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Duplex real-time fluorescent quantitative PCR detection kit for Seneca virus A and foot and mouth disease virus

A real-time fluorescent quantitative, foot-and-mouth disease virus technology, applied in the direction of microbial determination/inspection, recombinant DNA technology, biochemical equipment and methods, etc., can solve the difficulty of clinical diagnosis, inability to distinguish SVA infection and FMDV infection and other problems, to ensure safety The effect of high reliability and rationality, easy operation and broad application prospects

Inactive Publication Date: 2018-12-28
JINYUBAOLING BIO PHARMA CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] In recent years, due to the discovery of Seneca virus, it is impossible to distinguish SVA infection from FMDV infection only from clinical symptoms, which brings difficulties to clinical diagnosis and needs to be confirmed by laboratory testing techniques

Method used

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  • Duplex real-time fluorescent quantitative PCR detection kit for Seneca virus A and foot and mouth disease virus
  • Duplex real-time fluorescent quantitative PCR detection kit for Seneca virus A and foot and mouth disease virus
  • Duplex real-time fluorescent quantitative PCR detection kit for Seneca virus A and foot and mouth disease virus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Example 1. Design of primers and TaqMan probes for detection of Seneca virus (SVA) and foot-and-mouth disease virus (FMDV) using real-time fluorescent quantitative PCR technology

[0048] Retrieved from NCBI's Nucleic Acid Database GenBank (http: / / www.ncbi.nlm.nih.gov) to obtain the complete genome sequence of Seneca virus (GeneBank serial numbers: 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) and the sequence of the foot-and-mouth disease virus full genome (AF50 GeneBank, 6822.2) KX712091.1, HQ412603.1, AJ539141.1, AY390432.1, AY304994.1, GQ406249.1, KT968663.1, HQ632773.1), after comparison with DNA Star software, according to the design principles of primers and TaqMan probes , Respectively select the relatively conservative nucleotide sequence between different strai...

Embodiment 2

[0073] Example 2. Double real-time fluorescent quantitative PCR detection of Seneca virus and foot-and-mouth disease virus using primers of group 1 and TaqMan probe

[0074] 1. Extract the genomic RNA of Seneca virus and foot-and-mouth disease virus

[0075] Seneca virus cell culture (the cell-passage virus isolated and identified by our laboratory, used to obtain standard and positive control), foot-and-mouth disease virus cell culture (Jinyu Baoling company MYA98 vaccine strain, used to obtain standard And positive control) and the sample to be tested are used as the sample to be extracted, and the genomic RNA of the sample to be extracted is extracted. The specific extraction method refers to the AXYGEN kit (Axyprep TM The introduction of Body Fluid Viral DNA / RNA Miniprep Kit, AXYGEN Company) includes the following steps:

[0076] (1) According to the kit instructions, prepare isopropanol containing 1% glacial acetic acid in advance and add the specified concentration of absolut...

Embodiment 3

[0122] Example 3. Preparation of a dual real-time fluorescent quantitative PCR detection kit for Seneca virus and foot-and-mouth disease virus

[0123] Based on Example 1 and Example 2, the dual real-time fluorescent quantitative PCR detection kit for Seneca virus and foot-and-mouth disease virus of the present invention includes primers (SVA-F) for dual real-time fluorescent quantitative PCR detection of Seneca virus and foot-and-mouth disease virus. , SVA-R, FMDV-F and FMDV-R) and TaqMan probes (SVA-P and FMDV-P).

[0124] The 25μL dual real-time PCR detection system when using the kit is: real-time fluorescence quantitative one-step PCR reaction solution 2×One Step RT-PCR Buffer III 12.5μL, TaKaRa Ex Taq HS 0.5μL, PrimeScript RT Enzyme Mix II 0.5μL , SVA-F (10μM) 1.0μL, SVA-R (10μM) 1.0μL, SVA-P (10μM) 1μL, FMDV-F (10μM) 1.0μL, FMDV-R (10μM) 1.0μL, FMDV-P (10μM) ) 1.5μL, template RNA 2.0μL, RNA-free H 2 O 3.0μL.

[0125] To facilitate detection, the kit can also include a positi...

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Abstract

The invention provides a duplex real-time fluorescent quantitative PCR detection kit for Seneca virus A (SVA) and foot and mouth disease virus (FMDV) and special primers and TaqMan probes of the duplex real-time fluorescent quantitative PCR detection kit. The kit contains two pairs of primers and two probes; the primers and the probes are respectively designed for an SVA conservative gene region and an FMDV conservative gene region. The kit and the detection method provided by the invention have the advantages of simplicity and convenience in operation, high specificity, high sensitivity, highrepeatability and broad application prospect; accurate quantitation of the SVA and the FMDV can be realized; and the duplex real-time fluorescent quantitative PCR detection kit and the detection method play an important role in detection and vaccine production of the SVA and the FMDV, such as quality monitoring and reasonable seedling distribution in the production process of vaccines, and have wide application prospects.

Description

Technical field [0001] The invention belongs to the detection of veterinary animal pathogens in the technical field of biological detection, and particularly relates to a dual real-time fluorescent quantitative PCR detection kit for qualitative and quantitative detection of Seneca virus and foot-and-mouth disease virus, and its dedicated primers and TaqMan probes . Background technique [0002] Seneca Virus A (SVA) and Foot-and-Mouth Disease Virus (FMDV) belong to the Picornaviridae family, both of which are single-stranded positive-stranded RNA viruses without envelopes. Seneca virus belongs to the typical representative of Seneca Virus virus genus, and is the closest to Cardio Virus, while foot-and-mouth disease virus belongs to the foot-and-mouth disease virus genus virus. [0003] Seneca virus is an infectious disease that mainly occurs in pigs. Its clinical features are the formation of ulcers, anorexia, and lameness in the nose and mouth of infected pigs, leading to acute de...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/686C12N15/11
CPCC12Q1/686C12Q1/701
Inventor 王秀明陈君彦武瑾贤魏学峰刘国英关平原范秀丽张贵刚王艳杰刘建奇
Owner JINYUBAOLING BIO PHARMA CO LTD
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