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LAMP (Loop-Mediated Isothermal Amplification) kit for priA gene in acute hepatopancreas necrosis syndrome and application of LAMP kit

A technology of hepatopancreas and kits, applied in the field of microbial detection, can solve the problems that there is no guarantee of no cross-reaction, no guarantee of detection, and it is not suitable for grassroots and on-site detection.

Inactive Publication Date: 2017-01-25
GUANGXI ACADEMY OF FISHERY SCI
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  • 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
The PCR method is only used as a temporary detection tool

Method used

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  • LAMP (Loop-Mediated Isothermal Amplification) kit for priA gene in acute hepatopancreas necrosis syndrome and application of LAMP kit
  • LAMP (Loop-Mediated Isothermal Amplification) kit for priA gene in acute hepatopancreas necrosis syndrome and application of LAMP kit
  • LAMP (Loop-Mediated Isothermal Amplification) kit for priA gene in acute hepatopancreas necrosis syndrome and application of LAMP kit

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Effect test

Embodiment 1

[0063] The specificity result of embodiment 1LAMP detection method

[0064] Perform LAMP amplification on the PriA positive plasmid, 10 strains of negative control bacteria and water control, the results are as follows figure 1 As shown, the PriA 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

[0065] The sensitivity result of embodiment 2LAMP detection method

[0066] The initial concentration of the recombinant plasmid pMD18-T-PriA was 5.02×10ng / μL, after 10-fold dilution, LAMP and PCR amplification were performed, and the results were as follows figure 2 , image 3 As shown, the detection limit of the LAMP method is about 5.02×10 -6 ng / μL, while the detection limit of PCR method is 5.02×10 -5 ng / μL.

Embodiment 3

[0067] Fluorescence visualization detection result of embodiment 3 LAMP detection method

[0068] 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 PriA 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 priA gene in acute hepatopancreas necrosis syndrome and application of the LAMP kit. The kit comprises LAMP primers, 2*Reaction Mix, Bst DNA (Deoxyribonucleic Acid) Polymerase, a fluorescent visual detection reagent, a DNA template and Distilled Water, wherein the LAMP primers comprise outer primers of F3 and B3 and inner primers of FIP and BIP. Specific detection and sensitive detection prove that the LAMP detection method provided by the invention has the characteristics that the reaction can be monitored in real time, and the copy number of the priA gene in the acute hepatopancreas necrosis syndrome is quantitatively detected; a detection result is quickly and accurately obtained; the convenience is brought for simply, conveniently and quickly 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 priA 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, and the hepatopancreas of dead prawns is characterized by obvious lesions, which is currently a serious disease that endangers the shrimp industry in my country. . The disease broke out in Vietnam in 2009, followed by outbreaks in Malaysia and Thailand. Because of its proximity to Vietnam and other countries and the frequent trading of prawns, my country began to discover the prevalence of AHPNS in coastal aquaculture areas in 2010. The mortality rate of prawns in Hainan, Fujian, Guangdong and some parts of Guangxi was as high as 80%. According to statisti...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
CPCC12Q1/6848C12Q1/6851C12Q2531/119C12Q2563/107C12Q2545/114
Inventor 张彬罗洪林熊建华何苹萍李沐谢达祥陈晓汉
Owner GUANGXI ACADEMY OF FISHERY SCI
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