Primer pair for detecting enzootic nasal tumor virus gene and application of primer pair

A tumor virus, endemic technology, applied in the determination/inspection of microorganisms, biochemical equipment and methods, DNA/RNA fragments, etc., can solve the problems of time-consuming, laborious, long time, and inability to detect live infected sheep, To achieve the effect of good specificity, high sensitivity and high sensitivity

Inactive Publication Date: 2016-09-07
NORTHWEST A & F UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the laboratory diagnosis methods for sheep endemic intranasal tumors in my country are relatively backward, mainly based on clinical symptoms and autopsy observations, and lack of rapid and simple diagnostic methods
There have been reports abroad using RT-PCR method to detect sheep endemic intranasal tumor virus from tumor tissue, but this requires slaughtering sheep and extracting tumor tissue, which takes a long time, is time-consuming and laborious, and most importantly, it cannot be used to infect sheep in vivo. detection

Method used

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  • Primer pair for detecting enzootic nasal tumor virus gene and application of primer pair
  • Primer pair for detecting enzootic nasal tumor virus gene and application of primer pair
  • Primer pair for detecting enzootic nasal tumor virus gene and application of primer pair

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Experimental program
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Effect test

Embodiment 1

[0021] Embodiment 1: primer pair and the preparation of the kit that adopts primer pair of the present invention and relevant PCR reagent

[0022] Acquisition of sheep endemic intranasal tumor virus disease materials: The clinical disease materials were collected from Baoji City, Shaanxi Province and Yangling High-tech Agricultural Technology Demonstration Zone sheep farms in August 2015. A total of 21 nasal cavity swabs were collected from sheep. Add 300 μL of normal saline to soak for 20 minutes, transfer to a 1.5 mL EP tube, freeze and thaw repeatedly 2 to 3 times, and store at -20°C.

[0023] Primers: upstream primer P1 (ENTVS): 5'-AATATTTCTTTAGATCTTC-3', downstream primer P2 (ENTVA): 5'-CCTAAAAGCTCCATTAGC-3', the expected amplified fragment size is 314bp. Primers were synthesized by Shanghai Yingjun, 2OD / tube.

[0024] Other reagents for PCR amplification experiments used in the kit:

[0025] DNA extraction reagents: purchased from the virus nucleic acid extraction kit ...

Embodiment 2

[0037] Embodiment 2: detection method based on the present invention

[0038] The main instruments are as follows: PCR amplification instrument (Eppendorf), centrifuge (1-14, Sigma), UV gel imager (JS-780 automatic gel imaging analysis system, Shanghai Peiqing), electrophoresis tank (DYCP-31BN , Beijing Liuyi Biotechnology Co., Ltd.).

[0039] The specific method is as follows:

[0040] First, extract the DNA of the sample to be tested: use the virus nucleic acid extraction kit (blood tissue cell genome extraction kit DP304) produced by Tiangen Biochemical Technology (Beijing) Co., Ltd., and extract the DNA of the virus according to the instructions, specifically:

[0041] (1a) Take a sheep nasal cavity cotton swab and put it into an EP tube containing 200 μL of PBS solution, soak for 20 minutes, let the contents of the sheep nasal cavity enter the PBS solution, discard the cotton swab, and use the remaining liquid as a sample. If the sample is less than 200 μL, Add PBS to 200...

Embodiment 3

[0058] Embodiment 3: Sensitivity test of the detection method of the present invention.

[0059] Using the DNA extracted from sheep endemic intranasal tumor tissue as a template, starting from 5ng DNA, it was diluted by 10 times, and the DNA extracted from sheep endemic intranasal tumor tissue was used as a template for PCR reaction procedure to amplify, and the PCR product was amplified by 1% Agarose gel electrophoresis, the result shows that PCR method can detect the template DNA of 5pg, has higher sensitivity, the result is as follows figure 2 shown.

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Abstract

The invention discloses a primer pair for detecting an enzootic nasal tumor virus gene and an application of the primer pair. The primer pair disclosed by the invention consists of single-stranded DNA as shown in SEQ ID No. 1 and single-stranded DNA as shown in SEQ ID No. 2. Shown by experiments of the applicant, the primer pair disclosed by the invention can be used for judging whether the enzootic nasal tumor virus gene is contained in a nasal secretion sample of a diseased sample or not with specificity and high sensitivity.

Description

technical field [0001] The invention belongs to the technical field of virus detection, and in particular relates to a primer pair for detecting sheep endemic intranasal tumor virus and its application in detecting sheep endemic intranasal tumor virus. Background technique [0002] Enzootic nasal tumor (ENT) is a chronic, progressive, contagious neoplastic disease caused by enzootic nasal tumor virus (ENTV). Loss of appetite, extreme weight loss, dyspnea, rhinorrhea, unilateral or bilateral hyperplasia in the nose, most of the cases reported so far are adenocarcinoma, and a few are papillary adenomas. The disease is currently distributed worldwide, and the disease also occurs in Inner Mongolia, Hunan, Sichuan, Shaanxi and other places in China. After the cells are infected by ENTV, it can induce the non-acute transformation (that is, tumorigenicity) of sheep nasal mucosal epithelial cells, and induce the generation of tumors. ENT can occur throughout the year, and it is en...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12N15/11
CPCC12Q1/686C12Q1/70
Inventor 许信刚付明哲张琪何亚鹏
Owner NORTHWEST A & F UNIV
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