A sensitive and versatile multiplex PCR system for the rapid detection of enterotoxigenic (ETEC), enterohaemorrhagic (EHEC) and enteropathogenic (EPEC) strains of Escherichia coli

Although Escherichia coli is widely distributed in the marine environment, only a small percentage are pathogenic to humans. Nonetheless, the widespre ad occurrence of waterborne infections of E. coli origin in humans has beco me one of the major health problems worldwide. To date, several types of en terovirulent E. coli have been recognized as the aetiologic agents of vario us gastrointestinal infections in humans. The most commonly encountered are those belonging to the enterotoxigenic (ETEC), enteroinvasive (EIEC), ente rohaemorrhagic (EHEC) and enteropathogenic (EPEC) subtypes. In order to bet ter determine the health risks that are associated with exposure to some of these specific subtypes, we have developed a very sensitive multiplex PCR system for the rapid detection and typing of ETEC, EHEC and possibly EPEC s trains of E. coli in the aquatic environment. The target genes chosen for t his investigation included: the PHO-A housekeeping gene (present in all E. coli); the LT1, LT2 and STI genes of ETEC; the VT1 and VT2 verotoxin, and E AE virulence genes of EHEC and EPEC, respectively. Six pairs of oligonucleo tide primers were designed to simultaneously amplify internal fragments of these genes by multiplex PCR to generate PCR products that could be analyse d and confirmed with relative ease by gel electrophoresis and HincII enzyme digestion. The results showed that the six sets of PCR primers were highly specific for their target genes and produced specific amplimers of the exp ected size from several control strains of E. coli - ATCC 35401 (LT1+/ST1+) ; SA53 (LT2+/VT2+); and O157 (VT1+/VT2 +/EAE+). The detection sensitivity o f the multiplex PCR system for the six target genes in an E. coli cell mixt ure was optimized and enhanced by preincubating serially diluted cells in L uria-Bertani broth for 6 h prior to PCR analysis. The results obtained indi cated a detection sensitivity of 10 degrees CFU (of each strain) per 100 mu l reaction. Multiplex PCR analysis of seawater samples collected from four sewage-polluted sites in Hong Kong indicated the presence in all four samp les of E. coli bacteria that were positive for LT1, ST1, VT1 and EAE virule nce genes. Overall, the data indicated that the multiplex PCR system descri bed in this study is a potentially very useful and powerful method for rout ine monitoring and risk assessment of water quality. (C) 1999 Elsevier Scie nce Ltd. All rights reserved.