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- 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.
- Authors
Kurakawa, Takashi; Kubota, Hiroyuki; Tsuji, Hirokazu; Matsuda, Kazunori; Asahara, Takashi; Takahashi, Takuya; Ramamurthy, Thandavarayan; Hamabata, Takashi; Takahashi, Eizo; Miyoshi, Shin-ichi; Okamoto, Keinosuke; Mukhopadhyay, Asish K.; Takeda, Yoshifumi; Nomoto, Koji
- Abstract
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 103 cells/g stool by RT-qPCR, in marked contrast with conventional quantitative polymerase chain reaction (qPCR) at the detection limit of 105 to 106 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, RNA later®, even at 37oC. 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.
- Subjects
RIBOSOMAL RNA; REVERSE transcriptase polymerase chain reaction; VIBRIO cholerae; VIBRIO parahaemolyticus; CAMPYLOBACTER jejuni; DIAGNOSTIC microbiology; PATHOGENIC microorganisms; FECES; MICROBIOLOGY
- Publication
Microbiology & Immunology, 2012, Vol 56, Issue 1, p10
- ISSN
0385-5600
- Publication type
Article
- DOI
10.1111/j.1348-0421.2011.00405.x