Grass carp reovirus (GCRV) reverse transcription polymerase chain reaction nucleic acid detection method

A technology of reverse transcription polymerase and reovirus, which is applied in the field of virology, can solve the problems of poor sensitivity and specificity, time-consuming detection methods, and inability to adapt to clinical detection accuracy, speed, and sensitivity, and achieve strong practicability , the effect of improving the sensitivity

Inactive Publication Date: 2010-01-20
WUHAN INST OF VIROLOGY CHINESE ACADEMY OF SCI
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Problems solved by technology

[0004] At present, although there are serological detection methods such as ELISA and RT-PCR related detection methods in the detection of GCRV pathogens, the existing detection methods are tim...

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Embodiment Construction

[0032] The present invention will be further described below in conjunction with embodiment.

[0033] 1. Preparation of samples to be tested and extraction of total RNA from virus cells

[0034] 1. Preparation of samples to be tested:

[0035] Take 0.5-1ml of normal cells, virus-infected cells, normal fish tissue or a certain amount of diseased fish tissue (100-150mg) as the RNA extraction material of the sample to be tested. Wherein, diseased or normal fish body tissue is added 3 times volume 1 * PBS (phosphate-buffered saline, abbreviated PBS) in fish body tissue sample according to weight volume ratio, and its composition is: 137mMNaCl, 2.7mM KCl, 8.1mMNa2HPO4, 1.5mM KH2PO4, pH 7.5), repeated grinding. The ground tissue samples to be tested and the cells to be tested were repeatedly frozen and thawed 3 times, that is, they were quickly frozen in a low-temperature refrigerator at -20°C and quickly thawed at 37°C to prepare a homogenate to be tested. After the obtained hom...

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Abstract

The invention provides a grass carp reovirus (GCRV) reverse transcription polymerase chain reaction nucleic acid detection method, comprising the following steps: directly extracting the total RNA in the samples to be detected by adopting a RNA adsorption column centrifugal method; detecting the s10 gene segment of the structural protein VP 7 with the code of GCRV of the outer capsid by reverse transcription chain reaction. The sample to be detected comprises normal cells, cells infected by virus, normal fish tissues and tissues of infected fish. The detection method has the advantages that the total RNA can be extracted from the samples to be detected by the column centrifugal method and RT-PCR amplification can be carried out by only needing a small amount of tissue of the samples to be detected; the minimum detection sensitivity is 10<-6>ng, and the required time is only 4-5 hours; the detection method is simple and reliable, high in sensitivity and strong in specificity; and the result is prospective to be used for GCRV clinical quick diagnosis and entry-exit customs quarantine of fish fry.

Description

technical field [0001] The present invention relates to the field of virology, particularly a kind of grass carp reovirus (Grass carp reovirus, abbreviated GCRV, also known as Grass carp hemorrhagic virus, abbreviated GCHV) reverse transcription polymerase chain reaction (abbreviated RT-PCR ) nucleic acid detection method. The present invention is based on the research results of grass carp reovirus Hunan Shaoyang strain GCRV873 (also known as grass carp hemorrhagic disease virus Hunan Shaoyang strain GCHV873). date, and the deposit number is CCTCC NO: V200414. Background technique [0002] Aquatic animal viral diseases are the main pathogens that seriously endanger aquaculture and disease transmission. With the rapid development of aquaculture intensive farming, viral epidemics occur frequently, and these diseases have caused great harm to aquaculture worldwide. GCRV belongs to the genus Reovirus of aquatic animals in the Reoviridae family. The pathogen once caused a lar...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12R1/93
Inventor 方勤张岚岚丁清泉
Owner WUHAN INST OF VIROLOGY CHINESE ACADEMY OF SCI
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