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SYBR GreenI real-time quantitative polymerase chain reaction (PCR) detection method for S gene of transmissible gastroenteritis virus of swine

A real-time quantitative and detection method technology, applied in the determination/inspection of microorganisms, biochemical equipment and methods, fluorescence/phosphorescence, etc., can solve the problems of cumbersome operation, long cycle, insufficient sensitivity, etc. Precise, easy-to-use effects

Inactive Publication Date: 2011-06-22
ZHENGZHOU HOUYI PHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] In view of the problems existing in the current detection method for TGEV, the purpose of the present invention is to provide a real-time quantitative PCR detection method of S gene SYBR GreenⅠ of porcine transmissible gastroenteritis virus

Method used

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  • SYBR GreenI real-time quantitative polymerase chain reaction (PCR) detection method for S gene of transmissible gastroenteritis virus of swine
  • SYBR GreenI real-time quantitative polymerase chain reaction (PCR) detection method for S gene of transmissible gastroenteritis virus of swine
  • SYBR GreenI real-time quantitative polymerase chain reaction (PCR) detection method for S gene of transmissible gastroenteritis virus of swine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1 Total RNA extraction of gastroenteritis virus

[0044] Take 400 uL of the proliferated porcine transmissible gastroenteritis virus solution, add 400 ul of Trizol, shake vigorously for 1-2 min, and make it fully mixed; add 100 ul of chloroform / isoamyl alcohol (24:1), shake vigorously to mix Homogenize for 30 s, centrifuge at 12 000 rpm for 5 min; carefully transfer the supernatant to a sterile EP tube, add 200 ul absolute ethanol, and mix well; transfer all the above solution to the GenClean column in a 2 ml collection tube place at room temperature for 2 min, and centrifuge at 8 000 rpm for 1 min; carefully take out the column, discard the waste liquid in the collection tube, and put the column back into the collection tube; Ethanol), 10 000 rpm, centrifuge at room temperature for 30 s; discard the waste liquid, repeat washing once, carefully take out the column, discard the waste liquid in the collection tube, put the column back into the collection tube, a...

Embodiment 2

[0045] Example 2 Design primers

[0046]According to the S gene sequence of porcine transmissible gastroenteritis virus (NC002306) published in GenBank, a pair of primers were designed using Primer Premier 5.0 software, and the designed primers could amplify 250 bp of the partial fragment of the S gene (TGEV-S). The sequence is as follows:

[0047] Upstream primer p1: 5'-GTATTGGGATTATGCT-3'

[0048] Downstream primer p2: 5'-GGTGGTGGTAGTAGGT-3';

[0049] Primers were synthesized by Shanghai Bioengineering Technology Co., Ltd.

Embodiment 3

[0050] Example 3 S gene RT-PCR amplification of porcine transmissible gastroenteritis virus

[0051] Take 5ul RNA sample and add DEPC water to make up 11ul, then add 1 ul of reverse transcription primer, bathe in 70°C water for 10 min, then quickly place it on ice for 5 min, and centrifuge slightly; take it out and add 4 ul of 5×Buffer, 2 ul of dNTP, and 1 ul of RNasinhibiter in sequence ul, instantaneous centrifugation; 42°C water bath for 2 min, quickly add 1 ul reverse transcriptase, and seal it with parafilm; cDNA was used as template for PCR amplification,

[0052] The PCR amplification system is: Premix Ex Taq 12 ul, the concentration is 0.5 umoL / L, 0.5 uL each of primers P1 and P2, 1 ul of cDNA, and finally make up to 25 ul with sterilized double distilled water;

[0053] The PCR amplification reaction program is:

[0054] (1) Pre-denaturation at 95°C for 5 minutes;

[0055] (2) Denaturation at 95°C for 50 s, annealing at 48°C for 50 s, extension at 72°C for 1 mi...

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Abstract

The invention discloses an SYBR GreenI real-time quantitative PCR detection method for the S gene of transmissible gastroenteritis virus of swine, which comprises the following steps: extracting total RNA of viral gastroenteritis; designing primers; performing reverse transcription; performing PCR amplification of a fragment of the S gene of transmissible gastroenteritis virus of swine; cloning and identifying the S gene of transmissible gastroenteritis virus of swine; preparing a standard product template; performing fluorescence quantitative PCR amplification; obtaining a kinetic curve and a standard curve; and performing repeatability, sensibility and specificity tests. In the method of the invention, only a good S type amplification curve is shown, only specific single peak of the product of the amplification appears, and the Tm value of the product is uniform; the detection sensitivity reaches 3 copies / microliter, and the sensitivity is high; the method avoids cross reaction with other viruses and has high specificity; and the concentration of the starting template has a good linear relationship with a Ct value, and the reaction system has high accuracy and high stability.

Description

technical field [0001] The invention belongs to the field of virus detection, and in particular relates to a real-time quantitative PCR detection method for S gene SYBR Green I of porcine transmissible gastroenteritis virus. Background technique [0002] Transmissible gastroenteritis of swine (TGE) is a highly contagious intestinal disease caused by transmissible gastroenteritis virus (TGEV), and is an important virus that causes diarrhea in piglets. Sexually infectious agent. The main clinical symptoms of infected pigs are diarrhea, dehydration and vomiting, and the mortality rate of sick young piglets is extremely high, which can reach 100%. It has become one of the important porcine viral diarrhea diseases, causing huge economic losses to the pig industry. TGE belongs to the porcine infectious disease that must be inspected in the Class B epidemic disease of the World Organization for Animal Health (OIE) Code, and is a legal quarantine epidemic in my country. It is diff...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68G01N21/64
Inventor 吴红云李厚伟李建正
Owner ZHENGZHOU HOUYI PHARMA
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