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Streptococcus agalactiae loop-mediated isothermal amplification kit and application thereof

A ring-mediated isothermal and lactis streptococcus technology, applied in the field of microbial detection, can solve the problems of being unsuitable for grassroots and on-site detection, high cost, and complicated equipment, and achieve the effects of increasing test costs, easy operation, and avoiding pollution

Inactive Publication Date: 2015-06-24
GUANGXI ACADEMY OF FISHERY SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Although the PCR method is faster and more accurate than the conventional method, it requires complex instruments and equipment, and the cost is high, so it is not suitable for grassroots and on-site detection

Method used

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  • Streptococcus agalactiae loop-mediated isothermal amplification kit and application thereof
  • Streptococcus agalactiae loop-mediated isothermal amplification kit and application thereof
  • Streptococcus agalactiae loop-mediated isothermal amplification kit and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0077] The specificity result of embodiment 1 LAMP detection method

[0078] LAMP amplification was performed on 1 strain of Streptococcus agalactiae, 8 strains of negative control bacteria and water control, the results were as follows figure 1 As shown, the Streptococcus agalactiae reaction tube showed a rising curve of turbidity at about 37 minutes, which was a positive result, and the curves of the 8 negative control bacteria reaction tubes and the water control reaction tube showed no amplification, which was a negative result.

Embodiment 2

[0079] Example 2 Sensitivity results of LAMP detection method

[0080] Recombinant plasmid pMD18-T- Sip The starting concentration is 3.55×10 1 ng / μL, LAMP and PCR amplification were performed after 10-fold dilution, the results were as follows figure 2 and image 3 As shown, the results show that the detection limit of the LAMP method is about 3.55×10 -5 ng / μL, while the detection limit of PCR method is 3.55×10 -3 ng / μL.

Embodiment 3

[0081] Example 3 Fluorescence visualization detection results of LAMP detection method

[0082] According to the optimized conditions monitored by the turbidimeter, fluorescent dyes were added to the reactor, and after 60 minutes of reaction at 63°C, observed under ultraviolet light, Figure 4 In order to observe the results, the left tube is the negative control, and the right tube is the reaction with Streptococcus agalactiae as the template, which shows that the established method can be used at the grassroots level. It is only necessary to use the LAMP primer designed by this method in the kit, and after adding the sample, Use an inexpensive water bath to maintain 63°C for 60 minutes, and you can quickly observe the results without opening the cover, avoiding pollution.

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Abstract

The invention discloses a streptococcus agalactiae loop-mediated isothermal amplification kit and application thereof. The kit comprises an LAMP primer, a 2*reaction buffer solution, Bst DNA polymerase, a fluorescent visual detection reagent, ultrapure water and a streptococcus agalactiae DNA template, wherein the LAMP primer comprises outer primerS F3 and B3 and inner primers FIP and BIP. Specificity detection and sensitivity detection prove that the LAMP detection kit disclosed by the invention can be used for monitoring reaction in real time, quantitatively detecting the copy number of the streptococcus agalactiae, rapidly and accurately obtaining detection results and providing convenience for simply and rapidly detecting the streptococcus agalactiae.

Description

technical field [0001] The invention relates to the technical field of microbial detection, in particular to a loop-mediated isothermal amplification kit capable of rapid, visual and real-time quantitative detection of Streptococcus agalactiae and its application. Background technique [0002] Fish streptococcosis is a disease caused by streptococcal infection, and the pathogenic bacteria belong to 3 genera and 7 species. Pathogens of the genus Streptococcus include Streptococcus iniae ( Streptococcus iniae ), Streptococcus agalactiae ( Streptococcus agalactme ), Streptococcus paramastitis (Streptococcus parauberis ) and Streptococcus dysgalactiae ( Streptococcus dysgalactiae ); Lactococcus gordonii of the genus Lactococcus ( Lactococcus garvieae ) and Lactococcus fish ( Lactococcus piscium ); the genus of the genus Salmonella ( Vagococcus salmoninarum ). Among them, Streptococcus iniae, Streptococcus agalactiae, Streptococcus paramastitis, Streptococcus dysgal...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/14
CPCC12Q1/6844C12Q1/689
Inventor 罗洪林李军冯世文梁万文彭昊黄婷陈福艳张瑶瑶陈泽祥杨威潘艳
Owner GUANGXI ACADEMY OF FISHERY SCI
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