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Detection method of infectious bovine rhinotracheitis virus

A rhinotracheitis virus, infectious technology, applied in the detection field of bovine infectious rhinotracheitis virus, can solve the problems affecting animal health and productivity, low, error accuracy, etc., to facilitate the detection application and operation process at the grassroots level Simple, short detection time effect

Inactive Publication Date: 2021-06-18
XINJIANG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The virus can remain latent throughout the life of the host, the virus is self-reactivating and can be reactivated by stress and corticosteroid treatment leading to relapse, causing perpetuation of the virus and transmission in the environment, with serious implications for animal health and productivity, causing significant economic losses to the cattle industry due to abortion, mortality, slaughter demand and reduced milk production
The traditional virus isolation technology takes a long time and has certain technical requirements. The traditional PCR detection has certain errors and the accuracy is not high. Cattle with negative serological antibody test may also be virus carriers. Therefore, it is necessary to establish a sensitive, stable, and Rapid IBRV detection method is essential for accurate screening of infected animals and effective control of IBR transmission

Method used

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  • Detection method of infectious bovine rhinotracheitis virus
  • Detection method of infectious bovine rhinotracheitis virus
  • Detection method of infectious bovine rhinotracheitis virus

Examples

Experimental program
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Effect test

Embodiment 1

[0017] Embodiment 1, bovine infectious rhinotracheitis virus TaqMan probe fluorescent quantitative PCR detection kit composition This kit includes a pair of specific primers (Forward, Reverse) and a specific fluorescent probe of bovine infectious rhinotracheitis virus (Probe), Positive Control, Negative Control, 2×PerfectStart TM IIProbe qPCR SuperMix. 1. A pair of specific primers for bovine infectious rhinotracheitis virus (SEQ ID NO: 2, SEQ ID NO: 3); 2. A specific fluorescent probe for bovine infectious rhinotracheitis virus (SEQ ID NO: 3), the 5' end of the fluorescent probe is labeled with the fluorescent reporter group HEX, and the 3' end is labeled with the fluorescent quencher group BHQ-1; 3. The positive control is bovine infectious rhinotracheitis virus standard; 4. The negative control is Nuclease -free Water; 5, 2×Perfect Start TM ⅡProbe qPCR SuperMix (Beijing Quanshijin Biology).

Embodiment 2

[0018] Embodiment 2, bovine infectious rhinotracheitis virus TaqMan probe fluorescent quantitative PCR detection method:

[0019] 1. Preparation of target DNA template

[0020] Take a 200 μL sample and use Tiangen Blood / Cell / Tissue Genomic DNA Extraction Kit to operate according to the instructions.

[0021] 2. Fluorescence quantitative PCR amplification is 20 μL for each system

[0022] Table 1

[0023]

[0024] 3. Fluorescent quantitative PCR amplification program: pre-denaturation at 94°C for 30s; denaturation at 94°C for 5s; annealing and extension at 60°C for 30s, a total of 40 cycles.

[0025] 4. The present invention uses a Bio-Rad CFX96 fluorescent quantitative PCR instrument for detection.

[0026] 5. Judgment of the result: when Cq≤35, the sample is judged to be BITRV positive; when Cq>35 or there is no amplification curve, the sample is judged to be BITRV-negative.

Embodiment 3

[0027] Embodiment 3, performance measurement of bovine infectious rhinotracheitis virus TaqMan probe fluorescent quantitative PCR kit 1, preparation of infectious rhinotracheitis virus standard positive plasmid:

[0028] Take the BHV-1 virus liquid propagated in MDBK cells, and use Tiangen Blood / Cell / Tissue Genomic DNA Extraction Kit to operate according to the instructions.

[0029] The PCR amplification system (20 μL) was: ddH2O 10 μL, PCR Mixture 7 μL, template DNA 2 μL, upstream primer (SEQ ID NO:5) 0.5 μL, downstream primer (SEQ ID NO:6) 0.5 μL. The amplification program is: pre-denaturation at 94°C for 5 min, denaturation at 94°C for 30 s, annealing at 58°C for 30 s, extension at 72°C for 30 s, 35 cycles, and extension at 72°C for 10 min; PCR amplification products were electrophoresed on 1.5% agarose gel Detect the cutting target band (such as figure 1 ), was recovered using the Shanghai Shengxiang Glue Recovery Kit.

[0030] The above-mentioned purified target gene f...

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Abstract

The invention belongs to the technical field of biology, and particularly relates to a detection method of an infectious bovine rhinotracheitis virus. The detection method comprises a specific target sequence as shown in SEQ ID NO. 1, a kit comprises a fluorescent quantitative PCR detection primer pair and a probe, the primer pair is as shown in SEQ ID NO. 2 and SEQ ID NO. 3, and the sequence of the probe is as shown in SEQ ID NO. 4. The TaqMan probe fluorescent quantitative PCR kit for detecting the infectious bovine rhinotracheitis virus has high specificity and sensitivity, can be used for screening various clinical samples to detect infectious bovine rhinotracheitis positive samples, and has the characteristics of short detection time, high detection sensitivity and high stability.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular to a method for detecting bovine infectious rhinotracheitis virus. Background technique [0002] Infectious Bovine Rhinotracheitis (IBR), also known as "necrotizing rhinitis" or "red nose disease", is a disease caused by bovine herpes virus type 1 (BHV-1). An acute, febrile contact infectious disease that can cause severe respiratory infections in cattle, and sometimes conjunctivitis, encephalitis, reduced milk production, metritis, enteritis, contagious pustular vulvovaginitis and other symptoms. The virus can remain latent throughout the life of the host, the virus is self-reactivating and can be reactivated by stress and corticosteroid treatment leading to relapse, causing perpetuation of the virus and transmission in the environment, with serious implications for animal health and productivity, causing significant economic losses to the cattle industry due to abortion, mortality, d...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/6851C12N15/11C12R1/93
CPCC12Q1/705C12Q1/6851C12Q2531/113C12Q2561/101
Inventor 姚刚付强任强林马义诚张杨马雪连苏战强陈卓夏瑞阳李鑫李紫仟金肖叶郭雪萍
Owner XINJIANG AGRI UNIV
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