A primer set, a kit, a typing method and application for rapid typing of porcine senecavirus a

Abstract

The application discloses a primer group, a kit, a typing method and application for rapid typing of porcine Senecavirus A, and relates to the technical field of virus genome detection. The primer group comprises an upstream primer SVA-F, wherein the nucleotide sequence of the upstream primer SVA-F is shown as SEQ ID NO:1; and a downstream primer SVA-R, wherein the nucleotide sequence of the downstream primer SVA-R is shown as SEQ ID NO:2. The primer group is used for amplifying a core genome fragment obtained through screening, the fragment can represent the evolutionary topology structure of the whole genome, the typing resolution is significantly higher than that of a traditional single gene method, and information distortion is avoided; the fragment is short in length, only needs conventional PCR and Sanger sequencing, the cost is reduced by more than 80%, data analysis is simple, and a professional biological information platform is not needed; an operation process is standardized, the flux is high, and the application is suitable for clinical large-scale molecular epidemiological monitoring; and a typing result can truly reflect the virus evolution history and the transmission path, and can provide a reliable and scientific basis for epidemic prevention and control.

Description

Technical Field

[0001] This invention relates to the field of viral genome detection technology, specifically to a primer set, reagent kit, typing method, and application for rapid typing of porcine type A Seneca virus. Background Technology

[0002] Senecavirus A (SVA) is a significant emerging virus threatening the global swine industry in recent years. It causes vesicular lesions in piglets and leads to neonatal mortality, resulting in severe economic losses to pig production. Accurate virus typing is crucial for molecular epidemiological investigations, tracing the source of outbreaks, and developing control measures. Currently, whole-genome sequencing (WGS) is widely recognized as the "gold standard" for pathogen typing, providing the most complete genomic evolutionary information. However, WGS technology suffers from drawbacks such as high cost, long sequencing cycles, and complex subsequent bioinformatics analyses, hindering its widespread application in large-scale clinical surveillance and resource-limited areas.

[0003] On the other hand, while traditional PCR typing methods based on conventional single gene fragments (such as the VP1 gene) are simple to operate and low in cost, the phylogenetic resolution is often insufficient because the detected gene fragments constitute a very small proportion of the whole genome, making it difficult to accurately reflect the complete evolutionary history of the virus and the true genetic structure of the population. In particular, when viral genome recombination occurs or different gene regions have inconsistent evolutionary rates, single-gene typing results are prone to bias, misleading the tracing of the epidemic's origin and the inference of its transmission routes.

[0004] Therefore, there is an urgent need in this field to develop a novel typing scheme that can preserve the evolutionary topological information of the whole genome while being efficient, accurate, and low-cost. Summary of the Invention

[0005] This invention addresses the shortcomings of existing technologies by providing a primer set, reagent kit, typing method, and application for rapid typing of porcine Seneca virus type A. The aim is to screen core genomic fragments that perfectly represent the evolutionary information of the entire genome, establish a typing strategy based on the topological consistency of the entire genome's evolution, and provide a scientific basis for molecular epidemiological investigation and precise prevention and control of porcine Seneca virus type A.

[0006] Therefore, in a first aspect, the present invention provides a primer set for rapid typing of porcine type A Seneca virus, comprising: The upstream primer SVA-F has the nucleotide sequence shown in SEQ ID NO:1; The downstream primer SVA-R has the nucleotide sequence shown in SEQ ID NO:2.

[0007] A second aspect of the present invention provides a kit for rapid typing of porcine Seneca virus type A, the kit comprising the aforementioned primer set.

[0008] Furthermore, the kit also includes reverse transcription reagents and PCR amplification reaction solution.

[0009] A third aspect of the present invention provides a method for rapid typing of porcine Seneca virus type A, not for the purpose of disease diagnosis and treatment, comprising: rapidly typing porcine Seneca virus type A using the aforementioned kit.

[0010] Furthermore, the method includes: Viral nucleic acid was extracted from the sample to be tested and cDNA was obtained by reverse transcription; PCR amplification was performed using the cDNA as a template to obtain the amplified fragment; The amplified fragment was sequenced and analyzed to obtain the typing results of porcine Seneca virus type A.

[0011] Furthermore, the sample to be tested includes at least one of blister fluid, oral and nasal swab samples, blood samples, fecal samples, tissue samples, environmental samples, and virus cultures.

[0012] Furthermore, the PCR amplification reaction procedure includes: Pre-denaturation: 94℃~96℃ for 2~4 minutes; Denaturation at 94℃~96℃ for 28s~32s, annealing at 54℃~56℃ for 28s~32s, extension at 71℃~73℃ for 0.5min~1.5min, 35 cycles; The final extension temperature is 71℃~73℃ for 2min~4min.

[0013] Furthermore, the PCR amplification reaction system includes: The following reagents were prepared: cDNA template 0.5 μL to 1.5 μL, upstream primer SVA-F 0.5 μL to 1.5 μL, downstream primer SVA-R 0.5 μL to 1.5 μL, 2×Magic Green Taq SuperMix 11.5 μL to 13.5 μL, and Nuclease & Nucleic acid-free ddH2O 8.5 μL to 10.5 μL.

[0014] A fourth aspect of the invention provides the use of the primer set or the kit in the preparation of reagents for typing porcine Seneca virus type A.

[0015] A fifth aspect of the invention provides the application of the primer set or the kit in porcine Seneca virus type A typing and / or strain tracing.

[0016] Compared with the prior art, the present invention has at least the following beneficial effects: This invention provides a primer set and kit for rapid typing of porcine Seneca virus type A. The primer set includes upstream primer SVA-F and downstream primer SVA-R, whose nucleotide sequences are shown in SEQ ID NO:1 and SEQ ID NO:2. The primer set of this invention is used to amplify the core genomic fragment obtained through screening. This fragment represents the evolutionary topology of the entire genome, and the typing resolution is significantly higher than that of traditional single-gene methods, avoiding information distortion. Furthermore, this fragment is short (typically 1kb~3kb), requiring only routine PCR and Sanger sequencing, reducing costs by more than 80%. Data analysis is simple and does not require a professional bioinformatics platform. The operation process is standardized, with high throughput, suitable for large-scale clinical molecular epidemiological surveillance. The typing results can accurately reflect the virus's evolutionary history and transmission pathway, providing a reliable scientific basis for epidemic prevention and control. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments recorded in the embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings.

[0018] Figure 1 A flowchart of a genotyping method based on genome-wide evolutionary topological consistency provided in an embodiment of the present invention; Figure 2 Mantel correlation analysis diagram of SVA sliding window provided in embodiments of the present invention; Figure 3 This is a schematic diagram showing the location of the core genome in the SVA whole genome, as provided in an embodiment of the present invention. Figure 4 This is a comparison chart of genetic distance linear regression analysis provided in the embodiments of the present invention. The left chart shows the analysis results of the SVA core genome amplicon region of the present invention, and the right chart shows results from other studies (…). Korea Science Results of amplicon region analysis based on the VP1 gene in (2021); Figure 5 The phylogenetic topology diagram of the SVA whole genome (left) and the SVA core genome amplicon region (right) provided in the embodiments of the present invention; Figure 6 The SVA whole genome (left) provided for embodiments of the present invention and other studies ( Korea Science Phylogenetic tree topology diagram of the amplicon region based on the VP1 gene (right) in (2021); Figure 7The source tracing analysis results of clinical positive samples provided in the embodiments of the present invention. Detailed Implementation

[0019] To better understand the above technical solutions, the technical solutions of the embodiments of this application will be described in detail below with reference to the accompanying drawings and specific embodiments. It should be understood that the embodiments of this application and the specific features in the embodiments are detailed descriptions of the technical solutions of the embodiments of this application, rather than limitations on the technical solutions of this application. In the absence of conflict, the embodiments of this application and the technical features in the embodiments can be combined with each other.

[0020] A first aspect of this invention provides a primer set for rapid typing of porcine Seneca virus type A, comprising: The upstream primer SVA-F has the nucleotide sequence shown in SEQ ID NO:1; The downstream primer SVA-R has the nucleotide sequence shown in SEQ ID NO:2.

[0021] Specifically, the primer set in this embodiment of the invention can amplify the core genomic fragment representing the whole genome evolution information of porcine Seneca virus type A. The screening method for the core genomic fragment includes: obtaining the whole genome sequence of porcine Seneca virus type A and constructing a phylogenetic topology based on the whole genome sequence; and using an evolutionary topological consistency assessment algorithm to screen out the core genomic fragment that can perfectly represent the whole genome evolution information from the whole genome sequence.

[0022] These fragments, while preserving complete genomic evolutionary relationships, significantly reduce data complexity and sequencing costs, and can be directly applied to subsequent virus typing detection. Based on this method, high-throughput, low-cost, and high-resolution typing of porcine Seneca virus type A can be achieved, providing a reliable scientific basis for molecular epidemiological investigations, epidemic tracing, and the formulation of precise prevention and control measures.

[0023] A second aspect of this invention provides a kit for rapid typing of porcine type A Seneca virus, comprising a primer set.

[0024] In some embodiments, the kit further includes reverse transcription reagents and PCR amplification reaction solution.

[0025] A third aspect of the present invention provides a method for rapid typing of porcine Seneca virus type A, not for the purpose of disease diagnosis and treatment, comprising: rapidly typing porcine Seneca virus type A using a kit.

[0026] In some embodiments, the method includes: Viral nucleic acid was extracted from the sample to be tested and cDNA was obtained by reverse transcription; PCR amplification was performed using cDNA as a template to obtain the amplified fragment; The amplified fragments were sequenced and analyzed to obtain the typing results of porcine Seneca virus type A.

[0027] Specifically, Sanger sequencing was used, and bioinformatics analysis of the sequencing information was performed to obtain the core genome sequence, which in turn yielded the genotyping results for porcine Seneca virus type A. The bioinformatics analysis included manually assembling the core genome fragments to ultimately obtain the core genome sequence.

[0028] In some embodiments, the sample to be tested includes at least one of blister fluid, nasal and oral swab samples, blood samples, fecal samples, tissue samples, environmental samples, and virus cultures.

[0029] In some embodiments, the PCR amplification reaction procedure includes: Pre-denaturation: 94℃~96℃ for 2~4 minutes; Denaturation at 94℃~96℃ for 28s~32s, annealing at 54℃~56℃ for 28s~32s, extension at 71℃~73℃ for 0.5min~1.5min, 35 cycles; The final extension temperature is 71℃~73℃ for 2min~4min.

[0030] Preferably, the PCR amplification reaction procedure includes: Pre-denaturation at 95℃ for 3 minutes; Denaturation at 95℃ for 30 seconds, annealing at 55℃ for 30 seconds, extension at 72℃ for 1 minute, 35 cycles; Finally, extend the temperature to 72℃ for 3 minutes.

[0031] In some embodiments, the PCR amplification reaction system includes: The following reagents were prepared: cDNA template 0.5 μL to 1.5 μL, upstream primer SVA-F 0.5 μL to 1.5 μL, downstream primer SVA-R 0.5 μL to 1.5 μL, 2×Magic Green Taq SuperMix 11.5 μL to 13.5 μL, and Nuclease & Nucleic acid-free ddH2O 8.5 μL to 10.5 μL.

[0032] Preferably, the PCR amplification reaction system includes: The test sample contained 1 μL of cDNA template, 1 μL of upstream primer SVA-F, 1 μL of downstream primer SVA-R, 12.5 μL of 2×MagicGreen Taq SuperMix, and 9.5 μL of Nuclease & Nucleic acid-free ddH2O.

[0033] A fourth aspect of the present invention provides the use of primer sets or kits in the preparation of reagents for typing porcine type A Seneca virus.

[0034] A fifth aspect of this invention provides the application of primer sets or kits in the typing and / or tracing of porcine type A Seneca virus outbreaks.

[0035] The biological material used in the examples was sourced from 15 clinically positive cases of porcine type A Seneca virus, provided by Jiangsu Lihua Animal Husbandry Co., Ltd.

[0036] The reagents used in the examples were sourced from the following sources: QIAamp Viral RNA Mini Kit (52904) was purchased from Qiagen Biotechnology (Germany); PureScript II 1st StrandcDNA Synthesis Kit (Low Nucleic-acid Contamination, PR111) was purchased from Nanjing Novizan Biotechnology Co., Ltd.; and hot-start Taq DNA polymerase (2×Magic Green Taq SuperMix, 21502) was purchased from Tulu Harbour Biotechnology Co., Ltd.

[0037] Example 1: Screening of core genomes based on genome-wide evolutionary topological consistency Full-length SVA sequences were collected from the NCBI virus database and aligned using the bioinformatics software MAFFT. The core genome screening method of this invention is implemented through two self-written scripts, which respectively perform a sliding window Mantel topological consistency test and sequence consistency calculation. The overall logic of the two scripts is as follows: Figure 1 As shown. The script takes the aligned SVA whole-genome multiple sequence alignment file as input and outputs the Mantel correlation coefficients for each window and the top-ranked candidate fragment intervals, ultimately obtaining the region with the highest Mantel r value. Regions with high Mantel r values ​​are highly correlated with the genetic distance of the whole genome and can serve as the best representatives of the whole genome for accurate genotyping. Figure 2 As shown, primers were manually designed in this region, and the final primer sequences are shown in Table 1. The primers were then synthesized by Suzhou Genewiz Biotechnology Co., Ltd.

[0038] Table 1 Primer sequences for the SVA core genome

[0039] The primer set was validated by BLAST to achieve a matching coverage of ≤2 mismatches for 306 different SVA strains: 97.4% (298 / 306) for SVA-F and 92.8% (284 / 306) for SVA-R. The results indicate that the primer set of this invention has good universality and high adaptability to existing prevalent SVA strains.

[0040] The location of the core genome in this invention within the SVA whole genome is as follows: Figure 3 As shown. The SVA core genome amplicon region screened in this embodiment is compared with existing studies ( Korea Science In 2021, genetic distance correlation analysis and phylogenetic consistency analysis were performed on the amplicon region of the VP1 gene.

[0041] A schematic diagram of linear regression obtained from genetic distance correlation analysis is shown below. Figure 4 As shown, the left figure represents the amplicon region of the SVA core genome of this invention, and the right figure represents the amplicon region based on the VP1 gene. In the phylogenetic consistency analysis, the phylogenetic topology of the SVA core genome amplicon region (right) and the SVA whole genome (left) of this invention is as follows: Figure 5 As shown; the phylogenetic topology based on the amplicon region of the VP1 gene (right) and the entire SVA genome (left) is as follows. Figure 6 As shown. Calculations show that the Mantel r value of the core genome amplicon of this invention is 0.941, R... 2 The value was 0.902; while the Mantel r value for the VP1 gene amplicon was 0.874, R 2 The value is 0.903.

[0042] A comprehensive comparison shows that the core genome amplicon of this invention is significantly better than the VP1 gene amplicon in terms of Mantel correlation coefficient, indicating that it has higher phylogenetic topological consistency with the whole genome and can more accurately represent the genetic distance of the whole genome.

[0043] Example 2: Application of core genomic primers in clinical positive samples 2.1 Extraction of viral nucleic acid Nucleic acid was extracted from clinically positive SVA samples using the QIAamp Viral RNA Mini Kit. The specific steps are as follows: (1) Pipette 560 μL of Buffer AVL into a 1.5 mL centrifuge tube and add 140 μL of cell culture supernatant. Vortex for 15 seconds.

[0044] (2) Incubate at room temperature for 10 min, then centrifuge briefly to remove liquid droplets from the inner wall and inner cap.

[0045] (3) After adding 560 μL of ethanol, vortex for 15 seconds and then centrifuge briefly to remove liquid droplets from the inner wall and inner cap.

[0046] (4) Carefully aspirate 630 μL of the QIAamp Mini column obtained in the previous step, cap it, and centrifuge at 6000 × g for 1 min. Transfer the QIAamp Mini column to a new 2 ml collection tube.

[0047] (5) Repeat the previous step.

[0048] (6) Carefully open the cap and add 500 μL of Buffer AW1. After capping, centrifuge at 6000 × g for 1 min. Transfer the QIAamp Mini column to a new 2 ml collection tube and discard the old tube containing waste liquid.

[0049] (7) Carefully open the lid and add 500 μL of Buffer AW2. Close the lid and centrifuge at 12000 × g for 3 min.

[0050] (8) Transfer the QIAamp Mini column to a new 2ml collection tube and centrifuge at 12000 × g for 1 min to remove residual ethanol.

[0051] (9) Transfer the QIAamp Mini column to a new 1.5ml centrifuge tube, carefully open the cap, and add 60μL Buffer AVE onto the adsorption membrane. After capping, incubate at room temperature for 1 min. Centrifuge at 6000 × g for 1 min. Store the extracted viral RNA at -80℃.

[0052] 2.2 PCR amplification The PureScript II 1st Strand cDNA Synthesis Kit was used to reverse transcribe RNA viruses from nucleic acids into cDNA for amplification and detection of the RNA. The specific steps are as follows: (1) RNA template denaturation The reaction mixture consisted of 10 pg to 1 μg RNA, with enzyme-free water added to a final volume of 14 μL. The prepared mixture was heated at 65°C for 5 min, then rapidly cooled on ice and allowed to stand on ice for 2 min.

[0053] (2) cDNA synthesis reaction The reaction mixture consisted of 14 μL of the mixture from the previous step, 2 μL of 10 × RT Mix, 2 μL of PureScript Enzyme Mix, and 2 μL of Random hexamers. The mixed sample was placed in a PCR instrument and incubated at 25°C for 5 min, 37°C for 45 min, and 85°C for 5 s. The reverse transcription product was stored at -20°C.

[0054] (3) Conventional PCR amplification The cDNA obtained in the previous step was amplified by conventional PCR using primers SVA-F and SVA-R from Example 1.

[0055] The reaction system consisted of: 1 μL Input cDNA, 1 μL SVA-F, 1 μL SVA-R, 12.5 μL 2 × Magic GreenTaq SuperMix, and 9.5 μL Nuclease & Nucleic acid-free ddH2O.

[0056] The reaction program was as follows: pre-denaturation at 95℃ for 5 min; denaturation at 95℃ for 30 s, annealing at 55℃ for 30 s, extension at 72℃ for 3 min, for 35 cycles; final extension at 72℃ for 3 min.

[0057] 2.3 Sequencing and bioinformatics analysis After PCR, the amplification products were verified by agarose gel electrophoresis. Once confirmed as a single band, the products were sent to Suzhou Genewiz Biotechnology Co., Ltd. for Sanger sequencing.

[0058] According to the genomic location coordinates shown in SEQ ID NO:1 and SEQ ID NO:2, the full-length aligned SVA sequences collected in Example 1 were trimmed to obtain the sequences corresponding to the core genomic regions. The results returned by Sanger sequencing were manually assembled to obtain the consensus sequences of each sample, and then combined with the multiple SVA sequences obtained above to construct a phylogenetic tree.

[0059] The results of the phylogenetic tree are as follows Figure 7 As shown, the results indicate that the core genome primers in this invention successfully distinguished the actual samples into different evolutionary clusters, verifying their high resolution in resolving viral genetic differences.

[0060] It will be readily understood by those skilled in the art that the above-described advantageous methods can be freely combined and superimposed without conflict. The above are merely preferred embodiments of this application and are not intended to limit this application. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of this application should be included within the protection scope of this application. The above are merely preferred embodiments of this application. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the technical principles of this application, and these improvements and modifications should also be considered within the protection scope of this application.

Claims

1. A primer set for rapid typing of porcine Seneca virus type A, characterized in that, include: The upstream primer SVA-F has the nucleotide sequence shown in SEQ ID NO:1; The downstream primer SVA-R has the nucleotide sequence shown in SEQ ID NO:

2.

2. A kit for rapid typing of porcine Seneca virus type A, characterized in that, The kit includes the primer set as described in claim 1.

3. The reagent kit according to claim 2, characterized in that, The kit also includes reverse transcription reagents and PCR amplification reaction solution.

4. A method for rapid typing of porcine Seneca virus type A, not for the purpose of disease diagnosis and treatment, characterized in that, include: Rapid typing of porcine Seneca virus type A was performed using the kit described in claim 2 or 3.

5. The method according to claim 4, characterized in that, The method includes: Viral nucleic acid was extracted from the sample to be tested and cDNA was obtained by reverse transcription; PCR amplification was performed using the cDNA as a template to obtain the amplified fragment; The amplified fragment was sequenced and analyzed to obtain the typing results of porcine Seneca virus type A.

6. The method according to claim 5, characterized in that, The samples to be tested include at least one of the following: blister fluid, nasal and oral swab samples, blood samples, fecal samples, tissue samples, environmental samples, and virus cultures.

7. The method according to claim 5, characterized in that, The PCR amplification reaction procedure includes: Pre-denaturation: 94℃~96℃ for 2~4 minutes; Denaturation at 94℃~96℃ for 28s~32s, annealing at 54℃~56℃ for 28s~32s, extension at 71℃~73℃ for 0.5min~1.5min, 35 cycles; The final extension temperature is 71℃~73℃ for 2min~4min.

8. The method according to claim 5, characterized in that, The PCR amplification reaction system includes: The following reagents were used: cDNA template 0.5 μL to 1.5 μL, upstream primer SVA-F 0.5 μL to 1.5 μL, downstream primer SVA-R 0.5 μL to 1.5 μL, 2×Magic Green Taq SuperMix 11.5 μL to 13.5 μL, and Nuclease & Nucleic acid-free ddH2O 8.5 μL to 10.5 μL.

9. The use of the primer set of claim 1 or the kit of claim 2 or 3 in the preparation of reagents for typing porcine Seneca virus type A.

10. The application of the primer set of claim 1 or the kit of claim 2 or 3 in the typing and / or tracing of porcine type A Seneca virus outbreaks.

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