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A kind of koi herpes virus cpa detection primer and application

A koi herpes virus and detection primer technology, applied in the field of fish virus detection, can solve the problems of long time-consuming, complicated operation, virus detection, etc., and achieve the effect of simple determination, high sensitivity and low detection cost

Active Publication Date: 2019-11-08
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

The cell culture method cannot directly detect the virus. Only the lesion characteristics of the cell culture can be used for preliminary judgment and further virus detection to make an accurate diagnosis. This method is limited to the laboratory; although the electron microscope technology can directly observe the virus particles Existence, but its operation is complex, time-consuming and low accuracy
These methods have their own limitations. In order to detect and diagnose koi herpes virus disease early, it is necessary to establish an accurate, fast and sensitive detection method, which provides technical means for disease diagnosis and prevention, and also provides a new method for carp aquaculture industry. Provide technical support for the healthy development of

Method used

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  • A kind of koi herpes virus cpa detection primer and application
  • A kind of koi herpes virus cpa detection primer and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] A koi herpes virus CPA detection kit, comprising:

[0045] Reaction Buffer; Bst DNA polymerase (NEB); dNTPs; MgSO 4 Betaine (Sigma); Cross primers CPF, CPR; Detection primers DF, DR; Stripping primers DPF, DPR; Nucleic acid detection test strips (Hangzhou Ustar Company).

[0046] CPF: 5'-GGTCTATGGCGTGCTTCTATGCTGGAACTGGTGA-3'

[0047] CPR: 5'-CTATGCTGGAACTGGTGAGGTCTATGGCGTGCTT-3'

[0048] DF: 5'-(Biotin)CGGACCCAGGTTCGC-3'

[0049] DR:5'-(FITC)TCGCCCGCTGTAGGAC-3'

[0050] DPF:5'-TATCTCGCAGCCGCAC-3'

[0051] DPR: 5'-GCCACCTTGGACTCTTCG-3'

Embodiment 2

[0053] Optimization of different primer concentrations and ratios in a koi herpesvirus CPA detection kit:

[0054] 1. Take the sample to be tested and extract the virus DNA:

[0055] Koi herpes virus (Zhu Xia, Li Xinwei, Wang Hao, et al. Isolation and identification of a Koi herpes virus[J]. Chinese Journal of Preventive Veterinary Medicine, 2011,33(5):340-343. Infected Koi- Fin cell) cell culture medium 300μL, with Reagents or Viral DNA Kit were used to extract DNA according to the instructions, and finally dissolved in 30 μL of sterilized water, and stored at -20°C for future use.

[0056] 2. The reaction system of CPA amplification:

[0057] Using a 25 μL reaction system, add 1 μL of viral DNA template to the PCR tube, ReactionBuffer 2.5 μL, cross primers CPF, CPR, detection primers DF, DR, stripping primers DPF, DPR, MgSO 4 8.0mM, dNTPs1.2mM, Betaine 0.7M, Bst DNA polymerase 5.6U, nuclease-free water to make up to 25μL. At the same time set a blank control.

[0058...

Embodiment 3

[0070] Different concentrations of MgSO in a koi herpes virus CPA detection kit 4 Optimization:

[0071] 1. Take the sample to be tested and extract the virus DNA:

[0072] The preparation method is the same as in Example 2.

[0073] 2. The reaction system of CPA amplification:

[0074] Using a 25 μL reaction system, add 1 μL of viral DNA template to the PCR tube, ReactionBuffer 2.5μL, CPF / R 1.0μM, DF / R 0.6μM, DPF / DPR 0.4μM, MgSO 4 , dNTPs 1.2mM, Betaine 0.7M, Bst DNA polymerase 5.6U, nuclease-free water to make up to 25μL. At the same time set a blank control.

[0075] where MgSO 4 The concentrations were 2.0, 4.0, 6.0, 8.0, 10.0, 12.0 mM, respectively.

[0076] 3. Reaction conditions for CPA amplification:

[0077] The reaction tube was incubated at 63°C for 60min and then inactivated at 80°C for 2min.

[0078] 4. Judgment of test results:

[0079] Take 5 μL of the amplified product, electrophoresis with 2.0% agarose gel, and place it in a gel imaging system for i...

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Abstract

The invention discloses a KHV (koi herpes virus) CPA (cross priming amplification) detection primer and an application. A KHV CPA detection kit comprises following ingredients: 10*ThermoPol Reaction Buffer, Bst DNA polymerase, dNTPs, cross primers CPF and CPR, detection primers DF and DR, stripping primers DPF and DPR, MgSO4, Betaine and a nucleic acid test strip. The KHV CPA detection primer has the characteristics of simplicity, convenience, rapidness and high specificity and sensitivity and is objective and direct in determination of results, low in cost, convenient to use and very safe to people and the environment. The KHV CPA detection primer not only can be used in a special laboratory, but also can be applied to rapid onsite detection in the field, only one metal bath or water bath kettle is required, and a KHV in a sample can be accurately detected within 1.5 h.

Description

technical field [0001] The invention belongs to the technical field of fish virus detection, in particular to a detection primer for koi herpes virus CPA, and also relates to the application of the primer. Background technique [0002] Koi herpes virus disease (KHVD) was first discovered in Israel in May 1998, and its pathogen was not confirmed until 1999 as Koi herpes virus (KHV), also known as carp herpes virus type III (CyHV-3). The disease subsequently broke out in large areas in the United States, Indonesia, the United Kingdom, continental Europe, Japan and other regions. In 2002, it was first confirmed that the disease had spread to my country. KHV mainly infects koi, carp and their common variants. The range of infected hosts is very narrow. Grass carp, silver carp, bighead carp, tilapia, etc. are kept together with sick fish for a long time, and they will not get sick even if the temperature is suitable. Fish latently infected with KHV do not show clinical symptom...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12Q1/6844C12N15/11
Inventor 徐进曾令兵
Owner YANGTZE RIVER FISHERIES RES INST CHINESE ACAD OF FISHERY SCI
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