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

TitleA sensitive and versatile multiplex PCR system for the rapid detection of enterotoxigenic (ETEC), enterohaemorrhagic (EHEC) and enteropathogenic (EPEC) strains of Escherichia coli
Authors
KeywordsEnterohaemorrhagic
Enteropathogenic
Enterotoxigenic
Escherichia coli
Multiplex PCR
Virulence gene
Issue Date1999
PublisherPergamon. The Journal's web site is located at http://www.elsevier.com/locate/marpolbul
Citation
Marine Pollution Bulletin, 1999, v. 38 n. 12, p. 1207-1215 How to Cite?
AbstractAlthough Escherichia coli is widely distributed in the marine environment, only a small percentage are pathogenic to humans. Nonetheless, the widespread occurrence of waterborne infections of E. coli origin in humans has become one of the major health problems worldwide. To date, several types of enterovirulent E. coli have been recognized as the aetiologic agents of various gastrointestinal infections in humans. The most commonly encountered are those belonging to the enterotoxigenic (ETEC), enteroinvasive (EIEC), enterohaemorrhagic (EHEC) and enteropathogenic (EPEC) subtypes. In order to better 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 strains of E. coli in the aquatic environment. The target genes chosen for this investigation included: the PHO-A housekeeping gene (present in all E. coli); the LT1, LT2 and ST1 genes of ETEC; the VT1 and VT2 verotoxin, and EAE virulence genes of EHEC and EPEC, respectively. Six pairs of oligonucleotide primers were designed to simultaneously amplify internal fragments of these genes by multiplex PCR to generate PCR products that could be analysed 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 expected size from several control strains of E. coli - ATCC 35401 (LT1+/ST1+); SA53 (LT2+/VT2+); and O157 (VT1+/VT2+/EAE+). The detection sensitivity of the multiplex PCR system for the six target genes in an E. coli cell mixture was optimized and enhanced by preincubating serially diluted cells in Luria-Bertani broth for 6 h prior to PCR analysis. The results obtained indicated a detection sensitivity of 10°CFU (of each strain) per 100 μl reaction. Multiplex PCR analysis of seawater samples collected from four sewage-polluted sites in Hong Kong indicated the presence in all four samples of E. coli bacteria that were positive for LT1, ST1, VT1 and EAE virulence genes. Overall, the data indicated that the multiplex PCR system described in this study is a potentially very useful and powerful method for routine monitoring and risk assessment of water quality. Copyright (C) 1999 Elsevier Science Ltd.
Persistent Identifierhttp://hdl.handle.net/10722/92756
ISSN
2015 Impact Factor: 3.099
2015 SCImago Journal Rankings: 1.264
ISI Accession Number ID
References

 

DC FieldValueLanguage
dc.contributor.authorKong, RYCen_HK
dc.contributor.authorSo, CLen_HK
dc.contributor.authorLaw, WFen_HK
dc.contributor.authorWu, RSSen_HK
dc.date.accessioned2010-09-17T10:56:14Z-
dc.date.available2010-09-17T10:56:14Z-
dc.date.issued1999en_HK
dc.identifier.citationMarine Pollution Bulletin, 1999, v. 38 n. 12, p. 1207-1215en_HK
dc.identifier.issn0025-326Xen_HK
dc.identifier.urihttp://hdl.handle.net/10722/92756-
dc.description.abstractAlthough Escherichia coli is widely distributed in the marine environment, only a small percentage are pathogenic to humans. Nonetheless, the widespread occurrence of waterborne infections of E. coli origin in humans has become one of the major health problems worldwide. To date, several types of enterovirulent E. coli have been recognized as the aetiologic agents of various gastrointestinal infections in humans. The most commonly encountered are those belonging to the enterotoxigenic (ETEC), enteroinvasive (EIEC), enterohaemorrhagic (EHEC) and enteropathogenic (EPEC) subtypes. In order to better 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 strains of E. coli in the aquatic environment. The target genes chosen for this investigation included: the PHO-A housekeeping gene (present in all E. coli); the LT1, LT2 and ST1 genes of ETEC; the VT1 and VT2 verotoxin, and EAE virulence genes of EHEC and EPEC, respectively. Six pairs of oligonucleotide primers were designed to simultaneously amplify internal fragments of these genes by multiplex PCR to generate PCR products that could be analysed 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 expected size from several control strains of E. coli - ATCC 35401 (LT1+/ST1+); SA53 (LT2+/VT2+); and O157 (VT1+/VT2+/EAE+). The detection sensitivity of the multiplex PCR system for the six target genes in an E. coli cell mixture was optimized and enhanced by preincubating serially diluted cells in Luria-Bertani broth for 6 h prior to PCR analysis. The results obtained indicated a detection sensitivity of 10°CFU (of each strain) per 100 μl reaction. Multiplex PCR analysis of seawater samples collected from four sewage-polluted sites in Hong Kong indicated the presence in all four samples of E. coli bacteria that were positive for LT1, ST1, VT1 and EAE virulence genes. Overall, the data indicated that the multiplex PCR system described in this study is a potentially very useful and powerful method for routine monitoring and risk assessment of water quality. Copyright (C) 1999 Elsevier Science Ltd.en_HK
dc.languageengen_HK
dc.publisherPergamon. The Journal's web site is located at http://www.elsevier.com/locate/marpolbulen_HK
dc.relation.ispartofMarine Pollution Bulletinen_HK
dc.subjectEnterohaemorrhagicen_HK
dc.subjectEnteropathogenicen_HK
dc.subjectEnterotoxigenicen_HK
dc.subjectEscherichia colien_HK
dc.subjectMultiplex PCRen_HK
dc.subjectVirulence geneen_HK
dc.titleA sensitive and versatile multiplex PCR system for the rapid detection of enterotoxigenic (ETEC), enterohaemorrhagic (EHEC) and enteropathogenic (EPEC) strains of Escherichia colien_HK
dc.typeArticleen_HK
dc.identifier.emailWu, RSS: rudolfwu@hku.hken_HK
dc.identifier.authorityWu, RSS=rp01398en_HK
dc.description.naturelink_to_subscribed_fulltext-
dc.identifier.doi10.1016/S0025-326X(99)00164-2en_HK
dc.identifier.scopuseid_2-s2.0-0033400253en_HK
dc.relation.referenceshttp://www.scopus.com/mlt/select.url?eid=2-s2.0-0033400253&selection=ref&src=s&origin=recordpageen_HK
dc.identifier.volume38en_HK
dc.identifier.issue12en_HK
dc.identifier.spage1207en_HK
dc.identifier.epage1215en_HK
dc.identifier.isiWOS:000084587100031-
dc.publisher.placeUnited Kingdomen_HK
dc.identifier.scopusauthoridKong, RYC=7005290687en_HK
dc.identifier.scopusauthoridSo, CL=7102919929en_HK
dc.identifier.scopusauthoridLaw, WF=7103147825en_HK
dc.identifier.scopusauthoridWu, RSS=7402945079en_HK

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