Target-Enriched Multiplex PCR (Tem-PCR) Assay for Simultaneous Detection of S almonella spp., L isteria Monocytogenes and E scherichia Coli O157:H7 in Food

Abstract

A novel target-enriched multiplex polymerase chain reaction (Tem-PCR) assay for the simultaneous detection of Salmonella spp., Listeria monocytogenes and Escherichia coli O157:H7 was developed in the present study. The invA, hly and rfbE genes were selected as target genes for identifying Salmonella spp., L. monocytogenes and E. coli O157:H7, respectively. Three pairs of target-enriched long primers with a composition of pathogen-specific PCR primer combined with a universal oligonucleotide sequence at 5′ segment of each pathogen-specific PCR primer were designed. Following optimization of reaction conditions, the Tem-PCR assay for detecting Salmonella spp., L. monocytogenes and E. coli O157:H7 was successfully developed, which was able to simultaneously detect the three foodborne pathogens from pure cultures with an analytical detection limit of ca. <110 cfu/mL for each target. The Tem-PCR assay showed a high specificity to the target bacteria without specific priming observed in nontarget bacteria and also showed a potential diagnostic capability in practice. Compared with conventional multiplex PCR assay, the Tem-PCR assay does need not to optimize the concentration of each pair of primers, suggesting an efficient detection method for foodborne pathogens in laboratory diagnosis. Practical Applications Applying the target-enriched multiplex polymerase chain reaction assay to 355 food samples, including poultry meat, raw pork, raw milk, egg, sausage, raw beef, milk powder and frozen meat, revealed that 21 food samples were positive in this assay, in accordance with the testing results of conventional culture-based method, indicating a rapid and reliable tool for efficient screening of Salmonella spp., Listeria monocytogenes and Escherichia coli O157:H7 from food.