title: Development of TaqMan-based quantitative PCR for sensitive and selective detection of toxigenic Clostridium difficile in human stools.
abstract: BACKGROUND
  Clostridium difficile is the main cause of nosocomial diarrhea, but is also found in asymptomatic subjects that are potentially involved in transmission of C. difficile infection. A sensitive and accurate detection method of C. difficile, especially toxigenic strains is indispensable for the epidemiological investigation.
  
  
  METHODS
  TaqMan-based quantitative-PCR (qPCR) method for targeting 16S rRNA, tcdB, and tcdA genes of C. difficile was developed. The detection limit and accuracy of qPCR were evaluated by analyzing stool samples spiked with known amounts of C. difficile. A total of 235 stool specimens collected from 82 elderly nursing home residents were examined by qPCR, and the validity was evaluated by comparing the detection result with that by C. difficile selective culture (CDSC).
  
  
  RESULTS
  The analysis of C. difficile-spiked stools confirmed that qPCR quantified whole C. difficile (TcdA+TcdB+, TcdA-TcdB+, and TcdA-TcdB- types), TcdB-producing strains (TcdA+TcdB+ and TcdA-TcdB+ types), and TcdA-producing strains (TcdA+TcdB+ type), respectively, with a lower detection limit of 103 cells/g of stool. Of the 235 specimens examined, 12 specimens (5.1%) were C. difficile-positive by qPCR: TcdA+TcdB+ strain in six specimens and TcdA-TcdB- strain in the other six. CDSC detected C. difficile in 9 of the 12 specimens, and toxigenic types of the isolates from the 9 specimens were consistent with those identified by qPCR, supporting the validity of our qPCR method. Moreover, the qPCR examination revealed that the carriage rate of whole C. difficile and that of toxigenic strains in the 82 subjects over a 6-month period ranged from 2.4 to 6.8% and 1.2 to 3.8%, respectively. An average qPCR count of C. difficile detected was 104.5 cells/g of stool, suggesting that C. difficile constituted a very small fraction of intestinal microbiota.
  
  
  CONCLUSIONS
  Our qPCR method should be an effective tool for both clinical diagnosis and epidemiological investigation of C. difficile.
authors.affiliation: Yakult Honsha European Research Center for Microbiology ESV, Gent-Zwijnaarde, Belgium; Yakult Central Institute, Tokyo, Japan.
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Kubota
authors.affiliation: Yakult Central Institute, Tokyo, Japan.
authors.forename: Takafumi
authors.initials: T
authors.lastname: Sakai
authors.affiliation: Yakult Honsha European Research Center for Microbiology ESV, Gent-Zwijnaarde, Belgium.
authors.forename: Agata
authors.initials: A
authors.lastname: Gawad
authors.affiliation: Yakult Central Institute, Tokyo, Japan.
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Makino
authors.affiliation: Yakult Honsha European Research Center for Microbiology ESV, Gent-Zwijnaarde, Belgium; Yakult Central Institute, Tokyo, Japan.
authors.forename: Takuya
authors.initials: T
authors.lastname: Akiyama
authors.affiliation: Yakult Central Institute, Tokyo, Japan.
authors.forename: Eiji
authors.initials: E
authors.lastname: Ishikawa
authors.affiliation: Yakult Honsha European Research Center for Microbiology ESV, Gent-Zwijnaarde, Belgium; Yakult Central Institute, Tokyo, Japan.
authors.forename: Kenji
authors.initials: K
authors.lastname: Oishi
mesh_terms: D000368:Aged; D000369:Aged, 80 and over; D016360:Clostridioides difficile / Q000235:genetics / Q000302:isolation & purification*; D017931:DNA Primers / Q000378:metabolism; D005243:Feces / Q000382:microbiology*; D005260:Female; D005798:Genes, Bacterial; D006801:Humans; D007118:Immunoassay; D057230:Limit of Detection; D008297:Male; D009735:Nursing Homes; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D019914:Taq Polymerase / Q000378:metabolism*
chemical_list: D017931:DNA Primers; D012336:RNA, Ribosomal, 16S; D019914:Taq Polymerase