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Loop-mediated isothermal amplification kit of vibrio cholerae and application of loop-mediated isothermal amplification kit

A ring-mediated isothermal and Vibrio cholerae technology, applied in the field of microbial detection, can solve problems such as unsuitable for grassroots and on-site detection, complex equipment, laboratory pollution, etc., and achieve the effect of increasing test costs, easy operation, and rapid acquisition

Inactive Publication Date: 2017-05-10
GUANGXI VETERINARY RES INST
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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 conventional methods, it requires complex equipment and high cost, and is not suitable for grassroots and on-site detection. In addition, agarose gel electrophoresis is required to determine the results, and then use ultraviolet transilluminator irradiation for determination , and it has human subjective factors judged by naked eyes, and it is easy to cause laboratory pollution

Method used

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  • Loop-mediated isothermal amplification kit of vibrio cholerae and application of loop-mediated isothermal amplification kit
  • Loop-mediated isothermal amplification kit of vibrio cholerae and application of loop-mediated isothermal amplification kit
  • Loop-mediated isothermal amplification kit of vibrio cholerae and application of loop-mediated isothermal amplification kit

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Effect test

Embodiment 1

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

[0078] LAMP amplification was performed on 2 strains of Vibrio cholerae, 5 strains of negative control bacteria and water control, and the results were as follows figure 1 As shown, the Vibrio cholerae reaction tube showed a rising curve of turbidity at about 33 minutes, which was a positive result, and the curves of the 5 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] The initial concentration of Vibrio cholerae DNA template was 2.05×10 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 2.05×10 -2 ng / μL, while the detection limit of PCR method is 2.05×10 -1 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 Vibrio cholerae as the template, indicating that the established method can be conveniently used at the grassroots level. It is only necessary to use the LAMP primer designed in the kit with this method. After adding the sample, use The low-cost water bath can be kept at 63°C for 60 minutes, and the results can be quickly observed without opening the cover, avoiding pollution.

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Abstract

The invention discloses a loop-mediated isothermal amplification (LAMP) kit of vibrio cholerae and application of the loop-mediated isothermal amplification kit. The kit comprises LAMP primers, a 2*reaction buffering solution, Bst DNA (Deoxyribonucleic Acid) polymerase, a fluorescent visual detection reagent, ultrapure water and a vibrio cholera DNA template; the LAMP primers comprise outer primers F3 and B3 and inner primers FIP and BIP. Specificity detection and sensitivity detection prove that the LAMP detection kit can be used for monitoring reactions in real time and quantitatively detecting the vibrio cholerae; a detection result is rapidly and accurately obtained, and convenience is brought to simple, convenient and rapid detection of the vibrio cholerae.

Description

technical field [0001] The invention relates to the technical field of microbial detection, in particular to a fast, visualized and real-time quantitative detection of Vibrio cholerae loop-mediated isothermal amplification kit and its application. Background technique [0002] Vibrio cholerae (V.cholerae) in the genus Vibrio can cause a severe intestinal infectious disease with acute onset, strong infectivity and high fatality rate, which belongs to the international quarantine infectious disease. [0003] Human beings are the only susceptible persons to Vibrio cholerae under natural circumstances, and it is mainly transmitted orally through contaminated water or food. Under certain conditions, after Vibrio cholerae enters the small intestine, it relies on the movement of flagella to pass through the mucus layer on the mucosal surface, and may adhere to the epithelial cells of the intestinal wall through the action of pili, and multiply rapidly on the surface of the intestin...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04C12R1/63
CPCC12Q1/6844C12Q2531/119C12Q2563/107Y02A50/30
Inventor 冯世文李军潘艳彭昊陈泽祥胡帅杨威钟舒红马春霞陶立许力干柳锋谢永平
Owner GUANGXI VETERINARY RES INST
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