title: Development of quantitative real-time PCR for detection and enumeration of Enterobacteriaceae.
abstract: The family Enterobacteriaceae, members of which are widely distributed in the environment, includes many important human pathogens. In this study, a rapid real-time PCR method targeting rplP, coding for L16 protein, a component of the ribosome large subunit, was developed for enumerating Enterobacteriaceae strains, and its efficiency was evaluated using naturally contaminated food products. The rplP-targeted real-time PCR amplified Enterobacteriaceae species with Ct values of 14.0-22.8, whereas the Ct values for non-Enterobacteriaceae species were >30, indicating the specificity of this method for the Enterobacteriaceae. Using a calibration curve of Ct=-3.025 (log CFU/g)+37.35, which was calculated from individual plots of the cell numbers in different concentrations of 5 Enterobacteriaceae species, the rplP-targeted real-time PCR was applied to 51 food samples. A <1log difference between the real-time PCR and culture methods was obtained in a majority of the food samples (81.8%), with good correlation (r2=0.8285). This study demonstrated that the rplP-targeted real-time PCR method could detect and enumerate Enterobacteriaceae species in foods rapidly and accurately, and therefore, it can be used for the microbiological risk analysis of foods.
authors.affiliation: Department of Food Science and Technology, Faculty of Marine Science, Tokyo University of Marine Science and Technology, 4-5-7 Konan, Minato-ku, Tokyo 108-8477, Japan. Electronic address: hajime@kaiyodai.ac.jp.
authors.forename: Hajime
authors.initials: H
authors.lastname: Takahashi
authors.affiliation: Department of Food Science and Technology, Faculty of Marine Science, Tokyo University of Marine Science and Technology, 4-5-7 Konan, Minato-ku, Tokyo 108-8477, Japan.
authors.forename: Rumi
authors.initials: R
authors.lastname: Saito
authors.affiliation: Department of Food Science and Technology, Faculty of Marine Science, Tokyo University of Marine Science and Technology, 4-5-7 Konan, Minato-ku, Tokyo 108-8477, Japan.
authors.forename: Satoko
authors.initials: S
authors.lastname: Miya
authors.affiliation: Department of Food Science and Technology, Faculty of Marine Science, Tokyo University of Marine Science and Technology, 4-5-7 Konan, Minato-ku, Tokyo 108-8477, Japan.
authors.forename: Yuichiro
authors.initials: Y
authors.lastname: Tanaka
authors.affiliation: Department of Food Science and Technology, Faculty of Marine Science, Tokyo University of Marine Science and Technology, 4-5-7 Konan, Minato-ku, Tokyo 108-8477, Japan.
authors.forename: Natsumi
authors.initials: N
authors.lastname: Miyamura
authors.affiliation: Department of Food Science and Technology, Faculty of Marine Science, Tokyo University of Marine Science and Technology, 4-5-7 Konan, Minato-ku, Tokyo 108-8477, Japan.
authors.forename: Takashi
authors.initials: T
authors.lastname: Kuda
authors.affiliation: Department of Food Science and Technology, Faculty of Marine Science, Tokyo University of Marine Science and Technology, 4-5-7 Konan, Minato-ku, Tokyo 108-8477, Japan.
authors.forename: Bon
authors.initials: B
authors.lastname: Kimura
mesh_terms: D015169:Colony Count, Microbial; D017931:DNA Primers; D004755:Enterobacteriaceae / Q000302:isolation & purification*; D005516:Food Microbiology / Q000379:methods*; D006920:Hygiene; D016014:Linear Models; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
chemical_list: D017931:DNA Primers
keywords: Enterobacteriaceae; Hygiene indicator; Real-time PCR; rplP