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Duplex PCR method for detecting porcine transmissible gastroenteritis virus and porcine epidemic diarrhea virus

A porcine epidemic diarrhea and infectious technology, which is applied in biochemical equipment and methods, microbial determination/examination, etc., can solve the problems of inappropriate epidemiological investigation, time-consuming and laborious, inappropriate clinical rapid diagnosis, etc., and achieves high sensitivity and detection. Efficient and specific effects

Inactive Publication Date: 2014-07-30
SHANGHAI JIAO TONG UNIV +1
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  • Abstract
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  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Traditional diagnostic techniques such as virus isolation and immunofluorescence tests are time-consuming and laborious, and are not suitable for rapid clinical diagnosis or large-scale epidemiological investigations

Method used

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  • Duplex PCR method for detecting porcine transmissible gastroenteritis virus and porcine epidemic diarrhea virus
  • Duplex PCR method for detecting porcine transmissible gastroenteritis virus and porcine epidemic diarrhea virus
  • Duplex PCR method for detecting porcine transmissible gastroenteritis virus and porcine epidemic diarrhea virus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] 1 RNA extraction

[0050] The method is the same as in Comparative Example 1.

[0051] 2 cDNA synthesis

[0052] The method is the same as in Comparative Example 1.

[0053] 3 Establishment of Multiplex PCR

[0054] RNA was extracted from samples suspected of being infected with TGEV or (and) PEDV, and cDNA was transcribed to obtain cDNA. Using this cDNA as a template, the sequences were respectively SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, and SEQ ID NO. The 2 pairs of primers in 4 were added to the amplification system at the same time to establish a double PCR amplification reaction system. The double PCR reaction system is as follows: 1 μl of cDNA template, 0.5 μl of 2 pairs of upstream and downstream primers, 12.5 μl of PCR Mix, and 50 μl of ultrapure water. PCR reaction conditions: pre-denature at 95°C for 5 minutes, enter a cycle of 95°C for 60 seconds, 52°C for 1 minute, and 72°C for 1 minute, cycle 35 times, and finally extend at 72°C for 5 minutes. After PCR...

Embodiment 2

[0057] Example 2 Verifies the specificity experiment of multiplex PCR.

[0058] 1 RNA extraction

[0059] The method is the same as in Comparative Example 1.

[0060] 2 cDNA synthesis

[0061] The method is the same as that in Comparative Example 1. The RNA of porcine respiratory and reproductive syndrome virus (PRRSV), classical swine fever virus (CSFV) and porcine rotavirus (PRAV) is extracted and reverse-transcribed to obtain cDNA, which is carried out with the cDNA templates of PEDV and TGEV. Multiplex RT-PCR reactions.

[0062] 3 Multiplex PCR detection

[0063] Using the cDNA synthesized in step 2 as a template, add two pairs of primers whose sequences are SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, and SEQ ID NO.4 to the amplification system at the same time to establish a double PCR Amplification reaction system. The double PCR reaction system was as follows: 1 μl of cDNA template, 0.5 μl of each pair of upstream and downstream primers, 12.5 μl of PCRMix, and 50 μl of ...

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Abstract

The invention belongs to the technical field of biology, and concretely relates to a duplex PCR method for detecting porcine transmissible gastroenteritis virus (TGEV) and porcine epidemic diarrhea virus (PEDV). The method comprises: designing two primers respectively amplifying the specific sequences of TGEV and PEDV, extracting RNA from positive intestinal canal tissue infected TGEV and PEDV, performing inverse transcription to obtain cDNA, and further taking cDNA as a template, so as to establish a multiplex PCR detection method capable of directly detecting two kinds of diarrhoea viruses from a sample once. The beneficial effects comprise that compared with presently reported PCR detection methods, the duplex PCR detection method is improved in detection efficiency and strong in specificity.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular to a multiple PCR detection method, in particular to a double PCR method for detecting porcine transmissible gastroenteritis virus and porcine epidemic diarrhea virus in pig tissues. Background technique [0002] Porcine transmissible gastroenteritis (TGE) is a contagious disease of pigs caused by transmissible gastroenteritis virus (TGEV) of the Coronaviridae family. In the late 1960s, there were reports about the disease in our country. In recent years, the epidemic area has expanded, causing huge economic losses to the pig industry. The main clinical features are vomiting, severe diarrhea and dehydrating enteritis. Pigs of different ages and breeds are susceptible, especially for piglets less than 2 weeks old, the lethality rate is as high as 100%. TGE viruses belong to the genus Coronavirus in the family Coronaviridae. [0003] Porcine epidemic diarrhea (Porcine epidemic diarrhea...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68
CPCC12Q1/686C12Q1/70C12Q2531/113C12Q2537/143
Inventor 朱建国叶佳欣俞向前叶承荣万世平
Owner SHANGHAI JIAO TONG UNIV
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