Method for detecting pseudorabies virus

A technique for pseudorabies virus and detection method, which is applied in the field of molecular biology and can solve the problems of strict annealing temperature, inability to identify PRV, and long time consumption.

Pending Publication Date: 2021-06-04
GUANGXI VETERINARY RES INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

For example, Zhang Yueyong et al. (Establishment of a nano-PCR detection method for porcine pseudorabies virus [J]. China Animal Quarantine, 2017, 034(003): 102-105) designed a pair of primers for the PRV gB gene and established a nano-PCR for the detection of PRV method, but since this method can detect both wild strains of PRV and vaccine strains, it is impossible to identify whether the infection of PRV comes from wild strains

Method used

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  • Method for detecting pseudorabies virus
  • Method for detecting pseudorabies virus
  • Method for detecting pseudorabies virus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] The materials and reagents used in the following examples can be obtained from commercial sources unless otherwise specified. Embodiment 1, screening and optimization of PRV detection method

[0022] 1 Materials and methods

[0023] 1.1 Virus strains and sources of positive cDNA

[0024] Chicken infectious anemia virus GXC060821 strain, porcine pseudorabies virus PRV and chicken astrovirus type 1 positive cDNA (derived from clinical samples) were preserved by Guangxi Veterinary Research Institute; CIAV and PRV were extracted according to the instructions in EasyPure Viral DNA / RNA Kit Viral DNA; DNA and Chicken Astrovirus Type 1 positive cDNA template were stored at -20°C for future use.

[0025] 1.2 Main reagents and instruments

[0026] The general amplification nano-PCR kit was purchased from Shanghai Huzheng Biotechnology Co., Ltd., the PCR instrument was purchased from Bio-rad, USA; the DNA / RNA co-extraction kit EasyPure Viral DNA / RNA Kit was purchased from Beiji...

Embodiment 2

[0061] Embodiment 2, a kind of detection method of PRV

[0062] The method comprises the steps of:

[0063](1) Preparation of template DNA

[0064] The DNA of the PRV virus can be extracted according to the instructions in the EasyPure Viral DNA / RNA Kit.

[0065] (2) Nano-PCR reaction

[0066] Reaction system (20 μL): 1 μL cDNA / DNA template, 10 μL 2×Nano-QPCR buffer, 0.1 μL upstream and downstream primer mixture with a working concentration of 20 μmol / L (the final concentration of primers is 0.1 μmol / L), taq enzyme Mix ( 5U / uL) 0.32μL, add ddH 2 O to make up 20 μL.

[0067] The primer combination is PRV-1F / PRV-1R.

[0068] PCR reaction program: 95°C for 3min; 94°C for 5s, annealing temperature at 50°C for 5s, 72°C for 30s, 20 cycles; 72°C for 5min, 12°C to end the reaction.

[0069] (3) Detection of PCR products

[0070] The amplification results can be verified by 1.2% agarose gel electrophoresis.

Embodiment 3

[0071] Embodiment 3, a kind of detection method of PRV

[0072] The method comprises the steps of:

[0073] (1) Preparation of template DNA

[0074] The DNA of the PRV virus can be extracted according to the instructions in the EasyPure Viral DNA / RNA Kit.

[0075] (2) Nano-PCR reaction

[0076] Reaction system (20 μL): 1 μL cDNA / DNA template, 10 μL 2×Nano-QPCR buffer, 1 μL upstream and downstream primer mixture with a working concentration of 20 μmol / L (the final concentration of primers is 1 μmol / L), taq enzyme Mix (5 U / L uL)0.32μL, add ddH 2 O to make up 20 μL.

[0077] The primer combination is PRV-2F / PRV-2R.

[0078] PCR reaction program: 95°C for 3min; 94°C for 5s, annealing temperature at 50°C for 5s, 72°C for 30s, 20 cycles; 72°C for 5min, 12°C to end the reaction.

[0079] (3) Detection of PCR products

[0080] The amplification results can be verified by 1.2% agarose gel electrophoresis.

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Abstract

The invention provides a method for detecting pseudorabies virus. The method is characterized in that PRV detection is carried out by using nano PCR, and the detection is carried out by adopting nucleotide sequences shown in SEQ ID No. 1-2 in a sequence table, the concentration of upstream and downstream primers being 0.1 micromole/L, and the annealing temperature being 50 DEG C, and nucleotide sequences shown in SEQ ID No. 3-4, the concentration of upstream and downstream primers being 1 micromole/L, and the annealing temperature being 50 DEG C. The method has the advantages of high sensitivity, high specificity, loose amplification conditions, specific detection of wild strains and the like.

Description

technical field [0001] The present application relates to the technical field of molecular biology, in particular, to a method for detecting pseudorabies virus. Background technique [0002] Pseudorabies virus (PRV) belongs to the family Herpesviridae (Herpesviridae) α-herpesvirus subfamily (Alphaerpesvirinae) Varicellovirus (Varicellovirus), can infect pigs, sheep, dogs, cattle, cats, rabbits and mice, etc. Animals, which mainly cause symptoms such as fever, itching (except pigs) and encephalomyelitis. [0003] Nano-PCR (nano-PCR) technology is to add nanomaterials (such as gold nanoparticles, silver nanoparticles, carbon nanoparticles and zinc oxide nanoparticles, etc.) To achieve special effects, it provides new ideas and technologies for optimizing conventional PCR. In 2005, Li H et al. (NanoparticlePCR: Nanogold-assisted PCR with enhanced specificity [J] Angewandte chemie-international edition. 2005, 44:2-5) found that gold nanoparticles can improve the activity and a...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/686C12R1/93
CPCC12Q1/705C12Q1/686C12Q2565/125C12Q2563/155
Inventor 谢芝勋张民秀张艳芳谢丽基谢志勤李孟邓显文罗思思范晴曾婷婷黄娇玲王盛李丹刘加波
Owner GUANGXI VETERINARY RES INST
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