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Kit and detection method for quickly detecting porcine transmissible gastroenteritis virus by adopting isothermal amplification technology

A detection kit and isothermal amplification technology are applied in the field of temperature amplification technology detection kits to achieve the effects of high sensitivity, high sensitivity and wide application.

Active Publication Date: 2013-09-11
重庆海关技术中心 +1
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These three enzymes generate reverse transcription and transcription processes that result in the replication of the target sequence itself
However, there is no report on the detection kit and detection method of porcine transmissible gastroenteritis virus by isothermal amplification gene technology (NASBA) at present.

Method used

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  • Kit and detection method for quickly detecting porcine transmissible gastroenteritis virus by adopting isothermal amplification technology

Examples

Experimental program
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Effect test

Embodiment 1

[0041] Embodiment 1, design and screening of primers of the present invention

[0042] Based on the full-length genome sequence of the known porcine transmissible gastroenteritis virus nucleic acid sequence, multiple alignments were performed using ClustW software, and the conserved region of the S gene of TGEV was selected using the primer design software Primer5.0 software to design specific primers and probes. The needles are respectively marked as: SEQ ID NO1...SEQ ID NO4, wherein the 5' end of the universal probe is labeled with biotin, and the 5' end of the detection probe is labeled with digoxin. All primers were synthesized by Bao Biological Engineering (Dalian) Co., Ltd. Extract porcine transmissible gastroenteritis virus RNA with commercially available viral nucleic acid extraction kit, adopt SEQ ID NO1, SEQ ID NO2, SEQ ID NO3, SEQ ID NO4 to amplify the RNA respectively, get rid of non-specific amplification primers and probes Needle. The primers and probes were ob...

Embodiment 2

[0048] Embodiment 2, the preparation of positive control substance

[0049] The viral RNA of the standard strain of porcine transmissible gastroenteritis virus was extracted with a commercially available nucleic acid extraction kit, and PCR amplification was performed. The amplified length was about 195 bp. The amplified target band was recovered, ligated with the pMD19-T vector, ligated overnight at 4°C, transformed into JM109 competent cells, and the recombinant plasmid was extracted from those positive for resistance screening, shaking bacteria, and PCR. Sequence verification, obtain clones containing target fragments, extract virus-positive plasmids, aliquot 50 μL each, and control the concentration at 80-100 ng / μL.

Embodiment 3

[0050] Embodiment 3, the preparation of negative control substance

[0051] The RNA of porcine transmissible gastroenteritis virus negative sera were extracted using commercially available nucleic acid extraction kits. Dilute with sterilized deionized water, control the concentration at 80-100ng / μL, and pack into 50μL each.

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Abstract

The invention relates to the field of molecular-biological detection methods and detection reagents of animal epidemics, in particular to a primer, a kit and a detection method for quickly detecting a porcine transmissible gastroenteritis virus by adopting an isothermal amplification technology (NASBA (Nucleic Acid Sequence-based Amplification)). In the invention, gene sequences of the porcine transmissible gastroenteritis virus are subjected to multiple alignments through ClustalW; conserved areas of the sequences are analyzed; primers and probes are respectively designed by primer 5.0 design software; and the porcine transmissible gastroenteritis virus can be quickly and specifically detected and distinguished. Various reaction solutions are prepared to form a corresponding assay kit. The kit provided by the invention has the advantages of simplicity and convenience for operation, high specificity, high sensitivity, high repeatability, no complex after treatment, wide applicability and the like.

Description

technical field [0001] The porcine transmissible gastroenteritis virus detection kit related to the invention belongs to the field of animal epidemic molecular biology detection methods and detection reagents, and in particular is a porcine transmissible gastroenteritis virus detection kit using isothermal amplification technology. The invention also applies the kit to a biological detection method for isothermal amplification detection of porcine transmissible gastroenteritis virus. Background technique [0002] Transmissible gastroenteritis of swine virus (TGEV) belongs to the genus Coronaviridae of the Coronaviridae family and is an important pathogen that causes viral diarrhea in piglets, causing transmissible gastroenteritis of swine (TGE). TGE is an acute, highly contagious disease characterized by vomiting, severe diarrhea, dehydration, and high mortality in piglets up to 2 weeks of age. It was first reported in Illinois, USA in 1933. At present, the disease has bec...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12Q1/68C12R1/93
Inventor 聂福平徐自忠李应国肖进文王国民杨俊李贤良陈瑞王昱
Owner 重庆海关技术中心
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