Novel method for estimating viable Salmonella cell counts using real-time PCR.
A novel method for estimating viable Salmonella Enteritidis cell counts with 5'-nuclease real-time PCR was developed in this study. Our method was based on the increase kinetics of the target DNA region (invA) of the microorganism growing in a food/clinical sample in a culture medium during incubation. The index of increase in the target DNA region studied here was threshold cycle, CT. A test Salmonella strain was grown in buffered peptone water at the optimal temperature (39 degrees C). As Salmonella cells were grown, the value of CT decreased with time, generating a downward sigmoidal curve. The slope of the curve was constant at various initial cell concentrations. With higher initial cell concentration, the CT value evaluated from the slope at a given time was lower. With this relationship, a novel method for estimating the initial viable cell concentration of a sample was developed. Dead Salmonella cells or bacteria other than the target cell caused deviation in the CT curve. Incubation in a selective media suppressed the deviation caused by other bacterial cells. We think that this method could be applied to many other microorganisms cultivable in a suitable medium.
Fujikawa
Hiroshi
H
Department of Microbiology, Tokyo Metropolitan Institute of Public Health: 3-24-1, Hyakunin-cho, Shinjuku-ku, Tokyo 169-0073, Japan.
Shimojima
Yukako
Y
Yano
Kazuyoshi
K
D015169:Colony Count, Microbial / Q000379:methods*; D003199:Computer Systems; D003470:Culture Media; D004269:DNA, Bacterial / Q000032:analysis; D005506:Food Contamination / Q000032:analysis; D005516:Food Microbiology; D016133:Polymerase Chain Reaction / Q000379:methods*; D012477:Salmonella enteritidis / Q000166:cytology / Q000235:genetics / Q000254:growth & development*
D003470:Culture Media; D004269:DNA, Bacterial