Multiplex-direct PCR assay for foodborne pathogen identification: An application in forensic investigation

Abstract

Foodborne pathogens present serious concerns to human health and can even lead to fatalities. Microbial forensic science thus plays an important role in consumer protection, food security, and even in litigation. The gold standard for pathogen identification – bacterial culture – is costly and time-consuming. A cheaper and quicker alternative will benefit both forensic science and medical diagnosis. In this study, we developed and validated a molecular-based method termed ‘multiplex-direct PCR assay’ to simultaneously detect three common foodborne pathogens – Escherichia coli O157:H7, Campylobacter jejuni, and Listeria monocytogenes. Three previously reported species-specific primer pairs were modified and used to directly amplify samples without DNA extraction. The assay was also validated for its specificity, sensitivity, and applied to test several samples obtained from a local market and clinical samples. The results showed the expected PCR fragments of approximately 490, 343, and 209bp for E. coli O157:H7, C. jejuni, and L. monocytogenes, respectively. The assay was specific to the targeted pathogens and was sufficiently sensitive and robust to effectively analyze market samples. The whole process took less than 1h to complete indicating that the assay is suitable for reliable, rapid, and inexpensive identification of these three foodborne pathogens, which could be useful in microbial forensic investigation.