Method for detecting FMDV (Foot and Mouth Disease Virus) through real-time fluorescent quantitative RT-PCR (Reverse Transcription-Polymerase Chain Reaction)

A real-time fluorescent quantitative and specific technology, applied in the field of detection, can solve the problems that specificity and sensitivity cannot meet the needs of detection, the process is cumbersome, and it takes a long time, so as to shorten the time required for testing and reduce pollution.

Inactive Publication Date: 2011-10-19
珠海出入境检验检疫局检验检疫技术中心
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Problems solved by technology

These methods are cumbersome and time-consuming to operate, and their specificity and sensitivity cannot meet the current detection needs.
In the virus isolation test, the process of culturing cells is particularly carefu

Method used

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  • Method for detecting FMDV (Foot and Mouth Disease Virus) through real-time fluorescent quantitative RT-PCR (Reverse Transcription-Polymerase Chain Reaction)
  • Method for detecting FMDV (Foot and Mouth Disease Virus) through real-time fluorescent quantitative RT-PCR (Reverse Transcription-Polymerase Chain Reaction)
  • Method for detecting FMDV (Foot and Mouth Disease Virus) through real-time fluorescent quantitative RT-PCR (Reverse Transcription-Polymerase Chain Reaction)

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Embodiment 1

[0050] In view of the history of FMDV prevalence in some areas of my country in 2005, the dynamic inspection laboratory of our technology center collected disease materials from relevant units in Shandong and Jiangsu once FMDV epidemic areas, and used the method of the present invention to identify and verify.

[0051] 1. Sample pretreatment: number the collected samples 1-10 respectively. Take a small amount of samples and mix penicillin and streptomycin double-antibody solutions, homogenate, take supernatant, and keep overnight at 4°C.

[0052] 2. RNA extraction: use Invitrogen's TRIzol reagent to extract RNA from the collected samples, and the operation steps are carried out according to the kit instructions.

[0053] 3. Real-time fluorescent quantitative RT-PCR amplification of FMDV: Use specific primers and probes of FMDV group, type O, and type I of Asia respectively, and use the RNA extracted from the sample as a template to amplify, and the specific primers and probes ...

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Abstract

The invention discloses a method for detecting an FMDV (Foot and Mouth Disease Virus) group and O type and AsiaI type thereof through real-time fluorescent quantitative RT-PCR (Reverse Transcription-Polymerase Chain Reaction). By carrying out specificity and sensitivity verification on the method established by the invention, the result shows that the FMDV can be well set apart from other Artiodactyla infectious viruses as well as O type and AsiaI type, and the sensitivity can be up to 10 copies of a template. The method established by the invention is characterized in that the whole reaction process is a closed pipe operation, thereby maximally reducing the pollution to the system. The whole process sequentially including the acquisition of a sample to be detected, the extraction of nucleic acid, the preparation of a fluorescent quantitative RT-PCR system and the representation of a reaction result can be completed within 2.5 hours, and the time required for detection is greatly shortened on the premise of ensuring the detection result.

Description

technical field [0001] The invention relates to the technical field of detection, in particular to a method capable of accurately, specifically and rapidly detecting foot-and-mouth disease virus group specificity, type O specificity and AsiaI specificity. Background technique [0002] Foot and mouth disease (FMD) is an acute, febrile and contact infectious disease caused by cloven-hoofed animals caused by FMDV (Foot and mouth disease virus) infection. The World Organization for Animal Health (OIE) classifies the disease as a Category A disease due to its rapid onset and high contagiousness, which can have severe impacts. Once the disease is detected, the transportation and export of corresponding livestock and livestock products will be prohibited, causing huge economic and political impact. In 2005, my country announced the outbreak of Asia I type FMD. In addition to the previous reports of FMD, there have been two serotypes of FMD in China. The outbreak and prevalence of...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12R1/93
Inventor 罗宝正王振全薄清如沙才华徐海聂廖秀云陈伟生李红星
Owner 珠海出入境检验检疫局检验检疫技术中心
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