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PCR amplification primer for rapid detection of bovine infectious rhinotracheitis virus and applications thereof

A technology for rhinotracheitis virus and amplification primers, which is applied in the determination/inspection of microorganisms, biochemical equipment and methods, DNA/RNA fragments, etc. It can solve the problems of indetermination of pathogens, time-consuming and laborious, false positives, etc., and achieve sensitivity High, time-consuming and low-cost effects

Inactive Publication Date: 2018-04-06
GUANGXI VETERINARY RES INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] So far, there are many methods for diagnosing bovine infectious rhinotracheitis virus at home and abroad, such as histopathological diagnosis, virus cell culture isolation and identification, neutralization test, indirect hemagglutination test, ELISA, colloidal gold technology and other means. There are the following disadvantages, such as time-consuming and labor-intensive, or the pathogen cannot be determined, or false positives occur

Method used

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  • PCR amplification primer for rapid detection of bovine infectious rhinotracheitis virus and applications thereof
  • PCR amplification primer for rapid detection of bovine infectious rhinotracheitis virus and applications thereof
  • PCR amplification primer for rapid detection of bovine infectious rhinotracheitis virus and applications thereof

Examples

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Effect test

Embodiment 1

[0038] Example 1 Establishment of a PCR method for rapid detection of bovine infectious rhinotracheitis virus

[0039] 1. Preparation of materials

[0040] Bovine infectious rhinotracheitis virus, Cryptobacterium cryptica, Mannella hemolytica, Mycoplasma bovis, bovine parainfluenza virus type 3, and Klebsiella pneumoniae were isolated, identified and preserved by Guangxi Veterinary Research Institute, and tissue samples were obtained from veterinary clinics. 10×PCR Buffer, dNTPs, ES-Taq DNA polymerase, DNA / RNA extraction kit, bacterial genome DNA extraction kit were purchased from Kangwei Century Biotechnology Co., Ltd.

[0041] 2. Design and synthesis of PCR primers

[0042] According to the homology comparative analysis of the gB gene sequence of bovine infectious rhinotracheitis virus in GenBank, the conserved sequence region was selected as the amplification region, and specific amplification primers were designed by using Oligo 7.0 primer design software and BLAST softwa...

Embodiment 2

[0073] Example 2 Rapidly detects the annealing temperature test of bovine infectious rhinotracheitis virus PCR method

[0074] Perform PCR amplification at annealing temperatures of 50 °C, 52 °C, 54 °C, 56 °C, 58 °C, 60 °C, and 62 °C to determine the optimal annealing temperature. The results showed that the designed primers had a large tolerance to annealing temperature, and could amplify well under the reaction programs with annealing temperatures of 50 ℃, 52 ℃, 54 ℃, 56 ℃, 58 ℃, 60 ℃ and 62 ℃. Add a single destination strip ( figure 1 ). To this end, the reaction program used in this PCR method was: 95°C for 5 min; followed by 30 cycles of 95°C for 35 s, 58°C for 40 s, and 72°C for 45 s; and finally 72°C for 10 min.

Embodiment 3

[0075] Embodiment 3 detects the specific detection result of bovine infectious rhinotracheitis virus PCR method

[0076] Extraction of genomic DNA / RNA from bovine infectious rhinotracheitis virus, Cryptobacterium crypticus, Mannella hemolyticus, Mycoplasma bovis, bovine parainfluenza virus type 3, and Klebsiella pneumoniae (RNA viruses are first reverse-transcribed into cDNA) , using the optimized reaction system and reaction program to carry out PCR amplification to detect the specificity of the detection method of the present invention, the results show that only the bovine infectious rhinotracheitis virus sample has amplified the target fragment band, which is a positive result, 5 There was no amplification in the reaction tube of the strain control strain and the water control reaction tube, which was a negative result ( figure 2 ), indicating that the method has good specificity.

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Abstract

The invention discloses a PCR amplification primer for the rapid detection of a bovine infectious rhinotracheitis virus and applications thereof, and belongs to the fields of animal bacteriology and molecular biology. The PCR amplification primer is composed of a forward primer having a nucleotide sequence represented by SEQ ID No.1 and a reverse primer having a nucleotide sequence represented bySEQ ID No.2. The PCR amplification primer is used to prepare a PCR amplification kit for detecting the bovine infectious rhinotracheitis virus. The provided PCR detection method using the kit has theadvantages of high specificity, high sensitivity, good repeatability, and high credibility. The bovine infectious rhinotracheitis virus can be detected specifically. The detection results can be obtained rapidly and accurately. At the same time, the cost is lower, the operation is simple, and the kit is suitable for being used in grassroots units; and a rapid, accurate and simple detection tool isprovided for laboratory rapid identification and large scale epidemiological investigation of bovine infectious rhinotracheitis virus.

Description

technical field [0001] The invention relates to the technical fields of animal bacteriology and molecular biology, in particular to a rapid, simple and low-cost PCR amplification primer for detecting bovine infectious rhinotracheitis virus and its application. Background technique [0002] Infectious bovine rhinotracheitis virus (IBRV) is an acute, febrile, and contagious infectious disease caused by bovine herpesvirus type 1. The clinical symptoms of sick cattle are cow abortion, metritis, and respiratory tract infection. , enteritis and pneumonia. Cattle infected with the virus have increased susceptibility to pathogens such as Pasteurella multocida, Mannella hemolyticus, and parainfluenza virus. At present, the pathogen is distributed all over the world, and it is also widely distributed in China, causing huge economic losses and becoming one of the important pathogens threatening the cattle breeding industry. [0003] So far, there are many methods for diagnosing bovin...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/686C12N15/11
CPCC12Q1/686C12Q1/705C12Q2565/125
Inventor 潘艳李军吴翠兰彭昊冯世文陶立李常挺钟舒红杨威陈泽祥谢永平许力干马春霞胡帅贺会利
Owner GUANGXI VETERINARY RES INST
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