Compositions, kits, methods of detecting sars-cov-2 and uses thereof

Abstract

The present application belongs to the field of molecular biology detection; specifically, it relates to the detection of SARS-CoV-2; more specifically, it relates to the detection and typing of SARS-CoV-2 variants. The present application provides a kit comprising the composition, the use of the composition, and a method for detecting SARS-CoV-2 variants and typing. The present application simultaneously realizes the detection and typing of SARS-CoV-2 variants in the reaction system by detecting different characteristic functional mutation sites on SARS-CoV-2 variants, thereby enabling different strains to be treated differently, thus making treatment and prevention more efficient. The composition of the present application is low in cost and high in throughput. Moreover, the operation is simple, and the result reading process can be determined through different channels. The whole detection process is carried out in a closed condition, avoiding false positives and environmental pollution caused by cross-contamination between samples.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology detection; specifically, it relates to the detection of SARS-CoV-2; more specifically, it relates to the detection and typing of SARS-CoV-2 variants. Background Technology

[0002] The novel coronavirus (2019-nCoV, SARS-CoV-2) was named SARS-CoV-2 by the International Committee on Taxonomy of Viruses on February 11, 2020. Coronaviruses are a large family of viruses. Previously, six species were known to infect humans, such as those causing the common cold, Middle East Respiratory Syndrome (MERS), and Severe Acute Respiratory Syndrome (SARS). SARS-CoV-2 is a novel coronavirus strain that had never been found in humans before. Taxonomically, this virus belongs to the β-type coronavirus of the genus Coronavirus in the family Coronaviridae. It is characterized by its envelope and spike cell cytology, and its genome is a linear single-stranded positive-sense RNA virus ((+)ssRNA).

[0003] Common signs and symptoms of COVID-19 include respiratory symptoms such as fever, cough, shortness of breath, and difficulty breathing. In more severe cases, the infection can lead to pneumonia, severe acute respiratory syndrome, kidney failure, and ultimately death. Currently, there is no specific treatment for the disease caused by the 2019 novel coronavirus, but many symptoms can be managed with symptomatic and supportive care.

[0004] As the spread of the virus intensifies, more and more variants are emerging, with significant differences in virulence and transmissibility. According to the World Health Organization's website, hundreds of variants of the novel coronavirus have been identified worldwide, each with varying virulence and transmissibility. Therefore, to conserve medical resources and alleviate the burden on the healthcare system, a reagent is needed that can accurately detect various variants, enabling targeted prevention and treatment measures for greater efficiency. Furthermore, the test should be quick and highly sensitive.

[0005] Therefore, there is a need in this field for a reagent that can accurately detect multiple variants of SARS-CoV-2, enabling targeted prevention and treatment measures for a more efficient response. Simultaneously, the reagent should be quick and highly sensitive. Summary of the Invention

[0006] In view of this, in a first aspect, the present invention provides a composition capable of detecting and typing SARS-CoV-2 variants, said composition comprising:

[0007] First nucleic acid composition:

[0008] The upstream primer of mutant Q954H shown in SEQ ID NO:1, the downstream primer of mutant Q954H shown in SEQ ID NO:2, and the probe of mutant Q954H shown in SEQ ID NO:3;

[0009] Second nucleic acid composition:

[0010] The upstream primer of mutant S982A shown in SEQ ID NO:4, the downstream primer of mutant S982A shown in SEQ ID NO:5, and the probe of mutant S982A shown in SEQ ID NO:6;

[0011] The upstream primer for mutant K417N shown in SEQ ID NO:7, the downstream primer for mutant K417N shown in SEQ ID NO:8, and the probe for mutant K417N shown in SEQ ID NO:9; and

[0012] The upstream primer of mutant E484K shown in SEQ ID NO:10, the downstream primer of mutant E484K shown in SEQ ID NO:11, and the probe of mutant E484K shown in SEQ ID NO:12;

[0013] Third nucleic acid composition:

[0014] The upstream primer of the mutant T478K shown in SEQ ID NO:13, the downstream primer of the mutant T478K shown in SEQ ID NO:14, and the probe of the mutant T478K shown in SEQ ID NO:15;

[0015] The upstream primer for mutation 69-70del shown in SEQ ID NO:16, the downstream primer for mutation 69-70del shown in SEQ ID NO:17, and the probe for mutation 69-70del shown in SEQ ID NO:18; and

[0016] The upstream primer of the mutant L452R shown in SEQ ID NO:19, the downstream primer of the mutant L452R shown in SEQ ID NO:20, and the probe of the mutant L452R shown in SEQ ID NO:21; and

[0017] Fourth nucleic acid composition:

[0018] The upstream primer for the mutant K417T shown in SEQ ID NO:22, the downstream primer for the mutant K417T shown in SEQ ID NO:23, and the probe for the mutant K417T shown in SEQ ID NO:24; and

[0019] The upstream primer of mutant T76I shown in SEQ ID NO:25, the downstream primer of mutant T76I shown in SEQ ID NO:26, and the probe of mutant T76I shown in SEQ ID NO:27.

[0020] The kit for detecting and typing SARS-CoV-2 variants provided by this invention mainly utilizes multiplex fluorescent PCR analysis. It types the variants by detecting different characteristic functional variant sites on the SARS-CoV-2 variants, thus simultaneously detecting SARS-CoV-2 variant typing within the reaction system. This allows for differentiated treatment of different strains, leading to more efficient treatment and prevention. The composition of this invention is low-cost, high-throughput, and easy to operate; results can be read through different channels. The entire detection process is performed under closed conditions, avoiding false positives and environmental contamination caused by cross-contamination between samples.

[0021] In some specific embodiments, the first nucleic acid composition may further include an upstream primer of ORF1ab as shown in SEQ ID NO:28, a downstream primer of ORF1ab as shown in SEQ ID NO:29, and an ORF1ab probe as shown in SEQ ID NO:30.

[0022] In some specific embodiments, the first nucleic acid composition may further include an upstream primer of the N gene as shown in SEQ ID NO:31, a downstream primer of the N gene as shown in SEQ ID NO:32, and an N gene probe as shown in SEQ ID NO:33.

[0023] In some specific embodiments, each nucleic acid composition of the present invention may include an upstream primer of an internal reference gene as shown in SEQ ID NO:34, a downstream primer of an internal reference gene as shown in SEQ ID NO:35, and an internal reference gene probe as shown in SEQ ID NO:36.

[0024] Furthermore, in some embodiments, the composition of the present invention may simultaneously comprise one or more pairs of the primers and probes described above. In the present invention, a "pair" refers to a mutually matched upstream and downstream primer and probe for detecting a mutation.

[0025] For example, it may include only the first nucleic acid composition; it may include only the second nucleic acid composition; it may include only the third nucleic acid composition; it may include only the fourth nucleic acid composition.

[0026] For example, it may also include only some primer and probe pairs of different nucleic acid compositions, such as the upstream primer of mutant Q954H shown in SEQ ID NO:1, the downstream primer of mutant Q954H shown in SEQ ID NO:2, and the mutant Q954H probe shown in SEQ ID NO:3; the upstream primer of mutant S982A shown in SEQ ID NO:4, the downstream primer of mutant S982A shown in SEQ ID NO:5, and the mutant S982A probe shown in SEQ ID NO:6; and the upstream primer of mutant T478K shown in SEQ ID NO:13, the downstream primer of mutant T478K shown in SEQ ID NO:14, and the mutant T478K probe shown in SEQ ID NO:15.

[0027] Furthermore, the fluorescent groups of the probes within each of the first, second, third, and fourth nucleic acid compositions are different from each other and do not interfere with each other.

[0028] In this article, "dissimilar and non-interfering" means that each probe in the composition uses a different fluorescent group, and these groups do not affect each other's detection; that is, different channels can be used for detection. For example, FAM, HEX, ROX, and CY5 can be used. These groups have different absorbance values, and different channels can be selected, so they will not interfere with each other.

[0029] In some specific embodiments, the compositions of the present invention are used for fluorescent PCR.

[0030] In this invention, the fluorescent reporter group may be selected from FAM, HEX, ROX, VIC, CY5, 5-TAMRA, TET, CY3 and JOE, but is not limited thereto.

[0031] In one specific embodiment, the fluorescent reporter group of SEQ ID NO:3, 9, 18 and 27 is ROX; the fluorescent reporter group of SEQ ID NO:6, 15 and 24 is FAM; and the fluorescent reporter group of SEQ ID NO:12, 21 and 27 is CY5.

[0032] Furthermore, the 3' end of the probe also has a quenching group, such as BHQ1, BHQ2, or MGB.

[0033] In one specific implementation, the 3' end of the probe is BHQ1.

[0034] Further, the amount of primers used in the composition is 0.1–0.3 μM; the amount of probes used in the composition is 0.1–0.3 μM.

[0035] In one specific embodiment, each nucleic acid composition of the present invention is contained in a separate package.

[0036] In one specific embodiment, the nucleic acid compositions of the present invention are contained in the same package.

[0037] Furthermore, the components in each nucleic acid composition of the present invention exist in a mixed form.

[0038] Secondly, the present invention provides the use of the above-described composition of the present invention in the preparation of a kit for detecting and typing SARS-CoV-2 variants.

[0039] Thirdly, the present invention provides a kit for detecting and typing SARS-CoV-2 variants, the kit comprising the composition of the present invention as described above.

[0040] Furthermore, the kit also includes negative and positive controls.

[0041] In one specific implementation scheme, the negative control is at least one of DEPC H2O, physiological saline, and a pseudovirus containing the internal reference gene. The positive control is at least one of plasmids containing the S target gene of the novel coronavirus, the N target gene of the novel coronavirus, various mutation sites of the novel coronavirus, and the target fragment of the internal reference gene, fragment RNA, and pseudovirus.

[0042] Furthermore, the kit also includes dNTPs, PCR buffer, and Mg. 2+ At least one of them.

[0043] Furthermore, the kit also includes at least one of the following: a nucleic acid release agent, a nucleic acid extraction reagent, a reverse transcriptase, a uracil glycosylation enzyme, and a DNA polymerase.

[0044] Furthermore, the kit also includes nucleic acid release reagents, nucleic acid extraction reagents, dNTPs, reverse transcriptase, uracil glycosylase, DNA polymerase, PCR buffer, and Mg2+. 2+ At least one of them.

[0045] Furthermore, the concentration of the reverse transcriptase is 5 U / reaction to 15 U / reaction, for example, the reverse transcriptase can be murine leukemia reverse transcriptase (MMLV) or Tth enzyme; the concentration of the DNA polymerase is 3 U / reaction to 15 U / reaction, for example, the DNA polymerase can be Taq enzyme.

[0046] In one specific embodiment, the kit of the present invention includes: reverse transcriptase, Taq enzyme, uracil glycosylation enzyme, and Mg... 2+ Mn 2+Rnasin, dNTPs, primers, probes, and PCR buffer.

[0047] Common PCR buffers consist of buffer systems such as Tris-HCl, MgCl2, KCl, and Triton X-100. The total volume in a single PCR reaction tube is typically 20-100 μl.

[0048] In one specific implementation, the kit of the present invention is compatible with digital PCR amplification systems, that is, it can be directly used for amplification on a digital PCR instrument.

[0049] Fourthly, a method for detecting and typing SARS-CoV-2 variants is provided, the method comprising the following steps:

[0050] 1) Extract or release nucleic acid from the sample to be tested;

[0051] 2) Perform quantitative real-time PCR analysis on the nucleic acid obtained in step 1) using the composition of the present invention as described above or the kit of the present invention as described above;

[0052] 3) Obtain and analyze the results.

[0053] In this invention, the samples used for detection can be pharyngeal swabs, oropharyngeal swabs, nasopharyngeal swabs, sputum, bronchoalveolar lavage fluid, blood, etc., but are not limited to these.

[0054] Furthermore, the reaction conditions for the real-time PCR are as follows:

[0055] Reverse transcription: 50℃~60℃, 5~30 minutes, 1 cycle; cDNA pre-denaturation: 95℃, 1~10 minutes, 1 cycle; denaturation: 95℃, 5~20 seconds; annealing: 55~60℃, 20~60 seconds, 40~50 cycles; fluorescence collection.

[0056] In one specific implementation, a method is provided for detecting and typing SARS-CoV-2 variants for non-diagnostic purposes, the method comprising the following steps:

[0057] 1) Extract or release nucleic acid from the sample to be tested;

[0058] 2) Perform quantitative real-time PCR analysis on the nucleic acid obtained in step 1) using the composition or kit of the present invention as described above;

[0059] 3) Obtain and analyze the results.

[0060] Furthermore, the reaction conditions for the real-time PCR are as follows:

[0061] Reverse transcription: 50℃~60℃, 5~30 minutes, 1 cycle; cDNA pre-denaturation: 95℃, 1~10 minutes, 1 cycle; denaturation: 95℃, 5~20 seconds; annealing: 55~60℃, 20~60 seconds, 40~50 cycles; fluorescence collection.

[0062] In this article, the term "non-diagnostic purpose" refers to a method not intended to obtain information about whether an individual is infected with a SARS-CoV-2 variant and develops pneumonia. For example, this method could be used to detect and genotype SARS-CoV-2 variants in test cultures used in research experiments. Attached Figure Description

[0063] Figures 1-4 The results of detecting Alpha variants using the composition of this invention;

[0064] Figures 5-8 The results of detecting Beta variants using the composition of this invention;

[0065] Figures 9-12 The results of detecting Gamma mutant strains using the composition of this invention;

[0066] Figures 13-16 The results of detecting Delta variant strains using the composition of this invention;

[0067] Figures 17-20 The results of detecting the Omicron BA.1 mutant strain using the composition of this invention;

[0068] Figures 21-24 The results of detecting the Omicron BA.2 mutant strain using the composition of this invention;

[0069] Figures 25-28 The results of detecting Lambda variants using the composition of this invention;

[0070] Figures 29-32 The results of wild-type detection of the composition of the present invention;

[0071] Figures 33-40 The sensitivity test results of the compositions of the present invention are as follows (Alpha, Beta, Gamma, Delta, Omicron BA.1, Omicron BA.2, Lambda and wild type, respectively);

[0072] Figures 41-42 The results show the specificity of the composition of this invention.

[0073] Figures 43-45 The results are for the comparative composition of this invention. Detailed Implementation

[0074] In this invention, the terms "first," "second," "third," and "fourth," etc., are used for descriptive purposes only to distinguish the substances being defined, and do not in any way limit the order or importance of the substances.

[0075] The present invention will be described in detail below with reference to specific implementation schemes and embodiments, thereby making the advantages and various effects of the present invention more clearly apparent. Those skilled in the art should understand that these specific implementation schemes and embodiments are for illustrative purposes only and are not intended to limit the present invention.

[0076] Example 1: Primers and probes used in this invention

[0077] Table 1

[0078]

[0079]

[0080] Example 2: Method for detecting SARS-CoV-2

[0081] 1. Specimen type: oropharyngeal swab, nasopharyngeal swab.

[0082] 2. Nucleic acid extraction:

[0083] Commercial RNA extraction kits, such as those based on silica membrane centrifugation columns or magnetic beads, were used and operated according to the kit instructions. Finally, 80 μL of RNA solution was collected and directly detected.

[0084] Alternatively, store at -80℃. Both negative and positive quality controls must be extracted.

[0085] 3. System Configuration:

[0086] Based on the total number of reactions N required for the test, add 16.5 μl of RT-PCR amplification buffer (MgCl2 6 mM, dNTP 0.8 mM, upstream / downstream primer concentrations both 0.2 μM, probe concentration 0.25 μM, universal upstream primer 6 μM, universal downstream primer 6 μM) and 3.5 μl of enzyme mixture (hot-start Taq enzyme, reverse transcriptase, uracil glycosylation enzyme (UNG)) to each PCR tube. Calculate the required total volume, mix well, and then aliquot into specially designed PCR reaction tubes.

[0087] 4. Sample addition:

[0088] Add 20 μL of negative control, sample RNA solution, and positive control to the PCR reaction tubes that have been aliquoted with reagents. Tighten the caps, mix well, centrifuge, and collect the solution at the bottom of the tube.

[0089] 5. Amplification and detection on the instrument:

[0090] The RT-PCR amplification program settings are shown in Table 2 below:

[0091] Table 2

[0092]

[0093] 6. Results Analysis:

[0094] Provided that the amplification is effective, the criteria for judgment are shown in Tables 3 and 4:

[0095] Table 3

[0096]

[0097] A positive HV69-70del amplification curve [Ct(+)] indicates that the sample does not carry the HV69-70del mutation, while a negative HV69-70del amplification curve [Ct(-)] indicates that the sample carries the HV69-70del mutation.

[0098] The criteria for typing different variants of the novel coronavirus are as follows:

[0099] Table 4

[0100]

[0101] Example 3: Detection results of test samples of the composition of the present invention

[0102] The primers and probes shown in Example 1 were used to verify pseudovirus samples of wild-type SARS-CoV-2 and its variants Alpha, Beta, Gamma, Delta, Omicron BA.1, Omicron BA.2, and Lambda, following the method in Example 2. The results showed that the above variants were correctly genotyped and showed no cross-reactivity with other variants, indicating that the kit can accurately differentiate and diagnose the detected variants from other variants. The detection process is shown below. Figures 1-32 As shown.

[0103] Example 4: Sensitivity of the composition of the present invention

[0104] Various variants of SARS-CoV-2 and wild-type pseudoviruses were diluted with negative samples to a concentration of 100 copies / mL to verify the sensitivity of the reagent and detection method. The test results are as follows: Figures 33-40 As shown, all pseudovirus simulation samples with a concentration of 100 copies / mL could be accurately detected, indicating that the detection method has high sensitivity.

[0105] Example 5: Specificity of the composition of the present invention

[0106] Pseudoviruses of endemic human coronaviruses (HKU1, OC43, NL63, and 229E), SARS coronavirus, MERS coronavirus, influenza A virus, and influenza B virus were diluted to 1×10⁻⁶. 6 The composition described in Example 1 of this invention was used as a specific detection sample at a concentration of copies / mL. Experimental results showed no specific amplification. Some detection results are as follows: Figures 41-42 As shown.

[0107] The test results showed that all eight pathogens, including endemic human coronaviruses (HKU1, OC43, NL63 and 229E), SARS coronavirus, MERS coronavirus, influenza A virus and influenza B virus, were negative, indicating that the composition of the present invention has good specificity.

[0108] Comparative Example 1: Other primers and probes designed in this invention that do not perform well.

[0109] Due to the principle of complementary base pairing, primers and / or probes can form dimers, but this probability is low and can be eliminated at the initial design stage. However, when detecting multiple pathogens together, there are numerous primers and probes, and dimers can easily form between primers, probes, or between different primers and probes. To ensure the conservation of the design (conservatism is crucial for detection accuracy) while also considering the mutual interference between different primers and probes, careful primer and probe design is required.

[0110] Therefore, the inventors also designed additional primers and probes (sequences not shown) to form different detection systems 1, 2, and 3, which were also used to detect SARS-CoV-2 variants. Specific detection results are as follows: Figures 43-45 As shown in the figure, only some amplification curves are detected, and the amplification curves have low amplification and poor repeatability. Other targets do not even have amplification curves. Therefore, the overall detection effect is poor. sequence list <110> Shenzhen Center for Disease Control and Prevention Shenzhen United Medical Technology Co., Ltd. <120> Compositions, kits, methods and applications for detecting SARS-CoV-2 <160> 36 <170> PatentIn version 3.5 <210> 1 <211> twenty three <212> DNA <213> Artificial sequence <400> 1 actcactttc ttccacagca agt 23 <210> 2 <211> twenty four <212> DNA <213> Artificial sequence <400> 2 gtcaagacgt gaaaggatat catt 24 <210> 3 <211> 13 <212> DNA <213> Artificial sequence <400> 3 aaccataatg cac 13 <210> 4 <211> twenty four <212> DNA <213> Artificial sequence <400> 4 caattttggt gcaatttcaa gtgt 24 <210> 5 <211> twenty two <212> DNA <213> Artificial sequence <400> 5 caatttgcac ttcagcctca ac 22 <210> 6 <211> 14 <212> DNA <213> Artificial sequence <400> 6 atccttgcac gtct 14 <210> 7 <211> twenty three <212> DNA <213> Artificial sequence <400> 7 aggtgatgaa gtcagacaaa tcg 23 <210> 8 <211> twenty two <212> DNA <213> Artificial sequence <400> 8 ccaagctata acgcagcctg ta 22 <210> 9 <211> 12 <212> DNA <213> Artificial sequence <400> 9 tggaamtatt gc 12 <210> 10 <211> twenty two <212> DNA <213> Artificial sequence <400> 10 tgaaatctat caggccggta gc 22 <210> 11 <211> twenty four <212> DNA <213> Artificial sequence <400> 11 tggttggtaa ccaacaccat wagt 24 <210> 12 <211> 14 <212> DNA <213> Artificial sequence <400> 12 tggtgttaaa ggtt 14 <210> 13 <211> 25 <212> DNA <213> Artificial sequence <400> 13 aaccttttga gagagatatttcaac 25 <210> 14 <211> twenty one <212> DNA <213> Artificial sequence <400> 14 gtgggttgga aaccatatga t 21 <210> 15 <211> 14 <212> DNA <213> Artificial sequence <400> 15 cggtarcaaa cctt 14 <210> 16 <211> 25 <212> DNA <213> Artificial sequence <400> 16 gttacttggt tccatgctat acatg 25 <210> 17 <211> twenty four <212> DNA <213> Artificial sequence <400> 17 ccagcctctt atgttagact tctc 24 <210> 18 <211> 30 <212> DNA <213> Artificial sequence <400> 18 tggtactaag aggtttgata accctgtcct 30 <210> 19 <211> twenty three <212> DNA <213> Artificial sequence <400> 19 taccagatga ttttacaggc tgc 23 <210> 20 <211> 26 <212> DNA <213> Artificial sequence <400> 20 tctctcaaaa ggtttgagat tagact 26 <210> twenty one <211> 14 <212> DNA <213> Artificial sequence <400> twenty one ttaccggtat agat 14 <210> twenty two <211> twenty two <212> DNA <213> Artificial sequence <400> twenty two ggtgatgaag tcagacaaat cg 22 <210> twenty three <211> twenty four <212> DNA <213> Artificial sequence <400> twenty three cagcctgtaa aatcatctgg taat 24 <210> twenty four <211> 13 <212> DNA <213> Artificial sequence <400> twenty four tggaackatt gct 13 <210> 25 <211> 26 <212> DNA <213> Artificial sequence <400> 25 cttggttcca tgctatacat gtctct 26 <210> 26 <211> 26 <212> DNA <213> Artificial sequence <400> 26 ggaagcaaaa taaacaccat cattaa 26 <210> 27 <211> 13 <212> DNA <213> Artificial sequence <400> 27 aatggtatta aga 13 <210> 28 <211> twenty one <212> DNA <213> Artificial sequence <400> 28 gcgcacctgt tgtctatgtg a 21 <210> 29 <211> twenty three <212> DNA <213> Artificial sequence <400> 29 aaccccattg ttgaacatca atc ​​23 <210> 30 <211> 25 <212> DNA <213> Artificial sequence <400> 30 acgtgccaca tgcttttcca ctgct 25 <210> 31 <211> 19 <212> DNA <213> Artificial sequence <400> 31 cgaaggtgtg acttccatg 19 <210> 32 <211> twenty four <212> DNA <213> Artificial sequence <400> 32 agatcacatt ggcacccgca atcc 24 <210> 33 <211> 20 <212> DNA <213> Artificial sequence <400> 33 gaccccaaaa tcagcgaaat 20 <210> 34 <211> 20 <212> DNA <213> Artificial sequence <400> 34 gaaacctcgg ccatcagaag 20 <210> 35 <211> 25 <212> DNA <213> Artificial sequence <400> 35 ggctgatgaa ctataaaagg gaaga 25 <210> 36 <211> 26 <212> DNA <213> Artificial sequence <400> 36 aatgccccag tctctgtcag cactcc 26

Claims

1. A composition capable of detecting SARS-CoV-2 variants and typing, said composition comprising simultaneously: a first nucleic acid composition: a mutant Q954H upstream primer as set forth in SEQ ID NO: 1, a mutant Q954H downstream primer as set forth in SEQ ID NO: 2, and a mutant Q954H probe as set forth in SEQ ID NO: 3; a second nucleic acid composition: a mutant S982A upstream primer as set forth in SEQ ID NO: 4, a mutant S982A downstream primer as set forth in SEQ ID NO: 5, and a mutant S982A probe as set forth in SEQ ID NO: 6; a mutant K417N upstream primer as set forth in SEQ ID NO: 7, a mutant K417N downstream primer as set forth in SEQ ID NO: 8, and a mutant K417N probe as set forth in SEQ ID NO: 9; and a mutant E484K upstream primer as set forth in SEQ ID NO: 10, a mutant E484K downstream primer as set forth in SEQ ID NO: 11, and a mutant E484K probe as set forth in SEQ ID NO: 12; a third nucleic acid composition: a mutant T478K upstream primer as set forth in SEQ ID NO: 13, a mutant T478K downstream primer as set forth in SEQ ID NO: 14, and a mutant T478K probe as set forth in SEQ ID NO: 15; a mutant 69-70del upstream primer as set forth in SEQ ID NO: 16, a mutant 69-70del downstream primer as set forth in SEQ ID NO: 17, and a mutant 69-70del probe as set forth in SEQ ID NO: 18; and a mutant L452R upstream primer as set forth in SEQ ID NO: 19, a mutant L452R downstream primer as set forth in SEQ ID NO: 20, and a mutant L452R probe as set forth in SEQ ID NO: 21; and a fourth nucleic acid composition: a mutant K417T upstream primer as set forth in SEQ ID NO: 22, a mutant K417T downstream primer as set forth in SEQ ID NO: 23, and a mutant K417T probe as set forth in SEQ ID NO: 24; and a mutant T76I upstream primer as set forth in SEQ ID NO: 25, a mutant T76I downstream primer as set forth in SEQ ID NO: 26, and a mutant T76I probe as set forth in SEQ ID NO:

27.

2. The composition of claim 1, wherein, The first nucleic acid composition can further comprise an ORF1ab upstream primer as set forth in SEQ ID NO: 28, an ORF1ab downstream primer as set forth in SEQ ID NO: 29, and an ORF1ab probe as set forth in SEQ ID NO: 30, and / or an N gene upstream primer as set forth in SEQ ID NO: 31, an N gene downstream primer as set forth in SEQ ID NO: 32, and an N gene probe as set forth in SEQ ID NO:

33.

3. The composition of claim 1, wherein, The nucleic acid compositions each comprise an internal reference gene upstream primer as set forth in SEQ ID NO: 34, an internal reference gene downstream primer as set forth in SEQ ID NO: 35, and an internal reference gene probe as set forth in SEQ ID NO:

36.

4. The composition of claim 1, wherein, The fluorescent groups of the probes in each of the first, second, third, and fourth nucleic acid compositions are different from each other and do not interfere with each other.

5. The composition of claim 4, wherein, The fluorescent reporter groups of SEQ ID NOs: 3, 9, 18, and 27 are ROX; the fluorescent reporter groups of SEQ ID NOs: 6, 15, and 24 are FAM; and the fluorescent reporter groups of SEQ ID NOs: 12, 21, and 27 are CY5.

6. The composition according to any one of claims 1 to 5, wherein, Each of the nucleic acid compositions of the composition is present in a separate package.

7. Use of the composition of any one of claims 1-6 in the preparation of a kit for detecting SARS-CoV-2 variants and typing.

8. A kit for detecting SARS-CoV-2 variants and typing, the kit comprising the composition of any one of claims 1-6.

9. The kit of claim 8, wherein, The kit also includes at least one of a nucleic acid release reagent, a nucleic acid extraction reagent, dNTPs, reverse transcriptase, uracil glycosylase, DNA polymerase, PCR buffer, and Mg 2+ ​

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