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LAMP (Loop-Mediated Isothermal Amplification) detection kit and detection method for giant salamander iridovirus

A giant salamander iridescent virus and detection kit technology is applied in the giant salamander iridescent virus LAMP detection kit, and the giant salamander iridescent virus LAMP detection kit is used to detect the giant salamander iridescent virus field, which can solve the problems of economic loss of the giant salamander breeding industry, restricting the development of healthy breeding and the like , to achieve the effect of low detection cost, high sensitivity and good specificity

Active Publication Date: 2013-04-03
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

However, with the expansion of the breeding scale, the problem of giant salamander disease has become increasingly prominent, especially the outbreak of giant salamander viral hemorrhagic disease in recent years, which has caused huge economic losses to the giant salamander farming industry and seriously restricted the development of its healthy farming.

Method used

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  • LAMP (Loop-Mediated Isothermal Amplification) detection kit and detection method for giant salamander iridovirus
  • LAMP (Loop-Mediated Isothermal Amplification) detection kit and detection method for giant salamander iridovirus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] A detection kit for giant salamander iridescent virus LAMP, comprising the following components:

[0046] The kit includes the following components: 10×ThermoPol Reaction Buffer; Bst DNA polymerase (NEB); dNTPs; outer primers F3 and B3, inner primers FIP and BIP, Betaine (Sigma), MgCl 2, 1000 × SYBR Green I (Invitrogen).

[0047] F3: 5, -CGACTTGGCCACTTATGACA-3,;

[0048] B3: 5'-TTGACCTCGGGGGTCTTG-3';

[0049] FIP: 5,-GTGAACCACCCCACGGGGTAAAAACAATGTACGGGGGTTCAGAT-3,;

[0050] BIP: 5'-AAGATGTCGGGTAACCCGGCTTAAAAACCAGGCGTTGAGGATGT-3'.

Embodiment 2

[0052] Giant salamander iridescent virus LAMP detection kit with different concentrations of Mg 2+ Optimization

[0053] 1. Take the sample to be tested and extract the virus DNA:

[0054] Cultivate carp epithelial papilloma cells (EPC cells) to a confluent monolayer, suck out the culture medium, inoculate 1ml giant salamander iridescent virus cytotoxic material (Wuhan University Collection Center) with a dose of 0.1 multiplicity of infection and centrifuge at 4000r / min for 5min to remove cell debris Accession No. CCTCC V201134), add 10 μl of Polybrene (final concentration 10 μg / ml), and place in a 26°C incubator for 1 hour to allow the virus to adsorb to the cells. During the adsorption process, gently shake the culture bottle once every 20 minutes to allow the virus liquid to be mixed with the cells. The single layer is fully and evenly contacted. After 1 hour of adsorption, the virus liquid is discarded, and 5ml of 2% fetal bovine serum (V / V) culture solution is added, and...

Embodiment 3

[0063] Optimization of the reaction temperature of the LAMP detection method for giant salamander iridescent virus:

[0064] 1. Take the sample to be tested and extract the virus DNA:

[0065] Harvest the GSIV-infected EPC cells after 90% of the lesions appear (the preparation method is the same as in Example 2), freeze and thaw three times at -80°C to room temperature, centrifuge at 5000r / min for 30min, take 250μl of the supernatant, and use the Viral DNA Kit kit DNA was extracted according to the instructions, finally dissolved in 50 μl sterilized water, and stored at -20°C for later use.

[0066] 2. The reaction system of LAMP amplification:

[0067] A 25 μl reaction system was used, including: 0.8 μM each of the inner primers FIP and BIP, 0.1 μM each of the outer primers F3 and B3, dNTPs1 mM, Betaine0.5M, MgCl 2 8mM, 8U of Bst DNA polymerase, 5μl of template DNA, 2.5μl of 10×ThermoPol Reaction Buffer, and make up the rest with deionized water.

[0068] 3. Reaction condi...

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Abstract

The invention discloses an LAMP (Loop-Mediated Isothermal Amplification) detection kit and a detection method for giant salamander iridovirus. The LAMP detection kit for the andrias advidianus iridescent virus comprises the following ingredients: 10*Thermol Reaction buffer, BstDNA polymerase, dNTPs, outer primers F3 and B3, inner primers FIP and BIP, BEtaine, MGc12 and 1000*SYBRGreenI. The LAMP detection kit and the LAMP detection method have the characteristics of simplicity, convenience, high specificity and sensitivity, the GSIV (Giant Salamander Iridovirus) in a sample can be detected accurately within 2h by a water bath kettle or a metal bath kettle, the sick fish tissues infected by GSIV can be detected, and the cells infected by the GSIV can be detected, so that the detection kit and the detection method are very suitable for field rapid detection of GSIV.

Description

technical field [0001] The invention belongs to the technical field of fish virus detection, in particular to a giant salamander iridescent virus LAMP detection kit, and also relates to a method for detecting giant salamander iridescent virus LAMP detection kit. Background technique [0002] The giant salamander (Andrias davidianus), commonly known as the giant salamander, belongs to Amphibians, Cauracea, Cryptobranchidae, and the genus Giant salamander. It was listed as a second-class national protected animal in 1988. The 14th Conference of the Parties was listed as a Class I endangered species. Giant salamander not only has certain medicinal and scientific value, but also can be used as ornamental animals and edible delicacies. Since the 1980s, artificial breeding and breeding of giant salamanders have been carried out successively in various parts of my country, and it has become one of the excellent species of high-efficiency breeding in aquaculture. However, with the...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12R1/93
Inventor 曾令兵孟彦徐进张辉周勇范玉顶
Owner YANGTZE RIVER FISHERIES RES INST CHINESE ACAD OF FISHERY SCI
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