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Wild porcine pseudorabies strain and gene-deleted strain dual real-time fluorescence quantification PCR detection kit, primers and probe

A technology of real-time fluorescence quantification and porcine pseudorabies, which is applied in the direction of microbial determination/testing, microbial-based methods, biochemical equipment and methods, etc., to achieve the effect of simple operation, ensuring safety and rationality, and broad application prospects

Active Publication Date: 2016-10-26
内蒙古金迈诗生物科技有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there are few dual qPCR detection methods for gB gene and gE gene. Ma et al. designed primers and probes respectively according to PRV gB and gE genes, and established a fluorescent quantitative PCR method for diagnosing PRV wild strains. strains have a good amplification effect, while the detection of vaccine deletion strains and other common pig pathogens are negative [Document 1: Development of real-time polymerase chain reaction assays for rapid detection and differentiation of wild-typep seudorabies and gene-deleted vaccine viruses , MaW, LagerKM, Richt JA, et al. J Vet DiagnInvest, 2008, 20(4):440-447.]

Method used

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  • Wild porcine pseudorabies strain and gene-deleted strain dual real-time fluorescence quantification PCR detection kit, primers and probe
  • Wild porcine pseudorabies strain and gene-deleted strain dual real-time fluorescence quantification PCR detection kit, primers and probe
  • Wild porcine pseudorabies strain and gene-deleted strain dual real-time fluorescence quantification PCR detection kit, primers and probe

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Example 1. Design of primers and TaqMan probes for dual real-time fluorescent quantitative PCR detection of gB gene and gE gene of porcine pseudorabies wild strain and gene deletion strain

[0045] The sequence of gB gene (GenBank number: NC_006151) and gE gene (GenBank number: AF207700) of porcine pseudorabies wild strain was retrieved from NCBI's nucleic acid database GenBank (http: / / www.ncbi.nlm.nih.gov), After comparison with DNA Man software, according to the principles of primer design, 3 pairs of primers were designed for the gB gene and the gE gene respectively for PCR amplification, and the primers with high primer specificity and good amplification effect were selected by electrophoresis results. Yes, the primer pair sequence to determine the specificity of the gB gene is the 17211-17574th base from the 5' end, and the primer pair sequence specific to the gE gene is the 443-1357th base from the 5' end; Using Primer Express 6.0 software, primers and TaqMan prob...

Embodiment 2

[0058] Embodiment 2, using the primers of the present invention and TaqMan probes to carry out double real-time fluorescent quantitative PCR detection on the gB gene and gE gene of the porcine pseudorabies wild strain and the gene deletion strain

[0059] 1. Extract the genomic DNA of the sample to be tested

[0060] Genomic DNA was extracted from the cell cultures of inactivated porcine pseudorabies field strains and gene-deleted strains and 12 collected clinical suspected serum disease materials. The specific method included the following steps:

[0061] (1) Take 200 μL of inactivated pseudorabies field strain and gene deletion strain cell culture and clinical serum into 1.5mL centrifuge tube, add 1mL DNAzol lysate, shake and mix, let stand for 10 minutes to lyse;

[0062](2) Centrifuge at 12000 rpm for 10 minutes, take about 800 μL of supernatant into a new 1.5 mL centrifuge tube, add 500 μL of absolute ethanol, mix gently and let stand for 5 minutes to precipitate DNA;

...

Embodiment 3

[0087] Embodiment 3, the specificity experiment of detection method of the present invention

[0088] According to the method described in embodiment 2, extract RNA to swine fever virus (CSFV), bovine viral diarrhea virus (BVDV) and carry out reverse transcription, directly to porcine circovirus type 2 (PCV2), rhinotracheitis virus (IBR) DNA was extracted, while the inactivated porcine pseudorabies wild strain cell culture was used as a positive control, and enzyme-free water was used as a negative control. Under the guidance of primers and TaqMan probes of the present invention, double real-time fluorescent quantitative PCR detection was carried out. PCR reaction The system and reaction conditions refer to Example 2 to verify the specificity of the method.

[0089] Test results such as Figure 5 As shown, the CT values ​​of the positive control gB gene and the gE gene were 20 / 21 (38, and no specific amplification curve appeared, and the results were negative. The detection ...

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Abstract

The invention discloses a dual real-time fluorescence quantification PCR detection kit, specific primers and TagMan probe for detecting a gB gene and a gE gene of a wild porcine pseudorabies strain and a gene-deleted strain. The method builds a gB gene and gE gene dual real-time fluorescence quantification PCR detection method. The method can fast identify a wild porcine pseudorabies strain (simultaneously containing a gB gene and a gE gene) and a gene-deleted strain (only containing the gB gene) and can realize accurate quantification of virus copy number. The kit and detection method can be operated simply, have good specificity and high sensitivity and produce effects in porcine pseudorabies detection, strain identification and vaccine production.

Description

technical field [0001] The invention belongs to the field of animal pathogen detection, and relates to a method for distinguishing wild strains of porcine pseudorabies virus and gene deletion strains, in particular to a double real-time fluorescence of gB gene and gE gene for distinguishing wild strains of porcine pseudorabies virus and gene deletion strains Quantitative PCR detection method and special primers thereof, TaqMan probe, detection kit and application of the primers and probes in detection of gB gene and gE gene in porcine pseudorabies wild strain and gene deletion strain. Background technique [0002] Pseudorabies (Pseudorabies, PR) is a Class B infectious disease (identified by OIE) caused by Pseudorabies Virus (PRV). Infection is the most serious, leading to abortion, stillbirth and mummified fetuses in pregnant sows, neurological symptoms, paralysis, exhaustion and death in suckling piglets, and the mortality rate is as high as 100%. At present, pseudorabies...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12N15/11C12R1/93
CPCC12Q1/6851C12Q1/705
Inventor 韩志玲张贵刚商晓桂郝鹏孔彩萍闫聪陈君彦刘国英范秀丽
Owner 内蒙古金迈诗生物科技有限公司
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