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

摘要

Aims. 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. Methods and Results. PMA treatment followed by quantitative PCR (qPCR) amplification of short DNA fragments (<200 bp) 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. 6 kb) 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. Conclusions. 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. Significance and Impact of the Study. 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