Optimization of blue light-assisted viability quantitative PCR for accurate detection of viable Erwinia amylovora

Accurate quantification of viable cells is crucial for reliable disease surveillance before and during outbreaks. However, conventional quantitative PCR (qPCR) often overestimates the inoculum potential because it amplifies DNA from non-viable cells. Viability-qPCR (v-qPCR) using propidium monoazide (PMA) can address this limitation, but inconsistent dye treatment and photoactivation protocols frequently compromise the assay's reproducibility. Here, we developed a standardized blue light (BL)-assisted v-qPCR method for the selective and quantitative detection of viable Erwinia amylovora. Through systematic optimization of PMA concentration, dark incubation time, and photoactivation parameters, we found that 5-20 µM PMA, combined with a 10-min dark incubation, effectively suppressed amplification from dead cells without affecting detection of viable cells. In pure bacterial suspensions in PBS, moderate BL photoactivation (300 µmol/m²s for 20 min) effectively suppressed dead-cell DNA (99.98%), comparable to the performance of a commercial PMA-Lite™ device (>1000 µmol/m2s), while exhibiting lower phototoxicity under our tested conditions. The optimized v-qPCR showed strong linearity (R2 = 0.999) from 104 to 108 CFU/mL with an amplification efficiency of 99.0% and maintained quantitative performance in mixtures of live and dead cells. Using replicated no-template controls, the assay demonstrated a clearly defined limit of blank (LoB), and limits of detection (LoD) and quantification (LoQ) of 5×104 CFU/mL under controlled conditions in PBS, including in the presence of high dead-cell DNA. Overall, these results indicate that light intensity is a key determinant of PMA activation efficiency and potential phototoxicity and provide an experimental basis for optimizing v-qPCR assays for viable E. amylovora, with further validation required for complex plant matrices.