Detecting and typing method for hoof and mouth disease virus RT-PCR

A foot-and-mouth disease virus, -GCCACTGTTGAGAGCTACGG-3 technology, which is applied in the field of serotype identification, can solve the problems of unavoidable scattered virus and low detection rate, and achieves the effect of satisfying clinical rapid diagnosis, strong specificity and high sensitivity

Inactive Publication Date: 2011-10-26
NORTHEAST AGRICULTURAL UNIVERSITY
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Problems solved by technology

[0004] At present, the commonly used methods for domestic quarantine and diagnosis of the disease include virus isolation, agar diffusion test, complement fixation test, enzyme-linked immunosorbent assay, etc. These methods may take a long time to obtain the test results, the detection rate is low, or in the process The risk of poisoning is unavoidable

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Embodiment Construction

[0020] The nucleotide sequence of 2 pairs of primers of detection method of the present invention is as follows:

[0021] P1: 5-AATTTTGAACAACATCTACGTGCT-3 (24bp)

[0022] P2: 5-CAGGTAAAGTGATCTGTAGCTTGG-3 (24bp)

[0023] S1: 5-GCCACTGTTGAGAGCTACGG-3 (20bp)

[0024] S2: 5-ACGTCGGAGAAGAAGAAGGG-3 (20bp)

[0025] The detection method process of the present invention is as follows:

[0026] 1) Treatment of sick materials

[0027] Treatment of animal tissue: add 5 times the volume (m / v) of PBS (pH 7.4), and grind the tissue with a tissue grinder. Repeat freeze-thawing 3 times, centrifuge at 8000rpm for 5min, take the supernatant for RNA extraction; blister fluid and salivation fluid: directly centrifuge the collected blister fluid or salivation fluid at 8000r / nim for 5min, and remove the supernatant for RNA extraction.

[0028] 2) Extraction and reverse transcription of viral RNA

[0029] The specific steps of virus RNA extraction are as follows: take 425 μl of the treated dise...

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Abstract

The present invention relates to foot-and-mouth disease virus RT-PCR detecting and typing method. The method includes detecting foot-and-mouth virus with primer P1 / P2 and S1 / S2 and identifying foot-and-mouth disease virus type by means of nucleotide homology comparison of PCR product of primer S1 / S2. The detection process includes: 1. designing foot-and-mouth disease virus detecting specific primer P1 / P2 and S1 / S2; 2. providing foot-and-mouth disease virus RNA; 3. synthesizing cDNA with extracted RNA as template and with reverse transcription primer and reverse transcriptase; 4. performing the foot-and-mouth disease virus nucleic acid amplification by means of PCR technology; and 5. cloning the PCR product of primer S1 / S2 to pMD18-The vector for nucleotide sequence determination and comparing nucleotide homology with biological software for serotype identification. The method is fast, stable, sensitive, specific and without virus diffusing risk.

Description

(1) Technical field [0001] The invention relates to a method for detecting foot-and-mouth disease virus by means of reverse transcription-polymerase chain reaction (RT-PCR), and identifying the serotype of the detected virus strain. (2) Background technology [0002] Foot-and-Mouth Disease (FMD) is an acute, febrile and contact infectious disease caused by Foot-and-Mouth Disease Virus (FMDV). It has the characteristics of fast transmission, strong infectivity, wide host spectrum, and easy mutation of antigens. There are seven serotypes of FMDV, O, A, C, Asia I, SAT I, SAT II, ​​and SATIII, and there is no cross-protection among the serotypes. Therefore, the rapid diagnosis and serotype identification of FMD is an important basis for effectively controlling the occurrence and spread of the disease. [0003] FMD has many transmission routes, is highly contagious, and is a common disease of many animals. Seriously endanger the productivity of livestock and the quality of liv...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12Q1/25
Inventor 王君伟曲哲会
Owner NORTHEAST AGRICULTURAL UNIVERSITY
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