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Cyprinid herpes virus type 2 CPA detection primer and application

A carp herpes virus and detection primer technology, which is applied in the field of fish virus detection, achieves the effects of good specificity, simple determination and low detection cost

Active Publication Date: 2016-11-23
YANGTZE RIVER FISHERIES RES INST CHINESE ACAD OF FISHERY SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This type of technology relies on expensive experimental instruments such as PCR machines and real-time fluorescent quantitative PCR machines. Although the detection results are accurate and reliable, they are only suitable for pathogen detection in professional laboratories

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] A detection kit for carp herpesvirus type 2 CPA, comprising:

[0038] Reaction Buffer; Bst DNA Polymerase (NEB); dNTPs (Takara); Cross Primer 1S; Probe Primer 2A, 3A; Stripping Primer 4S, 5A; MgSO 4 (Promega); Betaine (Sigma); nucleic acid detection test strips (Hangzhou Ustar Company).

[0039] 1S:5'-GTCCCTGGCAGAAATAAGCTGCAGCCATAGGACCTTCCACAT-3'

[0040] 2A: 5'-(Biotin)GTCCCTGGCAGAAATAAG-3'

[0041] 3A: 5'-(FITC)TGCCGTGCTCCAGTTTCA-3'

[0042] 4S: 5'-ATGTGTTGTGTTGTGTTTGTG-3'

[0043] 5A: 5'-ACTCGGTTTGATGGGACT-3'

Embodiment 2

[0045] Optimization of different primer concentrations and ratios in a carp herpesvirus type 2 CPA detection kit:

[0046] 1. Take the sample to be tested and extract the virus DNA:

[0047] Taking carp herpes virus type 2 (CyHV-2) (Xu Jin, Zeng Lingbing, Yang De, et al. Isolation and identification of CyHV-2 Wuhan strain[J]. Chinese Fisheries Science, 2013,20(6):1303- 1309) infected crucian carp disease fish gills, spleen, kidney and other tissue homogenate 300μL, with Reagents or Viral DNA Kit kits were used to extract DNA according to the instructions, and finally dissolved in 30 μL of sterilized water, and stored at -20°C for later use.

[0048] 2. The reaction system of CPA amplification:

[0049] Using a 25 μL reaction system, add 1 μL of viral DNA template to the PCR tube, ReactionBuffer 2.5μL, cross primer 1S, probe primer 2A, 3A, stripping primer 4S, 5A, MgSO 4 6.0mM, dNTPs 0.8mM, Betaine 0.7M, Bst DNA polymerase 8U, nuclease-free water to make up to 25μL. At t...

Embodiment 3

[0061] Different concentrations of MgSO in a carp herpesvirus type 2 CPA detection kit 4 Optimization:

[0062] 1. Take the sample to be tested and extract the virus DNA:

[0063] The preparation method is the same as in Example 2.

[0064] 2. The reaction system of CPA amplification:

[0065] Using a 25 μL reaction system, add 1 μL of viral DNA template to the PCR tube, ReactionBuffer 2.5μL, 1S 1.0μM, 2A / 3A 0.4μM, 4S / 5A 0.4μM, MgSO 4 , dNTPs 0.8mM, Betaine 0.7M, Bst DNA polymerase 8U, nuclease-free water to make up to 25μL. At the same time set a blank control.

[0066] where MgSO 4 The concentrations were 4.0, 6.0, 8.0, 10.0, 12.0 mM, respectively.

[0067] 3. Reaction conditions for CPA amplification:

[0068] The reaction tube was incubated at 63°C for 60min and then inactivated at 80°C for 2min.

[0069] 4. Judgment of test results:

[0070] Take 5 μL of the amplified product, electrophoresis with 2.0% agarose gel, and place it in a gel imaging system for imagi...

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Abstract

The invention discloses a cyprinid herpes virus type 2 CPA detection primer and application. A cyprinid herpes virus type 2 CPA detection kit comprises 10*ThermoPol Reaction Buffer, Bst DNA polymerase, dNTPs, a cross primer 1S, probe primers 2A and 3A, stripping primers 4S and 5A, MgSO4, Betaine and nucleic acid test strips. The primer has the advantages that of being easy and convenient to use, rapid, high in specificity and sensitivity, objective and visual in result judgment, low in cost, convenient to use and quite safe to people and environment. The cyprinid herpes virus type 2 CPA detection primer not only can be used in a special laboratory, but also can be applied in wild on-site rapid detection, and the cyprinid herpes virus type 2 in a sample can be accurately detected within 1.5 h through only one metal bath or water bath pot.

Description

technical field [0001] The invention belongs to the technical field of fish virus detection, and in particular relates to a detection primer for carp herpesvirus type 2 CPA, and also relates to the application of the primer. Background technique [0002] Carp herpesvirus type 2 (CyHV-2) is the pathogen of herpesviral haematopoietic necrosis (HVHN) that causes a large number of deaths in goldfish, so it is also called goldfish haematopoietic necrosis virus (GFHNV). In accordance with the systematic naming rules of the International Committee on Taxonomy of Viruses, it was officially named Cyprinid herpesvirus 2 (CyHV-2). The virus was first discovered in the fall of 1992 and the spring of 1993 as it caused mass mortality of farmed goldfish in western Japan, with a lethality rate as high as 100%. The disease subsequently broke out in the United States, Taiwan, Australia and the United Kingdom, causing huge economic losses to goldfish farming. In recent years, crucian carp (h...

Claims

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Application Information

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IPC IPC(8): C12N15/11C12Q1/68C12Q1/70
Inventor 徐进曾令兵
Owner YANGTZE RIVER FISHERIES RES INST CHINESE ACAD OF FISHERY SCI
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