Nucleic acid detection primer set, kit and detection method for SARS-CoV-2 virus mutant strains

Abstract

This invention discloses a set of primers, a kit, and a detection method for nucleic acid detection of SARS-CoV-2 virus mutant strains. The invention selects the gene sequence encoding the nucleocapsid protein N of the SARS-CoV-2 virus and designs a specific set of nucleic acid detection primers targeting the same mutation site R203M in the B.1.617 and Delta Plus mutant strains. This site is mutated in B1.617.1 (94%), B1.617.2 (99%), B1.617.3 (85%), AY.1 (99%), and AY.2 (93%). Specific fluorescence detection targeting this mutation site allows for rapid differentiation between SARS-CoV-2 virus mutant strains (B.1.617 and Delta Plus) with the R203M mutation and 2019-nCoV (WIV04) or other mutant strains without the R203M mutation. This invention has high specificity, high sensitivity, and no cross-reactivity. It can quickly and accurately screen mutant strains B.1.617 and Delta Plus mutant strains. It is simple to operate and provides accurate detection results, and has good application advantages in the field of viral nucleic acid detection.

Description

Technical Field

[0001] This invention relates to the field of viral nucleic acid detection technology, specifically to a nucleic acid detection primer set, kit, and detection method for a SARS-CoV-2 virus mutant strain. Background Technology

[0002] COVID-19 is an acute infectious pneumonia caused by the novel coronavirus (SARS-CoV-2). Currently, COVID-19 has resulted in nearly 200 million infections and 4 million deaths globally. Since the start of the COVID-19 pandemic in early 2020, a series of variants of the novel coronavirus have emerged worldwide. Currently, four variants of the novel coronavirus have been classified as "variants of concern" by the World Health Organization: the B.1.1.7 variant discovered in the UK last December, the B.1.351 variant discovered in South Africa last December, the P.1 variant discovered in Brazil this January, and the B.1.617 mutant strain discovered in India last October.

[0003] According to Elena Quinonez's research, the structure of B.1.617S-RBD, compared to the wild-type structure, has a 110% increased affinity score for human angiotensin-converting enzyme 2 (hACE2), making it highly infectious and enhancing its self-replication ability. Almost all surveyed countries demonstrated that the variants of concern designated by the World Health Organization rapidly replaced the previously circulating lineage, with an estimated 97% increase in B.1.617 transmission rate (95% CI: 76-117). Currently, according to the latest estimates from British scientists, the Indian mutant strain has three mutations or subclades—B.1.617.1 (Kappa mutant), B.1.617.3, and B.1.617.2 (Delta mutant), accounting for over 90% of the total. The B.1.617.2 (Delta mutant) spreads 40-80% faster than the Alpha mutant (B.1.1.7) previously observed in the UK.

[0004] For mutant strains, sequencing technology is the gold standard for diagnosis, currently mainly third-generation sequencing technology. This technology can directly sequence RNA without base bias, making continuous reading of the full-length genome of the novel coronavirus possible, significantly reducing subsequent genome assembly and annotation work. However, sequencing is expensive and time-consuming, making it unsuitable for point-of-care testing (POCT). For mutant strains B.1.617 and Delta Plus, there is an urgent need to develop a rapid nucleic acid detection method that is accurate, time-efficient, highly sensitive, and specific. This is crucial for the rapid diagnosis of suspected cases and the implementation of appropriate isolation measures, ensuring timely treatment for patients and the safety of others. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of existing technologies and provide a set of nucleic acid detection primers for SARS-CoV-2 mutant strains to achieve rapid detection of SARS-CoV-2 virus mutant strains (B.1.617 and Delta Plus).

[0006] The objective of this invention is achieved through the following technical solution: providing a nucleic acid detection primer set for a SARS-CoV-2 virus mutant strain, wherein the nucleic acid detection primer set includes outer primers F3 and B3, inner primers FIP and BIP, and a loop primer LF1, the nucleotide sequences of which are shown below:

[0007] F3: ACGTAGTCGCAACAGTTC;

[0008] B3: CtTAGAAGCCtCAGCAGC;

[0009] FIP:AAGCAAGAGCAGCATCACCG-GAAATTCAACtCCAGGCAGC;

[0010] BIP: CtGCtTGACAGATTGAACCAGC-GTGACAGTTTGGCCtTGTT;

[0011] LF1:CATTCtAGCAGGAGAAGTTCCCA.

[0012] To address the aforementioned technical problems, this invention also provides another set of nucleic acid detection primers for a SARS-CoV-2 virus mutant strain. This set includes outer primers F3 and B3, inner primers FIP and BIP, and a loop primer LF2, the nucleotide sequences of which are shown below:

[0013] F3: ACGTAGTCGCAACAGTTC;

[0014] B3: CtTAGAAGCCtCAGCAGC;

[0015] FIP:AAGCAAGAGCAGCATCACCG-GAAATTCAACtCCAGGCAGC;

[0016] BIP: CtGCtTGACAGATTGAACCAGC-GTGACAGTTTGGCCtTGTT;

[0017] LF2:TCtAGCAGGAGAAGTTCCCATAC.

[0018] The present invention also provides a nucleic acid detection kit for a SARS-CoV-2 virus mutant strain, comprising the nucleic acid detection primer set as described above.

[0019] Furthermore, the kit comprises the following components: reaction premix A, reaction premix B, two positive controls, and a negative control;

[0020] The reaction premix A includes 2-10 U Bst DNA large fragment polymerase, 2-10 U AMV reverse transcriptase, and 1 μL fluorescent dye.

[0021] The reaction premix B includes 10× reaction buffer, a set of nucleic acid detection primers as described above, 1.6 mM dNTPs, 8 mM MgSO4, 0.8 mM betaine, and DEPC water;

[0022] The two positive quality controls were at a concentration of 10. 5 The gene fragment encoding the nucleocapsid protein N gene of the SARS-CoV-2 virus mutant strain (copies / μL) and the gene fragment of the unmutated original virus strain 2019-nCoV (WIV04);

[0023] The negative control was DEPC water.

[0024] Furthermore, in the nucleic acid detection primer set, the molar ratio of the outer primer, inner primer, and loop primer is 1:4:2.

[0025] Further, the reaction buffer consists of the following components: 200 mM Tris-HCl, pH 8.8, 100 mM (NH4)2SO4, 100 mM KCl, 20 mM MgSO4, and 0.1% Triton-X.

[0026] Furthermore, the fluorescent dye is SYTO 9 fluorescent dye.

[0027] The present invention also provides a detection method for a nucleic acid detection kit for SARS-CoV-2 virus mutant strains as described above. By comparing the Ct values ​​of two sets of positive quality control samples, mutant strains (B.1.617 and Delta Plus) and 2019-nCoV (WIV04) or other mutant strains without the R203M mutation are identified. The nucleic acid detection primer set designed using the R203M mutation site on the N gene enables rapid detection and screening of SARS-CoV-2 virus mutant strains (B.1.617 and Delta Plus).

[0028] The detection method includes the following steps:

[0029] (1) Prepare the RT-LAMP reaction system: Add 25 μL of the reaction system to the test tube: 3 μL of reaction premix A, 17 μL of reaction premix B and 5 μL of the sample to be tested. At the same time, set up two positive quality control and negative control.

[0030] (2) Gene amplification reaction: Place the detection tube into the fluorescence amplification instrument, react at 65℃ for 60 min, and interrupt the reaction at 85℃;

[0031] (3) Result judgment: Observe the amplification curve through the fluorescence amplification instrument to determine the detection result and record the amplification Ct value;

[0032] If an "S"-shaped amplification curve is observed, the test result is determined to be positive for SARS-CoV-2 virus; when the copy number concentration of the sample to be tested is 10... 5 When the number of copies / μL is reached, the amplified Ct value is compared with the amplified Ct values ​​of two positive quality controls with the same copy number concentration. If the amplified Ct value of the sample to be tested is less than the amplified Ct value of the positive quality control of the N gene without mutation, and the error between the amplified Ct value of the sample and the positive quality control of the mutant N gene is within 5, then the sample is judged to be a SARS-CoV-2 virus mutant strain.

[0033] If the amplified Ct value of the sample to be tested is greater than the amplified Ct value of the positive quality control of the N gene without mutation, it is judged to be the original virus strain 2019-nCoV without mutation or other mutant strain.

[0034] If no normal amplification occurs, the result is considered negative for SARS-CoV-2 virus.

[0035] Further, the 25 μL reaction system consists of the following components: 3 μL reaction premix A: 4 U of Bst DNA large fragment polymerase, 8 U of AMV reverse transcriptase and 1 μL of SYTO 9 fluorescent dye;

[0036] 17 μL reaction premix B: 2.8 μL primer set, 2.5 μL 10× reaction buffer, 1.6 mM dNTP, 0.8 mM betaine, 8 mM MgSO4, and DEPC water to a final volume of 17 μL.

[0037] Compared with the prior art, the present invention has the following beneficial effects:

[0038] 1. This invention uses the original SARS-CoV-2 virus strain WIV04 (MN996528.1) published in Gene Bank, with a full length of 29891 bp, as the original sequence. The mutation site was determined by comparing the snap gene with a large number of SARS-CoV-2 virus mutant strains (B1.167 and Delta Plus). The R203M mutation encoding the N gene of the viral nucleocapsid in the B1.1.617 mutant strain was finally selected. This site was mutated in B1.617.1 (94%), B1.617.2 (99%), B1.617.3 (85%), AY.1 (99%), and AY.2 (93%). Based on this mutation site, two sets of specific nucleic acid detection primers were designed to achieve a rapid, accurate, and specific detection of SARS-CoV-2 mutant strains (B1.167 and Delta Plus) by matching the amplification time interval with the appearance of the unmutated 2019-nCoV. Plus) does not cross-react with SARS-CoV, Mers-CoV, EB virus, HCMV virus, common respiratory viruses (influenza A / B virus, respiratory syncytial virus and human parainfluenza virus, etc.) and enteroviruses, and has high sensitivity.

[0039] 2. This invention designs two sets of RT-LAMP primers based on the target sequence, including inner and outer primers. The SNP site, i.e., the mutation site, is flanked by two regions (F1C and F2C) of the inner primer to ensure that the SNP allele is located in the loop domain of the RT-LAMP product. Single-base mismatches lead to unstable hybridization between the LP and loop at the reaction temperature, which cannot effectively enhance the reaction containing the wild-type template, resulting in differences in amplification rates. Mutants are detected by time intervals.

[0040] 3. The RT-LAMP fluorescence detection method for detecting SARS-CoV-2 virus mutant strains (B.1.617 and Delta Plus) provided by this invention has simple and accurate results. The amplification curve can be observed through a fluorescence amplification instrument to determine the detection results. It can be used in hospitals, clinics and testing laboratories to simultaneously and rapidly screen carriers of SARS-CoV-2 virus mutant strains (B.1.617 and Delta Plus) with the R203M mutation, as well as carriers of 2019-nCoV (WIV04) or other mutant strains without the R203M mutation. It is of great significance for the rapid diagnosis of suspected cases and the implementation of corresponding isolation measures to ensure that patients receive timely treatment and the safety of others.

[0041] Additional aspects and advantages of the invention will be set forth in part in the description which follows, and will become apparent from the description or may be learned by practice of the invention. Attached Figure Description

[0042] The accompanying drawings, which are included to provide a further understanding of the invention and form part of this application, do not constitute an undue limitation of the invention. In the drawings:

[0043] Figure 1 This is a schematic diagram of the detection results of the nucleic acid detection primer set P1 specificity experiment in this invention;

[0044] Figure 2 This is a schematic diagram of the detection results of the P2 specificity experiment of the nucleic acid detection primer set in this invention;

[0045] Figure 3 This is a schematic diagram showing the Ct value determination results of the nucleic acid detection primer set P1 in this invention for the amplification of SARS-CoV-2 virus mutant strains and 2019-nCoV without the R203M mutation;

[0046] Figure 4 This is a schematic diagram showing the Ct value determination results of the nucleic acid detection primer set P2 in this invention for the amplification of SARS-CoV-2 virus mutant strains and 2019-nCoV without the R203M mutation.

[0047] Figure 1 and Figure 2 In the study, P- mutated to the N gene recombinant cloning plasmid of the SARS-CoV-2 virus mutant strain (B.1.617 and Delta Plus), and P- not mutated to the N gene recombinant cloning plasmid of the original virus strain 2019-nCoV (WIV04) without R203M mutation; N was the negative control, which was DEPC water; 2019-nCoV (WIV04) was a pseudovirus. Detailed Implementation

[0048] To better understand the technical content of this invention, the invention will be further described and explained below with reference to the accompanying drawings and specific embodiments. Obviously, the described embodiments are only some embodiments of this invention, and not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0049] Example 1

[0050] This embodiment uses the original SARS-CoV-2 virus strain WIV04 (MN996528.1), with a full length of 29891 bp, published in Gene Bank as the original sequence. The mutation site was determined by comparing the snap gene with a large number of SARS-CoV-2 virus mutant strains (B1.167 and Delta Plus), and finally, R203M of the N gene sequence was selected. Two sets of specific nucleic acid detection primers based on this mutation site were designed for the specific detection of SARS-CoV-2 mutant strains.

[0051] The two sets of nucleic acid detection primers are P1 and P2, respectively;

[0052] The nucleic acid detection primer set P1 includes a pair of outer primers F3 and B3, a pair of inner primers FIP and BIP, and a circular primer LF1;

[0053] The nucleotide sequences of F3, B3, FIP, BIP, and LF1 are as follows:

[0054] F3: ACGTAGTCGCAACAGTTC;

[0055] B3: CtTAGAAGCCtCAGCAGC;

[0056] FIP:AAGCAAGAGCAGCATCACCG-GAAATTCAACtCCAGGCAGC;

[0057] BIP: CtGCtTGACAGATTGAACCAGC-GTGACAGTTTGGCCtTGTT;

[0058] LF1:CATTCtAGCAGGAGAAGTTCCCA;

[0059] The nucleic acid detection primer set P2 includes one pair of outer primers F3 and B3, one pair of inner primers FIP and BIP, and one circular primer LF2;

[0060] The nucleotide sequences of F3, B3, FIP, BIP, and LF2 are as follows:

[0061] F3: ACGTAGTCGCAACAGTTC;

[0062] B3: CtTAGAAGCCtCAGCAGC;

[0063] FIP:AAGCAAGAGCAGCATCACCG-GAAATTCAACtCCAGGCAGC;

[0064] BIP: CtGCtTGACAGATTGAACCAGC-GTGACAGTTTGGCCtTGTT;

[0065] LF2:TCtAGCAGGAGAAGTTCCCATAC.

[0066] The two sets of nucleic acid detection primers P1 and P2 obtained in this embodiment can specifically detect SARS-CoV-2 virus mutant strains (B.1.617 and Delta Plus). They have been developed into an RT-LAMP kit and detection method that provides faster Ct values ​​for mutant strains (B.1.617 and Delta Plus) and slower Ct values ​​for 2019-nCoV (WIV04) or other mutant strains without the R203M mutation, when tested with the same concentration of sample template.

[0067] Example 2

[0068] This embodiment provides a nucleic acid detection kit for a mutant strain of SARS-CoV-2 virus.

[0069] This kit includes the following components: reaction premix A, reaction premix B, two positive controls, and a negative control;

[0070] The reaction premix A includes 2-10 U Bst DNA large fragment polymerase, 2-10 U AMV reverse transcriptase, and 1 μL SYTO 9 fluorescent dye;

[0071] The reaction premix B includes 10× reaction buffer, nucleic acid detection primer set P1 or P2, 1.6 mM dNTPs, 8 mM MgSO4, 0.8 M betaine, and DEPC water;

[0072] The two positive quality controls were at a concentration of 10. 5 Gene fragments encoding the nucleocapsid protein N gene from a SARS-CoV-2 viral mutant strain (copies / μL) and gene fragments from the unmutated original viral strain 2019-nCoV;

[0073] The negative control was DEPC water.

[0074] In the nucleic acid detection primer set, the molar ratio of the outer primer, inner primer, and loop primer is 1:4:2.

[0075] The reaction buffer consists of the following components: 200 mM Tris-HCl, pH 8.8, 100 mM (NH4)2SO4, 100 mM KCl, 20 mM MgSO4, and 0.1% Triton-X.

[0076] Example 3

[0077] This embodiment provides an RT-LAMP method for detecting SARS-CoV-2 viral mutant strains using the kit from Example 2, comprising:

[0078] Preparation of RT-LAMP reaction system: 25μL reaction system: 5μL sample to be tested, 3μL reaction premix A (4U Bst DNA large fragment polymerase, 8U AMV reverse transcriptase, 1μL SYTO 9 fluorescent dye), 17μL reaction premix B (2.8μL nucleic acid detection primer set P1 or P2, 2.5μL 10× reaction buffer, 1.6mM dNTP, 0.8mM betaine, 8mM magnesium sulfate, DEPC water to 17μL);

[0079] In the nucleic acid detection primer set P1, the molar ratio of the outer primer, inner primer, and loop primer is 1:4:2; in the nucleic acid detection primer set P2, the molar ratio of the outer primer, inner primer, and loop primer is 1:4:2.

[0080] The nucleic acid detection primer set provided in this invention is validated below through specificity verification and clinical sample detection amplification Ct value experiments.

[0081] Specificity verification

[0082] This experiment used the RT-LAMP fluorescence detection method for detecting SARS-CoV-2 virus mutant strains provided in Example 3 for specificity verification. Specificity verification was performed using nucleic acid detection primer sets P1 and P2 from Example 1.

[0083] (a) Samples to be tested: The samples to be tested consist of different types of RNA, including: concentrations of 10 5 Positive controls were obtained from SARS-CoV-2 viral mutant strains (B.1.617 and Delta Plus) at a concentration of 10 copies / μL. 5 Positive controls for the original 2019-nCoV (WIV04) (pseudovirus) strain without R203M mutation, copies / μL; SARS-CoV; Mers-CoV; inactivated influenza A virus H1N1 / H3N2; influenza B virus FluB; respiratory syncytial virus (RSV); human parainfluenza virus types 1 / 2 / 3; adenoviruses B / E; Mycoplasma pneumoniae; Chlamydia pneumoniae; enterovirus EV; enterovirus 71 EV71; Coxsackievirus A16 CA16; Epstein-Barr virus; and human cytomegalovirus (HCMV); all of the above samples were RNA.

[0084] (b) Reaction system and conditions (25 μL reaction system, same as in Example 3):

[0085] 3 μL of reaction premix A, 17 μL of reaction premix B, and 5 μL of the sample to be tested were used. The reaction system was reacted at 65℃ for 60 min and the reaction was stopped at 85℃.

[0086] (c) Judgment of test results:

[0087] The test result is determined by observing the amplification curve using a fluorescence amplification instrument to determine whether it exhibits an "S" shape. An "S"-shaped amplification curve indicates a positive result. If the amplification Ct value is between 10 and 15 minutes, the sample contains a positive control of the SARS-CoV-2 virus mutant strain (B.1.617 and Delta Plus). If the amplification Ct value is between 20 and 25 minutes, the sample contains a positive control of 2019-nCoV without the R203M mutation. If no "S"-shaped amplification curve is observed, the test result is negative, meaning the sample does not contain the SARS-CoV-2 virus.

[0088] Specifically, through repeated experiments, this invention has determined that when the amplified Ct value of the sample to be tested is less than the amplified Ct value of the positive quality control of the N gene without mutation, and the error between the amplified Ct value and the positive quality control of the mutant N gene is within 5, the sample is judged to be a SARS-CoV-2 virus mutant strain.

[0089] Specifically, after the reaction is completed, the amplification curve is observed using a fluorescence amplification instrument. The specificity verification results of nucleic acid detection primer sets P1 and P2 are as follows: Figure 1 and Figure 2 As shown.

[0090] Table 1 shows the RT-LAMP detection results of different viruses using nucleic acid detection primer sets P1 and P2.

[0091] Table 1:

[0092]

[0093]

[0094] In Table 1, ("-" indicates a negative reaction, "+" indicates a positive reaction), P- mutated to the SARS-CoV-2 virus mutant strain (B.1.617 and Delta Plus) N gene recombinant clone plasmid, P- not mutated to the original virus strain 2019-nCoV (WIV04) N gene recombinant clone plasmid without R203M mutation; N is the negative control, which is DEPC water; 2019-nCoV (WIV04) (a pseudovirus without the mutation site fragment).

[0095] Depend on Figure 1 , Figure 2As shown in Table 1, the nucleic acid detection primer sets P1 and P2 of the present invention can specifically detect SARS-CoV-2 virus mutant strains (B.1.617 and Delta Plus) and do not cross-react with other viruses to be detected.

[0096] Clinical sample amplification Ct value experiment

[0097] This experiment used the RT-LAMP fluorescence detection method for detecting SARS-CoV-2 virus mutant strains (B.1.617 and DeltaPlus) provided in Example 3 to compare and verify the Ct values ​​of clinical samples and positive control amplifications. The nucleic acid detection primer sets P1 and P2 from Example 1 were used for verification.

[0098] (a) Sample to be tested: The sample to be tested has a concentration of 10 5 SARS-CoV-2 viral mutant strains (B.1.617 and Delta Plus) RNA at a concentration of 10 copies / μL. 5 RNA of the original 2019-nCoV (WIV04) strain without the R203M mutation, at a concentration of 10 copies / μL. 5 Positive control 1: Gene fragment encoding nucleocapsid protein N gene from SARS-CoV-2 viral mutant strains (B.1.617 and Delta Plus), at a concentration of 10 copies / μL. 5 Positive control 2: Gene fragments of the N gene from the original viral strain 2019-nCoV (WIV04) or other mutant strains that have not undergone the R203M mutation, and negative control DEPC water.

[0099] (b) Reaction system and conditions (25 μL reaction system, same as in Example 3):

[0100] 3 μL of reaction premix A, 17 μL of reaction premix B, and 5 μL of the sample to be tested were used. The reaction system was reacted at 65℃ for 60 min and the reaction was stopped at 85℃.

[0101] (c) Judgment of test results:

[0102] Specifically, after the reaction is complete, observe the amplification curve using a fluorescence amplification instrument. The detection results are as follows: Figure 3 and Figure 4 As shown, Figure 3 and Figure 4 After amplification using the two sets of nucleic acid detection primers P1 and P2 from Example 1, the concentration of each primer was 10. 5 A schematic diagram showing the differences in amplification curves and Ct values ​​between two clinical samples (copies / μL) and two positive quality controls.

[0103] according to Figure 3 and Figure 4 It can be concluded that: the negative control (control) did not amplify; the mutation-positive control had a smaller Ct value than the non-mutation-positive control, indicating faster amplification; the amplification Ct value of the mutation-positive clinical sample was faster than that of the non-mutation-positive control, while the amplification Ct value of the non-mutation-positive clinical sample was slower than that of the non-mutation-positive control. This indicates that the nucleic acid detection primer sets P1 and P2 can distinguish 10... 5 By comparing mutated and non-mutated samples at a concentration of copies / μL with positive control samples, mutant strains with the R203M mutation and non-mutated strains without the R203M mutation can be identified.

[0104] The technical solutions provided by the embodiments of the present invention have been described in detail above. Specific examples have been used to illustrate the principles and implementation methods of the embodiments of the present invention. The descriptions of the embodiments above are only for helping to understand the principles of the embodiments of the present invention. At the same time, for those skilled in the art, there will be changes in the specific implementation methods and application scope based on the embodiments of the present invention. Therefore, the content of this specification should not be construed as a limitation of the present invention. sequence list <110> South China University of Technology <120> Nucleic acid detection primer set, kit and detection method for SARS-CoV-2 virus mutant strains <141> 2021-08-26 <160> 6 <170> SIPOSequenceListing 1.0 <210> 1 <211> 18 <212> DNA <213> Artificial Sequence <400> 1 acgtagtcgc aacagttc 18 <210> 2 <211> 18 <212> DNA <213> Artificial Sequence <400> 2 cttagaagcc tcagcagc 18 <210> 3 <211> 40 <212> DNA <213> Artificial Sequence <400> 3 aagcaagagc agcatcaccg gaaattcaac tccaggcagc 40 <210> 4 <211> 41 <212> DNA <213> Artificial Sequence <400> 4 ctgcttgaca gattgaacca gcgtgacagt ttggccttgt t 41 <210> 5 <211> twenty three <212> DNA <213> Artificial Sequence <400> 5 cattctagca ggagaagttc cca 23 <210> 6 <211> twenty three <212> DNA <213> Artificial Sequence <400> 6 tctagcagga gaagttccca tac 23

Claims

1. A set of primers for detecting a mutant strain of SARS-CoV-2 virus, characterized in that, The nucleic acid detection primer set includes outer primers F3 and B3, inner primers FIP and BIP, and a loop primer LF1, the nucleotide sequences of which are shown below: F3: ACGTAGTCGCAACAGTTC; B3: CtTAGAAGCCtCAGCAGC; FIP:AAGCAAGAGCAGCATCACCG-GAAATTCAACtCCAGGCAGC; BIP: CtGCtTGACAGATTGAACCAGC-GTGACAGTTTGGCCtTGTT; LF1:CATTCtAGCAGGAGAAGTTCCCA.

2. A set of primers for detecting a mutant strain of SARS-CoV-2 virus, characterized in that, The nucleic acid detection primer set includes outer primers F3 and B3, inner primers FIP and BIP, and a loop primer LF2, the nucleotide sequences of which are shown below: F3: ACGTAGTCGCAACAGTTC; B3: CtTAGAAGCCtCAGCAGC; FIP:AAGCAAGAGCAGCATCACCG-GAAATTCAACtCCAGGCAGC; BIP: CtGCtTGACAGATTGAACCAGC-GTGACAGTTTGGCCtTGTT; LF2:TCtAGCAGGAGAAGTTCCCATAC.

3. A nucleic acid detection kit for a SARS-CoV-2 virus mutant strain, characterized in that, Includes the nucleic acid detection primer set as described in claim 1 or 2.

4. The nucleic acid detection kit for the SARS-CoV-2 virus mutant strain according to claim 3, characterized in that, The kit includes the following components: reaction premix A, reaction premix B, two positive controls and a negative control; The reaction premix A includes 2-10 U Bst DNA large fragment polymerase, 2-10 U AMV reverse transcriptase, and 1 μL fluorescent dye; The reaction premix B comprises 10× reaction buffer, the nucleic acid detection primer set as described in claim 1 or 2, 1.6 mM dNTPs, 8 mM MgSO4, 0.8 mM betaine, and DEPC water; The two positive quality controls were at a concentration of 10. 5 Gene fragments encoding the nucleocapsid protein N gene from a SARS-CoV-2 viral mutant strain (copies / μL) and gene fragments from the unmutated original viral strain 2019-nCoV; The negative control was DEPC water.

5. The nucleic acid detection kit for the SARS-CoV-2 virus mutant strain according to claim 4, characterized in that, In the nucleic acid detection primer set, the molar ratio of the outer primer, inner primer, and loop primer is 1:4:

2.

6. The nucleic acid detection kit for the SARS-CoV-2 virus mutant strain according to claim 4, characterized in that, The reaction buffer consists of the following components: 200 mM Tris-HCl, pH 8.8, 100 mM (NH4)2SO4, 100 mM KCl, 20 mM MgSO4, and 0.1% Triton-X.

7. The nucleic acid detection kit for the SARS-CoV-2 virus mutant strain according to claim 4, characterized in that, The fluorescent dye is SYTO 9 fluorescent dye.

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