Detection And Identification Of Food Borne Pathogens Using Real Time PCR And Gene Chip Assay

The food borne pathogens are the primary reasons that lead to food poisoning and are capable of causing significant illness and represent a serious public heath threat worldwide. Most of food borne pathogens are difficult to be incubated in the testing laboratories. Current routine techniques for identification and detection of food borne pathogens are still based on biochemical and physiological methods. Several molecular methods are also used for the detection and identification of the pathogens such as real time PCR, ELISA, and southern blotting. The rapid and accurate methods for detection and identification of food borne pathogens are the precondition of preventing and controlling pathogens. In the paper, the rapid and accurate methods for detection and identification of food borne pathogens were established using real time PCR, RT-real time PCR and DNA chip. In the methods using real time PCR, the eight food borne pathogens were specially detected through different primers and probes, which were Listeria monocytogenes, Escherichia coli 0157:H7, Vibrio parahaemolyticus, Salmonella, Campy lobacter jejuni. Vibrio cholerae, vibrio vulnificus, Shigella. Listeria monocytogenes was regarded as a case, three kinds of stardard curves (genomic DNA, cell, plasmid) were appliedto quantitive detection, the sensitivity was approximately l0CFU/mL; At the same time, an assay using RT-real time PCR was applied to detect or distinguish between viable and nonviable Listeria monocytogenes. Furthermore, another assay for detection and identification of food borne pathogens was also established using DNA chip technique. The detected "targe" pathogens were Vibrio cholerae, Escherichia coli 0157 :H7, Vibrio parahaemolyticus, Yersinia enterocolitica, Listeria monocytogenes, Shigella flexneri, Salmonella typhimurium, Campylobacter j'ejuni. The DNA fragments of pathogens were amplified by 16S rDNA and 23S rDNA universal primers under the same conditions respectively. The PCR products were then applied to the DNA chip for hybridization. The results showed that the selected oligonucleotide probes were very specific. The process of whole operation was easier and more efficient than conventional methods. The repeatability and stability were eligible. The limit of detection was approximately 155CFU/mL. All above results indicated four assays for detection and identification of food borne pathogens were accurate, reliable, and feasible. Those assays would lead to an innovation in the field of detection and identification of food borne pathogens. Eight food borne pathogens were detected using DNA chip technique simultaneously in one operating process. The establishment of four methods in this paper provided a firm technical and theoretical basis for further research and use of wider scope in the field of detecting and identifying food borne pathogens.