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One-step triple real-time fluorescent quantitative PCR detection primer and probe for SVA, type O FMDV and type A FMDV

A real-time fluorescence quantitative and SVA-F technology, applied in the field of animal disease pathogen detection, can solve the problems of indistinguishability, difficulty in clinical diagnosis, and inability to quickly distinguish raw materials for vaccine production, etc., achieving broad application prospects and reducing workload and cost.

Pending Publication Date: 2019-10-08
JINYUBAOLING BIO PHARMA CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

[0005] It is impossible to distinguish between O-type FMDV and A-type FMDV infection or SVA infection only from clinical symptoms, which brings difficulties to clinical diagnosis and needs to be confirmed by laboratory testing techniques; in addition, it is impossible to quickly distinguish between the quality control of the vaccine production process What type of virus and virus pathogen content are in the raw materials, semi-finished products or finished products of vaccine production to manage vaccine production
However, there is no report of a detection kit for distinguishing O-type FMD virus, A-type FMD virus, and Seneca virus for simultaneous differential detection and accurate quantification

Method used

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  • One-step triple real-time fluorescent quantitative PCR detection primer and probe for SVA, type O FMDV and type A FMDV
  • One-step triple real-time fluorescent quantitative PCR detection primer and probe for SVA, type O FMDV and type A FMDV
  • One-step triple real-time fluorescent quantitative PCR detection primer and probe for SVA, type O FMDV and type A FMDV

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Embodiment 1: Design detects primers and TaqMan probes of Seneca virus, O-type foot-and-mouth disease virus and A-type foot-and-mouth disease virus with real-time fluorescent quantitative PCR technology

[0045] From the NCBI nucleic acid database GenBank (http: / / www.ncbi.nlm.nih.gov), the sequence of the whole genome of Seneca virus, the sequence of the whole genome of type O foot-and-mouth disease virus and type A foot-and-mouth disease virus (GeneBank number: NC_011349 、 KT321458、KX173339、KX173338、KX173340、KX751943、KX751944、KY747510、KY038016、 KY747511、KY747512、KX751945、KX751946、KX377924、KY419132、DQ641257、KU051392、 KT757280、KU051391、KY486158、KY486165、KC667560、KR063109、KR063107、KY368743、 AF506822 .2, KX712091.1, HQ412603.1, AJ539141.1, AY390432.1, AY304994.1, GQ406249.1, KT968663.1, HQ632773.1, KY421683.1, KY421686.1, KY07763.210.68, KY07763.210.64 , KF450794.1, AJ131665.1), compared with DNA Star software, according to the design principles of primers and TaqMan probe...

Embodiment 2

[0075] Embodiment 2: Triple real-time fluorescent quantitative PCR detection of Seneca virus, O-type foot-and-mouth disease virus and A-type foot-and-mouth disease virus

[0076] 1. Genomic RNA extraction of Seneca virus, type O foot-and-mouth disease virus and type A foot-and-mouth disease virus

[0077] With Seneca virus cell culture (Jinyu Baoling company vaccine strain, for obtaining standard substance and positive control substance), O type foot-and-mouth disease virus cell culture (Jinyu Baoling company vaccine strain, for obtaining standard substance and Positive control substance) and type A foot-and-mouth disease virus cell culture (Jinyu Baoling company vaccine strain, used to obtain standard substance and positive control substance) and the sample to be tested as the sample to be extracted, extract the genomic RNA of the sample to be extracted, and specifically extract The method refers to the introduction of AXYGEN kit (AxyprepTMBody Fluid Viral DNA / RNA Miniprep Ki...

Embodiment 3

[0119] Example 3, Sensitivity, specificity and repeatability of the triple real-time fluorescent quantitative PCR detection method for Seneca virus, O-type foot-and-mouth disease virus and A-type foot-and-mouth disease virus of the present invention

[0120] 3.1. Sensitivity detection

[0121] Recombinant plasmids pCR-4TOPO-SVA, pCR-4TOPO-O-FMDV and pCR carrying the Seneca virus nucleotide detection gene, O-type foot-and-mouth disease virus nucleotide detection gene and A-type foot-and-mouth disease virus nucleotide detection gene respectively -4 TOPO-A-FMDV was used as a standard (see Example 2), and was diluted to 3×10 according to a 10-fold gradient 8 , 3×10 7 , 3×10 6 , 3×10 5 , 3×10 4 , 3×10 3 , 3×10 2 , 3×10 1 copies / μL were mixed to obtain a standard concentration of 1×10 8 , 1×10 7 , 1×10 6 , 1×10 5 , 1×10 4 , 1×10 3 , 1×10 2 , 1×10 1 10-fold gradient mixture in copies / μL; using different concentrations of standards as templates, primers SVA-F, SVA-R, FM...

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Abstract

The invention provides a one-step triple real-time fluorescent quantitative PCR detection primer and probe capable of simultaneously detecting and identifying Seneca virus, type O foot and mouth disease virus, and type A foot and mouth disease virus, and belongs to the technical field of biological detection. A kit is developed based on the provided primer and probe and used for simultaneous and rapid detection of the Seneca virus, the type O foot and mouth disease virus, and type A foot and mouth disease virus. According to the kit and the detection method of the one-step triple real-time fluorescent quantitative PCR detection primer and probe capable of simultaneously detecting and identifying the Seneca virus, the type O foot and mouth disease virus, and the type A foot and mouth disease virus, one-time sample loading and one-time quantification analysis can be realized, and the purpose of simultaneously and rapidly detecting and identifying and accurately quantifying the Seneca virus, the type O foot and mouth disease virus, and the type A foot and mouth disease virus can be achieved, the workload and cost of detection are reduced, and the detection can be completed in the shortest time. The one-step triple real-time fluorescent quantitative PCR detection primer and probe capable of simultaneously detecting and identifying the Seneca virus, the type O foot and mouth diseasevirus, and the type A foot and mouth disease virus play an important role in the detection and vaccine production of the Seneca virus, the type O foot and mouth disease virus, and the type A foot andmouth disease virus and have broad application prospects.

Description

technical field [0001] The invention belongs to the detection of animal disease pathogens in the technical field of biological detection, and specifically relates to a one-step triple real-time fluorescent quantitative PCR detection reagent capable of simultaneously detecting and distinguishing Seneca virus, type O foot-and-mouth disease virus and type A foot-and-mouth disease virus cassettes and their specific primers and probes. Background technique [0002] Seneca virus (Seneca virus A, SVA, or SVV) and foot-and-mouth disease virus (FMDV) belong to the Picornaviridae family and are non-enveloped, single-stranded positive-sense RNA viruses. SVA is a typical representative of the genus Senecavirus, which is closest to the genus Cardiovirus, while FMDV belongs to the genus Aphrovirus. [0003] Seneca virus disease is an infectious disease that mainly occurs in pigs. Its clinical features are nasal and oral ulcers, anorexia, lameness, and acute death of newborn piglets. Ear...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/6851C12N15/11
CPCC12Q1/701C12Q1/6851C12Q2600/16C12Q2600/166C12Q2531/113C12Q2537/143C12Q2561/101C12Q2563/107C12Q2545/114
Inventor 陈君彦王秀明魏学峰刘国英关平原王艳杰刘建奇武瑾贤谢雪岑陈九连
Owner JINYUBAOLING BIO PHARMA CO LTD
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