Figure 1.
Effect of incubation time and PCR primer on detection of live and dead cells by real-time PCR.
Live or isopropanol-inactivated dead cells of E. coli O157:H7 ATCC 700728 (5×107 CFU) were incubated in the dark with or without 40 µM PMA for 15, 30, or 60. Real-time PCR was performed using the lpfA or hlyA primer sets [20], [21]. Each bar represents the mean ± stdev for three independent replicates.
Figure 2.
Interference of dead cells in the enumeration of live E. coli O157:H7 by PMA real-time PCR.
Cell amounts were quantified by real-time PCR (total; black bars) or PMA real-time PCR (viable; open bars) in mixtures of 103 or 104 live cells mixed with 106 isopropanol-inactivated dead cells. Samples were treated with 40 µM PMA for 30 min. Estimates of E coli O157:H7 cell amounts were based on comparisons to standard curves constructed using E. coli O157:H7 ATCC 700728 genomic DNA. Each bar represents the mean ± stdev for three independent replicates.
Figure 3.
Survival of E. coli O157:H7 ATCC 700728 and EC4045 on inoculated lettuce in a growth chamber.
E. coli O157:H7 cell amounts were measured by plate count (culturable), real-time PCR (total), and PMA real-time PCR (viable) methods. Plants were inoculated with a spray bottle (A) or by drop (B) and incubated at a relative humidity of 30%. The mean ± stdev of 15 lettuce plants or leaves at each sampling time for three replicate experiments is shown.
Figure 4.
E. coli O157:H7 ATCC 700728 survival on lettuce in the field.
E. coli O157:H7 amounts were measured by plate count (culturable), real-time PCR (total), and PMA real-time PCR (viable). Viable amounts were below the detection limit (3.7 log CFU/plant or leaf) at 2 and 48 h. The mean ± stdev of 12 lettuce plants at each sampling time is shown.