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

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.

A new PMA-qPCR method for rapid and accurate detection of viable bacteria and spores of marine-derived Bacillus velezensis B-9987.

Marine-derived Bacillus velezensis B-9987 is an important biocontrol bacterium with a broad-spectrum antibacterial effect. The traditional plate counting method is widely used for quantitative detection of viable bacteria and spores but has some disadvantages such as being laborious and time-consuming (at least 24-48 h). This study aimed to develop a new PMA-qPCR method for rapid and accurate detection of viable bacteria and spores of B-9987. The specific primers were designed for qPCR amplification based on the conserved region of the bmmA gene (encoding a malonyl CoA-ACP transacylase) of B-9987. According to the characteristic that propidium monoazide (PMA) dye can distinguish viable and dead bacteria, the optimal PMA concentration of 10 µg/ml and optimal exposure time of 10 min were achieved under PMA treatment conditions. The B-9987 spores' genomic DNA was successfully extracted after the spore coat was removed and spore germination was induced. The quantification limits of the PMA-qPCR method were determined for viable B-9987 bacteria, spores in pure culture, and spores in marine Bacillus wettable powder (marine Bacillus WP) and were 1.5 × 103 CFU/ml, 6.5 × 102 CFU/ml, and 103 CFU/ml, respectively. Compared with the qPCR method, the PMA-qPCR method could sensitively detect viable bacteria in the viable/dead bacterial mixture. In this study, the developed PMA-qPCR method was found to have excellent sensitivity and specificity in the context of a pure culture of B-9987 strain, which could accurately and rapidly detect viable B-9987 bacteria within 3-4 h and viable B-9987 spores in marine Bacillus WP within 4-6 h.