Carp herpes virus detection kit and detection method thereof

A detection kit and carp herpes virus technology, applied in the carp herpes virus detection kit and its detection field, can solve problems such as lack and troublesome detection, achieve a high positive detection rate, increase the positive detection rate, and reduce false detection. negative effect

Active Publication Date: 2016-01-20
ZHEJIANG INST OF FRESH WATER FISHERIES
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Since the above three carp herpes viruses can infect most of the cyprinidae fish, and because there is currently no detection method that can detect the above three carp herpes viruses at the same time, this will cause trouble for the simultaneous detection of the above three pathogens

Method used

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  • Carp herpes virus detection kit and detection method thereof
  • Carp herpes virus detection kit and detection method thereof
  • Carp herpes virus detection kit and detection method thereof

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Experimental program
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Effect test

Embodiment

[0031] Embodiment 1: PCR method detects the genomic DNA of carp herpes virus positive template

[0032] A. Two pairs of common primers for carp herpes virus were synthesized and submitted to Nanjing GenScript Biotechnology Co., Ltd.:

[0033] a: CYHVP1F / P1R

[0034] Swimming primer P1F is 5'-CGCTGAGMATGTCGTCRTTGTC-3', the sequence is shown in SEQ ID NO: 1,

[0035] The downstream primer P1R is 5'-GGSACCAAYCAGATTCACATCA-3', the sequence of which is shown in SEQ ID NO: 2;

[0036] b:CYHVP2F / P2R

[0037] The primer P2F is 5'-CTTSACCRTRTTCATCTGRGCCA-3', the sequence is shown in SEQ ID NO: 3,

[0038] The downstream primer P2R is 5'-CTACAAGCCCGACCARATAGAGA-3', the sequence of which is shown in SEQ ID NO:4.

[0039] B, a carp herpes virus detection kit, comprising 6 centrifuge tubes of 1.5mL, respectively equipped with 10 × reaction buffer, Taq enzyme (1U / μL), each 10 μ M of primer P1F / P1R, each 10 μ M of primer P1F / P1R , pure water, positive DNA (15 μg / mL).

[0040] The formula...

Embodiment 2

[0053] Embodiment 2: PCR method detects CYHV-2DNA in repeatedly freezing and thawing samples

[0054] Take virus-positive samples and pack them into EP tubes, then freeze and thaw repeatedly at -80°C for 30 minutes / room temperature for 30 minutes. DNA was extracted from samples with different freezing and thawing times, and nested amplification was performed on each extracted nucleic acid sample by using new CYHV primers (P1F / P1R, P2F / P2R).

[0055] 1. DNA extraction

[0056] Take 0.1 g of the repeated freeze-thaw sample, use the total DNA extraction kit (QIA&DNAMiniKit), and extract the total DNA according to the kit instructions.

[0057] 2. Nested PCR reaction

[0058] The first round of PCR: Take 1 μL of the above-mentioned total DNA and add it to a reaction system with a total volume of 50 μL, which contains 1UTaq DNA polymerase, 5 μL of 10× reaction buffer and 10 pmol of specific primers (P1F / P1R). After mixing well, put it into the PCR amplification instrument for re...

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Abstract

The invention relates to a Cyprinid herpesvirus detection kit and a detection method thereof, belonging to the technical field of virus genome detection. The detection kit comprises a 10* reaction buffer solution, a 1U / mu L Taq enzyme, a primer set composed of 10 mu M of P1F and 10 mu M of P1R, a primer set composed of 10 mu M of P2F and 10 mu M of P2R, pure water and a 15 mu g / mL positive DNA. The kit can be used for simultaneously detecting genomes of carp pox herpesvirus, haematopoietic necrosis herpesvirus of goldfish and Koi herpesvirus. The method has the advantages of excellent visuality, favorable sensitivity and high specificity, has favorable effects on detecting trace amounts of Cyprinid herpesvirus genome in the long-storage-time specimen, and can be used for Cyprinid-herpesvirus-infected laboratory detection and Cyprinid herpesvirus molecule epidemiological survey.

Description

technical field [0001] The invention belongs to the technical field of virus genome detection, and in particular relates to a detection kit for Cyprinidherpesvieus (CYHV) and a detection method thereof. Background technique [0002] Herpes viruses are linear double-stranded DNA viruses that can cause a variety of fatal diseases in mammals, poultry and aquatic animals. Among them, the herpes viruses that cause serious diseases in fish mainly include catherpes virus, carppoxherpes virus, and necrosis of hematopoietic tissue in goldfish. Virus (haematopoietic necrosisherpesvirus of goldfish, HNHV) and Koiherpesvirus (Koiherpesvirus), the latter three belong to carp herpesvirus, carp poxvirus belongs to Cyprinidherpesvirus 1 (Cyprinidherpesvirus 1, CYHV-1), goldfish hematopoietic necrosis virus belongs to carp herpesvirus Type 2 (Cyprinidherpesvirus 2, CYHV-2), Koi herpesvirus belongs to Cyprinidherpesvirus 3 (Cyprinidherpesvirus 3, CYHV-3). CYHV-1 can cause carp pox disease, w...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12Q1/68
CPCC12Q1/6848C12Q1/686C12Q1/70C12Q2531/113C12Q2549/119
Inventor 潘晓艺沈锦玉蔺凌云袁雪梅郝贵杰徐洋姚嘉赟尹文林
Owner ZHEJIANG INST OF FRESH WATER FISHERIES
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