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Grass carp reovirus type I type II type III type rt-lamp visual detection kit and its detection method

A technology of RT-LAMP and reovirus, applied in the field of detection of target DNA fragments, can solve the problems of immunological methods such as poor sensitivity and specificity, carcinogenicity, cumbersome operation, etc., to reduce background influence, enhance specificity, Amplify fast and efficient effects

Active Publication Date: 2017-01-04
ZHEJIANG INST OF FRESH WATER FISHERIES
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  • Abstract
  • Description
  • Claims
  • 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.

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  • Grass carp reovirus type I type II type III type rt-lamp visual detection kit and its detection method
  • Grass carp reovirus type I type II type III type rt-lamp visual detection kit and its detection method

Examples

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Embodiment 1

[0052] 1) Extract RNA with RNA extraction reagent:

[0053] 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 (containing 8-12 mM Tris, 0.8-1.2 mM EDTA, 0.6-1% NP-40 , 0.7~0.9% SDS, 3~5mol / L guanidine isothiocyanate, 40~44 mM sodium acetate, 0.2 mM β-mercaptoethanol, pH 4.0), continue to grind fully, and after standing at room temperature for 10 minutes, 11000g Centrifuge for 10 minutes, take the supernatant into a new centrifuge tube, then add 900 μL of isopropanol to mix, room temperature for 5 minutes, then centrifuge at 11,000 g for 10 minutes, remove the supernatant, add 800 μL of 50% isopropanol to the pellet Cold isopropanol, centrifuged at 11000g for 3 minutes, removed the supernatant, washed the precipitate twice with 70% ethanol, dried at room temperature for 5-10min, and then resuspended in 30 μL deionized water.

[0054] 2) RT-LAMP visualization amplification of grass carp ...

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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 visualization 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, an RT-LAMP visualization reaction reagent, a 1.5 ml centrifugal tube and an RT-LAMP reaction tube, wherein the RT-LAMP visualization reaction reagent contains an RT-LAMP amplification used nucleic acid primer group and a visualization dye for detecting grass carp reovirus types I, II and III. The kit provided by the invention has the advantages that the kit is high in detection specificity, high in sensitivity and short in detection time; the required instruments are simple; the detection result can be visually determined; the kit has huge advantages and innovativeness.

Description

technical field [0001] The invention relates 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 visual 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, near...

Claims

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Application Information

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