CN114480742A - A qRT-PCR method for identifying BA-1 branches of novel coronavirus Omicron variant strain - Google Patents

Abstract

The invention belongs to the technical field of biology, and particularly relates to a qRT-PCR method for identifying branches of a novel coronavirus Omicron variant BA-1. In the method, in order to improve the detection sensitivity and specificity, a TaqMan probe is introduced; in order to reduce the cost, 2 pairs of primers are changed into 1 pair of half primers, namely: 2 upstream primers respectively target mutation sites and original non-mutation sites, and 1 downstream primer is shared by 2 upstream primers. By reducing the degree of matching between the variant primers and the non-variant viral nucleic acids and between the non-variant primers and the variant viral nucleic acids in the reaction system, the amplification curve for amplifying the variant viral nucleic acids by the variant primers in the reaction system is earlier than the amplification curve for amplifying the non-variant nucleic acids, and similarly, the amplification curve for amplifying the non-variant nucleic acids by the non-variant primers is earlier than the amplification curve for amplifying the variant nucleic acids.

Description

A qRT-PCR method for identifying branches of novel coronavirus Omicron variant strain BA-1

technical field

The invention belongs to the field of biotechnology, and in particular relates to a qRT-PCR method for identifying branches of novel coronavirus Omicron variant strain BA-1.

Background technique

Gene sequencing is currently the most commonly used technology for identifying new coronavirus variants. This technology has the following shortcomings ^[1] : 1. It takes a long time from sample to result, and there are many uncontrollable influencing factors. It takes 6-8 hours from sample processing to result reporting. In addition, most virus detection units do not have the conditions for gene sequencing, so they need to be handed over to third-party sequencing companies for testing. The speed is slow, and there are many uncontrollable influencing factors; 2. The detection sensitivity is low. Gene amplification needs to be performed before gene sequencing is tested on the computer, and the amplification effect of low-abundance nucleic acid samples may affect the sequencing; 3. The detection cost is high. The new coronavirus gene sequencing includes tedious steps such as sample nucleic acid extraction, RT-PCR, PCR product purification, and on-board testing, resulting in high testing materials and labor costs, and multi-step operations are prone to inadvertent operations leading to deviations in test results. The above shortcomings make gene sequencing, when applied to the prevention and control of the new coronary pneumonia epidemic, will affect the detection time, throughput and cost of virus variants, and may affect the test results, which will have an important impact on the prevention and control of the new coronary pneumonia epidemic ^[2] .

The Omicron variant of the new coronavirus (also known as the B.1.1.529 branch) has 42 mutations such as T19I in the virus Spike protein ^[3] , which has higher replication and transmissibility. Since 2022, it has rapidly replaced the Delta variant as the world's major new coronavirus epidemic variant ^[3] . The new crown epidemic prevention and control work urgently needs a rapid detection method for the new coronavirus Omicron variant. Omicron mutants rapidly evolved into branches such as BA-1, BA.2 and BA.3 after their emergence, and had different etiological characteristics, so each branch should be monitored and monitored separately ^[4] . The Omicron variant BA-1 branch has unique mutations such as A67V, Δ143-145, N211I, Δ211, 215EPEins, S371L, G446S, G496S, T547K, N856K and L981F in the viral Spike protein, which can be used to distinguish the BA-1 of the Omicron variant. 1 and BA.2 branch and Omicron variants and Alpha, Beta, Gamma, Delta variants.

references:

[1] Bezerra et al, A Sanger-based approach for scaling up screening of SARS-CoV-2 variants of interest and concern. Infection, Genetics and Evolution, 2021, 92:104910.

[2] Vega-Magana, et al. RT-qPCR Assays for Rapid Detection of the N501Y, 69-70del, K417N, and E484K SARS-CoV-2 Mutations: A Screening Strategy to Identify Variants with Clinical Impact. Frontiers in Cellular and Infection Microbiology, 2021, 11:672562.

[3] Gowrisankar A, Priyanka TM, Banerjee S "Omicron: a mysterious variant of concern". The European Physical Journal Plus, 2022, 137(1):100.

[4] Kumar S, Karuppanan K, Subramaniam G. Omicron (BA-1) and Sub-Variants (BA-1, BA.2 and BA.3) of SARS-CoV-2 Spike Infectivity and Pathogenicity: AComparative Sequence and Structural-based Computational Assessment, bioRxiv, 2022, doi: https://doi.org/10.1101/2022.02.11.480029.

SUMMARY OF THE INVENTION

In view of the above problems, the present invention provides a qRT-PCR method for identifying the BA-1 branch of the novel coronavirus Omicron variant strain.

In order to achieve the above object, the present invention has adopted the following technical solutions:

Using an improved PCR method named amplification refractory mutation system (ARMS), viral nucleic acid extraction by viral nucleic acid extraction-qRT-PCR instead of gene sequencing-RT-PCR amplification-PCR product purification-on the machine For tedious operation steps such as testing, the accurate identification of virus variants can be completed by using the fluorescence quantitative PCR instruments equipped by most detection units, overcoming the dependence on expensive sequencers, and increasing the universality of the detection method for new coronavirus variants. Through the 96-well or 384-well qRT-PCR reaction plate, and with the help of the high sensitivity and low cost of TaqMan probe method qRT-PCR, the high-sensitivity, high-throughput, and low-cost detection of new coronavirus variants can be achieved.

The technical system of the basic method consists of two pairs of primers, one pair of primers binds to the variant site, and the other pair of primers binds to the original unmutated site, and determines whether there is variation according to the presence or absence of amplified fragments and the length of the fragments.

In order to improve the detection sensitivity and specificity, TaqMan probe was introduced in this method; in order to reduce the cost, the two pairs of primers were changed to one pair of half-primers, that is, the two upstream primers targeted the mutation site and the original unmutated site respectively, 1 The downstream primer is shared by the two upstream primers. By reducing the matching degree between the mutated primer and the unmutated viral nucleic acid and the unmutated primer and the mutated viral nucleic acid in the reaction system, the amplification curve of the mutated primer in the reaction system for amplifying the mutated viral nucleic acid is earlier than the amplification curve for amplifying the non-mutated nucleic acid , Similarly, the amplification curve of the non-variant primer for amplifying the non-variant nucleic acid is earlier than the amplification curve for amplifying the variant nucleic acid. Two sets of experimental systems, mutated primers and non-mutated primers, were used to detect samples. The amplification curve of the mutated primer system appeared earlier than that of the non-mutated primers, and the detection sample was determined to contain the mutated virus.

A qRT-PCR method for identifying branches of novel coronavirus Omicron variant strain BA-1, comprising the following steps:

Step 1, use the Trizol method to extract the new coronavirus RNA;

Step 2, ARMS-based qRT-PCR primer probe design:

Step 2.1, primer design: According to the general principles of primer design, two pairs of primers are designed for each mutation site in combination with the nucleotide sequences near the mutation site of the new coronavirus Indian variant, in which the 3' ends of the upstream primers match the mutation and non-mutation sites respectively. Variation point, i.e. mutated upstream primer and non-variant upstream primer, two pairs of upstream primers share one downstream primer;

When the variant site to be identified is A67V, the nucleotide sequence of the variant upstream primer is shown in SEQ ID NO.1: 5'-CCAATGTTACTTGGTTCCATGT-3', and the nucleotide sequence of the non-variant upstream primer is shown in SEQ ID NO. 2: 5'-CCAATGTTACTTGGTTCCATGC-3', the downstream primer is shown in SEQ ID NO.3: 5'-TGTTAGACTTCTCAGTGGAAGC-3';

When the variant site to be identified is N211I, the nucleotide sequence of the variant upstream primer is shown in SEQ ID NO.4: 5'-AATATATTCTAAGCACACGCCTATTAT-3', and the nucleotide sequence of the non-variant upstream primer is shown in SEQ ID NO. Shown in 5: 5'-AATATATTCTAAGCACACGCCTATTAA-3', the downstream primer is shown in SEQ ID NO.6: 5'-GAGTCAAATAACTTCTATGTAAAGCA AG-3';

When the variant site to be identified is S371L, the nucleotide sequence of the variant upstream primer is shown in SEQ ID NO.7: 5'-GTTGCTGATTATTCTGTCCTATATAATTTA-3', and the nucleotide sequence of the non-variant upstream primer is shown in SEQ ID NO.8 Shown: 5'-GTTGCTGATTATTCTGTCCTATATAAT TCC-3', the downstream primer is shown in SEQ ID NO.9: 5'-GTCTGACTTCATCACCTCTAATTAC A-3';

When the variant site to be identified is G446S, the nucleotide sequence of the variant upstream primer is shown in SEQ ID NO.10: 5'-TGGAATTCTAACAATCTTGATTCTAAGGTTA-3', and the nucleotide sequence of the non-variant upstream primer is shown in SEQ ID NO.11 Shown: 5'-TGGAATTCTAACAATCTTGA TTCTAAGGTTG-3', the downstream primer is shown in SEQ ID NO.12: 5'-GGAAACCATATGATT GTAAAGGAAAGTAAC-3';

Step 2.2, TaqMan probe design: Design a TaqMan probe according to the general principles of TaqMan probe design, combined with the sequence characteristics of the viral genome region defined by the upstream and downstream primers in Step 2.1, and the two pairs of primers for each variant site share one TaqMan probe;

When the variant site to be identified is A67V, the nucleotide sequence of the TaqMan probe is shown in SEQ ID NO.13: 5'-CATGTCTCTGGGACCAATGGTACTAAGAGG-3';

When the variant site to be identified is N211I, the nucleotide sequence of the TaqMan probe is shown in SEQ ID NO.14: 5'-CGTGATCTCCCTCAGGGTTTTTCGGC-3';

When the variant site to be identified is S371L, the nucleotide sequence of the TaqMan probe is shown in SEQ ID NO.15: 5'-CGCATCATTTTCCACTTTTAAGTGTTATGGAGTGTCTCC-3';

When the variant site to be identified is G446S, the nucleotide sequence of the TaqMan probe is shown in SEQ ID NO. 16: 5'-CAGGCCGGTAGCACACCTTGTAATGGTGTT-3'.

Step 3, carry out qRT-PCR experiment based on ARMS;

Step 4, result interpretation:

Step 4.1, when the amplification curve of the variant primer system appears more than 2 cycles earlier than that of the non-variant primer system, it is determined that there is variation in the detected sample;

In step 4.2, when the amplification curve of the non-variant primer system appears more than 2 cycles earlier than that of the variant primer system, it is determined that there is no mutation in the test sample.

Further, described step 3 carries out qRT-PCR experiment based on ARMS, and concrete steps are:

Step 3.1, prepare the reaction system: the volume of each reaction system is 20 μL, which contains 10 μL of 2X reaction solution, 0.2 μL of 50 μM upstream primer, 0.2 μL of 50 μM downstream primer, 0.1 μL of 50 μM TaqMan probe, 7.5 μL of sterile RNase-free water, RNA The template is 2 μL, and the detection of each variant site consists of two reaction systems, one contains the variant upstream primer, the other contains the non-variant downstream primer, and the other components are the same;

Step 3.2, qRT-PCR reaction conditions: perform the following reaction program on a real-time PCR instrument: 95°C, 3 minutes; 45 cycles of 95°C, 30 seconds-60°C, 30 seconds.

Compared with the prior art, the present invention has the following advantages:

1. Improve the detection speed and reduce the dependence on valuable instruments. By applying the method of the present invention, the identification speed of the new coronavirus variant is shortened from 4-6 hours to 2 hours, and the method does not depend on an expensive sequencer, and only needs an ordinary fluorescent quantitative PCR instrument to complete, reducing the need for sequencers. Reliance on other valuable instruments enables virus detection users to complete virus variant identification within the unit, saving time such as sample transfer, and minimizing uncontrollable factors;

2. Improve detection sensitivity. Based on TaqMan probe method qRT-PCR to detect viral nucleic acid variation, the detection sensitivity can reach 1-15 copies/microliter, qRT-PCR can realize the detection of viral nucleic acid variation with amplification signal, avoiding weak amplification signal to gene-based Impact of sequencing on nucleic acid variant detection.

3. Reduce the detection cost and improve the detection throughput. The entire experimental process of this technology only includes two steps of viral nucleic acid extraction and qRT-PCR. There are few experimental steps, and the required material and labor costs are greatly reduced, and it can be passed through 96-well or 384-well reaction plates, which is convenient to achieve high throughput of samples.

4. The probability of operation pollution is small, and it is easy to operate with high throughput. Existing next-generation sequencing technology to detect virus mutation requires multiple steps such as virus RNA extraction, RT-PCR amplification, PCR product purification, and on-machine sequencing. Batch operation is easy to introduce operational pollution. This method only has two methods: virus RNA extraction and qRT-PCR. Step operation, the corresponding operation contamination probability is also reduced, and fewer operation steps are easy to detect a large number of samples at one time.

Description of drawings

Fig. 1 is the sensitivity result graph that the present invention identifies A67V variation site;

Fig. 2 is the sensitivity result diagram of the present invention to identify N211I variation site;

Fig. 3 is the sensitivity result diagram of the present invention to identify S371L variation site;

Fig. 4 is a graph showing the sensitivity results of identifying G446S variant sites according to the present invention.

Detailed ways

Example 1

A qRT-PCR method for identifying branches of novel coronavirus Omicron variant strain BA-1, comprising the following steps:

Step 1, use the TrizoL method to extract the new coronavirus RNA;

Step 2, ARMS-based qRT-PCR primer probe design:

Step 2.1, primer design: According to the general principles of primer design, two pairs of primers are designed for each mutation site in combination with the nucleotide sequences near the mutation site of the new coronavirus Indian variant, in which the 3' ends of the upstream primers match the mutation and non-mutation sites respectively. Variation point, i.e. mutated upstream primer and non-variant upstream primer, two pairs of upstream primers share one downstream primer;

When the variant site to be identified is A67V, the nucleotide sequence of the variant upstream primer is shown in SEQ ID NO.1: 5'-CCAATGTTACTTGGTTCCATGT-3', and the nucleotide sequence of the non-variant upstream primer is shown in SEQ ID NO. 2: 5'-CCAATGTTACTTGGTTCCATGC-3', the downstream primer is shown in SEQ ID NO.3: 5'-TGTTAGACTTCTCAGTGGAAGC-3';

When the variant site to be identified is N211I, the nucleotide sequence of the variant upstream primer is shown in SEQ ID NO.4: 5'-AATATATTCTAAGCACACGCCTATTAT-3', and the nucleotide sequence of the non-variant upstream primer is shown in SEQ ID NO. Shown in 5: 5'-AATATATTCTAAGCACACGCCTATTAA-3', the downstream primer is shown in SEQ ID NO.6: 5'-GAGTCAAATAACTTCTATGTAAAGCA AG-3';

When the variant site to be identified is S371L, the nucleotide sequence of the variant upstream primer is shown in SEQ ID NO.7: 5'-GTTGCTGATTATTCTGTCCTATATAATTTA-3', and the nucleotide sequence of the non-variant upstream primer is shown in SEQ ID NO.8 Shown: 5'-GTTGCTGATTATTCTGTCCTATATAAT TCC-3', the downstream primer is shown in SEQ ID NO.9: 5'-GTCTGACTTCATCACCTCTAATTAC A-3';

When the variant site to be identified is G446S, the nucleotide sequence of the variant upstream primer is shown in SEQ ID NO.10: 5'-TGGAATTCTAACAATCTTGATTCTAAGGTTA-3', and the nucleotide sequence of the non-variant upstream primer is shown in SEQ ID NO.11 Shown: 5'-TGGAATTCTAACAATCTTGAT TCTAAGGTTG-3', the downstream primer is shown in SEQ ID NO.12: 5'-GGAAACCATATGATT GTAAAGGAAAGTAAC-3';

Step 2.2, TaqMan probe design: Design a TaqMan probe according to the general principles of TaqMan probe design, combined with the sequence characteristics of the viral genome region defined by the upstream and downstream primers in Step 2.1, and the two pairs of primers for each variant site share one TaqMan probe;

When the variant site to be identified is A67V, the nucleotide sequence of the TaqMan probe is shown in SEQ ID NO.13: 5'-CATGTCTCTGGGACCAATGGTACTAAGAGG-3';

When the variant site to be identified is N211I, the nucleotide sequence of the TaqMan probe is shown in SEQ ID NO.14: 5'-CGTGATCTCCCTCAGGGTTTTTCGGC-3';

When the variant site to be identified is S371L, the nucleotide sequence of the TaqMan probe is shown in SEQ ID NO.15: 5'-CGCATCATTTTCCACTTTTAAGTGTTATGGAGTGTCTCC-3';

When the variant site to be identified is G446S, the nucleotide sequence of the TaqMan probe is shown in SEQ ID NO. 16: 5'-CAGGCCGGTAGCACACCTTGTAATGGTGTT-3'.

Step 3, perform qRT-PCR experiment based on ARMS, the specific steps are:

Step 3.1, prepare the reaction system: the volume of each reaction system is 20 μL, which contains 10 μL of 2X reaction solution, 0.2 μL of 50 μM upstream primer, 0.2 μL of 50 μM downstream primer, 0.1 μL of 50 μM TaqMan probe, 7.5 μL of sterile RNase-free water, RNA The template is 2 μL, and the detection of each variant site consists of two reaction systems, one contains the variant upstream primer, the other contains the non-variant downstream primer, and the other components are the same;

Step 3.2, qRT-PCR reaction conditions: perform the following reaction program on a real-time PCR instrument: 95°C, 3 minutes; 45 cycles of 95°C, 30 seconds-60°C, 30 seconds.

Step 4, result interpretation:

Step 4.1, when the amplification curve of the variant primer system appears more than 2 cycles earlier than that of the non-variant primer system, it is determined that there is variation in the detected sample;

In step 4.2, when the amplification curve of the non-variant primer system appears more than 2 cycles earlier than that of the variant primer system, it is determined that there is no mutation in the test sample.

Example 2

Sensitivity experiment (template dilution 10 ⁶ -10 ^-1 copy/microliter) is done to the method of the present invention, and the experimental results are as follows:

As shown in Figure 1, when the mutation site to be identified is A67V, the detection sensitivity is 4.55 copies/µL; as shown in Figure 2, when the mutation site to be identified is N211I, the detection sensitivity is 6.82 copies/µL liter; as shown in Figure 3, when the mutation site to be identified is S371L, the detection sensitivity is 1.20 copies/microliter; as shown in Figure 4, when the mutation site to be identified is G446S, the detection sensitivity is 12.32 copies /µl.

Example 3

The precision of the repeatability test evaluation method refers to the closeness of a series of single measured values obtained by repeated determination of the same sample under certain conditions, and is an indicator of the size of the random error. Inter-batch repeatability (intra-day, inter-day repeatability), operator/instrument repeatability, etc.

We investigated the repeatability (precision) of this method, which was measured by the coefficient of variation. The experimental results are shown in Table 1.

Table 1. Repeatability (precision) of this method

The coefficient of variation is calculated as:

Coefficient of variation (%)=(Ct standard deviation/Ct mean)×100%.

It can be seen from Table 1 that:

The intra-assay coefficient of variation of A67V ranges from 0.66% to 2.77%, and the inter-assay coefficient of variation is 0.51% to 3.48%; the intra-assay coefficient of variation of N211I ranges from 0.53% to 2.45%, and the inter-assay coefficient of variation is .61%-2.33%; the intra-assay coefficient of variation of S371L is in the range of 0.23%-2.48%, and the inter-assay coefficient of variation is in the range of 0.48%-2.35%; the intra-assay coefficient of variation of G446S is in the range of 0.39%-1.84%, The inter-assay coefficient of variation ranged from 0.74% to 2.82%;

The intra-assay coefficient of variation of each mutation detection primer in this method was less than 3%, and the inter-assay coefficient of variation was less than 4%. Therefore, the method has good repeatability (precision).

Example 3

Linearity and range (linearity and range) The linearity of the analytical method is the ability to obtain an experimental result that is proportional to the concentration of the test substance in the sample within a given range, that is, the range of the test substance concentration that is linearly related to the experimental result. It is a reaction method. An important indicator of detection performance.

We investigated the linear detection range of this method, and the experimental results are shown in Table 2. The slope shows that each system of this method can obtain a detection signal that is linearly related to the amount of template at 10 ⁶ -10 ¹ copies/microliter of template, indicating that this method has a wide linear detection range.

Table 2. Linearity detection range of this method

sequence listing

SEQ ID NO. 1: 5'-CCAATGTTACTTGGTTCCATGT-3'

SEQ ID NO. 2: 5'-CCAATGTTACTTGGTTCCATGC-3'

SEQ ID NO. 3: 5'-TGTTAGACTTCTCAGTGGAAGC-3'

SEQ ID NO. 4: 5'-AATATATTCTAAGCACACGCCTATTAT-3'

SEQ ID NO. 5: 5'-AATATATTCTAAGCACACGCCTATTAA-3'

SEQ ID NO. 6: 5'-GAGTCAAATAACTTCTATGTAAAGCAAG-3'

SEQ ID NO. 7: 5'-GTTGCTGATTATTCTGTCCTATATAATTTA-3'

SEQ ID NO. 8: 5'-GTTGCTGATTATTCTGTCCTATATAATTCC-3'

SEQ ID NO. 9: 5'-GTCTGACTTCATCACCTCTAATTACA-3'

SEQ ID NO. 10: 5'-TGGAATTCTAACAATCTTGATTCTAAGGTTA-3'

SEQ ID NO. 11: 5'-TGGAATTCTAACAATCTTGATTCTAAGGTTG-3'

SEQ ID NO. 12: 5'-GGAAACCATATGATTGTAAAGGAAAGTAAC-3'

SEQ ID NO. 13: 5'-CATGTCTCTGGGACCAATGGTACTAAGAGG-3'

SEQ ID NO. 14: 5'-CGTGATCTCCCTCAGGGTTTTTCGGC-3'

SEQ ID NO. 15: 5'-CGCATCATTTTCCACTTTTAAGTGTTATGGAGTGTCTCC-3'

SEQ ID NO. 16: 5'-CAGGCCGGTAGCACACCTTGTAATGGTGTT-3'

sequence listing

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Claims (2)

1. a qRT-PCR method for identifying the branch of novel coronavirus Omicron variant strain BA-1, is characterized in that, comprises the following steps: Step 1, use the TrizoL method to extract the new coronavirus RNA; Step 2, ARMS-based qRT-PCR primer probe design: Step 2.1, primer design: According to the general principles of primer design, two pairs of primers are designed for each mutation site in combination with the nucleotide sequences near the mutation site of the new coronavirus Indian variant, in which the 3' ends of the upstream primers match the mutation and non-mutation sites respectively. Variation point, i.e. mutated upstream primer and non-variant upstream primer, two pairs of upstream primers share one downstream primer; When the variant site to be identified is A67V, the nucleotide sequence of the variant upstream primer is shown in SEQ ID NO.1: 5'-CCAATGTTACTTGGTTCCATGT-3', and the nucleotide sequence of the non-variant upstream primer is shown in SEQ ID NO. 2: 5'-CCAATGTTACTTGGTTCCATGC-3', the downstream primer is shown in SEQ ID NO.3: 5'-TGTTAGACTTCTCAGTGGAAGC-3'; When the variant site to be identified is N211I, the nucleotide sequence of the variant upstream primer is shown in SEQ ID NO.4: 5'-AATATATTCTAAGCACACGCCTATTAT-3', and the nucleotide sequence of the non-variant upstream primer is shown in SEQ ID NO. Shown in 5: 5'-AATATATTCTAAGCACACGCCTATTAA-3', the downstream primer is shown in SEQ ID NO.6: 5'-GAGTCAAATAACTTCTATGTAAAGCA AG-3'; When the variant site to be identified is S371L, the nucleotide sequence of the variant upstream primer is shown in SEQ ID NO.7: 5'-GTTGCTGATTATTCTGTCCTATATAATTTA-3', and the nucleotide sequence of the non-variant upstream primer is shown in SEQ ID NO. 8: 5'-GTTGCTGATTATTCTGTCCTATATAAT TCC-3', the downstream primer is shown in SEQ ID NO.9: 5'-GTCTGACTTCATCACCTCTAATTAC A-3'; When the variant site to be identified is G446S, the nucleotide sequence of the variant upstream primer is shown in SEQ ID NO.10: 5'-TGGAATTCTAACAATCTTGATTCTAAGGTTA-3', and the nucleotide sequence of the non-variant upstream primer is shown in SEQ ID NO.11 Shown: 5'-TGGAATTCTAACAATCTTGA TTCTAAGGTTG-3', the downstream primer is shown in SEQ ID NO.12: 5'-GGAAACCATATGAT TGTAAAGGAAAGTAAC-3'; Step 2.2, TaqMan probe design: according to the general principles of TaqMan probe design, design a TaqMan probe based on the sequence characteristics of the viral genome region defined by the upstream and downstream primers in Step 2.1, and the two pairs of primers at each variant site share one TaqMan probe; When the variant site to be identified is A67V, the nucleotide sequence of the TaqMan probe is shown in SEQ ID NO.13: 5'-CATGTCTCTGGGACCAATGGTACTAAGAGG-3'; When the variant site to be identified is N211I, the nucleotide sequence of the TaqMan probe is shown in SEQ ID NO.14: 5'-CGTGATCTCCCTCAGGGTTTTTCGGC-3'; When the variant site to be identified is S371L, the nucleotide sequence of the TaqMan probe is shown in SEQ ID NO.15: 5'-CGCATCATTTTCCACTTTTAAGTGTTATGGAGTGTCTCC-3'; When the variant site to be identified is G446S, the nucleotide sequence of the TaqMan probe is shown in SEQ ID NO. 16: 5'-CAGGCCGGTAGCACACCTTGTAATGGTGTT-3'. Step 3, carry out qRT-PCR experiment based on ARMS; Step 4, result interpretation: Step 4.1, the amplification curve of the variant primer system appears more than 2 cycles earlier than that of the non-variant primer system, and it is determined that there is a mutation in the detected sample; In step 4.2, the amplification curve of the non-variant primer system appears more than 2 cycles earlier than the variant primer system, and it is determined that there is no mutation in the test sample. 2. a kind of qRT-PCR method of identifying novel coronavirus Omicron variant strain BA-1 branch according to claim 1, is characterized in that, described step 3 carries out qRT-PCR experiment based on ARMS, and concrete steps are: Step 3.1, prepare the reaction system: the volume of each reaction system is 20 μL, which contains 10 μL of 2X reaction solution, 0.2 μL of 50 μM upstream primer, 0.2 μL of 50 μM downstream primer, 0.1 μL of 50 μM TaqMan probe, 7.5 μL of sterile RNase-free water, RNA The template is 2 μL, and the detection of each variant site consists of two reaction systems, one contains the variant upstream primer, the other contains the non-variant downstream primer, and the other above-mentioned components are the same; Step 3.2, qRT-PCR reaction conditions: perform the following reaction program on a real-time PCR instrument: 95°C, 3 minutes; 45 cycles of 95°C, 30 seconds-60°C, 30 seconds.

Images