Abstract:

The combination of ethidium monoazide (EMA) and PCR was used for detecting viable cells of Salmonella
enteritis. The optimization of experimental parameters showed that a final EMA concentration of 50 μg/mL and exposure
time of 10 min could restrain DNA amplification of dead bacteria in 107 CFU/mL of Salmonella enteritis. EMA-PCR
and direct PCR methods showed the same detection limit of 27.5 CFU/mL for viable cells, suggesting that neither DNA
amplification from viable cells nor PCR sensitivity is affected by the addition of EMA. As we observed for mixtures of
viable and dead cells by the EMA-PCR method, the brightness of DNA stripes turned darker with reducing the proportion
of viable cells, and when all bacteria were dead, no target stripe appeared. As for the control group without added EMA,
the brightness of DNA stripe had no change, and even though the bacteria were all dead, the target stripe remained distinct.
Therefore, the EMA-PCR method can distinguish dead bacteria from live ones, thereby avoiding false positive detection.

Key words: ethidium monoazide bromide (EMA), polymerase chain reaction (PCR), Salmonella enteritidis