On-site rapid high-sensitivity detection kit and detection method of taura syndrome virus (TSV)

A detection kit and technology for Taola virus, which are applied in the directions of microorganism-based methods, biochemical equipment and methods, and microbial determination/inspection, etc., can solve the limitation of the popularization and application of RT-PCR detection methods, which are time-consuming and difficult to quickly Detection and other issues to achieve the effect of eliminating pollution risks, eliminating pollution, rapid and highly sensitive detection

Inactive Publication Date: 2011-02-09
YELLOW SEA FISHERIES RES INST CHINESE ACAD OF FISHERIES SCI
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  • Summary
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  • 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 TSV by antibody detection method is faster than pathological section method, its sensitivity is low, and it is difficult to detect by antibody method before TSV has caused infection or the very early stage of infection; the RT of TSV -PCR detection method, although it overcomes the shortcomings of the previous methods, it can achieve relatively fast and accurate detection of TSV under laboratory conditions, but because conventional RT-PCR detection requires expensive PCR machines, gel electrophoresis and imaging system, making it difficult for RT-PCR detection method to be used for rapid detection on the spot, which greatly limits the application of RT-PCR detection method in production

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1: On-site Rapid and Highly Sensitive Detection Kit for Shrimp Taura Virus

[0035] Test kit of the present invention is made up of following parts (can detect the packing of 4 samples):

[0036] (1) Sampling tubes, 4 pieces, used to hold and grind samples to be tested;

[0037] (2) Rinsing tubes, 6 pieces, each tube is equipped with 1ml of distilled water;

[0038] (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);

[0039] (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 (bet...

Embodiment 2

[0046] Embodiment 2 isothermal amplification detection method of prawn taura virus of the present invention

[0047] Use the detection kit in embodiment 1, carry out according to the following steps:

[0048] (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;

[0049] (2) Dip the slurry sample with a grinding rod to fully wet the FTA diaphragm;

[0050] (3) Draw the quick-drying liquid onto the above-mentioned FTA membrane, and let the FTA membrane stand at room temperature for 10 minutes;

[0051] (4) Transfer the above-mentioned FTA membrane to the rinse tube with a toothpick, and shake the rinse tube vigorously for 3 minutes;

[0052] (5) Use a toothpick to transfer the FTA membrane in the above-mentioned nucleic acid denaturation tube to the amplification detection tube, and place the amplification detection tube at 57° C. for 60 min;

[0053] (6) Shake the amplification detectio...

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Abstract

The invention relates to an on-site rapid high-sensitivity detection kit and a detection method of taura syndrome virus (TSV). The on-site rapid high-sensitivity detection kit comprises a sampling tube, a rinsing tube filled with distilled water, a nucleic acid denaturation tube filled with TE buffer liquid, an amplification detection tube filled with amplification reaction liquid and nucleic acid dye, a negative control tube, a positive control tube, a FTA membrane, quick drying liquid, and the like. The detection method has higher specificity, sensitivity and convenience than the TR-PCR detection method and has the advantages of low cost, no toxicity, high safety, convenient use, more accurate and quicker detection and high sensitivity and can replace the prior related detection methods, such as a pathological section method, an electron-microscopic observation method, an antibody detection method and a TR-PCR detection method. The on-site rapid high-sensitivity detection kit and the detection method can be both used in an indoor laboratory and a production site in the field, have significance for strengthening the TSV epidemiological monitoring and the disease control and prevention and have high popularization 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 prawn Taura syndrome virus (TSV for short) and a detection method thereof. Background technique [0002] Shrimp taura virus is a single-stranded RNA virus belonging to the Dicistroviridae family. The virus mainly infects Penaeus vannamei and Penaeus stylirostru, and can also infect Penaeus chinensis and Penaeus japonicus. (Penaeus japonicus), Penaeus monodon (Penaeus monodon), etc. Among them, Litopenaeus vannamei is the most sensitive, and the mortality rate of prawns after infection with the virus is as high as 40-95%. The virus once caused huge losses in the American shrimp farming industry. In recent years, with the rapid expansion of the culture scale of Litopenaeus vannamei in my country, the risk of large-scale occurrence of TSV has gradually increased. During the national epidemiological investiga...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12Q1/68C12R1/93
Inventor 张庆利黄倢杨冰宋晓玲
Owner YELLOW SEA FISHERIES RES INST CHINESE ACAD OF FISHERIES SCI
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