Primer group and kit for carrying out spot quick detection on shrimp covert mortality nodavirus

A Nodamura virus and primer set technology, applied in microorganisms, recombinant DNA technology, microorganism-based methods, etc., can solve problems such as non-detection, and achieve the effect of less error-prone, standardized operation, and realization of programming.

Inactive Publication Date: 2014-12-17
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 features of the disease are similar to those caused by shrimp myonecrosis virus (Infectious myonecrosis virus, IMNV), vannamei nodavirus (Penaeus vannamei nodavirus, PvNV) and Macrobrachium rosenbergii nodavirus (Macrobrachium rosenbergii nodavirus, MrNV) The pathological features were similar, but the presence of IMNV, PvNV and MrNV could not be detected in the affected shrimp

Method used

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  • Primer group and kit for carrying out spot quick detection on shrimp covert mortality nodavirus
  • Primer group and kit for carrying out spot quick detection on shrimp covert mortality nodavirus
  • Primer group and kit for carrying out spot quick detection on shrimp covert mortality nodavirus

Examples

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Effect test

Embodiment 1

[0040] Example 1 Design and screening of on-site rapid detection primers for shrimp stealing dead wild field virus

[0041] First of all, for the CMNV protein A gene cloned from Shandong, Hebei, Fujian and other places in my country, the CMNV protein A gene was compared with the variation of the above sequence using the NCBI online program Blastn and the molecular biology software BioEitd 7.0, and the CMNV protein A gene was selected. Amplification primers were designed with the software Lamp Designer 1.02 for the conserved region sequences, and a total of 4 sets of primers were designed (Table 1).

[0042] Table 1: Amplification primers designed based on CMNV protein A gene

[0043]

[0044]

[0045] Using the 4 sets of primer combinations designed and synthesized above, the reaction system was formulated according to the following components and the amplification reaction was carried out (25 μL / reaction, referred to as method 1): each of the upstream primer 1 and downst...

Embodiment 2

[0049] Embodiment 2 Detection kit of the present invention is made up of following parts (can detect the packing of 4 samples):

[0050] (1) Sampling tubes, 4 pieces, used to hold and grind samples to be tested;

[0051] (2) Rinsing tubes, 6 pieces, each tube is equipped with 1ml of distilled water;

[0052] (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);

[0053] (4) Amplification detection tubes, 6 pieces, each containing 24 μL of amplification reaction solution and 1 μL of dye. 0.8 μM each for melting primers 1 and 2 of the amplification primers, 0.2 μM each for upstream primer 2 and downstream primer 2 of the amplification primers, 1.4 mM each for dATP, dTTP, dGTP, and dCTP, MgCl 2 2mM, Betaine (betaine) 1.2M, Tris-HCl 20mM, KCl 10mM, MgSO 4 6mM, (NH 4 ) 2 SO 4 10mM, Triton X-100 0.1%, reverse transcriptase 5U, Bst DNA polymerase 8U; the dye is a complex formed by 50μM calcein and ma...

Embodiment 3

[0060] Embodiment 3 The detection kit of the present invention can also be made up of following parts (can detect the packing of 4 samples):

[0061] (1) Sampling tubes, 4 pieces, used to hold and grind samples to be tested;

[0062] (2) Rinsing tubes, 6 pieces, each tube is equipped with 1ml of distilled water;

[0063] (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);

[0064] (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, Melting primers 1 and 2 of amplification primers are 0.8 μM each, amplification primers upstream primer 2 and downstream primer 2 are each 0.2 μM, dATP, dTTP, dGTP and dCTP are each 1.4 mM, MgCl 2 8mM, Betaine (betain...

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Abstract

The invention relates to a primer group and a high-sensitivity detection kit for carrying out spot quick detection on shrimp covert mortality nodavirus. The primer group comprises six primers. The high-sensitivity detection kit comprises a sampling tube, a rinsing tube internally filled with distilled water, a nucleic acid denaturation tube internally filled with a TE buffer solution, an amplification detection tube internally filled with an amplification reaction liquid and a dye, a negative control tube, a positive control tube, an FTA membrane and a quick drying fluid thereof. The primer group can be used for specifically distinguishing and efficiently amplifying target nucleic acid of detected CMNV (covert mortality nodavirus). The detecting method adopting the high-sensitivity detection kit has specificity, sensitivity and convenience which are higher than those of an electron microscope detecting method, a histopathology detecting method, a virus isolation detecting method and the like, is low in cost, convenient to use, relatively accurate and quick in detection, and extremely high in sensitivity.

Description

technical field [0001] The invention belongs to the technical field of detection of marine biological pathogens, and in particular relates to a primer set and a kit for field rapid detection of covert mortality nodavirus (CMNV) in shrimps. Background technique [0002] Since 2009, a large number of farmed prawns have died in succession in Vietnam, Malaysia and many southern provinces of my country. The food intake of prawns in the affected ponds does not increase with the increase of the breeding time, and a considerable part of them decreases with the extension of the breeding time; observations on the breeding site usually show that there is no obvious abnormality in the activity of the affected prawns. Check the bait table Dead prawns can be found at the bottom of the pond and the bottom of the culture tank; when the disease is severe, the number of dead prawns can increase greatly in just a few days. Shrimp farmers call it "covert mortality disease", "covert mortality d...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12N15/11C12R1/93
CPCC12Q1/701C12Q1/6851C12Q1/70
Inventor 张庆利黄倢杨昊霖刘爽
Owner YELLOW SEA FISHERIES RES INST CHINESE ACAD OF FISHERIES SCI
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