title: Development of a sensitive rRNA-targeted reverse transcription-quantitative polymerase chain reaction for detection of Vibrio cholerae/mimicus, V. parahaemolyticus/alginolyticus and Campylobacter jejuni/coli.
abstract: A sensitive rRNA-targeted reverse transcription-quantitative polymerase chain reaction (RT-qPCR) method was developed for detection of Vibrio cholerae/mimicus, V. parahaemolyticus/alginolyticus and Campylobacter jejuni/coli by using specific primers. Counts of the enteric pathogens spiked in human stools were quantified at the lower detection limit of 10(3) cells/g stool by RT-qPCR, in marked contrast with conventional quantitative polymerase chain reaction (qPCR) at the detection limit of 10(5) to 10(6) cells/g stool. The bacterial counts determined by RT-qPCR were almost equivalent to those determined by the culture method and fluorescence in situ hybridization (FISH) during the course of in vitro culture. Bacterial rRNA in the stools was stable for at least 4 weeks when the stools were kept as the suspensions in RNA-stabilizing agent, RNAlater®, even at 37(o) C. These data suggested that the rapid and high sensitive rRNA-targeted RT-qPCR was applicable for the accurate quantification of viable enteric pathogens, such as V. cholerae/mimicus, V. parahaemolyticus/alginolyticus and C. jejuni/coli.
authors.affiliation: Yakult Central Institute for Microbiological Research, 1796 Yaho, Kunitachi, Tokyo 186-8650, Japan. takashi-kurakawa@yakult.co.jp
authors.forename: Takashi
authors.initials: T
authors.lastname: Kurakawa
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Kubota
authors.forename: Hirokazu
authors.initials: H
authors.lastname: Tsuji
authors.forename: Kazunori
authors.initials: K
authors.lastname: Matsuda
authors.forename: Takashi
authors.initials: T
authors.lastname: Asahara
authors.forename: Takuya
authors.initials: T
authors.lastname: Takahashi
authors.forename: Thandavarayan
authors.initials: T
authors.lastname: Ramamurthy
authors.forename: Takashi
authors.initials: T
authors.lastname: Hamabata
authors.forename: Eizo
authors.initials: E
authors.lastname: Takahashi
authors.forename: Shin-ichi
authors.initials: S
authors.lastname: Miyoshi
authors.forename: Keinosuke
authors.initials: K
authors.lastname: Okamoto
authors.forename: Asish K
authors.initials: AK
authors.lastname: Mukhopadhyay
authors.forename: Yoshifumi
authors.initials: Y
authors.lastname: Takeda
authors.forename: Koji
authors.initials: K
authors.lastname: Nomoto
mesh_terms: D058491:Bacterial Load / Q000379:methods; D017000:Campylobacter coli / Q000302:isolation & purification*; D016123:Campylobacter jejuni / Q000302:isolation & purification; D017931:DNA Primers / Q000235:genetics; D005243:Feces / Q000382:microbiology; D020459:Genes, rRNA; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D012329:RNA, Bacterial / Q000235:genetics; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D013048:Specimen Handling; D014734:Vibrio cholerae / Q000302:isolation & purification; D044164:Vibrio mimicus / Q000302:isolation & purification; D014736:Vibrio parahaemolyticus / Q000302:isolation & purification*
chemical_list: D017931:DNA Primers; D012329:RNA, Bacterial; D012336:RNA, Ribosomal, 16S