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

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.The traditional cultural techniques for the direct isolation and identification of food-or waterborne pathogens from stool specimens in food poisoning outbreaks are time-consuming and laborious. Efforts have been made in diagnostic laboratories to reduce the time required for identification of food-or waterborne pathogens. Real-time PCR is currently used for the diagnosis of infectious agents, and there are few reports of the application of real-time PCR for the direct detection of Escherichia coli strain O157:H7 (11) and Plesiomonas shigelloides (20) in stool specimens. However, traditional PCR is often used for the detection of infectious agents from stool specimens and food samples, and there are a number of previous reports of PCR use for detection of food-or waterborne pathogens, including E. coli (6,14,12,27,37,38,40), Salmonella spp. (26) (38). The published PCR protocols used for detection of food-or waterborne pathogens differ and include the use of different PCR cycler machines, annealing temperatures, and buffer systems. For routine laboratory analysis, the development of streamlined PCR methods would be ideal. In addition, not all of the published PCR methods are so sensitive or specific. Therefore, careful choice of PCR primers, modification of some existing PCR primers, and development of new PCR methods for detection and identification of a wide range of food-or waterborne pathogens are all necessary for the development of a standardized PCR protocol (38). Moreover, interference and inhibitors should be considered...

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