An Improved Real-Time Viability PCR Assay to Detect Salmonella in a Culture-Independent Era

Viability PCR (vPCR) uses a DNA intercalating dye to irreversibly bind double-stranded DNA from organisms with compromised cell membranes. This allows the selective amplification of DNA from intact cells. An optimized vPCR protocol should minimize false positives (DNA from compromised cells not fully removed) and false negatives (live cell DNA bound by the dye). We aimed to optimize a vPCR protocol using PMAxx (TM) as the intercalating agent and Salmonella Enteritidis as the target organism. To do this, we studied (1) single vs. sequential PMAxx (TM) addition; (2) a wash step post-PMAxx (TM) treatment; (3) a change of tube post-treatment before DNA extraction. The single vs. sequential PMAxx (TM) addition showed no difference. Results signified that PMAxx (TM) potentially attached to polypropylene tube walls and bound the released DNA from PMA-treated live cells when lysed in the same tube. A wash step was ineffective but transfer of the treated live cells to a new tube minimized these false-negative results. Our optimized protocol eliminated 10(8) CFU/mL heat-killed cell DNA in the presence of different live cell dilutions without compromising the amplification of the live cells, minimizing false positives. With further improvements, vPCR has great potential as a culture-independent diagnostic tool.