Field fast high-sensitive detecting kit of monodon baculovirus and detecting method

A detection kit and baculovirus technology, which are applied to the on-site rapid and highly sensitive detection kit for baculovirus of Penaeus monodon and the detection field thereof, can solve the problem of restricting the popularization and application of PCR detection method, the difficulty of rapid detection by PCR detection method, and the high cost. problems such as long time, to achieve the effect of programming and standardization, shortening preparation time, and less error-prone

Inactive Publication Date: 2010-01-13
YELLOW SEA FISHERIES RES INST CHINESE ACAD OF FISHERIES SCI
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  • Summary
  • Abstract
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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 MBV by antibody detection method is faster than the pathological section method, its sensitivity is low, and it is difficult to detect by antibody method when MBV has not yet caused infection or the very early stage of infection; PCR of MBV Although the detection method overcomes the shortcomings of the previous methods and can achieve relatively fast and accurate detection of MBV under laboratory conditions, because conventional PCR detection requires expensive PCR machines, gel electrophoresis and imaging systems, PCR The detection method is difficult to be used for on-site rapid detection, which greatly limits the popularization and application of PCR detection method in on-site production

Method used

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Examples

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Effect test

Embodiment 1

[0034] Example 1: On-site rapid and highly sensitive detection kit for baculovirus of Penaeus monodon

[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, ...

Embodiment 2

[0046] Embodiment 2 isothermal amplification detection method of Penaeus monodon baculovirus 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 rinsing tube to the nucleic acid denaturation tube, and incubate at 95°C for 3 minutes to allow the DNA double strands to fully melt, and then quickly place it at room t...

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PUM

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Abstract

The invention relates to a field fast high-sensitive detecting kit of monodon baculovirus and a detecting method. The detecting kit comprises a sampling pipe, a rinsing pipe internally filled with distilled water, a nucleic acid denaturation pipe internally filled with TE cushion fluid, an amplified detecting pipe internally filled with amplified reaction liquid and nucleic acid dye, a negative tube, a positive tube, an FTA membrane, a fast drying liquid thereof, etc. Compared with the common PCR detecting method, the detecting method has higher specificity, sensitivity and convenience, has extreme low cost, nontoxicity, safety, convenient use, more exact detecting, fastness and extreme high sensitivity, and can replace the relevant existing detecting method such as a pathological section method, an electronic speculum observation method, an antibody detecting method and a PCR detecting method. The detecting kit and the detecting method not only can be used for an indoor laboratory, but also can be used for a wild production field, have significant meaning for enhancing epidemiological surveillance and disease control as well as prevention to the monodon baculovirus, and have extreme high population 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 Monodon Baculovirus (MBV) and a detection method thereof. Background technique [0002] Penaeus monodon-type Baculovirus, also known as Grass Shrimp Baculovirus and Penaeus monodon-type Baculovirus, is a double-stranded DNA baculovirus. MBV can infect Penaeus monodon (Penaeus monodon), long-haired prawns (P.penicillatus), Moji prawns (P.merguiensis), short ditch prawns (P.semisulcatus), new prawns and other farmed and wild prawns. When the virus is seriously infected, it can cause a large number of prawn larvae to die, causing economic losses in prawn nursery and breeding, and affecting the stability and sustainable development of the aquaculture industry. Therefore, it is of great significance to develop new technologies to detect MBV quickly, accurately and sensitively, to strengthen the quarantine of s...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12R1/93
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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