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Method and kit for on-site rapid high sensitivity detection of fish nervous necrosis viruses

A technology for nerve necrosis and detection methods, applied in the direction of microorganism-based methods, biochemical equipment and methods, microorganism measurement/inspection, etc., can solve the problem that RT-PCR detection sensitivity is not high enough, cannot meet the requirements of disease prevention and control, and cannot be applied on-site and other problems, to achieve the effect of good detection effect, reliable detection results and guaranteed stability

Inactive Publication Date: 2015-10-28
YELLOW SEA FISHERIES RES INST CHINESE ACAD OF FISHERIES SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although these methods can detect NNV, there are some defects: conventional RT-PCR detection sensitivity is not high enough, up to 10 2 copy, which cannot meet the needs of disease prevention and control; fluorescent quantitative RT-PCR detection has high sensitivity, but requires expensive instruments and cannot be applied on-site; although LAMP detection has high sensitivity and can be used on-site, the dye method is used to detect the amplification product. Chromogenic, poor specificity, prone to false positives

Method used

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  • Method and kit for on-site rapid high sensitivity detection of fish nervous necrosis viruses
  • Method and kit for on-site rapid high sensitivity detection of fish nervous necrosis viruses
  • Method and kit for on-site rapid high sensitivity detection of fish nervous necrosis viruses

Examples

Experimental program
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Effect test

Embodiment 1

[0046] Example 1: Design and screening optimization of primer sets

[0047] Red spotted grouper nerve necrosis virus (RGNNV) is one of the main viral pathogens of marine fish, which is particularly harmful to the cultured grouper fry in Fujian, Guangdong, Hainan and Taiwan, and the mortality rate of diseased fish fry is as high as 60%. % to 100%. On the basis of analyzing the conserved region of the RGNNV genome sequence, the inventor finally determined 5 primers among more than 100 candidate primers after repeated attempts and condition optimization

[0048] CPA-LFD amplification primers not only ensure the specificity of detection, but also ensure its stability. The primer sequences are:

[0049] 5'-TGAGTACGCTCCTGGACCGTCTGCAGTTGTGGCAA

[0050] 1s:

[0051] GCTCGTTGG-3'

[0052] 2a: 5'-Fitc-TGAGTACGCTCCTGGACCGT-3'

[0053] 3a: 5'-Biotin-TGACTCAAACTGCTGTATTTCCAAG-3'

[0054] 4s: 5'-CCAGCAATACGACCGTCG-3'

[0055] 5a: 5'-GCAGGGCAAGTGTTACGCTCG-3'

[0056] In order to impro...

Embodiment 2

[0058] Embodiment 2: the sensitivity of CPA-LFD detection method

[0059] In order to determine the detection sensitivity of the CPA-LFD method and compare it with the existing conventional RT-PCR method, the following tests were carried out. Take 1 μL of 10-fold serial dilution (10 6 ~10 0 copies / μL) of RGNNV RNA was used as a template, and the two methods were used to amplify and detect respectively. Among them, CPA-LFD adopts the above-mentioned optimized reaction system and amplification temperature. Conventional RT-PCR adopts the method of Dalla Valle et al. (Dalla Valle L, Toffolo V, Lamprecht M, et al. Development of a sensitive and quantitative diagnostic assay for fish nervous necrosis virus based on two-target real-time PCR[J]. Vet Microbiol 2005,110:167-179.), using primers Q-RdRP-1 (5'-GTG TCC GGA GAG GTT AAG GAT G-3') and Q-RdRP-2 (5'-CTT GAA TTG ATC AAC GGT GAA CA-3'); reverse transcription at 42°C for 1h; PCR reaction program: 95°C for 1min; 95°C for 20sec, ...

Embodiment 3

[0061] Embodiment 3: detection kit of the present invention is made up of following reagent and and material (each kit can detect 4 samples)

[0062] (1) 4 pieces of diaphragms for sampling: respectively packed in 4 0.2ml plastic centrifuge tubes;

[0063] (2) 1 piece of positive control membrane: packed in a 0.2ml plastic centrifuge tube;

[0064] (3) One piece of negative control film: packed in one 0.2ml plastic centrifuge tube;

[0065] (4) 4 sample collection tubes;

[0066] (5) 1 cleaning tube: 0.8ml of absolute ethanol inside;

[0067] (6) 4 rinsing tubes: each with 1.0ml of distilled water;

[0068] (7) 6 NNV detection tubes: each contains 25 μl of amplification reaction solution;

[0069] (8) 4 grinding rods;

[0070] (9) 20 toothpicks;

[0071] (10) 1 straw;

[0072] (11) 6 fully enclosed observation boxes: each with a built-in nucleic acid detection test strip.

[0073] (12) 1 instruction manual.

[0074] (13) 1 packing box: put the above-mentioned material...

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Abstract

The invention provides a simple, fast, high sensitivity and easy operation method for detection of fish nervous necrosis viruses, and a portable on-site usage kit composed of detection reagents and devices. The primer set used by the method has nucleotide sequences of SEQ ID NO: 1-5. The method and kit are used for red-spotted grouper nervous necrosis virus detection for non-disease diagnosis and treatment, have the advantages of operation easiness, short time consumption and high sensitivity, are suitable for on-site detection and monitoring of fish nervous necrosis viruses in a culture enterprise and various-grade aquatic animal disease prevention and control parts, and have a high practical value and a good application prospect.

Description

technical field [0001] The invention belongs to the technical field of aquatic disease prevention and control, and in particular relates to an on-site rapid and highly sensitive detection method and a kit for fish nerve necrosis virus. Background technique [0002] Fish nerve necrosis virus (NNV) is a single-stranded RNA virus belonging to the family Nodaviridae and belonging to the genus Betanodavirus, and is the pathogen of viral neuronecrosis in fish. Red-spotted grouper nervous necrosis virus (RGNNV) is the most common fish neuronecrosis virus in my country, which can infect more than 30 kinds of fish such as grouper, tongue sole, and perch. The virus mainly infects larvae and juveniles, and the mortality rate can reach 100% in severe cases, causing serious economic losses. At present, there is no effective treatment for fish viral neuronecrosis, so rapid and highly sensitive detection of pathogens is particularly important for the prevention and treatment of the diseas...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12N15/11C12R1/93
CPCC12Q1/6844C12Q1/70C12Q1/701C12Q2531/119
Inventor 史成银粟子丹张庆利黄倢谢国驷杨冰李晨刘莉
Owner YELLOW SEA FISHERIES RES INST CHINESE ACAD OF FISHERIES SCI
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