A measles virus B3 / D8 / H1 gene typing detection kit and detection method

Abstract

The application relates to a primer probe set, a kit and a method for genotyping of a measles virus B3 / D8 / H1. The method for detecting the measles virus by using the primer probe set and the kit has good sensitivity, the sensitivity of the measles virus of different genotypes can reach 500 copies / mL, and the detection of low virus load can be realized.

Description

Technical Field

[0001] This invention relates to the field of viral nucleic acid detection technology, specifically to a measles virus B3 / D8 / H1 genotyping detection kit and detection method. Background Technology

[0002] Measles is an infectious disease caused by the measles virus, an airborne pathogen transmitted through inhaled respiratory droplets. The clinical presentation is characterized by a prodromal phase with fever, conjunctivitis, runny nose, and cough, followed by a maculopapular rash. Over the past two decades, reports indicate a resurgence of measles, with multiple outbreaks occurring globally in countries with high population indices. Measles virus (MeV) belongs to the genus Measlesvirus in the family Paramyxoviridae. Its genome is a single-stranded negative-sense RNA, and it has only one serotype.

[0003] Genotyping is used to confirm the origin of an outbreak and rule out endemic transmission; it is also the only method to distinguish between vaccine strains and wild-type strains. By analyzing the sequence of 450 nucleotides (nt) encoding the 150 amino acids at the carboxyl terminus of the nucleoprotein (N-450), the genetic characteristics of the measles virus can be analyzed. Currently, the World Health Organization (WHO) has identified 24 genotypes of measles virus. Statistical analysis of the Measles Nucleotide Surveillance (MeaNS) database from 2016 to 2018 shows that genotypes B3, D8, and H1 dominate global incidence rates. In 2020, the WHO released the "Global Measles and Rubella Strategic Framework 2021-2030," which clearly sets the goal of achieving and maintaining regional measles and rubella elimination by 2030. China began virological surveillance of measles virus in 1993, and nearly 30 years of data monitoring have revealed that the H1 subtype is the absolutely dominant endemic genotype for measles in my country. Therefore, establishing a rapid measles virus genotyping detection method is of great significance for disease tracing, genetic evolution analysis, epidemic prevention and control monitoring, and epidemiological surveillance.

[0004] Real-time quantitative PCR (qPCR) is a molecular biology technique that monitors the amount of DNA amplified in real time during a PCR reaction using fluorescence signals. With its advantages of high sensitivity, precise quantification, and high degree of automation, real-time quantitative PCR has become the gold standard technology for molecular diagnostics, life science research, and clinical testing.

[0005] Current detection technologies lack kits and methods for measles virus genotyping, resulting in the inability to rapidly and accurately identify common genotypes. Therefore, there is an urgent need in this field for a highly specific and sensitive method for rapid genotyping of measles virus. Summary of the Invention

[0006] The purpose of this invention is to provide a rapid detection kit for distinguishing and detecting measles virus.

[0007] Another objective of this invention is to provide a highly specific and sensitive method for detecting measles virus that enables rapid genotyping.

[0008] In a first aspect, the present invention provides a primer and probe set for measles virus B3 / D8 / H1 genotyping, the primer and probe set comprising: (1) A first primer-probe set targeting the measles virus B3 gene, the first primer-probe set comprising the upstream primer shown in SEQ ID NO:1, the downstream primer shown in SEQ ID NO:2, and the probe shown in SEQ ID NO:3; (2) A second primer-probe set targeting the measles virus D8 gene, comprising the upstream primer shown in SEQ ID NO:4, the downstream primer shown in SEQ ID NO:5, and the probe shown in SEQ ID NO:6; and (3) A third primer and probe set for the measles virus H1 gene, the third primer and probe set comprising the upstream primer shown in SEQ ID NO:7, the downstream primer shown in SEQ ID NO:8, and the probe shown in SEQ ID NO:9.

[0009] In another preferred embodiment, the probe is modified with a fluorescent group and a quenching group.

[0010] In another preferred embodiment, the fluorescent group is selected from the group consisting of ATTO425, FAM, VIC / HEX, ROX, CY5, CY5.5, CY7, AF405, or combinations thereof.

[0011] In another preferred embodiment, the quenching group is selected from the group consisting of BHQ1, BHQ2, BHQ3, MGB, or combinations thereof.

[0012] In another preferred embodiment, the fluorescent group is modified at the 5' end of the probe; the quenching group is modified at the 3' end of the probe.

[0013] In another preferred embodiment, the fluorescent groups modified on the probe may be the same or different.

[0014] In another preferred embodiment, the probe contains 1-5 bases modified with locked nucleic acids, preferably 1-3, and more preferably 2-3.

[0015] In another preferred embodiment, the primer-probe set includes: [T*] / [C*] indicates locked nucleic acid modification.

[0016] In a second aspect, the present invention provides a PCR amplification system comprising a buffer system for amplification and a primer and probe set as described in the first aspect of the present invention.

[0017] In another preferred embodiment, the PCR amplification system further includes an enzyme mixture for nucleic acid amplification.

[0018] In another preferred embodiment, the concentration of each primer in the PCR amplification system is 10-1000 nM, more preferably 50-1000 nM, and even more preferably 100-900 nM; The concentration of each probe is 10-800 nM, preferably 30-500 nM, and even more preferably 50-450 nM.

[0019] In another preferred embodiment, the buffer system comprises Tris-HCl, MgCl2, dNTPs, DMSO, betaine, bovine serum albumin, KCl, Tween20, and TMAC.

[0020] In another preferred embodiment, the enzyme mixture includes Taq enzyme, reverse transcriptase, and UNG enzyme.

[0021] In a third aspect, the present invention provides a kit comprising a container and a primer-probe set as described in the first aspect of the present invention or a PCR amplification system as described in the second aspect of the present invention, located within the container.

[0022] In another preferred embodiment, the kit further includes positive and negative standards.

[0023] In another preferred embodiment, the positive standard is a plasmid containing fragments of measles virus type B3 N-450, measles virus type D8 N-450 and / or measles virus type H1 N-450, preferably a mixture of plasmids containing fragments of measles virus type B3 N-450, measles virus type D8 N-450 and measles virus type H1 N-450.

[0024] In another preferred embodiment, the nucleotide sequence of the measles virus B3 type N-450 fragment is shown in SEQ ID NO:10.

[0025] In another preferred embodiment, the nucleotide sequence of the measles virus D8 type N-450 fragment is shown in SEQ ID NO:11.

[0026] In another preferred embodiment, the nucleotide sequence of the measles virus H1 type N-450 fragment is shown in SEQ ID NO:12.

[0027] In another preferred embodiment, the negative standard is physiological saline.

[0028] In a fourth aspect, the present invention provides a method for genotyping measles virus B3 / D8 / H1, the method comprising the steps of: (s1) Provide the nucleic acid from the sample to be tested; (s2) Using the primer and probe set as described in the first aspect of the present invention, the PCR amplification system as described in the second aspect of the present invention, or the kit as described in the third aspect of the present invention, the nucleic acid of the sample to be tested is subjected to a nucleic acid amplification reaction, and the fluorescence signal is detected to obtain a fluorescent PCR amplification curve; and (s3) Analyze the fluorescence PCR amplification curve to determine the typing result of the sample to be tested.

[0029] In another preferred embodiment, the reaction procedure for the nucleic acid amplification reaction is: reverse transcription at 50°C for 2 min; pre-denaturation at 95°C for 20 s; denaturation at 95°C for 5 s; annealing / extension at 55°C for 30 s; 40-45 cycles.

[0030] In another preferred embodiment, the criterion for determining the method is: A sample with a Ct value > 38 or no detection is considered negative; a sample with a typical S-curve amplification curve and a Ct value ≤ 35 is considered positive; a sample with a typical S-curve amplification curve and 35 < Ct value ≤ 38 needs to be retested. If the retest results are consistent, the result is considered positive; if the Ct value > 38 or no detection, the result is considered negative.

[0031] In another preferred embodiment, the quality control criteria for the method are: negative control Ct value > 38 or not detected, positive control amplification curve showing a typical S-curve, and Ct value ≤ 35.

[0032] In another preferred embodiment, the method is non-diagnostic and non-therapeutic.

[0033] In another preferred embodiment, the method is in vitro.

[0034] In another preferred embodiment, the detection limit of the method is ≤800 copies / mL, more preferably ≤600 copies / mL, and even more preferably ≤500 copies / mL.

[0035] In another preferred embodiment, in step (s2), a fluorescence signal is detected during the annealing / extension process of the nucleic acid amplification reaction.

[0036] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0037] Figure 1 The kit demonstrates the specificity test of the measles virus B3 primer probe.

[0038] Figure 2 The kit demonstrates the specificity test of the measles virus D8 primer probe.

[0039] Figure 3 The kit demonstrates the specificity test of the measles virus H1 type primer probe.

[0040] Figure 4 The kit sensitivity test - B3 sample is shown.

[0041] Figure 5 The kit sensitivity test - D8 sample is shown.

[0042] Figure 6 The kit sensitivity test - H1 sample is shown.

[0043] Figure 7 The results of testing a standard B3 / D8 RNA sample of 500 copies / mL are shown.

[0044] Figure 8 The results of testing a standard B3 / H1 RNA sample of 500 copies / mL are shown.

[0045] Figure 9 The results of testing a standard D8 / H1 RNA sample of 500 copies / mL are shown.

[0046] Figure 10 The results of testing a standard B3 / D8 / H1 RNA sample at 500 copies / mL are shown.

[0047] Figure 11 This diagram shows the positions of the measles virus B3, D8, and H1 primer probes on N-450. Detailed Implementation

[0048] Through extensive and in-depth research, and after numerous experiments and screenings, the inventors unexpectedly discovered for the first time a primer-probe set and kit for measles virus B3 / D8 / H1 genotyping. The probes of this invention are modified with locked nucleic acids, improving the distinguishability between different measles virus genotypes and thus enhancing specificity. The method for detecting measles virus using the primer-probe set and kit of this invention exhibits good sensitivity, reaching 500 copies / mL for different measles virus genotypes, and enabling the detection of low viral loads. This invention was completed based on this discovery.

[0049] the term To facilitate understanding of the invention, certain technical and scientific terms are specifically defined below. Unless otherwise expressly defined herein, all other technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this invention pertains. Before describing the invention, it should be understood that the invention is not limited to the specific methods and experimental conditions described, as such methods and conditions can vary. It should also be understood that the terminology used herein is intended only to describe particular embodiments and is not intended to be restrictive; the scope of the invention will be limited only by the appended claims.

[0050] As used herein, the term “comprising” or its variations such as “including” or “comprising” are understood to include the said element or component without excluding other elements or other components.

[0051] The term “about” can refer to a value or composition within an acceptable margin of error for a particular value or composition as determined by a person skilled in the art, depending in part on how the value or composition is measured or determined. For example, as used herein, the expression “about 100” includes all values ​​between 99 and 101 (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0052] As used herein, unless otherwise stated, any concentration range, percentage range, proportion range, or integer range shall be understood to include any integer value within the range and, where appropriate, its fractional value (e.g., one-tenth and one-hundredth of an integer).

[0053] As used herein, the term “and / or” refers to and covers any and all possible combinations of one or more of the related listed items.

[0054] Measles virus B3 / D8 / H1 gene Measles virus (MeV) is a single serotype, classified by the WHO into 8 evolutionary branches (Clade AH) and 24 genotypes. Among them, B3 (Clade B), D8 (Clade D), and H1 (Clade H) are the three most prevalent genotypes globally in the past decade. Currently, the C-terminal 450 bp (N-450) of the measles virus N gene is the gold standard for measles genotyping. However, the N-450 gene sequences of different genotypes show extremely high homology; N gene analysis of measles isolates shows nucleotide similarity ranging from 89.1% to 100%, posing a significant challenge to direct quantitative real-time PCR for genotyping. The nucleotide homology of some measles virus B3, D8, and H1 studies is shown in Table A.

[0055] Table A Figure 11This diagram shows the positions of the primers and probes of the present invention on N-450.

[0056] The primer probe set of the present invention The primer and probe set of this invention includes: (1) A first primer-probe set targeting the measles virus B3 gene, the first primer-probe set comprising the upstream primer shown in SEQ ID NO:1, the downstream primer shown in SEQ ID NO:2, and the probe shown in SEQ ID NO:3; (2) A second primer-probe set targeting the measles virus D8 gene, comprising the upstream primer shown in SEQ ID NO:4, the downstream primer shown in SEQ ID NO:5, and the probe shown in SEQ ID NO:6; and (3) A third primer and probe set for the measles virus H1 gene, the third primer and probe set comprising the upstream primer shown in SEQ ID NO:7, the downstream primer shown in SEQ ID NO:8, and the probe shown in SEQ ID NO:9.

[0057] In a preferred embodiment, the probe is modified with a fluorescent group and a quenching group. In a preferred embodiment, the fluorescent group is selected from the group consisting of ATTO425, FAM, VIC / HEX, ROX, CY5, CY5.5, CY7, AF405, or combinations thereof. In a preferred embodiment, the quenching group is selected from the group consisting of BHQ1, BHQ2, BHQ3, MGB, or combinations thereof. In a preferred embodiment, the fluorescent group is modified at the 5' end of the probe; the quenching group is modified at the 3' end of the probe.

[0058] In a preferred embodiment, the primer-probe set includes: [T*] / [C*] indicates locked nucleic acid modification.

[0059] Measles virus B3 and H1 show high homology with other subtypes. By modifying the probe with locked nucleic acid, the probe Tm value is increased, enhancing probe binding ability and single-base discrimination ability. The primer-probe set of this invention exhibits excellent measles virus typing ability.

[0060] The reagent kit of the present invention The kit of the present invention is used for measles virus B3 / D8 / H1 genotyping, comprising a container and a primer and probe set as described in the first aspect of the present invention or a PCR amplification system as described in the second aspect of the present invention located in the container.

[0061] In a preferred embodiment, the PCR amplification system further includes an enzyme mixture for nucleic acid amplification. In a preferred embodiment, the enzyme mixture includes Taq enzyme, reverse transcriptase, and UNG enzyme.

[0062] In a preferred embodiment, the concentration of each primer in the PCR amplification system is 10-1000 nM, more preferably 50-1000 nM, and even more preferably 100-900 nM; The concentration of each probe is 10-800 nM, preferably 30-500 nM, and even more preferably 50-450 nM.

[0063] In a preferred embodiment, the buffer system comprises Tris-HCl, MgCl2, dNTPs, DMSO, betaine, bovine serum albumin, KCl, Tween20, and TMAC.

[0064] In a preferred embodiment, the kit further includes positive and negative standards. In a preferred embodiment, the positive standard is a plasmid containing fragments of measles virus B3 N-450, measles virus D8 N-450, and / or measles virus H1 N-450, preferably a mixture of plasmids containing fragments of measles virus B3 N-450, measles virus D8 N-450, and measles virus H1 N-450. In a preferred embodiment, the negative standard is physiological saline.

[0065] The method of the present invention The method of the present invention refers to a method for genotyping measles virus B3 / D8 / H1, the method comprising the following steps: (s1) Provide the nucleic acid from the sample to be tested; (s2) Using the primer and probe set as described in the first aspect of the present invention, the PCR amplification system as described in the second aspect of the present invention, or the kit as described in the third aspect of the present invention, the nucleic acid of the sample to be tested is subjected to a nucleic acid amplification reaction, and the fluorescence signal is detected to obtain a fluorescent PCR amplification curve; and (s3) Analyze the fluorescence PCR amplification curve to determine the typing result of the sample to be tested.

[0066] In a preferred embodiment, the criterion for determining the method is: A sample with a Ct value > 38 or no detection is considered negative; a sample with a typical S-curve amplification curve and a Ct value ≤ 35 is considered positive; a sample with a typical S-curve amplification curve and 35 < Ct value ≤ 38 needs to be retested. If the retest results are consistent, the result is considered positive; if the Ct value > 38 or no detection, the result is considered negative.

[0067] In a preferred embodiment, the quality control criteria for the method are: negative control Ct value > 38 or not detected, positive control amplification curve showing a typical S-curve, and Ct value ≤ 35.

[0068] In a preferred embodiment, in step (s2), a fluorescence signal is detected during the annealing / extension process of the nucleic acid amplification reaction.

[0069] The main advantages of this invention include The detection kit described in this invention is based on the gold standard method for molecular diagnostics—real-time quantitative PCR. Conserved primer and probe sequences are designed for specific regions of three measles virus genotypes: B3, D8, and H1. Specific base modifications are made to the probe sequences to enhance the differentiation between different genotypes and ensure specificity. Furthermore, this kit exhibits excellent detection sensitivity, reaching 500 copies / mL for different genotypes, enabling the detection of low viral loads and avoiding false negatives.

[0070] There is currently no method for genotyping measles virus based on real-time quantitative PCR. This invention relies on a professional bioinformatics analysis platform for thorough sequence alignment analysis, designs highly specific primers and probes, and thus develops a genotyping detection kit and method for measles virus, filling a gap in this detection field and greatly promoting measles virus surveillance nationwide and even globally.

[0071] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are by weight.

[0072] Example 1: Design of primers and probes for measles virus B3, D8, and H1 genotyping This invention utilizes multiple rounds of primer and probe screening to obtain primer and probe combinations that possess both specificity and sensitivity. Preferred primer and probe sets are shown in Table 1. All primers and probes were synthesized by Sangon Biotech (Shanghai) Co., Ltd. The 5' and 3' ends of each probe are labeled with fluorescent chromophores and fluorescence quenchers.

[0073] Table 1. Primer and probe sequence listing for measles virus B3, D8, and H1 genotyping Note: [T*] / [C*] both represent locked nucleic acid modified bases.

[0074] Example 2: Detection of measles virus B3, D8 and H1 using the primers and probes of the present invention. 1. Construction of standard sequences Gene fragments were synthesized according to Table 2 and constructed into the pUC57 vector. RNA was then transcribed using the T7 promoter. Both plasmid and RNA synthesis were performed by Hunan Aikerui Biotechnology Co., Ltd.

[0075] Table 2. Measles virus B3, D8, and H1 transcribed RNA sequence listing 2. Construction of Standard Sequences Prepare standard concentration samples according to Table 3 for sensitivity and specificity testing.

[0076] Table 3 Sample Information Required for Testing 3. Construction of the amplification reaction system Prepare qPCR reaction solutions for samples A / B3 / C1 / D8 / E / F / G1 / H1, positive control, and negative control as shown in Table 4. The total number of samples is denoted as n. Calculate the total volume of reaction solution prepared by performing three replicates for each sample and one replicate for each negative and positive control, resulting in a total volume of 4n. Prepare the qPCR reaction solution by mixing the amplification reaction solution, enzyme mixture, and primers / probes according to the calculated amounts of each component, ensuring thorough mixing before use. Aliquot the qPCR reaction solution into 8-tube strips at 20 μL / well. The final primer concentration is 100–900 nM, and the final probe concentration is 50–450 nM.

[0077] Table 4 qPCR reaction solution preparation 4. Specificity detection of the reagent kit Prepare eight different measles genotype samples according to Table 3, and prepare the qPCR reaction solution (excluding the template) according to Table 4. Use the kit to detect measles virus A, B3, C1, D8, E, F, G1, and H1 samples respectively. Run the real-time PCR instrument according to the amplification program in Table 5. The equipment used includes, but is not limited to, Hongshi series instruments (SLAN-96S, SLAN-96P), ABI series instruments (ABI7500, ABI Q5), and Tianlong series instruments (Gentier96E). When testing B3 samples, the kit should show a clear amplification curve in the FAM channel; when testing D8 samples, the kit should show a clear amplification curve in the VIC channel; and when testing H1 samples, the kit should show a clear amplification curve in the ROX channel. Otherwise, when testing A / C1 / E / F / G1 samples, the kit should not show amplification curves in the FAM, VIC, or ROX channels.

[0078] Table 5 PCR amplification program Test results are as follows Figures 1-3 As shown: The B3 type primers and probes described in this invention can only amplify B3 genotype samples, and there is no cross-reaction with other genotype samples. Figure 1 The D8 type primers and probes described in this invention can only amplify D8 genotype samples, and do not exhibit cross-reactivity with other genotype samples. Figure 2 The H1 type primers and probes described in this invention can only amplify H1 genotype samples, and do not exhibit cross-reactivity with other genotype samples. Figure 3 ).

[0079] 5. Reagent kit sensitivity testing Prepare measles virus B3, D8, and H1 samples according to Table 3. 1) Use RNase-free water to serially dilute measles virus B3, D8, and H1 to a final concentration of 500 copies / mL, respectively, as single-sensitivity samples; 2) Use RNase-free water to serially dilute measles virus B3 / D8, measles virus B3 / H1, and measles virus D8 / H1 to a final target concentration of 500 copies / mL, as dual-sensitivity samples; 3) Use RNase-free water to serially dilute measles virus B3 / D8 / H1 to a target concentration of 500 copies / mL, as triple-sensitivity samples. Use this kit to run the experimental amplification program according to Table 5.

[0080] Test results are as follows Figures 4-6 As shown: The kit described in this invention detects 500 copies / mL of B3 RNA standard samples with a detection rate of 100% (20 / 20). Figure 4The kit described in this invention detects 500 copies / mL of D8 RNA standard samples with a detection rate of 100% (20 / 20). Figure 5 The kit described in this invention detects 500 copies / mL of H1 RNA standard samples with a detection rate of 100% (20 / 20). Figure 6 ).

[0081] The kit described in this invention detects 500 copies / mL of B3 / D8 RNA standard samples with a detection rate of 100% (20 / 20). Figure 7 ).

[0082] The kit described in this invention detects 500 copies / mL of B3 / H1 RNA standard samples with a detection rate of 100% (20 / 20). Figure 8 ).

[0083] The kit described in this invention detects 500 copies / mL of D8 / H1 RNA standard samples with a detection rate of 100% (20 / 20). Figure 9 ).

[0084] The kit described in this invention detects 500 copies / mL of B3 / D8 / H1 RNA standard samples with a detection rate of 100% (20 / 20). Figure 10 ).

[0085] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A primer and probe set for measles virus B3 / D8 / H1 genotyping, characterized in that, The primer and probe set includes: (1) A first primer-probe set targeting the measles virus B3 gene, the first primer-probe set comprising the upstream primer shown in SEQ ID NO:1, the downstream primer shown in SEQ ID NO:2, and the probe shown in SEQ ID NO:3; (2) A second primer-probe set targeting the measles virus D8 gene, comprising the upstream primer shown in SEQ ID NO:4, the downstream primer shown in SEQ ID NO:5, and the probe shown in SEQ ID NO:6; and (3) A third primer and probe set for the measles virus H1 gene, the third primer and probe set comprising the upstream primer shown in SEQ ID NO:7, the downstream primer shown in SEQ ID NO:8, and the probe shown in SEQ ID NO:

9.

2. The primer-probe set as described in claim 1, characterized in that, The primer and probe set includes: [T*] / [C*] indicates locked nucleic acid modification.

3. A PCR amplification system, characterized in that, The PCR amplification system includes a buffer system for amplification and a primer and probe set as described in claim 1.

4. The PCR amplification system as described in claim 3, characterized in that, The PCR amplification system also includes an enzyme mixture for nucleic acid amplification.

5. The PCR amplification system as described in claim 3, characterized in that, In the PCR amplification system, the concentration of each primer is 10-1000 nM, preferably 50-1000 nM, and more preferably 100-900 nM; The concentration of each probe is 10-800 nM, preferably 30-500 nM, and even more preferably 50-450 nM.

6. The PCR amplification system as described in claim 4, characterized in that, The enzyme mixture includes Taq enzyme, reverse transcriptase, and UNG enzyme.

7. The PCR amplification system as described in claim 3, characterized in that, The buffer system includes Tris-HCl, MgCl2, dNTPs, DMSO, betaine, bovine serum albumin, KCl, Tween20, and TMAC.

8. A reagent kit, characterized in that, The kit includes a container and a primer and probe set as described in claim 1 or a PCR amplification system as described in claim 3, located within the container.

9. A method for genotyping measles virus B3 / D8 / H1, characterized in that, The method includes the following steps: (s1) Provide the nucleic acid from the sample to be tested; (s2) Using the primer and probe set as described in claim 1, the PCR amplification system as described in claim 3, or the kit as described in claim 8, the nucleic acid of the sample to be tested is amplified, and the fluorescence signal is detected to obtain a fluorescent PCR amplification curve; and (s3) Analyze the fluorescence PCR amplification curve to determine the typing result of the sample to be tested.

10. The method as described in claim 9, characterized in that, The reaction procedure for the nucleic acid amplification reaction is as follows: reverse transcription at 50℃ for 2 min; pre-denaturation at 95℃ for 20 s; denaturation at 95℃ for 5 s; annealing / extension at 55℃ for 30 s; 40-45 cycles.

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