Effect of Amplicon Length in Propidium Monoazide Quantitative PCR for the Enumeration of Viable Cells of Salmonella in Cooked Ham

摘要

Real-time PCR-based methods have been frequently used to detect and enumerate foodborne pathogens. However, these techniques have a major drawback since they cannot differentiate between DNA from live and dead cells. In this study, we developed a propidium monoazide (PMA)-based PCR method to detect and enumerate viable Salmonella cells in the presence of high number of dead cells (up to 108 CFU/g) in cooked ham. Three different specific PCR targets differing in length (95, 285, and 417 bp) were tested. We found that the inhibition effect was dependent on the PCR amplification product length, and only the longer product achieved suppression of 108 CFU/g of heat-killed cells. SYBR~R Green and TaqMan~R chemistries were compared to develop a highly efficient PMA-quantitative PCR system targeting the 417-bp fragment. Both chemistries showed similar detection (103 CFU/g) and quantification limits (104 CFU/g), but TaqMan~R assay showed higher efficiency (98.6 %) than SYBR~R Green assay (92.8 %). PMA-quantitative PCR assay developed is a rapid method for the selective detection and enumeration of viable Salmonella cells with further application in postprocessed meat products and safe shelf-life studies.