Multiplex PCR kit for detecting 11 common food-borne pathogenic bacteria and application thereof

Abstract

The invention discloses a common primer-mediated PCR kit for detecting 11 food-borne pathogenic bacteria and an application of the common primer-mediated PCR kit. A common primer-mediated multiplex PCR technology is utilized; specific genes of 11 common food-borne pathogenic bacteria (salmonella, listeria monocytogenes, shigella flexneri, enterohemorrhagic escherichia coli O157, vibrio parahaemolyticus, staphylococcus aureus, vibrio cholerae, clostridium botulinum A, bacillus cereus, clostridium perfringens and yersinia enterocolitica) are used as detection markers; an efficient, high-specificity and high-flux multiplex PCR detection system is established, and a nucleic acid detection kit suitable for detecting the common food-borne pathogenic bacteria is researched and developed. The sensitivity of the common primers reaches 10<3> copies / mu L, a UP-M-PCR system for simultaneously detecting the 11 food-borne pathogenic bacteria is constructed, single-template and two-template experiments prove that the UP-M-PCR system has good specificity, and the sensitivity of a bacterial genome DNA detection system is as high as 0.01 ng / mu L.

Description

technical field [0001] The invention belongs to the technical field of bacterial strain detection, in particular to the detection technology of food-borne pathogenic bacteria, more specifically to a PCR kit mediated by public primers for the detection of eleven kinds of food-borne pathogenic bacteria and its application. Background technique [0002] Foodborne diseases refer to diseases caused by pathogenic factors such as toxic and harmful substances (including biological pathogens) that enter the human body through ingestion. With the development of society and economy, foodborne diseases have increasingly become the focus of global public health, causing considerable morbidity and mortality every year, and causing a huge economic burden. In 2010, the World Health Organization estimated that there were about 2 billion cases of foodborne diseases, and even in industrialized countries, about 30% of people suffer from foodborne diseases every year. In England and Wales, abou...

AI Technical Summary

Problems solved by technology

However, conventional PCR can only detect a single pathogen at a time. Therefore, on this basis, a series of technologies such as multiplex PCR, real-time fluorescent quantitative PCR, nested PCR, and digital PCR have been developed. Among them, multiplex PCR can be added to the reaction system. Multiple pairs of primers can simultaneously detect multiple target DNAs in one reaction tube, while the detection throughput of other PCR techniques is limited due to various reasons. Therefore, multiplex PCR technology has become the most promising method today and has been adopted Widely used in the detection of foodborne pathogens

[0004] For multiplex PCR, in theory, as long as the PCR reaction conditions are optimized, the number of primer pairs in the multiplex PCR reaction system can be continuously increased. Therefore, the actual detection throughput of multiplex PCR generally does not exceed 7 multiplex

In order to increase the detection throughput of multiplex PCR, experts at home and abroad have proposed a variety of solutions and made some progress, such as optimizing PCR reaction conditions, improving DNA polymerase activity, etc., or combining multiplex PCR technology with other technologies, such as microdroplets Digital PCR, gene chip, liquid phase suspension chip technology, etc., but these solutions have not fundamentally reduced the concentration of primers, reduced the interaction between primers or complicated operations, and are not easy to promote

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