Application Of Probe-based Melting Curve Analysis In Foodborne Pathogens Detection And Typing

Food safety issue is a public concern all over the world. Food poisoning incidents occur frequently which are mainly caused by foodborne pathogens. In China, the under-report rate stays high, partially due to the poor diagnostic methods, highlighting the importance to develop rapid and reliable methods for the detection of foodborne pathogens. This dissertation focuses on the detection and typing of those common pathogens including virulent genes of diarrheagenic Escherichia coli (DEC) as well as enterotoxin of staphylococcus aureus, salmonella, shigella spp., yersinia enterocolitica, Listeria monocytogenes, campylobacter jejuni. To enable the rapid detection of multiple bacteria, a novel detection system was established based on the combined uses of ligation, PCR and melting curve analysis that allow more than10target genes detected in one reaction.In the first chapter, the foodborne pathogens and its clinical diagnosis, the principle of real-time PCR and melting curve analysis, were briefly reviewed. In the second chapter, the epidemiological status of DEC was summarized together with traditional identification methods and molecular detection methods. A multiplex PCR assay based on ligation, PCR and melting curve analysis was proposed. The established assay was able to detect14relevant genes of E. coli. including five virulent types (ETEC, EAEC, EIEC, EPEC, EHEC) and two serotype (o104:H4and ol57:H7) in a single reaction. The detection limit of the assay was106CFU/mL. When tested with302clinical samples,64(21.2%) positive DECs were detected and confirmed by an independent real-time PCR assay. By contrast,56(18.5%) positive DECs were detected by the reference method (k>0.75). In the third chapter, the similar strategy was used to detect six common foodborne pathogens involving13genes. This assay could differentiate staphylococcus aureus (SE-A, SE-B, SE-C, SE-D,SE-E, and nuc), yersinia enterocolitica (ail, foxA), shigella spp.(ipaH), salmonella (ssaR, invA), Listeria monocytogenes (hly) and campylobacter jejuni (gyrA). In summary, we established a universal foodborne pathogen detection system based on the combined use of ligation, PCR and melting curve analysis. The system is specific, sensitive, could detect a large number of foodborne pathogens in a single reaction, and is expectedly useful in clinical settings.