ccaacccact 1500

tatggtgttg gttaccaacc atacagagta gtagtacttt cttttgaact tctacatgca 1560

ccagcaactg tttgtggacc taaaaagtct actaatttgg ttaaaaacaa atgtgtcaat 1620

ttcaacttca atggtttaac aggcacaggt gttcttactg agtctaacaa aaagtttctg 1680

cctttccaac aatttggcag agacattgat gacactactg atgctgtccg tgatccacag 1740

acacttgaga ttcttgacat tacaccatgt tcttttggtg gtgtcagtgt tataacacca 1800

ggaacaaata cttctaacca ggttgctgtt ctttatcagg gtgttaactg cacagaagtc 1860

cctgttgcta ttcatgcaga tcaacttact cctacttggc gtgtttattc tacaggttct 1920

aatgtttttc aaacacgtgc aggctgttta ataggggctg aacatgtcaa caactcatat 1980

gagtgtgaca tacccattgg tgcaggtata tgcgctagtt atcagactca gactaattct 2040

catcggcggg cacgtagtgt agctagtcaa tccatcattg cctacactat gtcacttggt 2100

gtagaaaatt cagttgctta ctctaataac tctattgcca tacccataaa ttttactatt 2160

agtgttacca cagaaattct accagtgtct atgaccaaga catcagtaga ttgtacaatg 2220

tacatttgtg gtgattcaac tgaatgcagc aatcttttgt tgcaatatgg cagtttttgt 2280

acacaattaa accgtgcttt aactggaata gctgttgaac aagacaaaaa cacccaagaa 2340

gtttttgcac aagtcaaaca aatttacaaa acaccaccaa ttaaagattt tggtggtttt 2400

aatttttcac aaatattacc agatccatca aaaccaagca agaggtcatt tattgaagat 2460

ctacttttca acaaagtgac acttgcagat gctggcttca tcaaacaata tggtgattgc 2520

cttggtgata ttgctgctag agacctcatt tgtgcacaaa agtttaacgg ccttactgtt 2580

ttgccacctt tgctcacaga tgaaatgatt gctcaataca cttctgcact gttagcgggt 2640

acaatcactt ctggttggac ctttggtgca ggtgctgcat tacaaatacc atttgctatg 2700

caaatggctt ataggtttaa tggtattgga gttacacaga atgttctcta tgagaaccaa 2760

aaattgattg ccaaccaatt taatagtgct attggcaaaa ttcaagactc actttcttcc 2820

acagcaagtg cacttggaaa acttcaagat gtggtcaacc aaaatgcaca agctttaaac 2880

acgcttgtta aacaacttag ctccaatttt ggtgcaattt caagtgtttt aaatgatatc 2940

cttgcacgtc ttgacaaagt tgaggctgaa gtgcaaattg ataggttgat cacaggcaga 3000

cttcaaagtt tgcagacata tgtgactcaa caattaatta gagctgcaga aatcagagct 3060

tctgctaatc ttgctgctac taaaatgtca gagtgtgtac ttggacaatc aaaaagagtt 3120

gatttttgtg gaaagggcta tcatcttatg tccttccctc agtcagcacc tcatggtgta 3180

gtcttcttgc atgtgactta tgtccctgca caagaaaaga acttcacaac tgctcctgcc 3240

atttgtcatg atggaaaagc acactttcct cgtgaaggtg tctttgtttc aaatggcaca 3300

cactggtttg taacacaaag gaatttttat gaaccacaaa tcattactac acacaacaca 3360

tttgtgtctg gtaactgtga tgttgtaata ggaattgtca acaacacagt ttatgatcct 3420

ttgcaacctg aattagactc attcaaggag gagttagata aatattttaa gaatcataca 3480

tcaccagatg ttgatttagg tgacatctct ggcattaatg cttcagttgt aaacattcaa 3540

aaagaaattg accgcctcaa tgaggttgcc aagaatttaa atgaatctct catcgatctc 3600

caagaacttg gaaagtatga gcagtatata aaatggccat ggtacatttg gctaggtttt 3660

atagctggct tgattgccat agtaatggtg acaattatgc tttgctgtat gaccagttgc 3720

tgtagttgtc tcaagggctg ttgttcttgt ggatcctgct gcaaatttga tgaagacgac 3780

tctgagctag tgctcaaagg agtcaaatta cattacacat aa 3822

Claims

1. A set of RAA primers, wherein the set of RAA primers targets at least one mutation site of K417N, T478K, E484K, N501Y, D614G in a novel coronavirus variant.

2. The RAA primer set of claim 1, wherein the 5' end of the mutation site further comprises a PAM sequence.

3. The RAA primer set according to claim 1, wherein the nucleotide sequence of the RAA primer set is as set forth in SEQ ID NO. 1-11.

4. The RAA primer set according to claim 3, wherein the upstream primer in the RAA primer set is any one of SEQ ID NOS 1-2, 4-8 and 10, and the downstream primer in the RAA primer set is any one of SEQ ID NOS 3, 9 and 11.

5. A group of crRNAs, which comprises a stem-loop structure sequence at the 5 'end and a complementary sequence of a target gene at the 3' end, wherein the target gene is a novel coronavirus S protein coding gene containing any one site of K417N, T478K, E484K, N501Y and D614G.

6. The crRNA of claim 5, wherein the nucleotide sequence of the crRNA is shown in SEQ ID NO 12-30.

7. A novel coronavirus detection reagent, wherein the detection reagent comprises the RAA primer set according to any one of claims 1 to 4 and the crRNA according to any one of claims 5 to 6.

8. The detection reagent of claim 7, further comprising a single-stranded DNA probe and a Cas12a protein.

9. The detection reagent according to claim 8, wherein the nucleotide sequence of the single-stranded DNA probe is: 5 '-TTATT-3'.

10. Use of the RAA primer set of any one of claims 1 to 4 and/or the crRNA of any one of claims 5 to 6 for preparing a novel coronavirus detection kit.