On-site rapid high-sensitivity detection kit of prawn infectivity muscle necrosis virus and detection method thereof

A detection kit, a technique for muscle necrosis, applied in biochemical equipment and methods, microbial determination/inspection, etc., can solve the problems of difficult and rapid detection, low sensitivity, limiting the popularization and application of RT-PCR detection methods, etc. The effect of highly sensitive detection, shortened detection time, and elimination of contamination risks

Inactive Publication Date: 2010-12-22
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 of IMNV by the antibody detection method is faster than the pathological section method, its sensitivity is low, and it is difficult to detect by the antibody method bef

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Embodiment 1 The detection kit of the present invention is made up of following parts (can detect the packing of 4 samples):

[0033] (1) Sampling tubes, 4 pieces, used to hold and grind samples to be tested;

[0034] (2) Rinsing tubes, 6 pieces, each tube is equipped with 1ml of distilled water;

[0035] (3) Nucleic acid denaturation tubes, 6 pieces, each containing 20 μL of TE buffer (containing 10 mM Tris-HCl and 1 mM EDTA, pH 8.0);

[0036] (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-HCl 20mM, KCl10mM, MgSO 4 2mM, (NH 4 ) 2 SO 4 10...

Embodiment 2

[0043] Example 2 A method for detecting shrimp infectious myonecrosis virus using the detection kit of the present invention.

[0044] (1) Take about 0.1 g of the sample prawn tissue and place it in the sampling tube, and quickly grind the sample to a pulp with a grinding rod;

[0045] (2) Dip the slurry sample with a grinding rod to fully wet the FTA diaphragm;

[0046] (3) Draw the quick-drying liquid onto the above-mentioned FTA membrane, and let the FTA membrane stand at room temperature for 10 minutes;

[0047] (4) Transfer the above-mentioned FTA membrane to the rinse tube with a toothpick, and shake the rinse tube vigorously for 3 minutes;

[0048] (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;

[0049] (6) Shake the amplification detection tube up and down repeatedly for 2 minutes to fully mix the amplification reaction solution with the nucleic acid dye on t...

Embodiment 3

[0052] Example 3 Use the kit of the present invention to detect the presence of viruses in aquaculture water.

[0053] Use the detection kit in embodiment 1, carry out according to the following steps:

[0054] (1) Filter seawater with 80 mesh sieve silk, and quickly grind the sample to slurry with a grinding rod;

[0055] (2) Dip the slurry sample with a grinding rod to fully wet the FTA diaphragm;

[0056] (3) Draw the quick-drying liquid onto the above-mentioned FTA membrane, and let the FTA membrane stand at room temperature for 10 minutes;

[0057] (4) Transfer the above-mentioned FTA membrane to the rinse tube with a toothpick, and shake the rinse tube vigorously for 3 minutes;

[0058] (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;

[0059] (6) Shake the amplification detection tube up and down repeatedly for 2 minutes to fully mix the amplification reaction ...

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PUM

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Abstract

The invention relates to an on-site rapid high-sensitivity detection kit of prawn infectivity muscle necrosis virus and a detection method thereof. The detection kit comprises a sampling tube, a rinsing tube filled with distilled water, a nucleate degeneration tube filled with a TE buffer solution, an amplification detection tube filled with amplification reaction liquid and nucleic acid dye, a negative control tube, a positive control tube, an FTA membrane, rapid drying liquid, and the like. Compared with the RT-PCR detection method, the detection method in the invention has the advantages of higher specificity, sensitivity and convenience, low cost, convenient use, more accurate and rapid detection and extremely sensitivity, is safe to both human and the environment and can substitute the traditional relevant detection method. The invention can be used in both the indoor laboratory and outdoor production field, has great significance for strengthening the epidemiological monitoring of prawn infectivity muscle necrosis virus, prawn cultivation waterbody monitoring and disease prevention and control and has great promotion and application value.

Description

technical field [0001] The invention relates to a marine biological pathogen detection technology, in particular to an on-site rapid and high-sensitivity detection kit for shrimp infectious myonecrosis virus (IMNV) and a detection method thereof. Background technique [0002] Infectious myonecrosis is a new viral infectious disease that endangers the production of prawns found in Brazil in 2002. In 2004, Dr. Donald V. Lightner of the University of Arizona found that the disease was caused by a new virus and Name it Infectious myonecrosis virus (IMNV for short). The novel virus is a double-stranded RNA virus belonging to the family Holoviridae. The susceptible prawn species of prawn infectious myonecrosis virus is Penaeus vannamei, mainly juvenile shrimp infected 60-80 days old. The virus can cause muscle tissue necrosis in the whole body of susceptible prawns. Generally, pathological death occurs slowly and the mortality rate is not high, but there are deaths throughout th...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68
Inventor 张庆利黄倢宋晓玲刘莉
Owner YELLOW SEA FISHERIES RES INST CHINESE ACAD OF FISHERIES SCI
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