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Fluorescence quantitative PCR (Polymerase Chain Reaction) detection method for porcine transmissible gastroenteritis virus gene S and primer thereof

A fluorescence quantification and detection method technology, applied in microorganism-based methods, microbial determination/inspection, biochemical equipment and methods, etc., can solve problems such as S gene and dye method not involved, and achieve shortened detection time and specificity. High, not easy to contaminate effect

Inactive Publication Date: 2011-08-17
SHANGHAI JIAO TONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But the S gene and dye method are not involved in this method

Method used

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  • Fluorescence quantitative PCR (Polymerase Chain Reaction) detection method for porcine transmissible gastroenteritis virus gene S and primer thereof
  • Fluorescence quantitative PCR (Polymerase Chain Reaction) detection method for porcine transmissible gastroenteritis virus gene S and primer thereof
  • Fluorescence quantitative PCR (Polymerase Chain Reaction) detection method for porcine transmissible gastroenteritis virus gene S and primer thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] (1) Design and synthesis of primers and probes

[0036] According to the S gene sequence of the TGEV strain preserved in our laboratory, primers were designed with primer5.0 and other software. The target fragment size was 111bp, which was synthesized by Shanghai Sangon Bioengineering Co., Ltd.

[0037] The upstream primer is sequence listing 1; the downstream primer is sequence listing 2.

[0038] (2) Cloning of S gene

[0039] Inoculate 10% of the TGEV strain into a cell bottle that has grown to 80% of the cells, adsorb at 37°C for 1 hour, change 2% of the maintenance solution, and CO 2 Cultivate in an incubator at 37°C for 3-6 days, collect the virus after 80% of the lesions appear, freeze and thaw the virus cell liquid three times repeatedly, collect the virus, and store it at -80°C for later use. Viral RNA was extracted according to Reagent kit instructions for use.

[0040] TGEV DNA was synthesized according to the instructions of the one-step reverse transcrip...

Embodiment 2

[0054] (1) Collection of clinical stool samples, adding 2 times the volume of PBS to the clinical stool samples, freezing and thawing 3 times, taking the supernatant as the extraction stock solution and storing it at -80°C for later use. Viral RNA was extracted according to Reagent kit instructions for use.

[0055] (2) PCR system optimization

[0056] Element

[0057] (3) Optimal reaction conditions

[0058]

[0059] (4) Calculate the virus content in the sample according to the fluorescent quantitative PCR result and the standard curve.

[0060] (5) Fluorescent quantitative PCR results, the fluorescent quantitative PCR instrument will automatically display the concentration of the sample, the unit is Copies / 2μL.

[0061]

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Abstract

The invention discloses a fluorescence quantitative PCR (Polymerase Chain Reaction) detection method for a porcine transmissible gastroenteritis virus gene S and a primer thereof in the technical field of biotechnology. The method comprises the following steps of: cloning a PCR amplification target segment identified as a positive PCR product to a vector pMD18-T, transforming to a competent cell DH5alpha, selecting positive clone by screening blue and white spots and identifying sequencing; extracting a positive recombinant plasmid, quantifying by using an ultraviolet spectrophotometer, diluting a standard product series by 10 times of gradient until the final concentration is 1.0*10<3>-1.0*10<11> copies / mL, undergoing a fluorescence quantitative PCR by taking the standard product series as a template, and establishing a fluorescence quantitative PCR standard curve; and extracting virus RNA (Ribonucleic Acid) of a clinical excrement sample, undergoing a fluorescence quantitative PCR, and calculating the content of viruses in the sample according to a result and the standard curve, wherein the sequences of the primer are sequence 1 and sequence 2. The method and the primer have theadvantages that: a fluorescent probe does not need to be designed additionally, the cost is lowered, operation is easy and convenient, and detection can be completed within 2 hours. The detection method and the primer are suitable for any fluorescence quantitative PCR instrument, and can be applied to the detection of large-scale and high-flux samples.

Description

technical field [0001] The invention relates to a detection method and gene primers in the field of biotechnology, in particular to a fluorescent quantitative PCR detection method and primers for porcine transmissible gastroenteritis virus S gene. Background technique [0002] Porcine transmissible gastroenteritis (TGE) is a highly contagious infection caused by porcine transmissible gastroenteritis virus (TGEV), characterized by vomiting, watery diarrhea, and dehydration in sick pigs. The disease is highly lethal to piglets under 1 week of age. After recovery, the piglets were stunted and stunted. The disease can impair the disease resistance and immune function of recovered adult pigs, lose fat, reduce feed utilization, waste medicine and manpower, etc., resulting in serious economic losses. Therefore, it is necessary to establish a fast, sensitive, low-cost and easy-to-operate detection method. [0003] TGEV encodes four structural proteins, namely the filament (S) pro...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12R1/93
Inventor 杨志彪顾江萍袁聪俐崔立华修国
Owner SHANGHAI JIAO TONG UNIV
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