title: A novel single-step multiplex polymerase chain reaction assay for the detection of diarrheagenic Escherichia coli.
abstract: Escherichia coli that causes diarrhea in humans is referred to as diarrheagenic E. coli (DEC), and has been categorized into the following 5 groups: shigatoxin-producing E. coli (STEC), enteropathogenic E. coli (EPEC), enteroinvasive E. coli (EIEC), enteroaggregative E. coli (EAggEC), and enterotoxigenic E. coli (ETEC). In this study, we developed a novel one-step multiplex polymerase chain reaction (mPCR) for the rapid detection of 10 pathogenic genes (stx1, stx2, eae, bfpA, invE, aggR, esth, estp, elt, and astA) of DEC. Five categorized strains were used as positive controls for DEC harboring each pathogenic gene, and 828 DEC-like strains, isolated from diarrheal stool samples and assumed to be DEC on the basis of serotyping, were used in the mPCR-based detection of the pathogenic genes. To demonstrate the utility of mPCR, the 828 strains were subjected to our optimized protocol, and the results obtained were compared with those obtained by monoplex PCR. The results showed agreement for all strains. Using mPCR, we also detected 65 DEC and 41 astA-positive E. coli, and 7 of these DEC strains were "O antigen untypable" (OUT). This novel mPCR protocol allowed for rapid, convenient, and economical pathogenicity-based identification of the DEC.
authors.affiliation: Hirosaki University, Graduate School of Health Sciences, Hon-cho 66-1, Hirosaki, Aomori, 036-8564, Japan. mfujioka@cc.hirosaki-u.ac.jp
authors.forename: Miyuki
authors.initials: M
authors.lastname: Fujioka
authors.forename: Yoshimitsu
authors.initials: Y
authors.lastname: Otomo
authors.forename: Chowdhury Rafiqul
authors.initials: CR
authors.lastname: Ahsan
mesh_terms: D001431:Bacteriological Techniques / Q000191:economics / Q000379:methods*; D003967:Diarrhea / Q000175:diagnosis* / Q000382:microbiology; D004926:Escherichia coli / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D004927:Escherichia coli Infections / Q000175:diagnosis* / Q000382:microbiology; D029968:Escherichia coli Proteins / Q000235:genetics; D006801:Humans; D025202:Molecular Diagnostic Techniques / Q000191:economics / Q000379:methods*; D060885:Multiplex Polymerase Chain Reaction / Q000191:economics / Q000379:methods*; D013997:Time Factors; D037521:Virulence Factors / Q000235:genetics
chemical_list: D029968:Escherichia coli Proteins; D037521:Virulence Factors