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 antibac-terial 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 mu g/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 x 10(3) CFU/ml, 6.5 x 10(2) CFU/ml, and 10(3) 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.