Establishment and application of multiplex fluorescent RT-PCR detection method for foot-and-mouth disease, vesicular stomatitis and porcine vesicular disease

A technique for vesicular stomatitis and swine vesicular disease

Inactive Publication Date: 2015-08-12
ANIMAL & PLANT & FOOD INSPECTION CENT OF TIANJIN ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The 3 vesicular diseases are clinically indistinguishable and must be differentially diagnosed by laboratory testing
At present, pathogen isolation and identification and conventional serological methods are mostly used for the diagnosis of the three diseases, which takes a long time, and PCR technology can also be used for detection, but there is no stable and operable method that can simultaneously identify the three viruses method

Method used

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  • Establishment and application of multiplex fluorescent RT-PCR detection method for foot-and-mouth disease, vesicular stomatitis and porcine vesicular disease
  • Establishment and application of multiplex fluorescent RT-PCR detection method for foot-and-mouth disease, vesicular stomatitis and porcine vesicular disease
  • Establishment and application of multiplex fluorescent RT-PCR detection method for foot-and-mouth disease, vesicular stomatitis and porcine vesicular disease

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Experimental program
Comparison scheme
Effect test

preparation example Construction

[0065] 2.3.1.3 Preparation of standards for quantification

[0066] Using specific primers for the 3D protein encoding gene of foot-and-mouth disease virus, the viral RNA was amplified by PCR in a 50 μL system, and the PCR product obtained was purified and cloned into the pGEM-T easy vector and transformed into E. coli TOP10 competent cells. The plasmid was extracted from the purified and amplified recombinant bacteria, purified by the kit, and the plasmid concentration was measured by OD260 with a UV reflectometer, and the concentration was converted into gene copy number according to Avogadro constant. The formula for calculating plasmid copy number is as follows:

[0067] Copy number (copy / μL)=(plasmid concentration (g / μL)×6.02×10 23 ) / [Base Pair (bp)×660(g / mol] (Formula 1-1).

[0068] According to formula 1-1, the copy number of recombinant plasmid pGEM T-3D is calculated to be 1.21×10 10 Copy / μL.

[0069] The extraction process of recombinant plasmid DNA was performed according ...

Embodiment 1

[0136] Example 1 Establishment of a fluorescent RT-PCR detection method for foot-and-mouth disease virus

[0137] 1.1 Method establishment and specificity

[0138] Synthesize primers and probes for the 3D protein coding gene sequence of foot-and-mouth disease virus. After exploring the reaction conditions, a reaction system was established with a total of 50 μL. Take foot-and-mouth disease virus (FMDV) RNA as a positive control, take genomic RNA extracted from cells that are not infected with foot-and-mouth disease virus as a negative control, and use water as a blank control. Other viruses such as Akabane virus RNA, bovine leukemia virus cDNA, and bovine viral diarrhea virus RNA , Vesicular stomatitis virus RNA, swine vesicular disease virus RNA and Mycobacterium paratuberculosis DNA to identify the specificity of the fluorescent quantitative RT-PCR detection method. The test results are attached figure 1 .

[0139] 1.2 Establishment of standard curve

[0140] According to serial ...

Embodiment 2

[0145] Example 2 Establishment of a fluorescent RT-PCR detection method for vesicular stomatitis virus

[0146] 2.1 Method establishment and specificity

[0147] Design and synthesis of primers and probes according to the coding gene sequence of N protein of vesicular stomatitis virus. After exploring the reaction conditions, a reaction system was established with a total of 50 μL. Vesicular stomatitis virus (VSV) RNA was used as a positive control, genomic RNA extracted from cells not infected with vesicular stomatitis virus was used as a negative control, and water was used as a blank control. Other viruses such as Akabane virus RNA, bovine leukemia virus eDNA, Bovine viral diarrhea virus RNA, foot-and-mouth disease virus RNA, swine vesicular disease virus RNA and Mycobacterium paratuberculosis DNA were used to identify the specificity of the fluorescent quantitative RT-PCR detection method. The test results are attached Figure 5 .

[0148] 2.2 Establishment of standard curve

...

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Abstract

Based on a highly conserved domain of a foot-and-mouth disease virus 3D protein coding gene, a vesicular stomatitis virus N protein coding gene and a swine vesicular disease virus VP1 protein coding gene, the invention designs a specific primer and a probe and develops a multiple fluorescence RT-PCR detection method used for simultaneously detecting the three animal viruses. The detection method can detect 102 copied plasmids containing target amplification sequences in 1 h. The method has high sensitivity and good specificity, and can achieve rapid high-throughput fluorescence RT-PCR detection for single-virus infection or mixed infection by a plurality of viruses.

Description

Technical field [0001] The invention relates to the application field of biotechnology, in particular to the simultaneous detection and identification of foot and mouth disease, vesicular stomatitis and swine vesicular disease. Background technique [0002] In the past 10 years, under the promotion of the government, processing and consumer demand, China's dairy industry has made considerable progress, gradually developing from a poor milk country to a large dairy country. First of all, the number of imported dairy cows has increased year by year. From 1997 to 2007, the number of dairy cows in China increased by 4.84 times; in 2009, 45,000 dairy cows were imported, 80,000 dairy cows were imported in 2010, and 100,000 dairy cows were imported in 2011. Secondly, the scale of domestic dairy cattle breeding has increased substantially. In 2010, the national dairy cattle breeding stock of more than 100 cows accounted for 28.48% of the country, an increase of 8.7% over 2008. More and...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12Q1/68C12N15/11C12Q1/06G01N21/64C12R1/93
CPCC12Q1/686C12Q1/70C12Q2537/143C12Q2563/107C12Q2531/113
Inventor 王乃福吴冬雪王建华陈本龙黄晨董志珍赵祥平王玉玲
Owner ANIMAL & PLANT & FOOD INSPECTION CENT OF TIANJIN ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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