Premium
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
Author(s) -
Kurakawa Takashi,
Kubota Hiroyuki,
Tsuji Hirokazu,
Matsuda Kazunori,
Asahara Takashi,
Takahashi Takuya,
Ramamurthy Thandavarayan,
Hamabata Takashi,
Takahashi Eizo,
Miyoshi Shinichi,
Okamoto Keinosuke,
Mukhopadhyay Asish K.,
Takeda Yoshifumi,
Nomoto Koji
Publication year - 2012
Publication title -
microbiology and immunology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.664
H-Index - 70
eISSN - 1348-0421
pISSN - 0385-5600
DOI - 10.1111/j.1348-0421.2011.00405.x
Subject(s) - biology , vibrio parahaemolyticus , microbiology and biotechnology , vibrio cholerae , vibrio alginolyticus , campylobacter jejuni , campylobacter , ribosomal rna , polymerase chain reaction , vibrionaceae , reverse transcription polymerase chain reaction , real time polymerase chain reaction , bacteria , vibrio , gene , messenger rna , biochemistry , genetics
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, RNA later ®, 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 .