Quantification of Viable Plesiomonas shigelloides in a Mixture of Viable and Dead Cells Using Ethidium Bromide Monoazide and Conventional PCR

摘要

A rapid and efficient method for quantitative detection of viable Plesiomonas shigelloides in pure culture containing a mixture of viable and heat-killed cells was developed using ethidium bromide monoazide (EMA) in combination with the polymerase chain reaction (PCR). The addition of EMA (1 mug/ml) to mixtures of viable and heat-killed cells of P. shigelloides inhibited the PCR amplification of DNA derived from the dead cells, but did not inhibit the PCR amplification of DNA derived from the viablecells. EMA at 5 mug/ml or less had little or no inhibition on the PCR amplification of DNA derived from viable cells of P. shigelloides. After EMA treatment, the DNA from viable P. shigelloides cells in varying ratios of viable to dead cells could be selectively quantified by PCR. The minimum level of detection was DNA from 24 genomic targets per PCR reaction. A linear relationship was found between the relative fluorescent intensity of the DNA bands and the log of genomic targets derived from the viable cells in mixtures of viable and dead cells in the range of 2.4 x 10~1 to 2.4 x 10~4 DNA targets from viable cells per PCR.