Nucleic acid persistence in heat-killed Escherichia coli O157 : H7 from contaminated skim milk

Polymerase chain reaction (PCR) and reverse transcriptase (RT)-PCR using pr imers targeting 16S rRNA sequences in Escherichia coli O157:H7 were applied to monitor the stability of rDNA anal rRNA in cells killed by mild heat tr eatment (60 degrees C) in skim milk. Serial dilutions of purified RNA and D NA from E. coli O157:H7 in skim milk were amplified by RT-PCR or PCR, respe ctively, before heat treatment and at time points 0, 6, 12, 24, and 48 h af ter heating. In general, DNA-PCR provided stronger amplification signals co mpared to RT-PCR at the corresponding time points with the same PCR primer set, indicating a tower efficiency of RNA amplification compared to that of DNA. Ribosomal RNA. and rDNA could be amplified by RT-PCR or PCR from both viable and dead cells throughout the 48-h posttreatment holding period. Fo r RT-PCR, amplification signals decreased in intensity with increased holdi ng time, while the efficiency of amplification of DNA sequences from dead c ells remained fairly stable throughout the study. DNA persistence was great er than that of rRNA following cell death by mild heat treatment in skim mi lk. Skim milk did not appear to accelerate nucleic acid degradation. While rRNA was less stable than DNA, its detection by RT-PCR may not be appropria te as an exclusive indicator of cell viability in minimally processed foods .