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Macrobrachium rosenbergii Nodavirus RT-LAMP-LFD detection method and detection kit thereof

A technology of RT-LAMP-LFD and Nodamura virus, which is applied in the direction of biochemical equipment and methods, and the determination/inspection of microorganisms, can solve the problems that the detection method cannot be directly applied in production, and achieve simple and fast extraction methods and high detection efficiency. The effect of high sensitivity and short detection time

Active Publication Date: 2013-11-27
ZHEJIANG INST OF FRESH WATER FISHERIES
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Problems solved by technology

[0007] Another object of the present invention is to provide a detection kit for the above method, which has strong specificity, high sensitivity, and is easy and fast to operate, overcomes the problem that existing detection methods cannot be directly applied in production, and is suitable for Roche Screening and Prevention of Macrobrachium White Body Disease

Method used

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  • Macrobrachium rosenbergii Nodavirus RT-LAMP-LFD detection method and detection kit thereof
  • Macrobrachium rosenbergii Nodavirus RT-LAMP-LFD detection method and detection kit thereof
  • Macrobrachium rosenbergii Nodavirus RT-LAMP-LFD detection method and detection kit thereof

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[0080] 1. Materials

[0081] Shrimp samples infected with Macrobrachium rosenbergii Nodamura virus were collected from a farm in Xihu District, Hangzhou City, Zhejiang Province; the primers and probes used were synthesized by Shanghai Sangon Biotechnology Company; the large fragment of Bst DNA polymerase was purchased from New England BIPolabs Company; reverse transcription Enzymes AMV and dNTP were purchased from promega; betaine and magnesium sulfate were purchased from sigma.

[0082] 2. Extraction of viral RNA from Macrobrachium rosenbergii samples:

[0083] (1) Take 25 mg of juvenile Macrobrachium rosenbergii or adult shrimp muscle tissue, add 500 μL of lysate, and grind with a disposable grinding rod. After thoroughly homogenizing, centrifuge at 12,000 rpm for 2 minutes, and absorb the supernatant to a new centrifuge tube;

[0084] (2) Add an equal volume (equal volume to the supernatant) of 70% absolute ethanol to a new centrifuge tube containing the supernatant, and m...

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Abstract

The invention discloses a Macrobrachium rosenbergii Nodavirus RT-LAMP-LFD (reverse transcription-loop-mediated isothermal amplification-lateral flow dipstick) detection kit and a detection method thereof. The detection method includes: according to the conserved region of a Macrobrachium rosenbergii Nodavirus (MrNV) sequence published by GenBank, designing the primers and probe needed by an MrNVRT-LAMP-LDF reaction system; employing a virus RNA extraction reagent prepared by the inventor to extract MrNV virus RNA, using the MrNVRT-LAMP reaction system established by the invention to perform detection, and according to the detection probe added to the reaction system, utilizing nucleic acid rapid test paper to determine the result at the end of the reaction. Compared with common detection technologies of the virus, the method provided in the invention has the characteristics of simplicity and rapidness, good specificity, and high sensitivity, can be used for field service of technical personnel and farmers on the production front line, and is also in favor of diagnosis and prevention of white tail disease.

Description

technical field [0001] The invention relates to the field of aquatic animal virus detection, in particular to an RT-LAMP-LFD detection method for Macrobrachium rosenbergii Nodamura virus and a detection kit thereof. Background technique [0002] Macrobrachium rosenbergii white body disease (white tail disease, WTD), also known as white tail disease, is caused by Macrobrachium rosenbergii Nodamura virus (Macrobrachium rosenbergii Nodavirus, MrNV) is a major hazard to desalinated M. rosenbergii seedlings. It is an infectious disease characterized by white spots, turbidity or whitening of the muscles of the diseased shrimp. The mortality rate is up to 100%, which has caused great harm to the M. rosenbergii aquaculture industry. great harm. At present, WTD is a worldwide disease of Macrobrachium rosenbergii, which has been listed as an OIE declared disease and a second-class disease of aquatic animals in my country. [0003] Studies have shown that the Macrobrachium rosenberg...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68
Inventor 林锋沈锦玉刘莉曹铮郝贵杰盛鹏程吴颖蕾
Owner ZHEJIANG INST OF FRESH WATER FISHERIES
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