Development Of Multiplex PCR And Gene Chip Methods For Detection Of Foodborn Pathogens

Foodborn pathogens including Staphylococcus aureus, Vibrio parahaemolyticus, Listeria monocytogenes, Salmonella, Enterobacter sakazaki, Shigella, Escherichia coli O157:H7 and Campylobacter Jejuni are primary factors threatening food safety and human health. A genome comparison method was used to identify specific PCR target sequences and create detection database of foodborn pathogens. Other reported sequences and primers for the detection were selected and validated. Then multiplex PCR and gene chip methods based on selected primers were developed and optimized to detect eight mentioned foodborn pathogens.In this study, the multiplex PCR was established with primers including nuc-j, Vp2, prs, C20, SG, ial, hly-A and hipO1, and accomplished at an optimal Mg2+ concentration of 1.5 mmol/l and an annealing temperature of 56oC. The sensitivity of multiplex PCR method was approximately 10 pg of genomic DNA per reaction. Based on the multiplex PCR, the single base extension-tags microarray was developed with designed SBE-tags primers. The sensitivity of gene chip method was 0.1 pg of genomic DNA and 5×102 cfu/ml for cultures per reaction. The rate of accuracy for detection of separate strains was 100%. In addition, the bead-based mesofluidic chip was applied to detect eight general foodborn pathogens with a sensitivity of 6.2×102 cfu/ml for cultures. Compared to the single base extension-tags microarray, the detection time of bead-based mesofluidic chip is shorter with a higher degree of automation.