Cyprinid herpesvirus detection kit and detection method thereof

The technology of carp herpes virus and detection kit is applied in the field of carp herpes virus detection kit and its detection, which can solve the problems of detection trouble and shortage, achieve high positive detection rate, improve positive detection rate and good detection effect Effect

Active Publication Date: 2014-09-10
ZHEJIANG INST OF FRESH WATER FISHERIES
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  • Summary
  • 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 c

Method used

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  • Cyprinid herpesvirus detection kit and detection method thereof
  • Cyprinid herpesvirus detection kit and detection method thereof
  • Cyprinid herpesvirus 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: CYHV PlF / 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: CYHV P2F / P2R

[0037] Swimming 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, including six 1.5mL centrifuge tubes, respectively equipped with 10 × reaction buffer, Taq enzyme (1U / μL), primer P1F / P1R each 10 μ M, primer P1F / P1R each 10 μ M , pure water, positive DNA (15 μg / mL).

[0040] The fo...

Embodiment 2

[0053] Embodiment 2: PCR method detects CYHV-2 DNA 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 repeated freeze-thaw samples, and use the total DNA extraction kit (QIAamp DNA Mini Kit) to extract 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 1U Taq DNA polymerase, 5 μL 10× reaction buffer and 10 pmol of specific primers (P1F / P1R). After mixing well, put it into the PCR amplification instrument for rea...

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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 Cyprinid herpesvieus (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 severe diseases in fish mainly include catherpes virus, carp pox virus (carp pox herpesvirus), goldfish hematopoietic Haematopoietic necrosis herpesvirus of goldfish (HNHV) and Koi herpesvirus (Koi herpesvirus), the latter three all belong to carp herpesvirus, carp pox virus belongs to Cyprinid herpesvirus 1 (CYHV-1), Goldfish hematopoietic necrosis virus belongs to Cyprinid herpesvirus 2 (CYHV-2), and koi herpesvirus belongs to Cyprinid herpesvirus 3 (CYHV-3). CYHV-1 can cause carp pox disease, which mainly harms carp, crucian carp and round-bellied crocodile, etc., ...

Claims

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Application Information

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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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