title: [Multiplex real-time PCR detecting Salmonella, Shigella and diarrheagenic Escherichia coli].
abstract: OBJECTIVE
  To develop a multiplex real-time PCR for the detection of Salmonella invasion protein A gene (invA), enterotoxigenic Escherichia coli (ETEC) heat-labile I enterotoxin gene (elt), and Shigella or enteroinvasive E. coli (EIEC) invasive plasmid antigen H gene (ipaH).
  
  
  METHODS
  Under the optimized reaction conditions of the multiplex real-time PCR, invA, elt, and ipaH were determined in 10-fold series of dilution of DNA extracted from Salmonella enterica serovar Typhimurium, ETEC 44815 strain and Shigella F301 strain. The three genes were examined in 90 fecal samples from diarrhea patients using the multiplex real-time PCR. When PCR-positive samples were found, the target strains were isolated and identified.
  
  
  RESULTS
  The detectable concentration for this multiplex real-time PCR was 10 CFU/microl for Shigella F301 strain, 10(2) CFU/microl for S. enterica serovar Typhimurium and ETEC 44815 strain, respectively. Out of 90 fecal samples from diarrhea patients, thirteen were found positive for elt gene (14.4%), and five were found positive for ipaH gene (5.6%). Three E. coli strains positive for elt gene and four E. coli strains positive for ipaH gene were isolated successfully from the PCR-positive samples mentioned above. The detection of invA, elt and ipaH genes was completed in 10 h, which included an enrichment period of 6 h.
  
  
  CONCLUSIONS
  The multiplex real-time PCR assay can detect invA, elt, ipaH simultaneously in a single reaction, moreover, it can detect for virulence genes in strains of Salmonella, ETEC, and Shigella or EIEC and screen these pathogens in fecal specimens from patients with diarrhea with a high specificity.
authors.affiliation: Hangzhou Center for Disease Prevention and Control, Hangzhou 310006, China.
authors.forename: Xin-Fen
authors.initials: XF
authors.lastname: Yu
authors.forename: Jin-Cao
authors.initials: JC
authors.lastname: Pan
authors.forename: Dong-Mei
authors.initials: DM
authors.lastname: Meng
authors.forename: Hao-Qiu
authors.initials: HQ
authors.lastname: Wang
authors.forename: Wei
authors.initials: W
authors.lastname: Zhang
authors.forename: Wei
authors.initials: W
authors.lastname: Zheng
mesh_terms: D004269:DNA, Bacterial / Q000032:analysis*; D003967:Diarrhea / Q000382:microbiology*; D004926:Escherichia coli / Q000235:genetics; D005243:Feces / Q000382:microbiology*; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*; D012475:Salmonella / Q000235:genetics; D012760:Shigella / Q000235:genetics
chemical_list: D004269:DNA, Bacterial