Introduction
Culture-based detection methods are widely used in microbial and viral testing but are hindered by lengthy time-to-answer and inability to detect viable but non-culturable bacteria, fungi, and viruses. PCR-based approaches address these limitations but lack live-dead discrimination. The Viability PCR Reagent combines rapid PCR results with the ability to differentiate signals from viable and non-viable cells and defective viruses, providing an advanced molecular assay for microbial viability.
Objective
We demonstrate various applications of the Viability PCR Reagent for detecting intact bacteria, viruses, and fungi in diverse samples, including plant and water matrices.
Method
The Viability PCR Reagent uses a membrane/capsid impermeable molecule that binds and modifies nucleic acids from non-viable cells and damaged viruses, preventing PCR amplification. We tested the reagent with bacteria (Legionella), RNA viruses (Norovirus and SARS-CoV-2) and fungi (Colletotrichum). Assay performance was demonstrated with mixtures of live and inactivated microorganisms.
Findings
Viability PCR reduced or eliminated PCR signals from defective viruses, heat inactivated fungus and non-viable bacteria, while signals from intact microbial species remained unaffected. Heat or pressure-treated Norovirus, Legionella and Colletotrichum were clearly discriminated from control samples. Live Legionella bacteria spiked into water samples were differentiated from dead Legionella using a filter-based collection and treatment protocol. For both bacteria and viruses, Viability PCR results closely matched those obtained from corresponding culture-based assays. Live Colletotrichum fungus spiked into leaf extract samples were differentiated from dead Colletotrichum. Colletotrichum concentration was 10^2 cfu/mL, which was before the onset of illness.
Conclusion
The Viability PCR Reagent System offers a rapid, accurate alternative to culture-based assays for microbial monitoring. Its application can facilitate timely risk assessments and outbreak responses, improving the safety of food and water supplies globally.