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LAMP (Loop-mediated Isothermal Amplification) kit for priB gene in acute hepatopancreas necrosis syndrome and application thereof

A technology of hepatopancreas and kits, applied in the field of microbial detection, can solve the problems of high cost, no guarantee of no cross-reaction, complex instruments and equipment, etc.

Inactive Publication Date: 2017-03-22
GUANGXI ACADEMY OF FISHERY SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the PCR method is faster and more accurate than conventional methods, it requires complex equipment and high cost, and is not suitable for grass-roots and on-site detection, and this method cannot guarantee that it is compatible with every possible sample of non-acute hepatopancreatic necrosis syndrome bacteria or other organisms. There is no cross-reactivity and there is no guarantee that this method will detect every strain of bacteria that may cause AHPND in prawns

Method used

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  • LAMP (Loop-mediated Isothermal Amplification) kit for priB gene in acute hepatopancreas necrosis syndrome and application thereof
  • LAMP (Loop-mediated Isothermal Amplification) kit for priB gene in acute hepatopancreas necrosis syndrome and application thereof
  • LAMP (Loop-mediated Isothermal Amplification) kit for priB gene in acute hepatopancreas necrosis syndrome and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0062] The specificity result of embodiment 1 LAMP detection method

[0063] Perform LAMP amplification on the PriB positive plasmid, 10 strains of negative control bacteria and water control, the results are as follows figure 1 As shown, the PriB positive plasmid reaction tube showed a rising curve of turbidity in about 15 minutes, which was a positive result, and the curves of the 10 negative control bacteria reaction tubes and the water control reaction tube showed no amplification, which was a negative result.

Embodiment 2

[0064] Example 2 Sensitivity results of LAMP detection method

[0065] The initial concentration of the recombinant plasmid pMD18-T-PriB was 4.53×10 ng / μL. After 10-fold dilution, LAMP and PCR amplification were performed. The results showed that the detection limit of the LAMP method was about 4.53×10 -5 ng / μL, the detection limit of PCR method is 4.53×10 - 5 ng / μL.

Embodiment 3

[0066] Example 3 Fluorescence visualization detection results of LAMP detection method

[0067] According to the optimized conditions monitored by the turbidimeter, fluorescent dyes were added to the reactor, and after 40 minutes of reaction at 63 ° C, observed under ultraviolet light, Figure 4 To observe the results, the left tube is the reaction of the PriB positive plasmid as the template, which is a positive result, and the right tube is the reaction of the negative control, which is a negative result. The test results show that the method established by the present invention can be used conveniently at the grassroots level. You only need to use the kit with the LAMP primer designed by this method, add the sample, and use an inexpensive water bath to keep it at 63°C for 40 minutes, and the result can be quickly observed without the need for Open the lid to avoid contamination.

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Abstract

The invention discloses an LAMP (Loop-mediated Isothermal Amplification) kit for a priB gene in an acute hepatopancreas necrosis syndrome and application thereof and belongs to the technical field of microbiological testing. The kit comprises an LAMP primer, a 2*reaction buffering solution, Bst DNA (Deoxyribonucleic Acid) polymerase, a fluorescence visual detection kit, a DNA template and ultrapure water, wherein the LAMP primer comprises outer primers F3 and B3 and inner primers FIP (Forward Inner Primer) and BIP (Backward Inner Primer). Specific detection and sensitive detection prove that an LAMP detection method provided by the invention can be used for monitoring a reaction in time, quantitatively detecting a copy number of the priB gene in the acute hepatopancreas necrosis syndrome and rapidly and accurately obtaining a detection result, thus bringing convenience for simply and rapidly detecting the acute hepatopancreas necrosis syndrome.

Description

technical field [0001] The invention relates to the technical field of microbial detection, in particular to a LAMP kit capable of rapid and real-time quantitative detection of the priB gene in acute hepatopancreatic necrosis syndrome and its application. Background technique [0002] Acute Hepatopancreas Necrosis Syndrome (AHPNS) mainly causes a large number of diseased prawns in the stage of larvae and juveniles to die. The hepatopancreas of dead prawns is characterized by obvious lesions, which seriously endangers the shrimp industry in my country. According to statistics, the global shrimp production in 2013 decreased by 23% compared with 2012, while the shrimp production in my country decreased by about 17%. It is estimated that the direct economic loss caused to my country's shrimp industry exceeds 1 billion yuan per year. It has caused unprecedented blows and economic losses to shrimp farming and related industries in Southeast Asian countries and the world. [0003]...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
CPCC12Q1/6844C12Q1/6851C12Q2531/119C12Q2563/107C12Q2561/113C12Q2545/114
Inventor 罗洪林张彬张永德陈晓汉林勇余艳玲
Owner GUANGXI ACADEMY OF FISHERY SCI
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