Duplex Real-Time SYBR Green PCR Assays for Detection of 17 Species of Food- or Waterborne Pathogens in Stools

Abstract

ABSTRACT A duplex real-time SYBR Green LightCycler PCR (LC-PCR) assay with DNA extraction using the QIAamp DNA Stool Mini kit was evaluated with regard to detection of 8 of 17 species of food- or waterborne pathogens in five stool specimens in 2 h or less. The protocol used the same LC-PCR with 20 pairs of specific primers. The products formed were identified based on a melting point temperature ( T m ) curve analysis. The 17 species of food- or waterborne pathogens examined were enteroinvasive Escherichia coli , enteropathogenic E. coli , enterohemorrhagic E. coli , enterotoxigenic E. coli , enteroaggregative E. coli , Salmonella spp., Shigella spp., Yersinia enterocolitica , Yersinia pseudotuberculosis , Campylobacter jejuni , Vibrio cholerae , Vibrio parahaemolyticus , Vibrio vulnificus , Aeromonas spp., Staphylococcus aureus , Clostridium perfringens , and Bacillus cereus. No interference with the LC-PCR assay was observed when stool specimens were artificially inoculated with each bacterial species. The detection levels were approximately 10 5 food- or waterborne pathogenic bacteria per g of stool. The protocol for processing stool specimens for less than 10 4 food- or waterborne pathogenic bacteria per g of stool requires an overnight enrichment step to achieve adequate sensitivity. However, the rapid amplification and reliable detection of specific genes of greater than 10 5 food- or waterborne pathogenic bacteria per g in samples should facilitate the diagnosis and management of food- or waterborne outbreaks.