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.