Development of a Foodborne Bacterial Pathogen PCR Electrospray Ionization Mass Spectrometry Biosensor Detection Assay for Rapid, High-Throughput Food Testing

摘要

Methods that reduce the time of detection, identification and discrimination of pathogenic foodborne bacterial pathogens are in great demand by industry and regulators both domestically and internationally. Molecular methods that focus on multiple DNA targets may allow for highly specific identification of bacterial species. To this end, we are reporting on the development and initial evaluation of a PCR/ESI-MS (electrospray ionization mass spectrometry) based biosensor assay for rapid detection of 4 important foodborne pathogens. This assay uses 8 primer pairs to detect and differentiate Salmonella spp. Escherichia coli, Shigella spp. and Listeria spp. These primers bind to conservative regions in mutS, mdh in Enterobacteriaceae and prfA and iap in Listeria. Initial work built a DNA basecount database that contains over 140 Salmonella, 139 E. coli, 11 Shigella, 36 Listeria patterns and 18 other Enteorbacteriaceae organisms. Evaluations using FDA SAFE Salmonella panel confirmed the assay's ability to subspeciate Salmonella. Surprisingly, some differentiation of S. enterica subsp. enterica into individual serovars or clusters containing 2 to 6 serovars was also observed. Tomato spiking experiments investigated the limit of detection in a food matrix. At 24 hr incubation, the assay was able to detect Salmonella at a pre- pre-enrichment (PPE) spike of 1 cfu/ml, similar to a conventional multiplex PCR. When the incubation period was reduced to 6 hr the PCR/ESI-MS assay could detect Salmonella at 20 cfu/ml PPE spike, whereas conventional PCR did not detect Samonella until 2X10~(4) cfu/ml PPE spike. This work demonstrates that the foodborne bacterial pathogen PCR/ESI-MS assay is capable of detecting and subtyping Salmonella, E. coli, Shigella and Listeria. Also the assay can detect the presence of Salmonella in a food matrix, specifically tomatoes. Current work is focusing on detection of Salmonella in other food matrices and the use of this assay in environmental surveillance samples.