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

Abstract

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 degrees C or 4 degrees C but disappeared within 2 h at 37 degrees 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.