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Multiple reverse transcription polymerase chain reaction detection method for swine transmissible gastroenteritis

A technology of chain reaction and detection method is applied in the detection field of porcine infectious gastroenteritis, which can solve the problems of long cycle, inability to identify, and large interference, and achieves the effect of saving time, reducing product pollution and high sensitivity

Inactive Publication Date: 2012-07-18
郑世民
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Problems solved by technology

[0003]Enzyme-linked immunosorbent method ELISA needs to prepare antibodies, and the cycle is long; external factors interfere with the diagnostic method of enzyme-linked immunosorbent method ELISA; Diagnostic method does not differentiate porcine transmissible gastroenteritis virus TGEV from respiratory coronavirus PRCV

Method used

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  • Multiple reverse transcription polymerase chain reaction detection method for swine transmissible gastroenteritis

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Embodiment 1

[0045] A multiplex reverse transcription polymerase chain reaction detection method for porcine transmissible gastroenteritis, said method has eight steps, respectively designing and synthesizing gene primers for porcine transmissible gastroenteritis virus TGEV and porcine epidemic diarrhea virus PEDV , Porcine transmissible gastroenteritis virus TGEV and porcine epidemic diarrhea virus PEDV multiplex reverse transcription polymerase chain reaction RT-PCR diagnostic method, duplex reverse transcription polymerase chain reaction RT-PCR primer concentration optimization, Double reverse transcription polymerase chain reaction RT-PCR magnesium chloride MgCl2 concentration optimization, double reverse transcription polymerase chain reaction RT-PCR base triphosphate deoxynucleotide dNTP concentration optimization, double reverse transcription polymerase Determination of optimal annealing temperature for chain reaction RT-PCR, double reverse transcription polymerase chain reaction R...

Embodiment 2

[0047] Porcine transmissible gastroenteritis multiplex reverse transcription polymerase chain reaction detection method described in embodiment 1, described primer design and synthesis; Spike protein S protein gene sequence of epidemic diarrhea virus PEDV, comparative analysis of conserved sequences, according to reverse transcription polymerase chain reaction RT-PCR and multiple reverse transcription polymerase chain reaction RT-PCR primer design principles, through the European Lige Oligo6.0 and Pulaimo Primer5.0 software designed porcine transmissible gastroenteritis virus TGEV and porcine epidemic diarrhea virus PEDV gene primers, and the primer sequences and amplified fragment sizes of each gene are shown in Table 2-1.

Embodiment 3

[0049] Porcine transmissible gastroenteritis multiple reverse transcription polymerase chain reaction detection method described in embodiment 1, described porcine transmissible gastroenteritis virus TGEV and porcine epidemic diarrhea virus PEDV multiple reverse transcription polymerase chain reaction Establishment of RT-PCR diagnostic method;

[0050] The porcine transmissible gastroenteritis virus TGEV and porcine epidemic diarrhea virus PEDV duplex reverse transcription polymerase chain reaction RT-PCR reaction system

[0051] Establish the following double reverse transcription polymerase chain reaction RT-PCR reaction system with a total volume of 25 μL:

[0052] Ultrapure water (ddH20) 7.87μl

[0053] Buffer (10×PCR Buffer) 5.0μl

[0054] Deoxynucleotide triphosphate base [dNTP Mixture(2.5mmol / L)] 4.0μl

[0055] P3 (10μmol / L) 0.5μl

[0056] P4 (10μmol / L) 0.5μl

[0057] P5 (10μmol / L) 0.5μl

[0058] P6 (10μmol / L) 0.5μl

[0059] Porcine transmis...

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Abstract

The invention relates to a multiple reverse transcription polymerase chain reaction detection method for swine transmissible gastroenteritis. An antibody is needed to be prepared by an euzymelinked immunosorbent assay, so that the period is longer, and external factors have larger interference on the diagnostic method of the euzymelinked immunosorbent assay. The method comprises the following steps of: designing and synthesizing a transmissible swine gastroenteritis virus gene primer; establishing a multiple reverse transcription polymerase chain reaction diagnostic method of the transmissible swine gastroenteritis viruses and porcine epidemic diarrhea viruses; optimizing concentration of the primer of a bigeminy reverse transcription polymerase chain reaction; optimizing the concentration of magnesium oxide of the bigeminy reverse transcription polymerase chain reaction; optimizing the concentration of triphosphoric acid base deoxynucleotide of the bigeminy reverse transcription polymerase chain reaction; determining the optimal annealing temperature of the bigeminy reverse transcription polymerase chain reaction; testing the sensitivity of the bigeminy reverse transcription polymerase chain reaction; and testing the specificity of the bigeminy reverse transcription polymerase chain reaction. The multiple reverse transcription polymerase chain reaction detection method for swine transmissible gastroenteritis is suitable for detecting swine transmissible gastroenteritis.

Description

technical field [0001] The invention relates to a novel detection method for porcine transmissible gastroenteritis, in particular to a multiple reverse transcription polymerase chain reaction detection method for porcine transmissible gastroenteritis. Background technique [0002] The diseases caused by porcine transmissible gastroenteritis virus TGEV and porcine epidemic diarrhea virus PEDV are very similar in clinical symptoms; in terms of genome structure, porcine respiratory coronavirus PRCV and porcine transmissible gastroenteritis virus TGEV have high homology , which is difficult to distinguish by traditional diagnostic methods. At present, the diagnostic methods of porcine transmissible gastroenteritis TGE and porcine epidemic diarrhea PED mainly include virus neutralization test, immunofluorescence method, immunoelectron microscopy method, enzyme-linked immunosorbent method ELISA and reverse transcription polymerase chain reaction method. [0003] Enzyme-linked imm...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
Inventor 郑世民赵良友刘超男高雪丽吕晓萍
Owner 郑世民
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