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Streptococcus iniae loop-mediated isothermal amplification kit and application thereof

A Streptococcus iniae, ring-mediated isothermal technology, applied in the field of microbial detection, can solve the problems of being unsuitable for grassroots and on-site detection, complex equipment, high cost, etc., and achieve the effect of simple result interpretation

Inactive Publication Date: 2015-05-27
GUANGXI VETERINARY RES INST
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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 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 iniae loop-mediated isothermal amplification kit and application thereof
  • Streptococcus iniae loop-mediated isothermal amplification kit and application thereof
  • Streptococcus iniae loop-mediated isothermal amplification kit and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0076] The specificity result of embodiment 1 LAMP detection method

[0077] Perform LAMP amplification on 1 strain of Streptococcus iniae, 8 strains of negative control bacteria and water control, the results are as follows figure 1 As shown, the Streptococcus iniae reaction tube showed a rising curve of turbidity in about 30 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

[0078] Example 2 Sensitivity results of LAMP detection method

[0079] Recombinant plasmid pMD18-T- Sim The starting concentration is 1.7×10 2 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 result shows that the detection limit of LAMP method is about 1.7×10 -8 ng / μL, while the detection limit of PCR method is 1.7×10 -6 ng / μL.

Embodiment 3

[0080] Example 3 Fluorescence visualization detection results of LAMP detection method

[0081] 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 right tube is the reaction of Streptococcus iniae as a template, which is a positive result, and the left tube is a negative control reaction, which is a negative result, indicating that the established method can be used at the grassroots level, and only need to use the kit to cooperate with this method After the designed LAMP primers are added to the sample, use an inexpensive water bath to maintain 63°C for 60 minutes, and the results can be quickly observed without opening the cover, avoiding contamination.

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Abstract

The invention discloses a streptococcus iniae loop-mediated isothermal amplification kit and application thereof. The kit comprises LAMP primers, a 2*reaction buffer, a Bst DNA polymerase, a fluorescent visual detection reagent, ultra-pure water and a streptococcus iniae DNA template, wherein the LAMP primers comprise outer primers F3 and B33 and inner primers FIP and BIP. Specificity detection and sensitivity detection show that the LAMP detection method provided by the invention can be used for monitoring reaction in real time and detecting the copy number of streptococcus iniae quantitatively so that the detection result can be obtained rapidly and accurately and convenience is brought for simple and rapid detection of the streptococcus iniae.

Description

technical field [0001] The invention relates to the technical field of microbial detection, in particular to a loop-mediated isothermal amplification kit for rapid, visual and real-time quantitative detection of Streptococcus iniae 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. Streptococcus pathogens include Streptococcus iniae, Streptococcus agalactme, Streptococcus paramastitis ( Streptococcus parauberis) and Streptococcus dysgalactiae; Lactococcus garvieae and Lactococcus piscium of the genus Lactococcus; Among them, Streptococcus iniae, Streptococcus agalactiae, Streptococcus paramastitis, Streptococcus dysgalactiae and Lactococcus griseri are the pathogenic bacteria of streptococcosis in warm water fish, while Lactococcus piscine and Salmonella are cold-water fish chains. The causative bacteria of coccidiosis. [0003] At present, S...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/14C12R1/46
CPCC12Q1/6851C12Q2531/119C12Q2545/101C12Q2545/113
Inventor 李军罗洪林冯世文彭昊陈福艳黄婷杨威陈泽祥张瑶瑶潘艳
Owner GUANGXI VETERINARY RES INST
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