A photoactivation-free viability PCR reagent for rapid, culture-independent detection of live bacteria.

Detection of viable bacteria in complex samples is a long-standing challenge in microbiology and public health. Traditional culture methods are slow and often fail to capture viable but non-culturable cells, while PCR-based methods can overestimate bacterial loads by amplifying DNA from non-viable (dead) cells. To address this challenge, we evaluated a novel, photoactivation-free viability PCR (vPCR) reagent from Promega Corporation that selectively crosslinks DNA from non-viable cells, rendering it unamplifiable by qPCR. This approach enables accurate, culture-independent quantification of viable bacteria. We optimized key parameters in both gram-positive ( IMPORTANCE: Rapid and reliable detection of viable bacteria is essential for protecting public health, monitoring water quality, ensuring food safety, and advancing clinical diagnostics. Conventional culture methods underestimate viable cells and require days to deliver results, while conventional PCR-based tests cannot distinguish viable (live) from non-viable (dead) organisms. Here, we describe a photoactivation-free viability PCR reagent that overcomes these limitations by selectively blocking PCR amplification from non-viable cells. Using