A qPCR assay for Bordetella pertussis cells that enumerates both live and dead bacteria
Fig 4
The effect of dark and light exposure times on PMA-inhibition of PCR amplification.
A) Dark incubation B) Light Incubation. PMA and untreated heat-killed suspensions were incubated for 10, 20, 30, and 70 minutes in the dark followed by exposure to 5 minutes of light. 10 minutes or longer of incubation in the dark produced a ≥99% reduction in the PCR amplification signal. Optimal light incubation periods were determined by incubating untreated and PMA treated heat-killed suspensions in the dark for 10 minutes followed by light exposure for 5, 10, 20, 30, and 40 minutes. Incubating PMA treated heat-killed samples under light for periods of 5, 10, 20, and 30 minutes produced a 99% or greater reduction in the PCR amplification signal. Five minutes was selected as the standard light incubation period. Error bars represent standard deviation from two biological replicates. The experiment was repeated with the same result.