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CPA (cross priming amplification) detection primers for GSIV (giant salamander iridovirus) and application

A giant salamander iridescent virus and detection primer technology, which is applied in the directions of microorganisms, recombinant DNA technology, microorganism-based methods, etc., to achieve the effects of simple determination, good specificity and high sensitivity

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

It can be seen that the above methods have their own limitations. In order to detect and diagnose giant salamander iridescent virus disease early, it is necessary to establish a detection method that is not only accurate, fast and sensitive, but also objective and intuitive in judging the results. Provide technical means, and also provide technical support for the healthy development of the giant salamander breeding industry

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] A detection kit for giant salamander iridescent virus CPA, comprising:

[0040] 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).

[0041] CPF:5'-GCCTCAGCGAACAGCGTGGCACCACCCTCTACTCCTAT-3'

[0042] CPR: 5'-GGCACCACCTCTACTCCTATGCCTCAGCGAACAGCGT-3'

[0043] DF:5'-(Biotin)CCTCAGCCTACAGCACCC-3'

[0044] DR:5'-(FITC)CTGGCGTTGGTCAGTCCG-3'

[0045] DPF:5'-TCCATCCCAGTCAGCA-3'

[0046] DPR: 5'-TACCCAGAGTCGTCACCT-3'.

Embodiment 2

[0048] Optimization of different primer concentrations and ratios in a giant salamander iridescent virus CPA detection kit:

[0049] 1. Take the sample to be tested and extract the virus DNA:

[0050] Take 300 μL of spleen and kidney tissue homogenate of diseased salamander infected with GSIV (China Center for Type Culture Collection, CCTCC NO:V201134), and use Reagent or Viral DNA Kit kit, extract DNA according to the instructions, finally dissolve in 30 μL sterile water, and store at -20°C for later use.

[0051] 2. The reaction system of CPA amplification:

[0052] 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, dNTPs 1.0mM, Betaine 0.6M, Bst DNA polymerase 8U, nuclease-free water to make up to 25μL. At the same time set a blank control.

[0053] Among them, cross primers (CPF, CPR), detection primers (DF, DR), and strippin...

Embodiment 3

[0064] Different concentrations of MgSO in a giant salamander iridescent virus CPA detection kit 4 Optimization:

[0065] 1. Take the sample to be tested and extract the virus DNA:

[0066] The preparation method is the same as in Example 2.

[0067] 2. The reaction system of CPA amplification:

[0068] 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 0.4μM, DPF / DPR 0.4μM, MgSO 4 , dNTPs 1.0mM, Betaine 0.6M, Bst DNA polymerase 8U, nuclease-free water to make up to 25μL. At the same time set a blank control.

[0069] where MgSO 4 The concentrations were 4.0, 6.0, 8.0, 10.0, 12.0 mM, respectively.

[0070] 3. Reaction conditions for CPA amplification:

[0071] The reaction tube was incubated at 63°C for 60min and then inactivated at 80°C for 2min.

[0072] 4. Judgment of test results:

[0073] Take 5 μL of the amplified product, electrophoresis with 2.0% agarose gel, and place it in a gel imaging sy...

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Abstract

The invention discloses CPA (cross priming amplification) detection primers for GSIV (giant salamander iridovirus) and an application. A CPA detection kit for the GSIV comprises the following components: 10*ThermoPol Reaction Buffer, Bst DNA polymerase, dNTPs, cross primers CPF and CPR, detection primers DF and DR, bomper primers DPF and DPR, MgSO4, Betaine and a nucleic acid test strip. The CPA detection primers have the characteristics of being simple, convenient, rapid and high in specificity and sensitivity, result determination is objective and visual, the cost is low, and the primers are convenient to use and quite safe to human and environment. The CPA detection primers not only can be used in a specialized laboratory, but also can be applied to outdoor on-site quick detection, and the GSIV in a sample can be detected accurately within 1.5 h only with a metal bath or water bath.

Description

technical field [0001] The invention belongs to the technical field of amphibian virus detection, and in particular relates to a detection primer for giant salamander iridescent virus CPA, and also relates to the application of the primer. Background technique [0002] The giant salamander (Andrias davidianus), commonly known as the salamander, is a rare amphibian unique to my country. It was listed as a second-class national protected animal in 1988, and was listed as a category I endangered species in the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES) in 2007. species. Because giant salamanders have extremely high nutritional value and medicinal value, my country has started large-scale artificial breeding of giant salamanders since it broke through the artificial breeding technology of giant salamanders in the 1980s. At present, the giant salamander has been widely cultivated in many provinces and cities across the country as a key...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12N15/11C12R1/93
Inventor 徐进曾令兵周勇
Owner YANGTZE RIVER FISHERIES RES INST CHINESE ACAD OF FISHERY SCI
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