Development of PMA-qPCR assay to accurately and reproducible quantify viable bacteria of Paenibacillus polymyxa

Abstract

Abstract Paenibacillus polymyxa is an important biocontrol bacterium. The combination of propidium monoazide (PMA) and quantitative polymerase chain reactionq (qPCR) has proven effective in quantifying live bacteria from various microorganisms. The objective was to create a PMA-qPCR assay to precisely and consistently measure the number of living bacteria of biocontrol P. polymyxa. The primers were designed for the spo0A gene of P. polymyxa HY96-2. The optimal conditions for treating the target strain with PMA were a PMA concentration of 15 μg/mL, an incubation time of 5 min, and an exposure time of 10 min. The PMA-qPCR method had a limit of quantification (LOQ) of 1.0 × 103 CFU/mL for measuring the amount of viable P. polymyxa bacteria. The PMA-qPCR method is more sensitive than the qPCR method in detecting viable bacteria in the mixtures of viable and dead bacteria. The accuracy and reproducibility of quantifying viable P. polymyxa bacteria using the PMA-qPCR method were higher compared to the plate count method.