Effect of post-treatment holding conditions on detection of tufA mRNA in ethanol-treated Escherichia coli: implications for RT-PCR-based indirect viability tests

The PCR is a rapid and sensitive method for detecting and identifying low numbers of bacteria, but it does not discriminate between living and dead cells. Most messenger RNA (mRNA) molecules have a short half-life in the bacterial cell and their presence may therefore indicate viability. We have compared PCR and RT–PCR (targeted at tufA DNA or mRNA, respectively) for the detection of Escherichia coli, using healthy cells and those killed by exposure to different stress treatments. PCR gave a positive signal in live cells and those killed by autoclaving, boiling, or treatment with 50% ethanol, but was negative after exposure to pH 2·0 for 5 min. RT–PCR was positive in live cells but negative after all treatments except exposure to ethanol. The persistence of tufA mRNA was examined in ethanol-killed cells incubated in LB broth at different temperatures. The RT–PCR signal persisted for up to 16 h at 15 °C or 4 °C but disappeared within 2 h at 37 °C. RT–PCR thus has potential as an indicator of viability provided samples are pre-incubated under appropriate conditions that will ensure decay of any residual mRNA in dead cells.