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Pseudorabies wild virus and porcine hepatitis E virus qPCR kit and method

A technology for porcine hepatitis E and pseudorabies, applied in biochemical equipment and methods, methods based on microorganisms, DNA/RNA fragments, etc., can solve the problems of simultaneous detection of hepatitis E virus, etc., to prevent and control the spread of the epidemic , Strong practicability and short time-consuming effect

Pending Publication Date: 2020-08-21
LONGYAN UNIV +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] Invention patent "Double real-time fluorescent quantitative PCR detection kit, primers and probes for porcine pseudorabies wild strain and gene deletion strain (public number CN201610634845)", invention patent "Fluorescence quantitative PCR detection primer for porcine pseudorabies wild strain , probes and kits (public number CN201410217702)" describes a fluorescent quantitative PCR method for detecting pseudorabies virus wild strains, which can detect pseudorabies virus but cannot detect hepatitis E virus at the same time

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  • Pseudorabies wild virus and porcine hepatitis E virus qPCR kit and method
  • Pseudorabies wild virus and porcine hepatitis E virus qPCR kit and method
  • Pseudorabies wild virus and porcine hepatitis E virus qPCR kit and method

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Embodiment 1

[0031] A qPCR kit for pseudorabies wild virus and porcine hepatitis E virus, comprising an enzyme liquid and a reaction buffer, the enzyme liquid is composed of DNA polymerase and reverse transcriptase, and also includes a negative control and a positive control; The negative control is PBS buffered saline with 0.01mol / L and pH7.2; the positive control is that the PCR amplification products of pseudorabies wild virus and porcine hepatitis E virus are respectively connected with the cloning vector pMD-18-T vector, transformed into Escherichia coli competent for DH5α, obtained positive clone strains, and prepared plasmids pMD18-RPV and pMD18-HEV as positive controls. Also included are the following primer pairs and probes:

[0032] The primer pair and probe RPV-Pro for detecting pseudorabies wild virus, the primer pair for detecting pseudorabies wild virus includes the upstream primer RPV-F shown in SEQ ID NO: 1 and the downstream primer RPV-R shown in SEQ ID NO: 2, The sequenc...

Embodiment 2

[0050] A detection method for pseudorabies wild virus and porcine hepatitis E virus qPCR kit, for the detection of 4 strains of pseudorabies wild virus and 3 strains of porcine hepatitis E virus samples, the specific process is as follows:

[0051] (1) Sample virus nucleic acid extraction: 7 samples to be extracted, including 4 cases of pseudorabies wild virus and 3 cases of porcine hepatitis E virus tissue samples, were extracted with commercial virus nucleic acid extraction kits respectively. figure 1 The electrophoresis images of nucleic acid extraction from 7 samples, A1 to A4 are the electrophoresis images of pseudorabies wild virus nucleic acid, and B1 to B3 are the electrophoresis images of porcine hepatitis E virus nucleic acid. figure 1 It can be seen that the nucleic acids of the 7 samples were successfully extracted.

[0052] (2) Take 3 μL of the viral nucleic acid extracted in the previous step and 17 μL of the PCR amplification reaction solution described in Examp...

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Abstract

The invention belongs to the field of molecular biological detection methods and detection reagents for animal epidemic diseases, and particularly relates to a qPCR kit and a method for pseudorabies wild virus and porcine hepatitis E virus. The kit is used for quickly detecting porcine pathogens. The kit aims at conserved sequences of pseudorabies virus and porcine hepatitis E virus. A preparationmethod comprises steps: respectively designing a PCR primer and a fluorescent TaqMan probe to form a primer group and a probe group special for the pseudorabies virus and the hepatitis E virus; forming a double fluorescent quantitative PCR detection kit for detecting pseudorabies virus and porcine hepatitis E virus. The detection method has the advantages of being simple and convenient to operate, high in sensitivity and the like, the two pathogens of the pseudorabies virus and the porcine hepatitis E virus can be detected and identified at the same time according to an amplification curve judgment result after the reaction is finished, the time cost can be saved, pollution can be reduced, and the detection method has extremely high application value.

Description

technical field [0001] The invention belongs to the field of animal epidemic molecular biology testing methods and testing reagents, in particular to a qPCR kit and method for wild pseudorabies virus and porcine hepatitis E virus. Background technique [0002] Pseudorabies virus (Pseudorabies virus, PRV) is a highly contagious disease of the a-herpesvirus subfamily. Pigs are the only natural host. The disease can lead to symptoms such as abortion, stillbirth, mummified fetuses, and infertility in pregnant sows; suckling piglets often have neurological symptoms and death; adult pigs are generally subclinical or recessively infected, becoming a storage host for the virus and long-term detoxification. [0003] Hepatitis E (Hepatitis E) is an acute viral hepatitis caused by the hepatitis E virus (HEV), which is mainly transmitted through the fecal-oral gastrointestinal tract. Hepatitis E has a global distribution and exists in the form of outbreaks or sporadic infections. In t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/686C12N15/11C12R1/93
CPCC12Q1/707C12Q1/705C12Q1/686C12Q2600/16C12Q2537/143C12Q2563/107
Inventor 范克伟傅文源包银莉林开雄郑琳黄翠琴林炜明杨守深
Owner LONGYAN UNIV
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