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Grass carp reovirus type I type II type III type iii RT-lamp fluorescence detection kit and detection method

A technology of RT-LAMP and reovirus, applied in the field of detection of target DNA fragments, can solve the problems of complicated operation, human safety, carcinogenicity, etc., and achieves high specificity and sensitivity, rapid and efficient amplification, and detection sensitivity. high effect

Active Publication Date: 2016-10-05
ZHEJIANG INST OF FRESH WATER FISHERIES
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  • Application Information

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Problems solved by technology

Electron microscope observation and cell culture virus isolation are classic methods, but they are cumbersome and time-consuming, and cannot distinguish grass carp reovirus infection from other viruses; immunological methods are poor in sensitivity and specificity, and there are gaps. window period, cross-reaction, serotype difference and other issues; RT-PCR method has great advantages, but it needs to perform gel electrophoresis on the amplified product, and the nucleic acid EB dye used in it is carcinogenic, causing potential safety hazards to humans.

Method used

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  • Grass carp reovirus type I type II type III type iii RT-lamp fluorescence detection kit and detection method
  • Grass carp reovirus type I type II type III type iii RT-lamp fluorescence detection kit and detection method
  • Grass carp reovirus type I type II type III type iii RT-lamp fluorescence detection kit and detection method

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Embodiment 1

[0054] 1) Extract RNA with RNA extraction reagent:

[0055] Take 0.03-0.05 g of grass carp spleen, liver or kidney tissue, grind it with a grinding rod in a centrifuge tube on ice, add 600 μL of lysate (lysate contains 5-15 mM Tris, 120-150 mM NaCl, 0.6-1% NP -40, 0.1-0.2% SDS, 3-5mol / L guanidine isothiocyanate, 1% β-mercaptoethanol, pH 8.0), continue to grind fully, let stand at room temperature for 10 minutes, and then add 900 μL of Mix with isopropanol, room temperature for 5 minutes, then centrifuge at 11000g for 10 minutes, remove the supernatant, add 800 μL cold isopropanol with a percentage concentration of 50% to the pellet, centrifuge at 11000g for 3 minutes, remove the supernatant, and use 70 Wash twice with % ethanol, dry at room temperature for 5-10 minutes, and resuspend with 30 μL deionized water.

[0056] 2) RT-LAMP fluorescence amplification of grass carp reovirus:

[0057] According to the number of RNA to be detected, set the required number of RT-LAMP reac...

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Abstract

The invention discloses a kit for detecting grass carp reovirus types I, II and III based on an RT-LAMP fluorescence technology and a method for detecting grass carp reovirus types I, II and III, and relates to the technical field of target DNA fragment detection. The kit includes a virus RNA extracting reagent and an RT-LAMP fluorescence reaction reagent, wherein the RT-LAMP fluorescence reaction reagent contains an RT-LAMP amplification used nucleic acid primer group and a fluorescent dye for detecting grass carp reovirus types I, II and III; the fluorescent dye is SYTO9. The kit provided by the invention has the advantages of being high in detection specificity, high in sensitivity and short in detection time (the result can be obtained in 15 minutes at the soonest), with huge advantages and innovativeness.

Description

technical field [0001] The invention belongs to the technical field of detection of target DNA fragments, in particular to a rapid and simultaneous detection of grass carp using reverse-transcription loop-mediated isothermal amplification (reverse-transcription, Loop-mediated isothermal amplification, RT-LAMP) technology and fluorescence color development technology Kits and detection methods for three genotypes of reovirus. Background technique [0002] Grass carp hemorrhagic disease virus is the first fish virus isolated in China. It belongs to the family Reoviridae and belongs to the genus Reovirus of aquatic animals. Named Grass Carp Reovirus (GCRV for short). Different strains exist in different regions. So far, 10 isolates have been reported. The virus mainly causes hemorrhagic disease in the fingerling stage of grass carp, the main freshwater aquaculture species in China, with a mortality rate of over 90%, causing huge losses to the aquaculture industry. At present...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12Q1/68C12R1/93
CPCC12Q1/6844C12Q1/70C12Q1/701C12Q2531/119C12Q2563/107
Inventor 沈锦玉潘晓艺郝贵杰袁雪梅藺凌云姚嘉赟徐洋尹文林
Owner ZHEJIANG INST OF FRESH WATER FISHERIES
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