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Real-time fluorescent quantitative PCR detection kit for Seneca valley virus, and special primer and TaqMan probe thereof

A real-time fluorescence quantification, Seneca technology, applied in the determination/inspection of microorganisms, biochemical equipment and methods, DNA/RNA fragments, etc. Results and other issues, to achieve the effect of high specificity, improved sensitivity, and efficient qualitative

Pending Publication Date: 2019-09-20
JINYUBAOLING BIO PHARMA CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there are relatively few related researches on the detection technology of Seneca Valley virus. The commonly used methods include ordinary PCR detection technology and real-time fluorescent quantitative PCR detection technology. The problem is that the ordinary PCR detection technology only uses a pair of primers for detection. Due to the strong adaptability of the target sequence, viruses with high nucleic acid sequence homology also appear specific bands, resulting in false positive results, and the sensitivity of ordinary PCR detection technology is lower than that of real-time fluorescent quantitative PCR detection technology

Method used

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  • Real-time fluorescent quantitative PCR detection kit for Seneca valley virus, and special primer and TaqMan probe thereof
  • Real-time fluorescent quantitative PCR detection kit for Seneca valley virus, and special primer and TaqMan probe thereof
  • Real-time fluorescent quantitative PCR detection kit for Seneca valley virus, and special primer and TaqMan probe thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] Embodiment 1, design uses real-time fluorescent quantitative PCR technique to detect the primer and TaqMan probe of Seneca Valley virus (SVV)

[0059]The sequence of the whole genome of Seneca Valley virus was retrieved from NCBI's nucleic acid database GenBank (http: / / www.ncbi.nlm.nih.gov) (GenBank 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),用DNA Star软件进行比对后,根据引物、TaqMan The principle of probe design is to first select the 3C region gene that is relatively conserved among different strains of Seneca Valley virus. Regarding the 3C region gene of Seneca Valley virus, the literature published by Hales L M et al. "Complete genome sequence analysis of Seneca Valley virus-001, a novel oncolytic picornavirus [J]. Journal of General Virology, 2008, 89 (5): 1265-1275 It has been sta...

Embodiment 2

[0077] Embodiment 2, carry out real-time fluorescent quantitative PCR detection to Seneca Valley virus with primer of the present invention and TaqMan probe

[0078] 1. Genomic RNA extraction of Seneca Valley virus

[0079] Extract Seneca Valley virus cell culture (for obtaining standard substance and positive control substance) and the genome RNA of test sample, and concrete extraction method comprises the following steps (Axyprep TM Body Fluid Viral DNA / RNA MiniprepKit, AXYGEN Company):

[0080] (1) AXYGEN kit preparation: According to the kit instructions, pre-configure isopropanol containing 1% glacial acetic acid and add absolute ethanol of specified concentration to reagent Buffer W1A and Buffer W2;

[0081] (2) Add 200 μL of the sample to be tested into a 1.5 mL centrifuge tube, and add 200 μL of Buffer V-L, vortex to mix, and let stand for 5 minutes;

[0082] (3) Add 75 μL of Buffer V-N to the 1.5 mL sample and reagent mixing centrifuge tube in step (2), vortex and s...

Embodiment 3

[0123] Embodiment 3, the real-time fluorescent quantitative PCR detection kit of Seneca Valley virus

[0124] Based on Example 1 and Example 2, the real-time fluorescent quantitative PCR detection kit of Seneca Valley virus of the present invention includes primers (SVV-F and SVV-R) for carrying out real-time fluorescent quantitative PCR detection of Seneca Valley virus and TaqMan probe (SVV-P).

[0125] Specifically, the kit includes the following reagents for 25 μL real-time fluorescent quantitative PCR detection system: real-time fluorescent quantitative one-step PCR reaction solution 2×One Step RT-PCR Buffer III 12.5 μL, TaKaRa Ex Taq HS 0.5 μL, PrimeScript RT Enzyme Mix II 0.5μL, SVV-F (20μM) 0.5μL, SVV-R (20μM) 0.5μL, SVV-P (10μM) 1μL, RNA-free H 2 O 7.5 μL.

[0126] For the convenience of detection, a positive control and a negative control can also be included in the kit. The positive control is Seneca virus cell culture RNA, and the negative control is a reaction sy...

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Abstract

The invention discloses a real-time fluorescent quantitative PCR detection kit for detecting Seneca valley virus (SVV), and a special primer and a TaqMan probe thereof. The kit can rapidly detect the SVV, provides a basis for quality monitoring and rational vaccine distribution in a vaccine production process, and guides the production of SVV vaccines. The kit and the detection method thereof are simple to operate, high in specificity, high in sensitivity and good in repeatability, can accurately quantify SVV, and can play an important role in SVV detection and vaccine production.

Description

technical field [0001] The invention belongs to the detection of veterinary animal pathogens in the technical field of biological detection, in particular to a real-time fluorescent quantitative PCR detection kit for qualitative and quantitative detection of Seneca Valley virus, special primers and TaqMan probes. Background technique [0002] seneca valley virus [0003] Seneca Valley Virus (SVV) or Seneca Virus A (SVA) is a typical representative of the Seneca Virus virus genus in the Picornaviridae family (Picornaviridae), which is related to the Cardiovirus genus [0004] (Cardio Virus) is the closest, and it is an infectious disease that mainly occurs in pigs. Its clinical features are the formation of ulcers in the nose and mouth of infected pigs, anorexia, lameness, and acute death of newborn piglets. Early Seneca Valley cases were mainly in fattening pigs with vesicles on the nose, mouth and crown of the hooves, but some of the Seneca Valley positive cases have recen...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12N15/11
CPCC12Q1/701C12Q1/686C12Q2561/113C12Q2563/107C12Q2545/114C12Q2561/101
Inventor 王秀明陈君彦魏学峰刘国英关平原范秀丽张贵刚王艳杰王云凌张宸武瑾贤张燕红杜宇荣
Owner JINYUBAOLING BIO PHARMA CO LTD
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