Micro-drop type digital PCR method for detecting fish nervous necrosis virus and corresponding kit

A nerve necrosis, droplet technology, applied in microorganism-based methods, biochemical equipment and methods, microorganisms, etc., to achieve the effect of good application prospects and high sensitivity

Pending Publication Date: 2021-11-12
SUN YAT SEN UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This method is less traumatic to farmed fish, especially the parents, and ha

Method used

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  • Micro-drop type digital PCR method for detecting fish nervous necrosis virus and corresponding kit
  • Micro-drop type digital PCR method for detecting fish nervous necrosis virus and corresponding kit
  • Micro-drop type digital PCR method for detecting fish nervous necrosis virus and corresponding kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1 Micro-droplet digital PCR and RT-PCR sensitivity and accuracy comparison in NNV detection

[0035] (1) Design and specificity verification of droplet digital PCR primers and probes

[0036]The primers used in the experiment were designed and optimized by Primer 5.0, and the 5' and 3' ends of the probe carried luminescent groups and quenching groups, respectively. First, download the seabass genome data (NCBI accession number: PRJNA408177), establish the seabass genome database, and the NNV primer sequence designed by local BLAST analysis has no complementary sequence in the seabass genome. The laboratory isolated NNV virus from diseased sea bass juveniles in the Pearl River Estuary breeding area of ​​Guangdong Province. After infecting sea bass brain cells with NNV virus, the RNA was extracted, and the NNV template was obtained after reverse transcription. NNV was amplified by PCR. pc-RDRP was obtained by inserting the genome fragment of pcDNA3.1 (Invitrogen)...

Embodiment 2

[0045] Example 2 Detection of NNV-challenged sea bass blood samples and wild-cultured sea bass parent blood samples by micro-droplet digital PCR

[0046] (1) Laboratory sea bass challenge

[0047] NNV was isolated from diseased juvenile seabass in the Pearl River Estuary breeding area of ​​Guangdong Province. After infecting seabass brain cells with NNV, it was repeatedly frozen and thawed at -80°C three times, centrifuged and filtered through a 0.22 μm filter membrane to obtain NNV suspension. The prepared NNV suspension was stored at -80°C for future use. 100 species of sea bass, weighing about 40g, were purchased in Doumen District, Zhuhai. Firstly, it was detected that they did not carry NNV virus. They were cultured in laboratory circulating water for two weeks, during which they were fed normally, twice a day. Two weeks later, the sea bass were anesthetized with eugenol, and the prepared NNV suspension was injected into the sea bass's thorax at the pectoral fin. Two e...

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Abstract

The invention belongs to the technical field of detection of fish nervous necrosis virus, and particularly relates to a micro-drop type digital PCR method for detecting fish nervous necrosis virus and a corresponding kit. In order to develop a nervous necrosis virus detection method with high sensitivity, the invention establishes the micro-drop type digital PCR method for detecting fish nervous necrosis virus, namely after a blood sample of a fish to be detected is collected, total RNA of the blood sample is extracted, and cDNA is synthesized; and then the cDNA is used as a template, and micro-drop type digital PCR is adopted, so that whether the fish to be detected is infected or carries NNV can be accurately detected. The micro-drop type digital PCR detection method provided by the invention is high in sensitivity, and has a good application prospect in breeding or selection of high-quality parents of cultured fishes.

Description

technical field [0001] The invention belongs to the technical field of fish neuronecrosis virus detection, in particular to a droplet digital PCR method and a corresponding kit for detecting fish neuronecrosis virus. Background technique [0002] Fish neuronecrosis virus (Nervous necrosis virus, NNV) is a viral pathogen that can infect more than 40 important marine and freshwater economic fishes, such as sea bass, turbot and grouper. NNV infection can cause fish epidemic viral encephalopathy and retinopathy, and the lethal rate to larvae and juveniles is as high as 100%. The International Organization for Epizootics (OIE) has listed this disease as an important fish disease. [0003] NNV virus classification belongs to Nodaviridae (Nodaviridae) Beta Nodaviruses (Betanodaviruses), is a non-enveloped single-stranded positive-sense icosahedral RNA virus with a diameter of 23-25nm. The genome contains two RNA molecules, RNA1 and RNA2. Among them, RNA1 encodes RNA-dependent RNA...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/6851C12R1/93
CPCC12Q1/701C12Q1/6851C12Q2531/113C12Q2563/159
Inventor 贾坤同李勇卢小兵黄聪灵易梅生古群红
Owner SUN YAT SEN UNIV
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