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

Takashi Kurakawa, Hiroyuki Kubota, Hirokazu Tsuji, Kazunori Matsuda, Takashi Asahara, Takuya Takahashi, Thandavarayan Ramamurthy, Takashi Hamabata, Eizo Takahashi, Shin-ichi Miyoshi, Keinosuke Okamoto, Asish K. Mukhopadhyay, Yoshifumi Takeda, Koji Nomoto

Research output: Contribution to journalArticle

15 Citations (Scopus)

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 6cells/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 oC. 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.

Original languageEnglish
Pages (from-to)10-20
Number of pages11
JournalMicrobiology and Immunology
Volume56
Issue number1
DOIs
Publication statusPublished - Jan 2012

Fingerprint

Vibrio mimicus
Campylobacter coli
Campylobacter jejuni
Vibrio cholerae
Reverse Transcription
Polymerase Chain Reaction
Limit of Detection
Bacterial Load
Excipients
Fluorescence In Situ Hybridization
Suspensions
RNA

Keywords

  • Campylobacter jejuni
  • RT-qPCR
  • Vibrio cholerae
  • Vibrio parahaemolyticus

ASJC Scopus subject areas

  • Immunology
  • Microbiology
  • Virology

Cite this

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. / 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.

In: Microbiology and Immunology, Vol. 56, No. 1, 01.2012, p. 10-20.

Research output: Contribution to journalArticle

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. / 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. In: Microbiology and Immunology. 2012 ; Vol. 56, No. 1. pp. 10-20.
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