title: A duplex SYBR Green I real-time quantitative PCR assay for detecting Escherichia coli O157:H7.
abstract: PCR and hybridization assays are widely used for the detection and identification of Escherichia coli serogroups and serotypes. We used these techniques for the detection of E. coli O157:H7, a dominant serogroup among E. coli strains that are considered major public health problems worldwide. We developed a quantitative PCR assay using SYBR Green I, based on the published sequences of the rfbE and fliC genes from E. coli O157:H7. This method detected the E. coli O157:H7 O somatic antigen gene and the flagellar antigen gene simultaneously, with good specificity, sensitivity, and repeatability. The sensitivity of the assay was 2.95 x 10 copies/µL, which is 10(3) times more sensitive than obtained with a conventional PCR. The intra-assay and inter-assay coefficients of variation were less than 2%. We concluded that this duplex quantitative PCR assay is adequate for the identification and quantitative analysis of E. coli O157:H7. This provides a new identification method for clinical diagnosis of E. coli O157:H7 and for food safety analysis, as well as for molecular epidemiological studies of foodborne diseases.
authors.affiliation: Institute of Poultry Diseases, Henan Agricultural University, Zhengzhou, Henan, China.
authors.forename: X
authors.initials: X
authors.lastname: Yang
authors.forename: H W
authors.initials: HW
authors.lastname: Cheng
authors.forename: L
authors.initials: L
authors.lastname: Chen
authors.forename: J
authors.initials: J
authors.lastname: Zhao
authors.forename: H T
authors.initials: HT
authors.lastname: Chang
authors.forename: X W
authors.initials: XW
authors.lastname: Wang
authors.forename: H Y
authors.initials: HY
authors.lastname: Liu
authors.forename: H X
authors.initials: HX
authors.lastname: Yao
authors.forename: L X
authors.initials: LX
authors.lastname: Zhang
authors.forename: C Q
authors.initials: CQ
authors.lastname: Wang
mesh_terms: D052160:Benzothiazoles; D003959:Diamines; D019453:Escherichia coli O157 / Q000145:classification / Q000235:genetics*; D006801:Humans; D009930:Organic Chemicals; D011804:Quinolines; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
chemical_list: D052160:Benzothiazoles; D003959:Diamines; D009930:Organic Chemicals; D011804:Quinolines; C098022:SYBR Green I