A rapid detection kit for coxsackie virus a16 gene subtypes

Abstract

The application discloses a coxsackievirus A16 gene subtype detection kit, which comprises three groups of primer probe combinations and can be used for simultaneously detecting three different gene subtypes (B1a, B1b and B1c) of coxsackievirus A16. Compared with a traditional gene sequencing and phylogenetic tree analysis method, the kit has high throughput, high specificity and high sensitivity in detection of coxsackievirus A16 genotype, and can be used for enterovirus monitoring and prevention of potential extensive infection and disease burden of coxsackievirus A16.

Description

Technical Field

[0001] This invention relates to the field of molecular biology technology, and in particular to a rapid detection kit for the Coxsackievirus A16 subtype. Background Technology

[0003] A phylogenetic tree constructed based on the VP1 gene coding region shows that CV-A16 has three genotypes (A, B, and D). Genotype A consists of the prototype strain G-10 isolated in South Africa in 1951. Genotype B is divided into two subgenotypes (B1 and B2), with 12% genetic difference between them. Genotype D is a new genotype of CV-A16, showing 16.7%, 16.2%, and 31.2% genetic differences compared to subgenotypes B1, B2, and genotype A, respectively. Genotype B1 can be further divided into three subtypes: B1a, B1b, and B1c. In my country, the prevalent CV-A16 genotypes are mainly B1a and B1b; in recent years, the B1c subtype has gradually increased from sporadic transmission. Starting in 2024, CV-A16 will exhibit a co-circulation of all three subgenotypes: B1a, B1b, and B1c. Due to antigenic differences among subtypes, different CV-A16 subtypes exhibit significant differences in transmissibility, virulence / pathogenicity, antigenicity, and immune evasion, directly affecting the epidemic characteristics and disease severity of the virus. To further control the spread of CV-A16, it is necessary to monitor CV-A16 subtypes and provide a scientific basis for developing multivalent enterovirus vaccines with cross-protective immune responses, thereby improving the diagnosis of enteroviruses, monitoring enterovirus data, and preventing the spread of enteroviruses.

[0004] In existing technologies, determining the CV-A16 genotype requires determining the full-length VP1 gene sequence and constructing a phylogenetic tree based on the VP1 gene coding region sequence for genetic evolution analysis. Although this method is the "gold standard" for enterovirus genotyping, it is complex, time-consuming, and the low sensitivity of sequencing-based detection methods makes it unsuitable for rapid screening of large numbers of samples. To efficiently and rapidly distinguish the three common CV-A16 subgenotypes and assess their transmissibility and pathogenicity, this invention develops a genotyping kit using TaqMan probe technology to identify the three CV-A16 subgenotypes (B1a, B1b, and B1c). The aim is to obtain a method that is simpler, less time-consuming, and more sensitive than sequencing for genotyping. Summary of the Invention

[0005] The technical problem solved by this invention is to provide a CV-A16 genotyping detection kit that is simple to operate, inexpensive, time-saving, and highly sensitive, addressing the shortcomings of existing technologies. By providing a series of primer and probe combinations, it is used to specifically detect Coxsackie A16 virus VP1 gene mutations and can simultaneously identify different Coxsackie A16 virus genotypes (B1a, B1b, or B1c), preventing potential widespread infection and disease burden.

[0006] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows: A respiratory pathogen detection kit is provided, characterized in that it includes at least one of the following three sets of primer-probe combinations: the first set of primer-probe combinations is used to detect CV-A16 subgenotypes B1a and B1b, with upstream and downstream primers as shown in SEO ID NO:1 and SEO ID NO:2, and its probe as shown in SEO ID NO:3; the second set of primer-probe combinations is used to detect CV-A16 subgenotype B1b, with upstream and downstream primers as shown in SEO ID NO:4 and SEO ID NO:5, and its probe as shown in SEO ID NO:6; the third set of primer-probe combinations is used to detect CV-A16 subgenotype B1c, with upstream and downstream primers as shown in SEO ID NO:7 and SEO ID NO:8, and its probe as shown in SEO ID NO:9. Preferably, in these three primer and probe combinations, the concentrations of each primer and probe in the reaction system are the same.

[0007] Preferably, the 5' end of the probe sequences SEO ID NO:3, SEO ID NO:6 and SEO ID NO:9 is labeled with a fluorescent group, and the 3' end is labeled with a quenching group.

[0008] Preferably, the 5' end of the sequence SEO ID NO:3 is labeled with a CY5 fluorescent group and the 3' end is labeled with an NFQ-MGB quenching group; the 5' end of the sequence SEO ID NO:6 is labeled with a FAM fluorescent group and the 3' end is labeled with an NFQ-MGB quenching group; and the 5' end of the sequence SEO ID NO:9 is labeled with a FAM fluorescent group and the 5' end is labeled with an NFQ-MGB quenching group.

[0009] Preferably, in the three primer and probe combinations, the final concentration of each primer in the reaction system is 0.1-1.0 μM, and the final concentration of each probe in the reaction system is 50-250 nM.

[0010] Preferably, the kit further comprises reverse transcriptase, RNase inhibitor, Taq DNA polymerase, and a dNTP / dUTP mixture.

[0011] Preferably, the kit further includes negative and positive quality controls.

[0012] The beneficial effects of this invention are as follows: Compared with the prior art, the advantages of this invention are as follows: The kit provided by this invention is the first to use the TaqMan probe method, targeting the VP1 gene of Coxsackievirus A16. Based on the Coxsackievirus A16 VP1 gene sequence accumulated in our laboratory and the sequence downloaded from the GenBank database, BLAST comparison was performed to identify genotype-specific mutation sites for CVA16 B1a, B1b, and B1c. Mutant primers were designed targeting the G546A mutation site specific to the CVA16 B1a and B1b genotypes, mutant primers targeting the A265C and G267T mutation sites specific to the CVA16 B1b genotype, and mutant primers targeting the T126C mutation site specific to the CVA16 B1c genotype. The probes are labeled with different fluorescent agents, and qRT-PCR is used to detect different CVA16 genotypes. That is, genotype determination is performed based on mutation sites.

[0013] Compared with the prior art, the present invention has the following advantages: (1) High specificity: Validation through specificity experiments and a large number of samples shows that this method has high specificity and is consistent with the results of genetic evolution analysis based on the phylogenetic tree constructed by the traditional sequencing method using the coding region sequence of the VP1 gene.

[0014] (2) High sensitivity: The kit described in this invention has high sensitivity, which is much higher than that of traditional sequencing methods that construct phylogenetic trees based on VP1 sequences for genetic evolution analysis. The minimum detection limit is less than 50 copies / reaction.

[0015] (3) Low cost, high throughput, fast and time-saving, and simple operation: It can be completed using commonly used laboratory qPCR instruments, reducing the number of operation steps. It takes less time than sequencing, and the consistency between the detection results and sequencing results is as high as 100%; and it effectively reduces costs. Compared with sequencing, more samples can be analyzed at once. Attached Figure Description

[0016] Figure 1 This is a schematic diagram of the phylogenetic tree analysis results of the VP1 sequence of the CVA16 specimen used in this invention; ■ in the diagram, the B1a genotype is shown; ▲ indicates the B1b genotype; and ● indicates the B1c genotype.

[0017] Figure 2 The results of quantitative fluorescence detection of B1a genotype specimens using the rapid detection kit for the Coxsackievirus A16 subtype described in this invention.

[0018] Figure 3The results of quantitative fluorescence detection of B1b genotype specimens using the rapid detection kit for the Coxsackievirus A16 subtype described in this invention.

[0019] Figure 4 The results of quantitative fluorescence detection of B1c genotype specimens using the rapid detection kit for the Coxsackievirus A16 subtype described in this invention. Detailed Implementation

[0020] The present invention will be further described below with reference to specific embodiments.

[0021] Example 1: Primer and probe design in a rapid detection kit for Coxsackievirus A16 genotype 1. Sample Selection: Based on the Jiangsu Provincial Hand-Foot-and-Mouth Disease Surveillance Network Laboratory, 196 pharyngeal or anal swab specimens from patients infected with Coxsackievirus A16 were collected from CDC patients in 13 cities of Jiangsu Province between 2020 and 2025. All virus strains or samples were isolated and identified in our laboratory, nucleic acid was extracted, and stored at -80℃.

[0022] 2. CVA16 VP1 Gene Amplification and Sequencing: The VP1 gene of the CVA16 isolate was amplified using a one-step RT-PCR kit, with a negative control included. The primer sequences were: CVA16-VP1-F: 5'-AGGTACTACACCCAGTGGTCAG-3' (2030-2052 nt), and CVA-VP1-R: 5'-GCAAGGTGCCGATTCACTACCCT-3' (3400-3423 nt). The PCR reaction conditions were: reverse transcription at 50℃ for 30 min, denaturation at 95℃ for 15 min, 35 cycles at 95℃ for 30 s, at 52℃ for 45 s, at 72℃ for 90 s, followed by extension at 72℃ for 10 min. The amplified products were verified using a QIAxcel capillary electrophoresis system and then sent to Shanghai Sangon Biotech Co., Ltd. for sequencing.

[0023] 3. Bioinformatics Analysis: Representative full-length CVA16 VP1 sequences (891 nt) from both domestic and international sources were downloaded from GenBank. A phylogenetic tree of CVA16 VP1 sequences was constructed using the neighbor-joining method. The results are as follows: Figure 1 As shown: The 196 CVA16 samples collected in this study belong to three genotypes: B1a (85 samples), B1b (60 samples), and B1c (51 samples).

[0024] 4. Primer and Probe Design and Synthesis: Literature was searched, and the NCBI Genebank database was consulted to download the VP1 gene sequences for the three genotypes B1a, B1b, and B1c of CVA16. Sequence alignment was performed using MEGA 7.0 software, revealing the G546A site specific to the CVA16 B1a and B1b genotypes, the A265C and G267T sites specific to the CVA16 B1b genotype, and the T126C site specific to the CVA16 B1c genotype. Primers were designed upstream and downstream of these mutation sites, and probes specifically targeting these mutation sites were designed. The specific probes have a fluorescent group and a quencher group at both ends; the fluorescent group is selected from any one or more of FAM, HEX, VIC, TET, TAMRA, ROX, CY3.5, and CY5; the quencher group is selected from any one or more of BHQ1, BHQ2, BHQ3, DABCYL, and MGB. The sequences are shown in Table 1. All primers and probes were synthesized by Shanghai Sangon Biotech Co., Ltd.

[0025] Table 1 Primer and probe sequences

[0026] Example 2: Validation of the primers and probes in the kit described in this invention 1. Mutation Site Detection and Primer / Probe Validation: Using the primers and probes listed in Table 1, genotyping was performed on the nucleic acids of 196 CVA16 samples collected in this study. Following the instructions of the Vazyme HiScript one-step qRT-PCR probeKit, reaction systems were prepared for different primer / probe combinations, with a template amount of 200 ng. In each system, the final concentration of each primer was 0.2 μM, and the probe concentration was 0.1 μM. The reaction conditions were as follows: reverse transcription: 55℃ for 15 minutes; pre-denaturation: 95℃ for 30 seconds; amplification and fluorescence collection: denaturation: 95℃ for 10 seconds; annealing extension and fluorescence detection: 60℃ for 30 seconds (fluorescence signal was collected at this step; the detection channel was set to the FAM or CY5 channel according to the probe modification, and the quencher group was MGB). Amplification was performed for 40 cycles using a quantitative real-time PCR instrument. Positive results showed an S-shaped amplification curve with a Ct value <40; negative results showed no detection or a Ct value >40. The nucleic acids of the above 196 CVA16 samples were subjected to qRT-PCR reaction, and the results are as follows: Figure 2 As shown in Figure 4, the genotyping results of 196 CVA16 specimens using the kit described in this invention are completely consistent with the results of evolutionary analysis constructed by sequencing in Example 1.

[0027] Example 2: Specificity of the kit described in this invention for detecting other enteroviruses or gastrointestinal pathogens. Using an optimized rapid detection kit for the Coxsackievirus A16 genotype, nucleic acid samples of different Coxsackievirus A16 genotypes, Coxsackievirus A6, Coxsackievirus A10, Coxsackievirus A5, Coxsackievirus A4, enterovirus EV-A71, norovirus type II, and rotavirus were amplified. The reaction products were detected using the method described in Example 2. The results are shown in Table 2. When using the kit described in this invention, Coxsackievirus A16 amplification results showed an S-shaped curve indicating a positive result only when the corresponding primers and probes were used. Results for other pathogens were all negative, indicating that this method has high specificity for detecting the Coxsackievirus A16 genotype.

[0028] Table 2. Specificity detection results of the rapid detection kit for Kassachus virus A16 genotype described in this invention.

[0029] Note: √ indicates a positive test result, × indicates a negative test result. Example 3: Sensitivity of the kit described in this invention for detecting Coxsackievirus A16 genotype Using nucleic acids from CVA16 subtypes B1a, B1b, and B1c as templates, the VP1 gene in the three samples was amplified using a One-step RT-PCR Kit. The upstream primer contained the T7 RNA polymerase promoter, with the primer sequence: 5'-TAATAGACACTCACTATAGGGATTGGTGCTCCYACTACAGCRTAT-3', and the downstream primer: 5'-GCAAGGTGCCGATTCACTACCCT-3'. The PCR reaction conditions were: reverse transcription at 50 ℃ for 30 min, denaturation at 94 ℃ for 3 min, followed by 35 cycles of 94 ℃ for 30 s, 55 ℃ for 30 s, 72 ℃ for 90 s, and extension at 72 ℃ for 10 min. After purification and recovery, the amplified products were transcribed in vitro using T7 RNA polymerase. Following purification and quantification, the products were diluted to six concentration gradients: 10 copies / μL, 50 copies / μL, 100 copies / μL, 200 copies / μL, 500 copies / μL, and 1000 copies / μL. One μL of each dilution was used as template for reverse transcription, followed by detection using the kit provided in this invention. Results showed that the primer-probe combination provided in this invention achieved a minimum detection limit of 50 copies / reaction for the three CVA16 gene subtypes B1a, B1b, and B1c.

[0030] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the present invention as claimed. The scope of protection of this invention is defined by the appended claims and their equivalents.

Claims

1. A Coxsackievirus A16 Genotype Detection Kit, characterized in that, The kit includes at least one of the following three primer-probe combinations: The first set of primer and probe combinations is used to detect Coxsackievirus A16 subgenotypes B1a and B1b. The upstream and downstream primers are shown as SEO ID NO:1 and SEO ID NO:2, and the probe is shown as SEO ID NO:

3. The second set of primer and probe combinations is used to detect Coxsackievirus A16 subgenotype B1b. The upstream and downstream primers are shown as SEOID NO:4 and SEO ID NO:5, and the probe is shown as SEO ID NO:

6. The third set of primer-probe combination is used to detect Coxsackievirus A16 subgenotype B1c. The upstream and downstream primers are shown as SEOID NO:7 and SEO ID NO:8, and the probe is shown as SEO ID NO:

9.

2. The Coxsackievirus A16 Genotype Detection Kit according to claim 1, characterized in that, In the three primer and probe combinations, the concentrations of each primer and probe in the reaction system were the same.

3. The Coxsackievirus A16 Genotype Detection Kit according to claim 1, characterized in that, The probe sequences SEO ID NO:3, SEO ID NO:6, and SEO ID NO:9 are labeled with a fluorescent group at their 5' end and a quenching group at their 3' end.

4. The Coxsackievirus A16 Genotype Detection Kit according to claim 1, characterized in that, The probe sequences SEO ID NO:3, SEO ID NO:6, and SEO ID NO:9 are respectively equipped with a fluorescent group and a quenching group at both ends; the fluorescent group is selected from any one or more of FAM, HEX, VIC, TET, TAMRA, ROX, CY3.5, and CY5; the quenching group is selected from any one or more of BHQ1, BHQ2, BHQ3, DABCYL, and MGB.

5. The Coxsackievirus A16 Genotype Detection Kit according to claim 3, characterized in that, The 5' end of the sequence SEO ID NO:3 is labeled with a fluorescent group of CY5 and the 3' end with a quencher group of NFQ-MGB; the 5' end of the sequence SEO ID NO:6 is labeled with a fluorescent group of FAM and the 3' end with a quencher group of NFQ-MGB; the 5' end of the sequence SEO ID NO:9 is labeled with a fluorescent group of FAM and the 5' end with a quencher group of NFQ-MGB.

6. The Coxsackievirus A16 Genotype Detection Kit according to claim 3, characterized in that, In the three primer and probe combinations, the final concentration of each primer in the reaction system was 0.1-1.0 μM, and the final concentration of each probe in the reaction system was 50-250 nM.

7. The Coxsackievirus A16 Genotype Detection Kit according to claim 3, characterized in that, The kit also contains reverse transcriptase, RNase inhibitor, Taq DNA polymerase, and a dNTP / dUTP mixture.

8. A Coxsackievirus A16 Genotype Detection Kit according to claim 3, characterized in that, The kit also includes negative and positive controls for each genotype.

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