title: Detection and identification of enterohemorrhagic Escherichia coli O157:H7 and Vibrio cholerae O139 using oligonucleotide microarray.
abstract: BACKGROUND
  The rapid and accurate detection and identification of the new subtype of the pathogens is crucial for diagnosis, treatment and control of the contagious disease outbreak. Here, in this study, an approach to detect and identify Escherichia coli O157:H7 and Vibrio cholerae O139 was established using oligonucleotide microarray. We coupled multiplex PCR with oligonucleotide microarray to construct an assay suitable for simultaneous identification of two subtypes of the pathogens.
  
  
  RESULTS
  The stx1, stx2 gene and uidA gene having the specific mutant spot were chosen as the targets for Escherichia coli O157:H7, and meanwhile the ctxA, tcpA, and LPSgt gene for Vibrio cholerae O139. The oligonucleotide microarray was composed of eight probes including negative control and positive control from 16S rDNA gene. The six primers were designed to amplify target fragments in two triplex PCR, and then hybridized with oligonucleotide microarray. An internal control would be to run a PCR reaction in parallel. Multiplex PCR did not produce any non-specific amplicons when 149 related species or genera of standard bacteria were tested (100% specificity). In addition, Escherichia coli O157:H7 and Escherichia coli O157:non-H7, Vibrio cholerae O139 and Vibrio cholerae O1 had been discriminated respectively. Using recombinant plasmid and target pathogens, we were able to detect positive hybridization signals with 102 copies/muL and 103 cfu/mL per reaction.
  
  
  CONCLUSIONS
  The DNA microarray assay reported here could detect and identify Escherichia coli O157:H7 and Vibrio cholerae O139, and furthermore the subtype was distinguished. This assay was a specific and sensitive tool for simultaneous detection and identification of the new subtype of two pathogens causing diarrhea in human.
authors.affiliation: Beijing Institute of Radiation Medicine, Beijing, 100850, China.
authors.forename: Da-Zhi
authors.initials: DZ
authors.lastname: Jin
authors.affiliation: Beijing Institute of Radiation Medicine, Beijing, 100850, China.
authors.forename: Xiao-Jing
authors.initials: XJ
authors.lastname: Xu
authors.affiliation: Beijing Institute of Radiation Medicine, Beijing, 100850, China.
authors.forename: Su-Hong
authors.initials: SH
authors.lastname: Chen
authors.affiliation: Beijing Institute of Radiation Medicine, Beijing, 100850, China.
authors.forename: Si-Yuan
authors.initials: SY
authors.lastname: Wen
authors.affiliation: College of Animal Science and Medicine, Inner Mongolia Agricultural University, Hohhot, 010018, China.
authors.forename: Xue-En
authors.initials: XE
authors.lastname: Ma
authors.affiliation: Zhejiang Provincial Center for Disease Control and Prevention, Hangzhou, 310009, China.
authors.forename: Zheng
authors.initials: Z
authors.lastname: Zhang
authors.affiliation: Wenzhou Medicine College affiliated Wenling First Hospital, Wenling, Zhejiang province, Wenling, 317500, China.
authors.forename: Feng
authors.initials: F
authors.lastname: Lin
authors.affiliation: Beijing Institute of Radiation Medicine, Beijing, 100850, China.
authors.forename: Sheng-Qi
authors.initials: SQ
authors.lastname: Wang