Kit and method for detecting Macrobrachium rosenbergii Nodavirus

A detection kit and technology for Noda virus, applied in biochemical equipment and methods, microbe determination/inspection, DNA/RNA fragments, etc. Rapid detection, long time-consuming and other problems, to achieve the effect of programming and standardization, shortening the preparation time, and reducing the cost of the experiment

Inactive Publication Date: 2010-11-03
YELLOW SEA FISHERIES RES INST CHINESE ACAD OF FISHERIES SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The pathological section method cannot directly detect the virus, but can only detect the histopathological signs of the disease, and it is also limited to the laboratory; although the electron microscope technology can directly observe the existence of virus particles, its operation is complicated and costly. The time is long and the accuracy is low; although the detection method of Nodavirus in Macrobrachium rosenbergii is faster than the pathological section method, its sensitivity is low, and the Nodavirus in Macrobrachium rosenbergii virus has not yet caused infection or is very e

Method used

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  • Kit and method for detecting Macrobrachium rosenbergii Nodavirus

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Experimental program
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Effect test

Embodiment 1

[0039] Detection kit of the present invention is made up of following parts (can detect the packing of 4 samples):

[0040] (1) Sampling tubes, 4 pieces, used to hold and grind samples to be tested;

[0041] (2) Rinsing tubes, 6 pieces, each tube is equipped with 1ml of distilled water;

[0042] (3) Nucleic acid denaturation tubes, 6 pieces, each filled with 20 μL of TE buffer solution (containing 10 mM Tris-HCl and 1 mM EDTA, pH 8.0);

[0043] (4) Amplification detection tubes, 6 pieces, each containing 24 μL of amplification reaction solution and 1 μL of dye. The upstream primer 2 and downstream primer 2 of the primers are each 0.2μM, dATP, dTTP, dGTP and dCTP are each 1.4mM, MgCl 2 8mM, Betaine (betaine) 1.2M, Tris-HCl 20mM, KCl 10mM, MgSO 4 2mM, (NH 4 ) 2 SO 4 10mM, Triton X-1000.1%, reverse transcriptase 5U, Bst DNA polymerase 8U; the dye is a complex formed by 50μM calcein and manganese chloride;

[0044] (5) Negative control tube, 1, containing FTA membrane witho...

Embodiment 2

[0050] Detection kit of the present invention also can be made up of following parts (can detect the packing of 4 samples):

[0051] (1) Sampling tubes, 4 pieces, used to hold and grind samples to be tested;

[0052] (2) Rinsing tubes, 6 pieces, each tube is equipped with 1ml of distilled water;

[0053] (3) Nucleic acid denaturation tubes, 6 pieces, each filled with 20 μL of TE buffer solution (containing 10 mM Tris-HCl and 1 mM EDTA, pH 8.0)

[0054] (4) Amplification detection tubes, 6 pieces, each tube is equipped with 25 μL of amplification reaction solution and 1 μL of nucleic acid dye, the components of the amplification reaction solution are as follows: each of the upstream primer 1 and downstream primer 1 of the amplification primers is 1.6 μM, 0.2 μM each of upstream primer 2 and downstream primer 2 of the amplification primers, 1.4 mM each of dATP, dTTP, dGTP, and dCTP, MgCl 2 8mM, Betaine (betaine) 1.2M, Tris-HCl20mM, KCl10mM, MgSO 4 2mM, (NH 4 ) 2 SO 4 10mM,...

Embodiment 3

[0061] The method using detection kit of the present invention to detect Macrobrachium rosenbergii Nodavirus is as follows:

[0062] (1) Take about 0.1 g of the sample tissue and place it in the sampling tube, and quickly grind the sample to a slurry state with a grinding rod;

[0063] (2) Dip the slurry sample with a grinding rod to fully wet the FTA diaphragm;

[0064] (3) Draw the quick-drying liquid onto the above-mentioned FTA membrane, and let the FTA membrane stand at room temperature for 10 minutes;

[0065] (4) Transfer the above-mentioned FTA membrane to the rinse tube with a toothpick, and shake the rinse tube vigorously for 3 minutes;

[0066] (5) Use a toothpick to transfer the FTA membrane in the nucleic acid denaturation tube to the amplification detection tube, and incubate at 57°C for 60 min;

[0067] (6) Shake the amplification detection tube up and down repeatedly for 2 minutes;

[0068] (7) Then shake the amplification detection tube downwards vigorously...

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Abstract

The invention relates to a kit and a method for detecting Macrobrachium rosenbergii Nodavirus. The kit comprises a sampling tube, a rinsing tube filled with distilled water, a nucleic acid denaturation tube filled with TE buffer solution, an amplification test tube filled with amplification reaction liquid and dyes, a negative control tube, a positive control tube, an FTA membrane, quick drying liquid for shortening time of preparing a sample to be detected, namely nucleic acid, and the like. The kit combines the advantages of isothermal amplication and quick dye detection, does not need precious instruments in the detection process, and develops by adopting a dye filling method to improve the detection reliability. The kit has the advantages of low detection cost, convenient use and safety for human bodies and environment, and can replace the conventional related detection methods. The kit can be used for production field in the open field, and has important promotion and application value of enhancing the monitoring of the Macrobrachium rosenbergii Nodavirus and preventing the large-scale outbreak of the Macrobrachium rosenbergii Nodavirus.

Description

technical field [0001] The invention relates to a marine biological pathogen detection technology, in particular to a kit and a detection method for Macrobrachium rosenbergii Nodavirus (MrNV for short). Background technique [0002] Macrobrachium rosenbergii Nodavirus (MrNV) is a non-enveloped RNA virus with a size of about 26-27nm and an icosahedral shape. Its genome consists of two single-stranded RNAs; it is often accompanied by Macrobrachium rosenbergii Nodavirus There is also a satellite virus called Extra smallvirus (XSV for short), which is about 14-16nm in size and whose genome consists of a segment of RNA. The virus mainly infects Macrobrachium rosenbergii, and the infected parts are mainly the striated muscle of the shrimp and the connective tissue of the hepatopancreas. The body trunk of the infected shrimp is milky and opaque, so it is also called white tail disease. , referred to as WTD), muscle white disease (White muscledisease, referred to as WMD), the virus...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12N15/11
Inventor 张庆利黄倢王勤涛杨冰
Owner YELLOW SEA FISHERIES RES INST CHINESE ACAD OF FISHERIES SCI
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