Comparative evaluation of viability PCR reagents highlights the superior efficacy of PMAxx azo dye for bacterial and viral viability discrimination using real-time and digital PCR

Viability PCR (vPCR) distinguishes viable from nonviable microorganisms by suppressing amplification from structurally compromised cells or virions. This study compared three viability reagents-ethidium monoazide (EMA), propidium monoazide derivative (PMAxx), and platinum (IV) chloride (PtCl₄)-for differentiating viable and heat-inactivated <i>Staphylococcus aureus</i> (<i>S. aureus</i>), <i>Escherichia coli</i> (<i>E. coli</i>), human coronavirus OC43 (HCoV-OC43), and Enterovirus A71 (EV-A71) using real-time and digital PCR. EMA and PtCl₄ showed limited or inconsistent suppression of dead-cell or dead-virion signals, whereas PMAxx consistently demonstrated superior performance. Under optimal vPCR treatment conditions-100 µM PMAxx for <i>S. aureus</i>, 25 µM PMAxx with 0.01% sodium deoxycholate (DC) for <i>E. coli</i>, and 200 µM PMAxx for both HCoV-OC43 and EV-A71-the assays produced clear live-dead discrimination, with the highest ΔCq values reaching approximately 12 cycles in the most responsive organism-condition combinations. PMAxx completely suppressed amplification from heat-inactivated <i>S. aureus</i> (10⁶ CFU/100 µL) and EV-A71 (10² PFU/100 µL) while preserving signals from viable organisms, with concordant results across PCR platforms. Applying these optimized conditions to spiked matrices (milk, vegetable wash water, and nasopharyngeal transport medium) reduced ΔCq separation relative to culture suspensions, indicating matrix-associated interference, yet PMAxx maintained effective viability discrimination in most scenarios. This comparative analysis, with digital PCR serving as confirmatory evidence in representative high-contrast conditions, demonstrates that PMAxx provides the most reliable viability discrimination among the reagents evaluated and may be applicable to broader microbial viability studies and environmental surveillance contexts.IMPORTANCEMolecular diagnostic assays are indispensable for rapid pathogen detection; however, their inability to distinguish viable from nonviable microorganisms can result in an overestimation of infection risk. The vPCR approach overcomes this limitation by combining selective nucleic acid-intercalating reagents with amplification-based detection. In this study, we evaluated EMA, PMAxx, and PtCl₄ across four representative pathogens-<i>S</i>. <i>aureus</i>, <i>E. coli</i>, HCoV-OC43, and EV-A71-using both real-time and digital PCR platforms. The results reveal that PMAxx provides superior performance in eliminating false-positive signals from nonviable cells and virions while preserving amplification from viable targets. The incorporation of digital PCR further enhances quantitative accuracy and absolute measurement of pathogen viability. These findings underscore the translational potential of PMAxx-based vPCR as a practical and robust strategy for improving molecular diagnostics, monitoring environmental contamination, and strengthening public health surveillance.