Long-amplicon propidium monoazide-PCR enumeration assay to detect viable Campylobacter and Salmonella

Abstract

The effect of amplicon length on the ability of propidium monoazide-PCR (PMA-PCR) to reliably quantify viable cells without interference from dead cells was tested on heat- and ultraviolet (UV)-killed Salmonella enterica and Campylobacter jejuni, two important enteric pathogens of concern in environmental, food and clinical samples. PMA treatment followed by quantitative PCR (qPCR) amplification of short DNA fragments (<200bp) resulted in incomplete signal inhibition of heat-treated Salm.enterica (3 log reduction) and Camp.jejuni (1 log reduction), whereas PCR amplification of a long DNA fragment (1·5 and 1·6kb) completely suppressed the dead cell signal. PMA pretreatment of UV-irradiated cells did not affect PCR amplification, but long-amplicon PCR was shown to detect only viable cells for these samples, even without the addition of PMA. The long-amplicon PMA-PCR method was effective in targeting viable cells following heat and UV treatment and was applicable to enteric pathogens including Salmonella and Campylobacter that are difficult to enumerate using culture-based procedures. PCR amplicon length is important for effective removal of the dead cell signal in PMA pretreatment methods that target membrane-damaged cells, and also for inactivation mechanisms that cause direct DNA damage.