title: Sensitive quantification of Clostridium difficile cells by reverse transcription-quantitative PCR targeting rRNA molecules.
abstract: We established a sensitive and accurate quantification system for Clostridium difficile in human intestines, based on rRNA-targeted reverse transcription-quantitative PCR (RT-qPCR). We newly developed a species-specific primer set for C. difficile targeting 23S rRNA gene sequences. Both the vegetative cells and the spores of C. difficile in human feces were quantified by RT-qPCR, with a lower detection limit of 10(2.4) cells/g of feces. In an analysis of the feces of residents (n = 83; age, 85 ± 8 years) and staff (n = 19; age, 36 ± 10 years) at a care facility for the elderly, C. difficile was detected by RT-qPCR in 43% of the residents (average count, log(10) 4.0 ± 2.0 cells/g of feces) and 16% of the staff (average count, log(10) 2.2 ± 0.1 cells/g of feces); these rates were far higher than those detected by qPCR (residents, 19%; staff, 0%) or selective cultivation (residents, 18%; staff, 5%). Another analysis of healthy adults (n = 63; age, 41 ± 11 years) also revealed the significant carriage rate of C. difficile in the intestines (detection rate, 13%; average count, log(10) 4.9 ± 1.2 cells/g of feces). From these results, it was suggested that rRNA-targeted RT-qPCR should be an effective tool for analyzing population levels of C. difficile in the human intestine.
authors.affiliation: Yakult Central Institute for Microbiological Research, Tokyo, Japan. kazunori-matsuda@yakult.co.jp
authors.forename: Kazunori
authors.initials: K
authors.lastname: Matsuda
authors.forename: Hirokazu
authors.initials: H
authors.lastname: Tsuji
authors.forename: Takashi
authors.initials: T
authors.lastname: Asahara
authors.forename: Takuya
authors.initials: T
authors.lastname: Takahashi
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Kubota
authors.forename: Satoru
authors.initials: S
authors.lastname: Nagata
authors.forename: Yuichiro
authors.initials: Y
authors.lastname: Yamashiro
authors.forename: Koji
authors.initials: K
authors.lastname: Nomoto
mesh_terms: D000328:Adult; D000369:Aged, 80 and over; D016360:Clostridioides difficile / Q000235:genetics / Q000254:growth & development*; D015169:Colony Count, Microbial / Q000379:methods*; D017931:DNA Primers / Q000235:genetics; D004761:Enterocolitis, Pseudomembranous / Q000453:epidemiology* / Q000382:microbiology*; D005243:Feces / Q000382:microbiology; D006801:Humans; D007422:Intestines / Q000382:microbiology*; D007564:Japan / Q000453:epidemiology; D012338:RNA, Ribosomal, 23S / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*
chemical_list: D017931:DNA Primers; D012338:RNA, Ribosomal, 23S