title: Rapid genetic typing of diarrheagenic Escherichia coli using a two-tube modified molecular beacon based multiplex real-time PCR assay and its clinical application.
abstract: BACKGROUND
  Diarrheagenic Escherichia coli (DEC), including Enterotoxigenic E.coli (ETEC), Enteroaggregative E.coli (EAEC), Enteropathogenic E.coli (EPEC), Enterohemolysin E.coli (EHEC) and Enteroinvasive E.coli (EIEC) causes diarrhea or hemolytic uremic syndromes among infants and travelers around the world. A rapid, reliable and repeatable method is urgent for identifying DEC so as to provide the reference for responding to diarrheal disease outbreak and the treatment of the diarrheal patients associated with DEC.
  
  
  METHODS
  In this study, specific primers and modified molecular beacon probes of nine specific virulence genes, whose 5'end were added with homo tail sequence, were designed; and a two-tube modified molecular beacon based multiplex real-time PCR (rtPCR) assay for the identification of five Escherichia coli pathotypes, including ETEC, EAEC, EPEC, EHEC and EIEC was developed and optimized. Totally 102 bacterial strains, including 52 reference bacterial strains and 50 clinical strains were detected to confirm whether the target genes selected were specific. Then detection limits of the assay were tested. Lastly, the assay was applied to the detection of 11860 clinical samples to evaluate the specificity and sensitivity of the developed assay compared with the conventional PCR.
  
  
  RESULTS
  The target genes were 100% specific as assessed on 102 bacterial strains since no cross-reactions were observed. The detection limits ranged from 88 CFU/mL (EHEC) to 880 CFU/mL (EPEC). Compared with the conventional PCR, the specificity and sensitivity of the multiplex rtPCR was 100% and over 99%, respectively. The coefficient of variation (CV) for each target gene ranged from 0.45% to 1.53%. 171 positive clinical samples were mostly identified as ETEC (n = 111, 64.9%) and EPEC (n = 38, 22.2%), which were the dominating pathotypes of DEC strains.
  
  
  CONCLUSIONS
  The developed multiplex rtPCR assay for the identification of DEC was high sensitive and specific and could be applied to the rapid identification of DEC in clinical and public health laboratories.
authors.forename: Qingliang
authors.initials: Q
authors.lastname: Chen
authors.forename: Xiaolu
authors.initials: X
authors.lastname: Shi
authors.forename: Yinghui
authors.initials: Y
authors.lastname: Li
authors.forename: Yixiang
authors.initials: Y
authors.lastname: Jiang
authors.forename: Yiman
authors.initials: Y
authors.lastname: Lin
authors.forename: Yaqun
authors.initials: Y
authors.lastname: Qiu
authors.forename: Qingge
authors.initials: Q
authors.lastname: Li
authors.forename: Qinghua
authors.initials: Q
authors.lastname: Hu
mesh_terms: D017931:DNA Primers / Q000235:genetics; D003967:Diarrhea / Q000382:microbiology*; D004926:Escherichia coli / Q000145:classification* / Q000235:genetics* / Q000302:isolation & purification; D004927:Escherichia coli Infections / Q000382:microbiology*; D006801:Humans; D058889:Molecular Typing / Q000379:methods*; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods*; D015345:Oligonucleotide Probes / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D013997:Time Factors
chemical_list: D017931:DNA Primers; D015345:Oligonucleotide Probes