LAMP detection method for clostridium difficile AB toxins and special primer and kit thereof

Abstract

The invention discloses an LAMP detection method for clostridium difficile AB toxins and a special primer and a kit thereof. The LAMP detection method disclosed by the invention comprises the following steps: designing a primer according to the specific conserved genes tcdA and tcdB of clostridium difficile; then, by taking a genomic DNA of an object to be detected as a template, carrying out LAMP amplification under the guide of the obtained primer; and synchronously and qualitatively detecting toxigenic strains of clostridium difficile in a sample to be detected through the color changes and turbidity changes of a reaction liquid. According to the invention, toxigenic strains of clostridium difficile can be detected in a fast, convenient, synchronous, high efficiency, high specificity and high sensitivity mode under isothermal conditions without using complex instruments, so that a new technical platform is provided for the detection and toxin classification of clostridium difficile, therefore, the LAMP detection method disclosed by the invention can be used for screening and detecting clostridium difficile by grassroots medical health units and various disease prevention and control centers, has a broad market prospect and great economic and social benefits, and is suitable for large-scale popularization and application.

Description

technical field [0001] The invention relates to a LAMP detection method of Clostridium difficile AB toxin, a special primer and a kit thereof, and belongs to the technical field of bacterial molecular biology detection methods in the field of biotechnology. Background technique [0002] Clostridium difficile (Cd) is a Gram-positive anaerobic bacillus that is widely distributed in water, soil and other natural environments, as well as animal and human feces. Clostridium difficile is an opportunistic pathogenic bacterium that is not invasive itself. When the normal microenvironment of the human intestinal tract is damaged, some toxin-producing bacteria can cause antibiotic-associated diarrhea, colonic Inflammation and even fatal pseudomembranous colitis, collectively referred to as Clostridium difficile infection (CDI). Among the pathogens identified in hospital-acquired infectious diarrhea, Clostridium difficile is the most common; among the causes of antibiotic-associated d...

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Problems solved by technology

In 2005, Japan Haru Kato et al. (Kato, H. and T. Yokoyama, et al. (2005). Rapid and simple method for detecting the toxin B gene of Clostridium difficile in stool specimens by loop-mediated isothermal amplification. J Clin Microbiol 43(12): 6108-12.) proposed to use the LAMP method to design 6 primers for the tcdB gene to detect Clostridium difficile, and interpret the results by turbidimeter and polyacrylamide gel electrophoresis. The disadvantage is that it is impossible to detect whether Clostridium difficile carries the tcd A gene, which is not conducive to the toxin typing of Clostridium difficile. The result interpretation method is cumbersome and requires the use of relevant instruments, so it is impossible to quickly interpret the results with the naked eye

Liu Chang et al. in 2012 (Liu Chang, Jiang Dongneng, Pu Xiaoyun. Research on rapid detection of Clostridium difficile in feces [J]. International Journal of Laboratory Medicine. 2012 (20)) and 2013 (Liu, C. and D. N. Jiang, et al. al. (2013). "DNA detection of Clostridium difficile infection based on real-time resistance measurement." Genet Mol Res 12(3): 3296-304.) proposed: 4 primers were designed for the tcdA gene to detect Clostridium difficile, respectively Through real-time resistance measurement and chromogenic reagent interpretation, the LAMP method is specific, sensitive, and fast, and is suitable for on-site and grassroots promotion. The disadvantage is that it cannot detect the carrying status of Clostridium difficile tcdB

At present, there is a lack of specific primers designed for the tcdA gene and tcdB gene of Clostridium difficile, and the fast, simple, specific and sensitive simultaneous detection of Clostridium difficile tcdA and tcdB can be achieved by reading the results with chromogenic reagents

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