Viability PCR: An Evaluation of Propidium Monoazide for Live/Dead Differentiation of Microorganisms

Introduction: Nucleic acid detection methods such as real-time PCR (rt-PCR) provide fast and powerful tools to analyze samples for the presence of potentially harmful microbes, but also hold the risk of false positives by detecting nucleic acid from harmless dead cells. Viability PCR utilizes the DNA-masking compound propidium monoazide (PMA) which is able to enter dead and membrane-compromised cells, modifying the DNA on photo-activation, resulting in a strong inhibition of PCR amplification.

Purpose: The aim of this study was to evaluate the performance of viability PCR centering on PMA and a new illumination device with several pathogens and matrices

Methods: Defined mixtures of live-dead pathogens were prepared (Salmonella spp. and Listeria spp.) and treated with/without PMA. Pathogen mixtures were illuminated at a specific wavelength to irreversibly bind the reagent to dead cell DNA, rendering it no longer PCR visible. Efficient suppression of amplification of such modified DNA allows preferential rt-PCR detection of live cells. The performance in turbid and colored mixtures was also studied.

Results: The sensitivity of the method is determined with titrations of different live/dead pathogen ratios (100% dead to 100% live). The rt-PCR performance data from these cell mixtures with or without PMA treatment demonstrates the extent of the masking effect of PMA on dead cells. Dead cells treated with PMA show a ~15 higher Ct value their non-PMA treated counterpart (X²< 0.05). The data using turbid/colored matrices demonstrates that the PMA activating light is sufficient to penetrate such samples, to lead to successful DNA intercalation and allow live/dead detection.

Significance:  Live/dead differentiation can play an important role in procedures such as hygiene monitoring (success of decontamination processes), water testing (distinguishing between live and dead legionella for regulatory compliance) and human diagnostics (monitoring medication efficiency in pathogen killing).