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Primer for rapidly detecting carp edema virus and method thereof

A carp, rapid technology, applied in the direction of biochemical equipment and methods, microbiological determination/inspection, DNA/RNA fragments, etc., to achieve the effect of optimizing reaction conditions and avoiding pollution

Inactive Publication Date: 2019-03-19
广州动佰生物科技有限公司 +3
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

At present, there is no report on the use of realtime RPA to detect CEV in the world

Method used

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  • Primer for rapidly detecting carp edema virus and method thereof
  • Primer for rapidly detecting carp edema virus and method thereof
  • Primer for rapidly detecting carp edema virus and method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] 1. Virus extraction

[0032] DNA was extracted from the fish tissue infected with the carp edema virus according to the instructions of the TIANamp Genomic DNAKit kit from TIANGEN Company.

[0033] 2. Design of primers

[0034] According to the sequence published by CEV in GenBank (GenBank: MG550029.1), PCR primers were designed to amplify the P4a sequence. The reaction system is: rTaq enzyme 20 μL, upstream primer (SEQ ID NO: 1) 2 μL, downstream primer (SEQ ID NO: 2) 2 μL, template DNA (DNA extracted in step 1) 2 μL, dH 2 O 14 μL. The amplification program was: 95°C pre-denaturation for 5 min; 95°C denaturation for 30 sec, 55°C annealing for 30 sec, 72°C extension for 30 sec, a total of 35 cycles; 72°C extension for 10 min; 12°C storage.

[0035] The target product of PCR was recovered by agarose gel electrophoresis according to the E.Z.N.A. of OMEGA Company. GelExtraction Kit kit instructions for recovery.

[0036] 3. Cloning of the target gene

[0037] Mix 4.5...

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Abstract

The invention discloses a primer for rapidly detecting a carp edema virus and a method thereof. The invention aims to provide a primer and a method for rapidly detecting the carp edema virus (CEV) with advantages of high specificity, high sensitivity and excellent repeatability. According to the technical scheme, the primer comprises primers having nucleotide sequences shown as SEQ ID NO: 1 to SEQID NO: 3. The detection method comprises the following steps: 1) extracting viral genome DNA from a clinical sample; 2) taking the extracted sample DNA as a template, preparing an RPA (Recombinase Polymerase Amplification) reaction system by using a primer group according to Claim 1 and a probe according to Claim 2, and carrying out an RPA reaction by adopting a TwistAmp exo kit; and 3) placing areaction tube subjected to RPA in a fluorescence detector, and monitoring a fluorescence signal in real time, wherein the sample contains the CEV if an obvious fluorescence signal curve exists, otherwise the sample does not contain the CEV if no fluorescence signal curve exists. The invention belongs to the field of pathogen detection of animals.

Description

technical field [0001] The invention belongs to the field of animal pathogen detection, and in particular relates to a primer for rapid detection of carp edema virus and a method thereof. Background technique [0002] Carp edema virus disease (carp edema virus disease) is an important infectious disease infecting carp and koi carps, the pathogen is carp edema virus CEV, according to Oyamatsu and Hata et al. in 1997 and Miyazaki, Isshiki, and Katsuyuki et al proposed that the causative agent of the pathogen is assumed to be a double-stranded DNA virus of the Poxviridae family, clinically manifested as lethargy, skin hemorrhage with edema of the underlying tissues, enophthalmos and pale swollen gills, similar to Koi herpesvirus (Koi herpesvirus) herpesvirus KHV) clinical symptoms. [0003] Carp edema disease was first discovered in Japan in 1970. In recent years, CEV has occurred frequently, and explosive deaths have occurred in several European countries, India, Japan, China...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/6844C12N15/11
CPCC12Q1/6844C12Q1/701C12Q2531/119C12Q2563/107
Inventor 董建国胡明明王华南丛潇杨忠艳左晓
Owner 广州动佰生物科技有限公司
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