CN113215164B - Method for detecting novel coronavirus variant and subtype - Google Patents

Method for detecting novel coronavirus variant and subtype Download PDF

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CN113215164B
CN113215164B CN202110686862.4A CN202110686862A CN113215164B CN 113215164 B CN113215164 B CN 113215164B CN 202110686862 A CN202110686862 A CN 202110686862A CN 113215164 B CN113215164 B CN 113215164B
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唐时幸
梁源浩
赵建辉
王海鹰
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Abstract

A specific crRNA, a probe, a detection system and a method for detecting a novel coronavirus SARS-CoV-2 variant strain based on a CRISPR-Cas12a method are used for identifying Orf8-crRNA as the specific crRNA of an L subtype and an S subtype, and identifying the variant sites L5F, D80A, D215G, R246I, Y453F, N501Y, A570D, D614G, A701G, T G, S982G, P1263G, K417G, L452R and the specific crRNAs of E484Q are S‑L5F‑crRNA, S‑D80A‑crRNA, S‑D215G‑crRNA, S‑R246I‑crRNA, S‑Y453F‑crRNA, S‑N501Y‑crRNA, S‑A570D‑crRNA, S‑D614G‑crRNA, S‑A701V‑crRNA, S‑T716I‑crRNA, S‑S982A‑crRNA, S‑P1263L‑crRNA, S‑K417N‑crRNA, S‑L452R‑crRNA, S‑E484Q‑crRNA. The nucleotide sequence of the probe is SEQ ID NO. 14. The detection system comprises crRNA, a probe, ribozyme-free water and buffer 2.1. Can accurately detect the SARS-CoV-2 variant strain without depending on expensive instruments and professional technicians, has short detection time, and has the advantages of high specificity, high sensitivity, high speed and high efficiency, simple and convenient operation, easy reading of results and the like.

Description

Method for detecting novel coronavirus variant and subtype
Technical Field
The invention relates to the technical field of in-vitro virus detection, in particular to a specific crRNA, a probe, a detection system and a method for detecting a novel coronavirus SARS-CoV-2 variant based on a CRISPR-Cas12a method.
Background
At present, a plurality of variant strains and subtypes have evolved from the novel coronavirus SARS-CoV-2, which causes the virus pathogenicity and the transmission efficiency to change, influences the protection rate of the vaccine and becomes a new problem of epidemic situation prevention and control. Although the variant and subtype of SARS-CoV-2 can be found and diagnosed by virus genome sequence analysis, the requirement of on-site detection cannot be met, and the high-throughput detection of a large number of samples cannot be realized, which affects epidemic prevention and control.
Two types of the novel coronavirus SARS-CoV-2 are L-subtype and S-subtype, the two subtypes are different in 28144 th nucleotide of SARS-CoV-2 genome, L-subtype is T base (corresponding to leucine, Leu), and S-subtype is C base (corresponding to serine, Ser). The S subtype is more prevalent in the early stages of the SARS-CoV-2 outbreak, while the frequency of the L subtype increases gradually after the beginning of 1 month of 2020 and then becomes the dominant strain. Since the beginning of pandemics, an increasing number of SARS-CoV-2 variant sequences were detected, with the most important mutations being located in the gene encoding the spike protein (S protein), which may alter the infectivity and antigenicity of SARS-CoV-2 and thereby affect the spreading ability, pathogenic ability, immune escape ability, etc., of SARS-CoV-2. A variant containing the mutation at amino acid 614 in the spike protein (D614G) appeared early in a pandemic and spread rapidly in Europe and North America, and evidence has shown that SARS-CoV-2, which contains the D614G mutation, spreads more readily. Subsequently, a SARS-CoV-2 variant (B.1.1.298) associated with mink infection was found in Denmark and transmitted to humans. On day 14/12 of 2020, the UK Bureau announced that a new SARS-CoV-2 variant (B.1.1.7) has been identified by viral genome sequencing. Related studies found that the b.1.1.7 variant had a greater transmission and that patients infected with the b.1.1.7 variant had more disease, and that the neutralizing effect of sera from convalescent patients or vaccinees on the b.1.1.7 variant was slightly reduced. Subsequently, the south African authorities announced the discovery of one variant of SARS-CoV-2 (B.1.351) at 12/18/2020 and another variant of SARS-CoV-2 (P.1) was discovered in Brazil or travelers from Brazil by 1/2021. Some reports show that the variant strains B.1.351 and P.1 have stronger transmission capability and reinfection capability, and more importantly, the neutralizing effect of the serum of convalescent patients or vaccinees on the variant strains B.1.351 and P.1 is obviously reduced. The structural protein (especially spike protein) of different variants of SARS-CoV-2 can be mutated quickly, so that the virus can evade the immunity of organism induced by natural infection or vaccine inoculation, therefore, it is very important to realize the typing of SARS-CoV-2 and the identification of variants. The properties of the novel coronavirus variants are shown in Table I.
Watch 1
Figure BDA0003124883340000011
Figure BDA0003124883340000021
The detection of SARS-CoV-2 variant is mainly carried out by traditional sequencing, and whether the sequencing technology is the first generation sequencing technology, or the metagenome sequencing and the third generation high-throughput sequencing based on the second generation gene sequencing, the cDNA library is constructed by utilizing the PCR amplification technology, and then sequencing is carried out by utilizing expensive and professional instruments and professional operators. And (3) washing and analyzing sequencing data by using professional bioinformatics software to obtain a 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. However, the virus gene sequencing has high requirements on laboratory configuration, long time consumption, high cost and extremely high requirements on the technical and professional knowledge of operators, so that the virus gene sequencing is limited in the rapid identification application of SARS-CoV-2 virus variant strains.
Therefore, aiming at the defects of the prior art, it is necessary to provide a specific crRNA, a probe, a kit and a method for detecting a novel coronavirus SARS-CoV-2 variant based on the CRISPR-Cas12a method, which are short in time consumption and easy to operate, so as to overcome the defects of the prior art.
Disclosure of Invention
The invention aims to avoid the defects of the prior art and provides a specific crRNA, a probe, a detection system and a method for detecting a novel coronavirus SARS-CoV-2 variant based on a CRISPR-Cas12a method, and the detection system and the method have the characteristics of short detection time and simple and convenient operation.
The object of the invention is achieved by the following technical measures.
Provides a specific crRNA for identifying different variant nucleic acids of a novel coronavirus SARS-CoV-2, is detected based on CRISPR-Cas12a technology,
the specific crRNA for identifying the L subtype and the S subtype is Orf8-crRNA, and the nucleotide sequence is 5'-UAAUUUCUACUAAGUGUAGACCUUUUACAAUUAAUUGCCA-3' (SEQ ID NO 1);
the specific crRNA for identifying the mutation sites of S protein gene of SARS-CoV-2, including L5F, D80A, D215G, R246I, Y453F, N501Y, A570D, D614G, A701V, T716I, S982I, P1263I, K417I, L452I and E484I, is S-L5I-crRNA, S-D80I-crRNA, S-D215I-crRNA, S-R246I-crRNA, S-Y453I-crRNA, S-N501I-crRNA, S-A570I-crRNA, S-D614-I-crRNA, S-701A I-crRNA, S-T716 4-crRNA, S-S982-I-crRNA, S-P3-I-crRNA, S-I-sequence, S-I sequence, S-I sequence, S-6853-I sequence, S-6853 sequence, S-I sequence:
S-L5F-crRNA:5’-UAAUUUCUACUAAGUGUAGAUUUUAUUGCCACUAGUCUCU-3’(SEQ ID NO 2)；
S-D80A-crRNA:5’-UAAUUUCUACUAAGUGUAGACUAACCCUGUCCUACCAUUU-3’(SEQ ID NO.3)；
S-D215G-crRNA:5’-UAAUUUCUACUAAGUGUAGAGUGCGUGGUCUCCCUCAGGG-3’(SEQ ID NO.4)；
S-R246I-crRNA:5’-UAAUUUCUACUAAGUGUAGACAUAUAAGUUAUUUGACUCC-3’(SEQ ID NO.5)；
S-Y453F-crRNA:5’-UAAUUUCUACUAAGUGUAGAGAUUGUUUAGGAAGUCUAAU-3’(SEQ ID NO.6)；
S-N501Y-crRNA:5’-UAAUUUCUACUAAGUGUAGACAACCCACUAAUGGUGUUGG-3’(SEQ ID NO.7)；
S-A570D-crRNA:5’-UAAUUUCUACUAAGUGUAGAGCAGAGACAUUGAUGACACU-3’(SEQ ID NO.8)；
S-D614G-crRNA:5’-UAAUUUCUACUAAGUGUAGAUCAGGACGUUAACUGCACAG-3’(SEQ ID NO.9)；
S-A701V-crRNA:5’-UAAUUUCUACUAAGUGUAGUACACCAAGUGACAUAGUGU-3’(SEQID NO.10)；
S-T716I-crRNA:5’-UAAUUUCUACUAAGUGUAGAUGGGUAUGGCAAUAGAGUU-3’(SEQ ID NO.11)；
S-S982A-crRNA:5’-UAAUUUCUACUAAGUGUAGAACGUCUUGACAAAGUUGAGG-3’(SEQ ID NO.12)；
S-P1263L-crRNA:5’-UAAUUUCUACUAAGUGUAGAGCACUAGCUCAGAGUCGUC-3’(SEQ ID NO.13)；
S-K417N-crRNA:5’-UAAUUUCUACUAAGUGUAGAAACGAUUGCUGAUUAUAAUU-3’(SEQ IDNo.32)；
S-L452R-crRNA:5’-UAAUUUCUACUAAGUGUAGACCGAUAUAGAUUGUUUAGGA-3’(SEQ IDNo.33)；
S-E484Q-crRNA:5’-UAAUUUCUACUAAGUGUAGAAAUGGUGUACAAGGUUUUAA-3’(SEQ IDNo.34)。
preferably, the above-mentioned specific crRNA for identifying different coronavirus SARS-CoV-2 variant nucleic acids has the nucleotide sequence of 5 '-TTATT-3' (SEQ ID NO. 14).
Preferably, the probe is modified with a fluorescent reporter group FAM at the 5 'end and a fluorescent quencher group BHQ1 at the 3' end, and is used for identifying the specific crRNA of different coronavirus SARS-CoV-2 variant nucleic acids.
Preferably, the specific crRNA for identifying different coronavirus SARS-CoV-2 variant nucleic acids is characterized in that the nucleotide sequence of the specific primer group is:
orf8 upstream primer 5'-AGGAATCATCACAACTGTAGC-3' (SEQ ID No. 19);
orf8 downstream primer 5'-AGGAATCATCACAACTGTAGC-3' (SEQ ID No. 20);
s gene-1 upstream primer: 5'-CTAGTGATGTTCTTGTT-3' (SEQ ID No. 21);
s gene-1 downstream primer: 5'-TGCACAGTCTACAGCATC-3' (SEQ ID No. 22);
s gene-2 upstream primer: 5'-ACTTGTGCCCTTTTGGTGAAG-3' (SEQ ID No. 23);
s gene-2 downstream primer: 5'-GCTATTCCAGTTAAAGCACGGT-3' (SEQ ID No. 24);
s gene-3 upstream primer: 5'-AGACTCACTTTCTTCCACAGCAA-3' (SEQ ID No. 25);
s gene-3 downstream primer: 5'-GTATCGTTGCAGTAGCGCGA-3' (SEQ ID No. 26);
K417N-upstream primer 5'-ATCGCTCCAGGGCAAATTTGAAA-3' (SEQ ID No. 27);
K417N-downstream primer 5'-GCTATTCCAGTTAAAGCACGGT-3' (SEQ ID No. 28);
L452R-upstream primer: 5'-AGGTTGGTGGTAATTATATTTAC-3' (SEQ ID No. 29);
L452R/E484Q-downstream primer: 5'-AGTACTACTACTCTGTATGGTTGGT-3' (SEQ ID No. 30);
E484Q-upstream primer: 5'-AGCACACCTTTTAATGGTGTT-3' (SEQ ID No. 31);
the specific PCR amplification primers used by Orf8-crRNA are an Orf8 upstream primer and an Orf8 downstream primer;
specific PCR amplification primers used for S-L5F-crRNA, S-D80A-crRNA, S-D215G-crRNA and S-R246I-crRNA are an S gene-1 upstream primer (SEQ ID No.21), an S gene-1 downstream primer (SEQ ID No.22), S-Y453F-crRNA, S-A570D-crRNA, S-D614G-crRNA, S-A701V-crRNA and S-T716I-crRNA are an S gene-2 upstream primer (SEQ ID No.23), an S gene-2 downstream primer (SEQ ID No.24), S-S982A-crRNA and S-P1263L-crRNA are an S gene-3 upstream primer (SEQ ID No.25) and an S gene-3 downstream primer (SEQ ID No. 26);
activation and cleavage activity of Cas12a protein requires the presence of a T-rich PAM sequence at the 5 'end of the target sequence, but in practical assay applications, there is not a PAM sequence at the 5' end of every mutation site. The three mutations, K417N, L452R and E484Q, appeared in the b.1.618 variant that was recently prevalent in india. To solve the problem that the PAM sequence is absent at the 5 'end of each of the three mutation sites and cannot be conventionally used in Cas12a protein-mediated differential assay, the present study introduced the PAM sequence to the 5' end of the mutation site by designing a specific PCR primer. Specific PCR amplification primers with PAM sites used by S-K417N-crRNA are a K417N-upstream primer and a K417N-downstream primer; specific PCR amplification primers with PAM sites used by the S-L452R-crRNA are an L452R-upstream primer (SEQ ID No.29), an L452R/E484Q-downstream primer (SEQ ID No. 30); specific PCR amplification primers with PAM sites used for S-E484Q-crRNA are E484Q-upstream primer (SEQ ID No.31) and L452R/E484Q-downstream primer (SEQ ID No. 30).
The second purpose of the invention is to provide a probe, which is based on CRISPR-Cas12a technology and uses the specific crRNA to identify different coronavirus SARS-CoV-2 variant strains, and the nucleotide sequence is as follows: 5 '-TTATT-3'.
Preferably, the probe is modified with a fluorescent reporter group FAM at the 5 'end and a fluorescent quencher group BHQ1 at the 3' end.
The third purpose of the invention is to provide a CRISPR-Cas12a detection system for detecting SARS-CoV-2 variant, which comprises the specific crRNA, a probe, ribozyme-free water and buffer 2.1.
Preferably, the CRISPR-Cas12a detection system for detecting SARS-CoV-2 variant is detected by the following steps:
(1) placing the detection system in a constant-temperature fluorescence detector at 37 ℃ for incubation for 10min, adding SARS-CoV-2 genome DNA as a template to carry out CRISPR-Cas12a detection for 30min, and obtaining a detection result;
(2) and analyzing the fluorescence signal to judge whether the sample contains SARS-CoV-2 variant virus nucleic acid.
Preferably, in the CRISPR-Cas12a detection system for detecting the SARS-CoV-2 variant, the CRISPR-Cas12a detection method is combined with PCR amplification to detect the SARS-CoV-2 variant, genome DNA of a sample to be detected is used as a template, a specific primer group, specific crRNA and a probe are used for detecting the SARS-CoV-2 variant, and the result is judged according to a fluorescent signal.
The fourth purpose of the invention is to provide the application of the CRISPR-Cas12a detection system as a kit for identifying SARS-CoV-2 variant.
The invention aims at the variant and subtype of SARS-CoV-2, and can realize the rapid detection and diagnosis of the common variant and subtype of SARS-CoV-2 by designing specific crRNA for detecting the variant and subtype of SARS-CoV-2. The invention can accurately detect the SARS-CoV-2 variant strain by the CRISPR-Cas12a detection specific SARS-CoV-2 genome DNA technology. Compared with the existing method for identifying the SARS-CoV-2 variant strain and the subtype based on virus gene sequencing, the method does not need to rely on expensive instruments and professional technical personnel in the aspect of identifying the SARS-CoV-2 variant strain and the subtype, can greatly shorten the detection time, and has the advantages of high specificity, high sensitivity, rapidness, high efficiency, simple and convenient operation, easy result reading and the like.
Drawings
The invention is further illustrated by means of the attached drawings, the content of which is not in any way limiting.
FIG. 1 is a diagram of SARS-CoV-2-Orf8-L/S type plasmid, HIV-1 full-length plasmid and HCV full-length plasmid detected by Orf 8-crRNA.
FIG. 2, FIG. 3 and FIG. 4 are diagrams of wild-type/mutant DNA plasmids for detecting SARS-CoV-2-S protein by using S-L5F-crRNA, S-D80A-crRNA, S-D215G-crRNA, S-R246I-crRNA, S-Y453F-crRNA, S-N501Y-crRNA, S-A570D-crRNA, S-D614G-crRNA, S-A701V-crRNA, S-T716I-crRNA, S-S982A-crRNA and S-P1263L-crRNA.
Figure 5 is a graph of SARS-CoV-2-Orf8-L type (n.s. for P > 0.05;. for P < 0.01;. for P < 0.001;. for P <0.0001) detected by CRISPR-Cas12a direct detection in combination with PCR amplification.
Fig. 6, fig. 7, fig. 8 shows the detection of SARS-CoV-2-S protein wild-type/mutant DNA plasmids by PCR amplification in combination with CRISPR-Cas12a (n.s. represents P > 0.05;. represents P < 0.01;. represents P < 0.001;. represents P < 0.0001).
FIG. 9 is a DNA plasmid map of SARS-CoV-2-S protein S-K417N, S-L452R, S-E484Q variants detected by PCR amplification using specific primers with PAM sites in combination with CRISPR-Cas12a (n.s. represents P > 0.05;. represents P < 0.01;. represents P < 0.001;. represents P < 0.0001).
Detailed Description
The invention is further illustrated by the following examples.
Example 1.
A specific crRNA for discriminating the nucleic acids of different coronavirus SARS-CoV-2 variants is based on CRISPR-Cas12a technique,
the specific crRNA for identifying the L subtype and the S subtype is Orf8-crRNA, and the nucleotide sequence is 5'-UAAUUUCUACUAAGUGUAGACCUUUUACAAUUAAUUGCCA-3' (SEQ ID NO 1).
The specific crRNA for identifying the mutation sites of S protein gene of SARS-CoV-2, including L5F, D80A, D215G, R246I, Y453F, N501Y, A570D, D614G, A701V, T716I, S982I, P1263I, K417I, L452I and E484I, is S-L5I-crRNA, S-D80I-crRNA, S-D215I-crRNA, S-R246I-crRNA, S-Y453I-crRNA, S-N501I-crRNA, S-A570I-crRNA, S-D614-I-crRNA, S-701A I-crRNA, S-T716 4-crRNA, S-S982-I-crRNA, S-P3-I-crRNA, S-I-sequence, S-I sequence, S-I sequence, S-6853-I sequence, S-6853 sequence, S-I sequence:
S-L5F-crRNA:5’-UAAUUUCUACUAAGUGUAGAUUUUAUUGCCACUAGUCUCU-3’(SEQ ID NO 2)；
S-D80A-crRNA:5’-UAAUUUCUACUAAGUGUAGACUAACCCUGUCCUACCAUUU-3’(SEQ ID NO.3)；
S-D215G-crRNA:5’-UAAUUUCUACUAAGUGUAGAGUGCGUGGUCUCCCUCAGGG-3’(SEQ ID NO.4)；
S-R246I-crRNA:5’-UAAUUUCUACUAAGUGUAGACAUAUAAGUUAUUUGACUCC-3’(SEQ ID NO.5)；
S-Y453F-crRNA:5’-UAAUUUCUACUAAGUGUAGAGAUUGUUUAGGAAGUCUAAU-3’(SEQ ID NO.6)；
S-N501Y-crRNA:5’-UAAUUUCUACUAAGUGUAGACAACCCACUAAUGGUGUUGG-3’(SEQ ID NO.7)；
S-A570D-crRNA:5’-UAAUUUCUACUAAGUGUAGAGCAGAGACAUUGAUGACACU-3’(SEQ ID NO.8)；
S-D614G-crRNA:5’-UAAUUUCUACUAAGUGUAGAUCAGGACGUUAACUGCACAG-3’(SEQ ID NO.9)；
S-A701V-crRNA:5’-UAAUUUCUACUAAGUGUAGUACACCAAGUGACAUAGUGU-3’(SEQID NO.10)；
S-T716I-crRNA:5’-UAAUUUCUACUAAGUGUAGAUGGGUAUGGCAAUAGAGUU-3’(SEQ ID NO.11)；
S-S982A-crRNA:5’-UAAUUUCUACUAAGUGUAGAACGUCUUGACAAAGUUGAGG-3’(SEQ ID NO.12)；
S-P1263L-crRNA:5’-UAAUUUCUACUAAGUGUAGAGCACUAGCUCAGAGUCGUC-3’(SEQ ID NO.13)；
S-K417N-crRNA:5’-UAAUUUCUACUAAGUGUAGAAACGAUUGCUGAUUAUAAUU-3’(SEQ IDNo.32)；
S-L452R-crRNA:5’-UAAUUUCUACUAAGUGUAGACCGAUAUAGAUUGUUUAGGA-3’(SEQ IDNo.33)；
S-E484Q-crRNA:5’-UAAUUUCUACUAAGUGUAGAAAUGGUGUACAAGGUUUUAA-3’(SEQ IDNo.34)。
the specific crRNA used for identifying different coronavirus SARS-CoV-2 variant nucleic acids in this example corresponds to probe nucleotide sequence 5 '-TTATT-3' (SEQ ID NO. 14). The 5 'end of the probe is modified with a fluorescence reporter group FAM, and the 3' end of the probe is modified with a fluorescence quenching group BHQ 1.
The nucleotide sequence of the specific primer group is as follows:
orf8 upstream primer 5'-AGGAATCATCACAACTGTAGC-3' (SEQ ID No. 19);
orf8 downstream primer 5'-AGGAATCATCACAACTGTAGC-3' (SEQ ID No. 20);
s gene-1 upstream primer: 5'-CTAGTGATGTTCTTGTT-3' (SEQ ID No. 21);
s gene-1 downstream primer: 5'-TGCACAGTCTACAGCATC-3' (SEQ ID No. 22);
s gene-2 upstream primer: 5'-ACTTGTGCCCTTTTGGTGAAG-3' (SEQ ID No. 23);
s gene-2 downstream primer: 5'-GCTATTCCAGTTAAAGCACGGT-3' (SEQ ID No. 24);
s gene-3 upstream primer: 5'-AGACTCACTTTCTTCCACAGCAA-3' (SEQ ID No. 25);
s gene-3 downstream primer: 5'-GTATCGTTGCAGTAGCGCGA-3' (SEQ ID No. 26);
K417N-upstream primer 5'-ATCGCTCCAGGGCAAATTTGAAA-3' (SEQ ID No. 27);
K417N-downstream primer 5'-GCTATTCCAGTTAAAGCACGGT-3' (SEQ ID No. 28);
L452R-upstream primer: 5'-AGGTTGGTGGTAATTATATTTAC-3' (SEQ ID No. 29);
L452R/E484Q-downstream primer: 5'-AGTACTACTACTCTGTATGGTTGGT-3' (SEQ ID No. 30);
E484Q-upstream primer: 5'-AGCACACCTTTTAATGGTGTT-3' (SEQ ID No. 31);
the specific PCR amplification primers used by Orf8-crRNA are an Orf8 upstream primer and an Orf8 downstream primer;
specific PCR amplification primers used for S-L5F-crRNA, S-D80A-crRNA, S-D215G-crRNA, S-R246I-crRNA are an S gene-1 upstream primer (SEQ ID No.21), an S gene-1 downstream primer (SEQ ID No.22), S-Y453F-crRNA, S-A570D-crRNA, S-D614G-crRNA, S-A701V-crRNA, and S-T716I-crRNA are an S gene-2 upstream primer (SEQ ID No.23), an S gene-2 downstream primer (SEQ ID No.24), S-S982A-crRNA, and S-P1263L-crRNA are an S gene-3 upstream primer (SEQ ID No.25) and an S gene-3 downstream primer (SEQ ID No. 26);
activation and cleavage activity of Cas12a protein requires the presence of a T-rich PAM sequence at the 5 'end of the target sequence, but in practical assay applications, there is not a PAM sequence at the 5' end of every mutation site. The three mutations, K417N, L452R and E484Q, appeared in the indian epidemic b.1.618 variant. In order to solve the problem that the 5 'ends of the three mutation sites lack PAM sequences and cannot be conventionally used in a Cas12a protein-mediated identification test, the PAM sequences are introduced into the 5' ends of the mutation sites by designing specific PCR primers. Specific PCR amplification primers with PAM sites used by S-K417N-crRNA are a K417N-upstream primer and a K417N-downstream primer; specific PCR amplification primers with PAM sites used by the S-L452R-crRNA are an L452R-upstream primer (SEQ ID No.29), an L452R/E484Q-downstream primer (SEQ ID No. 30); specific PCR amplification primers with PAM sites used for S-E484Q-crRNA are E484Q-upstream primer (SEQ ID No.31) and L452R/E484Q-downstream primer (SEQ ID No. 30).
The embodiment designs and develops a detection method based on CRISPR-Cas12a technology to identify SARS-CoV-2 variant strains and subtypes so as to realize faster and more economical detection.
The CRISPR-Cas12a system is used for nucleic acid detection, and is a new technology for in vitro nucleic acid detection. According to the technology, the combination of specific crRNA and nucleic acid to be detected is designed, so that Cas12 enzyme is activated, a probe with a fluorescent label is sheared, a fluorescent signal is released, target nucleic acid which is not amplified can be directly detected, and the nucleic acid detection process is simplified; and the traditional detection after nucleic acid amplification can be combined, so that the sensitivity and the specificity of a detection result are improved. The technology can be combined with an immunochromatographic test paper technology to realize the on-site rapid detection of nucleic acid. By adopting the technologies such as microfluidics and the like, the integration and automation of sample treatment, nucleic acid amplification and detection can be integrated.
Based on the principle, the method aims at the variant and the subtype of SARS-CoV-2, and is used for detecting the variant and the subtype of SARS-CoV-2 by designing specific crRNA, so as to realize the rapid detection and diagnosis of the common variant and the subtype of SARS-CoV-2.
The invention aims at the variant and subtype of SARS-CoV-2, and can realize the rapid detection and diagnosis of the common variant and subtype of SARS-CoV-2 by designing specific crRNA for detecting the variant and subtype of SARS-CoV-2. The invention can accurately detect the SARS-CoV-2 variant strain by the CRISPR-Cas12a detection specific SARS-CoV-2 genome DNA technology. Compared with the existing method for identifying the SARS-CoV-2 variant strain and the subtype based on virus gene sequencing, the method does not need to rely on expensive instruments and professional technical personnel in the aspect of identifying the SARS-CoV-2 variant strain and the subtype, can greatly shorten the detection time, and has the advantages of high specificity, high sensitivity, rapidness, high efficiency, simple and convenient operation, easy result reading and the like.
Example 2.
A CRISPR-Cas12a detection system for detecting SARS-CoV-2 variant comprises the specific crRNA, probe, ribozyme-free water and buffer2.1 in example 1.
The detection system comprises the following steps:
(1) the detection system is adopted, the detection system comprises 0.4 mu L of crRNA with the concentration of 15 mu M, 1 mu L of Cas12 a1 with the concentration of 10 mu M, 1 mu L of report probe with the concentration of 10 mu M and 1 Xbuffer 2.127.6 mu L, the CRISPR-Cas12a detection is carried out by adding SARS-CoV-2 genome DNA as a template after the incubation for 10min in a constant-temperature fluorescence detector with the temperature of 37 ℃, and the detection time is 30min, thus obtaining the detection result;
(2) and analyzing the fluorescence signal to judge whether the sample contains SARS-CoV-2 variant virus nucleic acid. Detecting the fluorescent signal, judging the fluorescent signal to be positive, and indicating that the nucleic acid of the corresponding variant strain novel coronavirus exists.
The CRRNA and the probe of the SARS-CoV-2 virus provided by the invention have no cross reaction with HIV-1 virus, hepatitis C virus and hepatitis B virus and have strong specificity.
The detection system of the invention identifies different SARS-CoV-2 variant strain genes, does not need to sequence viral genome, only needs to use different epidemic strain specific crRNA to carry out CRISPR-Cas12a detection at 37 ℃, and can realize the identification of different SARS-CoV-2 variant strains within 30 minutes.
In addition, the CRISPR-Cas12a detection system for detecting the SARS-CoV-2 variant can also adopt a CRISPR-Cas12a detection method to detect the SARS-CoV-2 variant by combining PCR amplification, takes the genome DNA of a sample to be detected as a template, utilizes a specific primer group, specific crRNA and a probe to detect the SARS-CoV-2 variant, and judges the result according to a fluorescent signal.
The CRISPR-Cas12a detection system of the embodiment can be used as a kit for identifying SARS-CoV-2 variant strains.
The invention aims at the variant and subtype of SARS-CoV-2, and can realize the rapid detection and diagnosis of the common variant and subtype of SARS-CoV-2 by designing specific crRNA for detecting the variant and subtype of SARS-CoV-2. The technology for detecting specific SARS-CoV-2 genome DNA by CRISPR-Cas12a can accurately detect the SARS-CoV-2 variant strain. Compared with the existing method for identifying the SARS-CoV-2 variant strain and the subtype based on virus gene sequencing, the method does not need to rely on expensive instruments and professional technical personnel in the aspect of identifying the SARS-CoV-2 variant strain and the subtype, can greatly shorten the detection time, and has the advantages of high specificity, high sensitivity, rapidness, high efficiency, simple and convenient operation, easy result reading and the like.
Example 3.
The SARS-CoV-2-Orf8-L type/S type is identified by using the CRISPR-Cas12a detection method.
The CRISPR-Cas12a detection system for detecting SARS-CoV-2 variant of this example contains SARS-CoV-2 specific crRNA, universal probe, ribozyme-free water and buffer 2.1. Buffer2.1(New England BioLabs, Ipswich, M0653T), ribozyme-free water (Ecoyori organism, AG 11012). crRNA (synthesized by Oncki Biotechnology Ltd.) was used at a concentration of 15. mu. M, Cas12a (New England BioLabs, Ipswich, M0653T) at a concentration of 10. mu.M, and a probe (synthesized by Biotechnology Ltd., Shanghai) at a concentration of 10. mu.M, and 1 Xbuffer 2.1.
crRNA and probe sequences are as follows:
Orf8-crRNA:5’-UAAUUUCUACUAAGUGUAGACCUUUUACAAUUAAUUGCCA-3’(SEQ ID NO.1)
the probe sequence is as follows: 5 '-TTATT-3' (SEQ ID NO.14)
The Gendto bioengineering (Shanghai) company Limited synthesizes SARS-CoV-2-Orf8-S type DNA plasmid (SEQ ID NO.17) and SARS-CoV-2-Orf8-L type (SEQ ID NO.18), and the sizes of the plasmids are all 366 bp; the concentration of the synthesized plasmid was measured with an ultraviolet spectrophotometer, and after calculating the copy number, the plasmid was diluted to 1.0X 1010Copy/. mu.L for use.
The implementation method comprises the following steps: (1) according to the CRISPER detection system: mu.L of Cas12a, 0.4. mu.L of Orf8-crRNA (SEQ ID NO.1), 1. mu.L of universal probe (SEQ ID NO.14) and 27.6. mu.L of buffer2.1 were used to formulate 5 reaction units.
(2) The detection instrument adopts a constant temperature fluorescence detector RAA-F1620 produced by a Wuxi Tian instrument, and the two instruments are connected with a power supply for preheating.
(3) Preparing a CRISPR-Cas12a detection system, adding 138 mu L of 1 XBuffer 2.1 into a prepared 1.5mL EP tube, adding 5 mu L of Cas12a, 2 mu L of Orf8-crRNA (SEQ ID NO.1) and 5 mu L of a report probe (SEQ ID NO.14), fully mixing, subpackaging into 5 reaction units of 30ul, and placing in a constant-temperature fluorescence detector at 37 ℃ for incubation for 10 min.
(5) Sample adding and reacting, namely adding the 5 prepared reaction units into the reaction units with the concentration of 1.0 multiplied by 10 respectively10SARS-CoV-2-Orf8-L plasmid DNA 1. mu.L in copy/. mu.L, at a concentration of 1.0X 1010Copied/ul SARS-CoV-2-Orf8-S plasmid DNA 1uL, negative quality control material (no ribozyme water) 1uL, concentration 1010mu.L of full-length DNA plasmid 1. mu.L of human immunodeficiency virus type 1 in a concentration of 1010mu.L of hepatitis C virus full-length DNA plasmid is copied to be 1 mu.L, and each reaction tube is fully and uniformly mixed after the sample is added, wherein the total volume of each reaction tube is 31 mu.L. The reaction tube was placed in a RAA-F1620 fluorescence detector, and the reaction temperature was set at 37 ℃ for 20 minutes. The determination of the presence of an increase in the fluorescence signal is positive according to the positive determination method in the RAA-F1620 assay instrument; no increase in fluorescence signal was judged negative.
(6) The above experiment was repeated three times.
(7) The results of the detection are shown in FIG. 1. The result shows that the CRISPR-Cas12a method can realize the identification of SARS-CoV-2-Orf8-L type/S type.
Example 4.
The S protein variant of SARS-CoV-2 is identified by the CRISPR-Cas12a detection method of the invention.
The composition for detecting SARS-CoV-2, provided in this example, with CRISPR-Cas12a, contains SARS-CoV-2 specific crRNA, a universal probe, and buffer 2.1.
The probe was the same as in example 1, and the crRNA sequence was as follows:
S-L5F-crRNA:5’-UAAUUUCUACUAAGUGUAGAUUUUAUUGCCACUAGUCUCU-3’(SEQ ID NO 2)；
S-D80A-crRNA:5’-UAAUUUCUACUAAGUGUAGACUAACCCUGUCCUACCAUUU-3’(SEQ ID NO.3)；
S-D215G-crRNA:5’-UAAUUUCUACUAAGUGUAGAGUGCGUGGUCUCCCUCAGGG-3’(SEQ ID NO.4)；
S-R246I-crRNA:5’-UAAUUUCUACUAAGUGUAGACAUAUAAGUUAUUUGACUCC-3’(SEQ ID NO.5)；
S-Y453F-crRNA:5’-UAAUUUCUACUAAGUGUAGAGAUUGUUUAGGAAGUCUAAU-3’(SEQ ID NO.6)；
S-N501Y-crRNA:5’-UAAUUUCUACUAAGUGUAGACAACCCACUAAUGGUGUUGG-3’(SEQ ID NO.7)；
S-A570D-crRNA:5’-UAAUUUCUACUAAGUGUAGAGCAGAGACAUUGAUGACACU-3’(SEQ ID NO.8)；
S-D614G-crRNA:5’-UAAUUUCUACUAAGUGUAGAUCAGGACGUUAACUGCACAG-3’(SEQ ID NO.9)；
S-A701V-crRNA:5’-UAAUUUCUACUAAGUGUAGAUACACCAAGUGACAUAGUGU-3’(SEQID NO.10)；
S-T716I-crRNA:5’-UAAUUUCUACUAAGUGUAGAAUGGGUAUGGCAAUAGAGUU-3’(SEQ ID NO.11)；
S-S982A-crRNA:5’-UAAUUUCUACUAAGUGUAGAACGUCUUGACAAAGUUGAGG-3’(SEQ ID NO.12)；
S-P1263L-crRNA:5’-UAAUUUCUACUAAGUGUAGAAGCACUAGCUCAGAGUCGUC-3’(SEQ ID NO.13)。
the SARS-CoV-2S protein variant S-N501Y is specifically described as an example.
The wild type DNA plasmid (SEQ ID NO.15) of SARS-CoV-2-S protein and the mutant DNA plasmid (SEQ ID NO.16) of SARS-CoV-2-S protein are synthesized by the company of Weituo biological engineering (Shanghai), the sizes of the plasmids are 3822 bp; the synthesized plasmid was subjected to concentration measurement with an ultraviolet spectrophotometer, and after calculating the copy number, it was diluted to 1.0X 1010Copy/. mu.L until use.
The implementation method comprises the following steps:
(1) according to a CRISPR-Cas12a detection system: mu.L of Cas12a, 0.4. mu.L of crRNA (SEQ ID NO.7), 1. mu.L of universal probe (SEQ ID NO.14) and 27.6ul of buffer2.1 were used to formulate 3 reaction units.
(2) The detection instrument adopts a constant temperature fluorescence detector RAA-F1620 produced by a Wuxi Tian instrument, and the two instruments are connected with a power supply for preheating.
(3) Preparing a CRISPR-Cas12a detection system, adding 82.8 mu L of 1 XBuffer 2.1 into a prepared 1.5mL EP tube, adding 3 mu L of Cas12a, 3 mu L of a report probe (SEQ ID NO.14) and 1.2uL of S-N501Y-crRNA (SEQ ID NO.7), fully mixing, subpackaging into 3 30uL reaction units, and placing in a constant-temperature fluorescence detector at 37 ℃ for incubation for 10 min.
(4) Sample adding reaction, namely adding 1.0X 10 of the prepared reaction units into the prepared reaction units respectively10Copy/ul of SARS-CoV-2-S protein wild type DNA 1. mu.L, 1.0X 1010mu.L of SARS-CoV-2-S protein mutant DNA 1. mu.L and negative quality control material (ribozyme-free water) 1. mu.L are copied/L, and after the sample is added, each reaction tube is fully and uniformly mixed, and the total volume of each reaction tube is 31. mu.L. The reaction tube was placed in a RAA-F1620 fluorescence detector, and the reaction temperature was set at 37 ℃ for 30 minutes. The determination of the presence of an increase in the fluorescence signal is positive according to the positive determination method in the RAA-F1620 assay instrument; no increase in fluorescence signal was judged negative.
(5) The above experiment was repeated three times.
(6) The rest of the identification systems and steps for the mutation sites such as S-L5F-crRNA, S-D80A-crRNA, S-D215G-crRNA, S-R246I-crRNA, S-Y453F-crRNA, S-A570D-crRNA, S-D614G-crRNA, S-A701V-crRNA, S-T716I-crRNA, S-S982A-crRNA and S-P1263L-crRNA are the same as those of the above examples.
(7) The detection results are shown in fig. 2, 3, and 4. The result shows that the CRISPR-Cas12a method can realize the identification of SARS-CoV-2S protein variant.
Example 5.
The CRISPR-Cas12a detection method of the invention is combined with PCR amplification to detect SARS-CoV-2-Orf8-L variant strain
The composition for detecting SARS-CoV-2 by CRISPR-Cas12a combined with PCR amplification provided in this example contains SARS-CoV-2 specific primer, crRNA, universal probe and buffer 2.1.
The probe and crRNA were the same as in example 1, and the primer sequences were as follows:
orf8 upstream primer 5'-AGGAATCATCACAACTGTAGC-3' (SEQ ID No. 19);
orf8 downstream primer 5'-AGGAATCATCACAACTGTAGC-3' (SEQ ID No. 20).
The SARS-CoV-2-Orf8-L (SEQ ID NO.18) is synthesized by the Tortoise Biotechnology engineering (Shanghai) corporation, and the size of the plasmid is 366 bp; measuring the concentration of the synthesized plasmid with ultraviolet spectrophotometer, calculating copy number, diluting to obtain working standard substance containing 1.0 × 10 of 1-10 plasmids1-1.0×1010Copies/. mu.L of SARS-CoV-2-Orf8-L type DNA plasmid.
The implementation method comprises the following steps:
according to the PCR amplification system:
(1)25 mu L of 2X ApexHF FS PCR Master Mix (Aikery organism, AG12202), 1 mu L of Orf8 upstream primer, 1 mu L of Orf8 downstream primer and 21ul of ribozyme-free water are prepared into 11PCR reaction units, 2ul of working standard 1-10 is added into the 1-10 PCR reaction units respectively, and 2ul of ribozyme-free water is added into the 11 th PCR reaction unit.
(2) The PCR amplification reaction can be carried out by adopting a common PCR instrument, and the reaction parameters are set to be pre-denaturation at 94 ℃ for 30 seconds, denaturation at 98 ℃ for 10 seconds, annealing at 55 ℃ for 15 seconds, extension at 72 ℃ for 30 seconds, and denaturation-extension-annealing for 30 cycles.
(3) According to a CRISPR-Cas12a detection system: mu.L of Cas12a, 0.4. mu.L of Orf8-crRNA (SEQ ID NO.1), 1. mu.L of universal probe (SEQ ID NO.14) and 27.6ul of buffer2.1 were used to formulate 22 reaction units.
(4) The detection instrument adopts a constant temperature fluorescence detector RAA-F1620 produced by a Wuxi Tian instrument, and the two instruments are connected with a power supply for preheating.
(5) Preparing a CRISPR-Cas12a detection system, adding 607.2 mu L of 1 XBuffer 2.1 into a prepared 1.5mL EP tube, adding 22 mu L of Cas12a, 22 mu L of a report probe (SEQ ID NO.14) and 8.8uL of Orf8-crRNA (SEQ ID NO.1), fully mixing uniformly, subpackaging into 22 detection reaction units of 30uL, and placing in a constant-temperature fluorescence detector at 37 ℃ for incubation for 10 min.
(6) And (2) sample adding reaction, namely adding 1ul of 1-11PCR reaction unit products of which the samples are 1ul respectively into the 1-11 prepared detection reaction units, adding 1ul of working standard 1-10 and 1ul of ribozyme-free water into the 12-22 prepared detection reaction units, fully and uniformly mixing each reaction tube after adding the samples, wherein the total volume of each detection reaction tube is 31 mu L. The reaction tube was placed in a RAA-F1620 fluorescence detector, and the reaction temperature was set at 37 ℃ for 30 minutes. The determination of the presence of an increase in the fluorescence signal is positive according to the positive determination method in the RAA-F1620 assay instrument; no increase in fluorescence signal was judged negative.
(7) The above experiment was repeated three times.
(8) The results of the detection are shown in FIG. 5. The result shows that the CRISPR-Cas12a method can realize the detection of SARS-CoV-2-Orf8-L type DNA plasmid, and the detection limit is 1010The detection of SARS-CoV-2-Orf8-L type DNA plasmid can be realized by combining copy/. mu.L and CRISPR-Cas12a with PCR amplification, and the detection limit is 101Copies/. mu.L.
The invention can accurately detect the SARS-CoV-2 variant strain by the CRISPR-Cas12a detection specific SARS-CoV-2 genome DNA technology. Compared with the existing method for identifying the SARS-CoV-2 variant strain and the subtype based on virus gene sequencing, the method does not need to rely on expensive instruments and professional technical personnel in the aspect of identifying the SARS-CoV-2 variant strain and the subtype, can greatly shorten the detection time, and has the advantages of high specificity, high sensitivity, rapidness, high efficiency, simple and convenient operation, easy result reading and the like.
Example 6.
The differential report probe of S protein variant strain for SARS-CoV-2 by using CRISPR-Cas12a detection method and PCR amplification of the invention is the same as that in example 1, and the crRNA is the same as that in example 2.
The composition for detecting SARS-CoV-2 by CRISPR-Cas12a combined with PCR amplification provided in this example contains SARS-CoV-2 specific primer, crRNA, universal probe and buffer 2.1.
The primer sequences are as follows:
orf8 upstream primer 5'-AGGAATCATCACAACTGTAGC-3' (SEQ ID No. 19);
orf8 downstream primer 5'-AGGAATCATCACAACTGTAGC-3' (SEQ ID No. 20);
an upstream primer 5'-CTAGTGATGTTCTTGTT-3' of S gene-1 (SEQ ID No. 21);
s Gene-1 downstream primer 5'-TGCACAGTCTACAGCATC-3' (SEQ ID No. 22);
an upstream primer 5'-ACTTGTGCCCTTTTGGTGAAG-3' of the S gene-2 (SEQ ID No. 23);
downstream primer 5'-GCTATTCCAGTTAAAGCACGGT-3' of S gene-2 (SEQ ID No. 24);
an S gene-3 upstream primer 5'-AGACTCACTTTCTTCCACAGCAA-3' (SEQ ID No. 25);
the S gene-3 downstream primer 5'-GTATCGTTGCAGTAGCGCGA-3' (SEQ ID No. 26).
The SARS-CoV-2S protein variant S-Y453F is specifically described as an example.
The wild type DNA plasmid (SEQ ID NO.15) of SARS-CoV-2-S protein and the mutant DNA plasmid (SEQ ID NO.16) of SARS-CoV-2-S protein are synthesized by the company of Weituo biological engineering (Shanghai), the sizes of the plasmids are 3822 bp; measuring the concentration of the synthesized plasmid with ultraviolet spectrophotometer, calculating copy number, diluting to obtain working standard substance containing 1.0 × 10 of 1-91-1.0×109Copy/. mu.L protein wild type DNA plasmid, Standard 10-18 contained 1.0X 101-1.0×109Copies/. mu.L of mutant DNA plasmid of SARS-CoV-2-S protein.
The implementation method comprises the following steps:
according to the PCR amplification system:
(1)25 mu L of 2X ApexHF FS PCR Master Mix, 1 mu L of S gene-2 upstream primer (SEQ ID No.23), 1 mu L of S gene-2 downstream primer (SEQ ID No.24) and 21ul of ribozyme-free water are prepared into 20PCR reaction units, the 10 th PCR reaction unit and the 20 th PCR reaction unit are added with 2ul of ribozyme-free water, the 1 st PCR reaction unit and the 9 th PCR reaction unit are added with 2ul of working standard 1-9 respectively, and the 11 th PCR reaction unit and the 19 th PCR reaction unit are added with 2ul of working standard 10-18 respectively.
(2) The PCR amplification reaction can be carried out by adopting a common PCR instrument, and the reaction parameters are set to be pre-denaturation at 94 ℃ for 30 seconds, denaturation at 98 ℃ for 10 seconds, annealing at 55 ℃ for 15 seconds, extension at 72 ℃ for 30 seconds, and denaturation-extension-annealing for 30 cycles.
(3) According to a CRISPR-Cas12a detection system: mu.L of Cas12a, 0.4. mu.L of S-Y453F-crRNA (SEQ ID NO.6), 1. mu.L of universal probe (SEQ ID NO.14) and 27.6ul of buffer2.1 were used to formulate 20 reaction units.
(4) The detection instrument adopts a constant temperature fluorescence detector RAA-F1620 produced by a Wuxi Tian instrument, and the two instruments are connected with a power supply for preheating.
(5) Preparing a CRISPR-Cas12a detection system, adding 552 mu L of 1 XBuffer 2.1 into a prepared 1.5mL EP tube, adding 20 mu L of Cas12a, 20 mu L of a report probe (SEQ ID NO.14) and 8uL of S-Y453F-crRNA (SEQ ID NO.6), fully mixing, subpackaging into 20 detection reaction units of 30uL, and placing in a constant-temperature fluorescence detector at 37 ℃ for incubation for 10 min.
(6) And (3) sample adding reaction, namely adding 1ul of products of 1-20PCR reaction units of the samples into the 20 prepared detection reaction units, and fully and uniformly mixing the samples in each reaction tube, wherein the total volume of each detection reaction tube is 31 mu L. The reaction tube was placed in a RAA-F1620 fluorescence detector, and the reaction temperature was set at 37 ℃ for 30 minutes. The determination of the presence of an increase in the fluorescence signal is positive according to the positive determination method in the RAA-F1620 assay instrument; no increase in fluorescence signal was judged negative.
(7) The above experiment was repeated three times.
(8) The detection results are shown in fig. 6, 7, and 8. The result shows that the CRISPR-Cas12a method can realize the detection of the SARS-CoV-2S protein variant strain S-Y453F by combining with PCR amplification, and the detection limit is 102Copies/. mu.L.
(9) The other identification systems and steps aiming at the variation sites of S-L5F-crRNA, S-D80A-crRNA, S-D215G-crRNA, S-R246I-crRNA, S-A570D-crRNA, S-D614G-crRNA, S-A701V-crRNA, S-T716I-crRNA, S-S982A-crRNA, S-P1263L-crRNA and the like are consistent with the above embodiment, wherein the specific PCR amplification primers used by the S-L5F-crRNA, S-D80A-crRNA, S-D215G-crRNA and S-R246I-crRNA are the upstream primer (SEQ ID No.21) of the S gene-1, the downstream primer (SEQ ID No.22) of the S-A570-D-614 RNA, S-D6-crRNA, S-A V-crRNA and the upstream primer (SEQ ID No. 3-crRNA) used by the S-R246I-crRNA and the specific PCR amplification primers used by the S-L5-D11-crRNA (SEQ ID No. 3-3623-CRRNA) The specific PCR amplification primers used for the S gene-2 downstream primer (SEQ ID No.24), the S-S982A-crRNA and the S-P1263L-crRNA are an S gene-3 upstream primer (SEQ ID No.25) and an S gene-3 downstream primer (SEQ ID No. 26).
The invention can accurately detect the SARS-CoV-2 variant strain by the CRISPR-Cas12a detection specific SARS-CoV-2 genome DNA technology. Compared with the existing method for identifying the SARS-CoV-2 variant strain and the subtype based on virus gene sequencing, the method does not need to rely on expensive instruments and professional technical personnel in the aspect of identifying the SARS-CoV-2 variant strain and the subtype, can greatly shorten the detection time, and has the advantages of high specificity, high sensitivity, rapidness, high efficiency, simple and convenient operation, easy result reading and the like.
Example 7.
The CRISPR-Cas12a detection method is utilized to design PCR primers with PAM sequences, and the identification of the variants of SARS-CoV-2 such as K417N, L452R and E484Q is carried out by combining PCR amplification. The reporter probe was the same as in example 1.
The composition for detecting SARS-CoV-2 by CRISPR-Cas12a combined with PCR amplification provided in this example contains SARS-CoV-2 specific primer, crRNA, universal probe and buffer 2.1.
The primer sequences are as follows:
K417N-upstream primer 5'-ATCGCTCCAGGGCAAATTTGAAA-3' (SEQ ID No. 27);
K417N-downstream primer 5'-GCTATTCCAGTTAAAGCACGGT-3' (SEQ ID No. 28);
L452R-forward primer 5'-AGGTTGGTGGTAATTATATTTAC-3' (SEQ ID NO. 29);
L452R/E484Q-downstream primer 5'-AGTACTACTACTCTGTATGGTTGGT-3' (SEQ ID No. 30);
E484Q-forward primer 5'-AGCACACCTTTTAATGGTGTT-3' (SEQ ID No. 31);
S-K417N-crRNA 5’-UAAUUUCUACUAAGUGUAGAAACGAUUGCUGAUUAUAAUU-3’(SEQ IDNo.32)；
S-L452R-crRNA 5’-UAAUUUCUACUAAGUGUAGACCGAUAUAGAUUGUUUAGGA-3’(SEQ IDNo.33)；
S-E484Q-crRNA 5’-UAAUUUCUACUAAGUGUAGAAAUGGUGUACAAGGUUUUAA-3’(SEQ IDNo.34)。
the SARS-CoV-2S-protein variant K417N is specifically described as an example.
The wild type DNA plasmid (SEQ ID NO.15) of SARS-CoV-2-S protein synthesized by Competition Biotechnology engineering (Shanghai),SARS-CoV-2-S protein mutant DNA plasmid (SEQ ID NO.16), the size of the plasmid is 3822 bp; measuring the concentration of the synthesized plasmid with ultraviolet spectrophotometer, calculating copy number, diluting to obtain working standard substance containing 1.0 × 10 of 1-91-1.0×109Copies/. mu.L of protein wild-type DNA plasmid (SEQ ID NO.15), standards 10-18 contained 1.0X 10, respectively1-1.0×109Copies/. mu.L of SARS-CoV-2-S protein mutant DNA plasmid (SEQ ID NO. 16)). The designed primers with PAM sites are respectively K417N-upstream primer (the 17 th base is mutated from C to T, the 19 th base is mutated from G to T), L452R-upstream primer (the 19 th base is mutated from A to T) and E484Q-upstream primer (the 11 th base is mutated from G to T).
The implementation method comprises the following steps:
according to the PCR amplification system:
(1)25 mu L of 2X ApexHF FS PCR Master Mix, 1 mu L of K417N-upstream primer (SEQ ID No.27), 1 mu L of K417N-downstream primer (SEQ ID No.28) and 21ul of ribozyme-free water are prepared into 20PCR reaction units, the 10 th PCR reaction unit and the 20 th PCR reaction unit are added with 2ul of ribozyme-free water, the 1 st PCR reaction unit and the 9 th PCR reaction unit are added with 2ul of working standard products 1-9, and the 11 th PCR reaction unit and the 19 th PCR reaction unit are added with 2ul of working standard products 10-18.
(2) The PCR amplification reaction can be carried out by adopting a common PCR instrument, and the reaction parameters are set to be pre-denaturation at 94 ℃ for 30 seconds, denaturation at 98 ℃ for 10 seconds, annealing at 55 ℃ for 15 seconds, extension at 72 ℃ for 30 seconds, and denaturation-extension-annealing for 30 cycles.
(3) According to a CRISPR-Cas12a detection system: mu.L of Cas12a, 0.4. mu.L of S-K417N-crRNA (SEQ ID NO.32), 1. mu.L of universal probe (SEQ ID NO.14) and 27.6ul of buffer2.1 were used to formulate 20 reaction units.
(4) The detection instrument adopts a constant temperature fluorescence detector RAA-F1620 produced by a Wuxi Tian instrument, and the two instruments are connected with a power supply for preheating.
(5) Preparing a CRISPR-Cas12a detection system, adding 552 mu L of 1 XBuffer 2.1 into a prepared 1.5mL EP tube, adding 20 mu L of Cas12a, 20 mu L of a report probe (SEQ ID NO.14) and 8uL of S-K417N-crRNA (SEQ ID NO.32), fully mixing, subpackaging into 20 detection reaction units of 30uL, and placing in a constant-temperature fluorescence detector at 37 ℃ for incubation for 10 min.
(6) And (3) sample adding reaction, namely adding 1ul of products of 1-20PCR reaction units of the samples into the 20 prepared detection reaction units, and fully and uniformly mixing the samples in each reaction tube, wherein the total volume of each detection reaction tube is 31 mu L. The reaction tube was placed in a RAA-F1620 fluorescence detector, and the reaction temperature was set at 37 ℃ for 30 minutes. The determination of the presence of an increase in the fluorescence signal is positive according to the positive determination method in the RAA-F1620 assay instrument; no increase in fluorescence signal was judged negative.
(7) The above experiment was repeated three times.
(8) The detection results are shown in fig. 9. The result shows that the CRISPR-Cas12a method can realize the detection of the SARS-CoV-2S protein variant S-K417N by combining with PCR amplification, and the detection limit is 102Copies/. mu.L.
(9) The remaining identification systems and procedures for the S-L452R and S-E484Q mutation sites are the same as those of the above example, and the specific PCR amplification primers with PAM site are respectively L452R-forward primer (SEQ ID No.29), L452R/E484Q-reverse primer (SEQ ID No.30), E484Q-forward primer (SEQ ID No.31) and L452R/E484Q-reverse primer (SEQ ID No. 30).
The invention can accurately detect the SARS-CoV-2 variant strain by the CRISPR-Cas12a detection specific SARS-CoV-2 genome DNA technology. Compared with the existing method for identifying the SARS-CoV-2 variant strain and the subtype based on virus gene sequencing, the method does not need to rely on expensive instruments and professional technical personnel in the aspect of identifying the SARS-CoV-2 variant strain and the subtype, can greatly shorten the detection time, and has the advantages of high specificity, high sensitivity, rapidness, high efficiency, simple and convenient operation, easy result reading and the like.
Finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention and not for limiting the protection scope of the present invention, and although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.
Sequence listing
<110> southern medical university
<120> method for detecting novel coronavirus variants and subtypes
<130> GZZRH0504-21-1-1262
<160> 34
<170> SIPOSequenceListing 1.0
<210> 1
<211> 40
<212> RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
uaauuucuac uaaguguaga ccuuuuacaa uuaauugcca 40
<210> 2
<211> 40
<212> RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
uaauuucuac uaaguguaga uuuuauugcc acuagucucu 40
<210> 3
<211> 40
<212> RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
uaauuucuac uaaguguaga cuaacccugu ccuaccauuu 40
<210> 4
<211> 40
<212> RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
uaauuucuac uaaguguaga gugcgugguc ucccucaggg 40
<210> 5
<211> 40
<212> RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
uaauuucuac uaaguguaga cauauaaguu auuugacucc 40
<210> 6
<211> 40
<212> RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
uaauuucuac uaaguguaga gauuguuuag gaagucuaau 40
<210> 7
<211> 40
<212> RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 7
uaauuucuac uaaguguaga caacccacua augguguugg 40
<210> 8
<211> 40
<212> RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 8
uaauuucuac uaaguguaga gcagagacau ugaugacacu 40
<210> 9
<211> 40
<212> RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 9
uaauuucuac uaaguguaga ucaggacguu aacugcacag 40
<210> 10
<211> 40
<212> RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 10
uaauuucuac uaaguguaga uacaccaagu gacauagugu 40
<210> 11
<211> 40
<212> RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 11
uaauuucuac uaaguguaga auggguaugg caauagaguu 40
<210> 12
<211> 40
<212> RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 12
uaauuucuac uaaguguaga acgucuugac aaaguugagg 40
<210> 13
<211> 40
<212> RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 13
uaauuucuac uaaguguaga agcacuagcu cagagucguc 40
<210> 14
<211> 5
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 14
ttatt 5
<210> 15
<211> 3822
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 15
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> 16
<211> 3822
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 16
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 taccggttta gattgtttag gaagtctaat 1380
ctcaaacctt ttgagagaga tatttcaact gaaatctatc aggccggtag cacaccttgt 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
<210> 17
<211> 366
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 17
atgaaatttc ttgttttctt aggaatcatc acaactgtag ctgcatttca ccaagaatgt 60
agtttacagt catgtactca acatcaacca tatgtagttg atgacccgtg tcctattcac 120
ttctattcta aatggtatat tagagtagga gctagaaaat cagcaccttt aattgaattg 180
tgcgtggatg aggctggttc taaatcaccc attcagtaca tcgatatcgg taattataca 240
gtttcctgtt caccttttac aattaattgc caggaaccta aattgggtag tcttgtagtg 300
cgttgttcgt tctatgaaga ctttttagag tatcatgacg ttcgtgttgt tttagatttc 360
atctaa 366
<210> 18
<211> 366
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 18
atgaaatttc ttgttttctt aggaatcatc acaactgtag ctgcatttca ccaagaatgt 60
agtttacagt catgtactca acatcaacca tatgtagttg atgacccgtg tcctattcac 120
ttctattcta aatggtatat tagagtagga gctagaaaat cagcaccttt aattgaattg 180
tgcgtggatg aggctggttc taaatcaccc attcagtaca tcgatatcgg taattataca 240
gtttcctgtt taccttttac aattaattgc caggaaccta aattgggtag tcttgtagtg 300
cgttgttcgt tctatgaaga ctttttagag tatcatgacg ttcgtgttgt tttagatttc 360
atctaa 366
<210> 19
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 19
aggaatcatc acaactgtag c 21
<210> 20
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 20
aggaatcatc acaactgtag c 21
<210> 21
<211> 17
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 21
ctagtgatgt tcttgtt 17
<210> 22
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 22
tgcacagtct acagcatc 18
<210> 23
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 23
acttgtgccc ttttggtgaa g 21
<210> 24
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 24
gctattccag ttaaagcacg gt 22
<210> 25
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 25
agactcactt tcttccacag caa 23
<210> 26
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 26
gtatcgttgc agtagcgcga 20
<210> 27
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 27
atcgctccag ggcaaatttg aaa 23
<210> 28
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 28
gctattccag ttaaagcacg gt 22
<210> 29
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 29
aggttggtgg taattatatt tac 23
<210> 30
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 30
agtactacta ctctgtatgg ttggt 25
<210> 31
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 31
agcacacctt ttaatggtgt t 21
<210> 32
<211> 40
<212> RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 32
uaauuucuac uaaguguaga aacgauugcu gauuauaauu 40
<210> 33
<211> 40
<212> RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 33
uaauuucuac uaaguguaga ccgauauaga uuguuuagga 40
<210> 34
<211> 40
<212> RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 34
uaauuucuac uaaguguaga aaugguguac aagguuuuaa 40

Claims (7)

1. A detection system for identifying different variant nucleic acids of novel coronavirus SARS-CoV-2 is based on CRISPR-Cas12a technology detection, and is characterized in that:
the specific crRNA for identifying the L subtype and the S subtype is Orf8-crRNA, and the nucleotide sequence is 5'-UAAUUUCUACUAAGUGUAGACCUUUUACAAUUAAUUGCCA-3' (SEQ ID NO 1);
the specific crRNAs for identifying the mutation sites L452R and E484Q of S protein gene of SARS-CoV-2 are S-L452R-crRNA and S-E484Q-crRNA respectively, and the nucleotide sequences are respectively:
S-L452R-crRNA:5’-UAAUUUCUACUAAGUGUAGACCGAUAUAGAUUGUUUAGGA-3’(SEQ ID No.33)；
S-E484Q-crRNA:5’-UAAUUUCUACUAAGUGUAGAAAUGGUGUACAAGGUUUUAA-3’(SEQ ID No.34)；
the nucleotide sequence of the specific primer group is as follows:
L452R-upstream primer: 5'-AGGTTGGTGGTAATTATATTTAC-3' (SEQ ID No. 29);
L452R/E484Q-downstream primer: 5'-AGTACTACTACTCTGTATGGTTGGT-3' (SEQ ID No. 30);
E484Q-upstream primer: 5'-AGCACACCTTTTAATGGTGTT-3' (SEQ ID No. 31).
2. The detection system for identifying nucleic acids of different variants of the novel coronavirus SARS-CoV-2 according to claim 1, wherein:
specific PCR amplification primers with PAM sites used by the S-L452R-crRNA are L452R-upstream primer (SEQ ID No.29), L452R/E484Q-downstream primer (SEQ ID No. 30); specific PCR amplification primers with PAM sites used for S-E484Q-crRNA are E484Q-upstream primer (SEQ ID No.31) and L452R/E484Q-downstream primer (SEQ ID No. 30).
3. The detection system for identifying nucleic acids of different variants of the novel coronavirus SARS-CoV-2 as claimed in claim 1 or 2, wherein the nucleotide sequence of the probe is 5 '-TTATT-3' (SEQ ID NO. 14).
4. The detection system for identifying the nucleic acid of different variants of the novel coronavirus SARS-CoV-2 as claimed in claim 3, wherein the probe is modified with a fluorescence reporter group FAM at the 5 'end and a fluorescence quencher group BHQ1 at the 3' end.
5. Use of a test system for the identification of nucleic acids of different variants of the novel coronavirus SARS-CoV-2 according to any of claims 1 to 4 for a test for non-theranostic purposes, wherein the test for non-theranostic purposes is carried out by:
(1) placing the detection system in a constant-temperature fluorescence detector at 37 ℃ for incubation for 10min, adding SARS-CoV-2 genome DNA as a template to carry out CRISPR-Cas12a detection for 30min, and obtaining a detection result;
(2) and analyzing the fluorescence signal to judge whether the sample contains SARS-CoV-2 variant virus nucleic acid.
6. The use of claim 5 in detection of non-therapeutic diagnostic purposes of detection systems for the identification of nucleic acids of different variants of the novel coronavirus SARS-CoV-2, wherein the detection method of CRISPR-Cas12a is combined with PCR amplification to detect the SARS-CoV-2 variant, the genomic DNA of a sample to be detected is used as a template, a specific primer set, specific crRNA and a probe are used to detect the SARS-CoV-2 variant, and the result is determined according to a fluorescent signal.
7. Use of the detection system for the identification of nucleic acids of different variants of the novel coronavirus SARS-CoV-2 according to claim 1 as a kit for the identification of variants of SARS-CoV-2.
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