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Macrobrachium rosenbergii Nodavirus NASBA-LFD detection method and detection kit thereof

A technology of Nodamura virus and detection kit, which is applied in the field of NASBA-LFD detection method of Macrobrachium rosenbergii Nodamura virus and its detection kit, can solve the problem of insufficient sensitivity and achieve high detection sensitivity, good specificity, and short detection time. short effect

Active Publication Date: 2014-12-03
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, simple and fast operation, overcomes the problem of insufficient sensitivity of existing detection methods, and is suitable for the detection of Macrobrachium rosenbergii. The screening and prevention of disease, as well as the parental traceability screening of diseased shrimp seedlings

Method used

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  • Macrobrachium rosenbergii Nodavirus NASBA-LFD detection method and detection kit thereof
  • Macrobrachium rosenbergii Nodavirus NASBA-LFD detection method and detection kit thereof
  • Macrobrachium rosenbergii Nodavirus NASBA-LFD detection method and detection kit thereof

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Embodiment approach

[0078] 1. Materials

[0079] 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; RNaseH, reverse transcriptase AMV, and dNTP were purchased from Promega Company; DMSO, Dithiothreitol, potassium chloride, etc. were purchased from sigma; BSA, RNase inhibitor, T7 RNA polymerase, and NTP were purchased from Fermentas.

[0080] 2. Extraction of viral RNA from Macrobrachium rosenbergii samples:

[0081] (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;

[0082] (2) Add an equal volume (equal volume to the supernatant) of 70% absolute ethanol to a new centrifuge tube containing the su...

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Abstract

The invention discloses a Macrobrachium rosenbergii Nodavirus NASBA-LFD (nuclear acid sequence-based amplification-lateral flow dipstick) detection method and a detection kit thereof. The detection method includes: according to a Macrobrachium rosenbergii Nodavirus (MrNV) sequence published by GenBank, screening a conserved region, and designing the primers and probe needed by an MrNV NASBA-LFD reaction system; employing a tissue RNA extraction reagent prepared by the inventor to extract the RNA of a to-be-detected sample, then utilizing the MrNV NASBA reaction system established by the invention to perform detection, and at the end of the reaction, determining the result according to nucleic acid rapid test paper. Compared with common detection technologies of the virus, the method and the kit provided in the invention have the characteristics of simplicity and rapidity, good specificity and high sensitivity, can be used for field service by technical personnel and farmers at the production front line, and are in favor of diagnosis prevention and pathogen blocking of Macrobrachium rosenbergii white tail disease.

Description

technical field [0001] The invention relates to the field of aquatic animal virus detection, in particular to a method for detecting Macrobrachium rosenbergii Nodamura virus NASBA-LFD 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 rosenbergii ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12Q1/68
Inventor 林锋沈锦玉刘莉钱冬曹铮王军毅郝贵杰盛鹏程袁雪梅吴颖蕾
Owner ZHEJIANG INST OF FRESH WATER FISHERIES
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