文献类型：期刊文献

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

作者：Guo, Jiacai[1];Wang, Weiliang[1];Zhao, Haoyu[1];Luo, Yuanchan[1];Wan, Minxi[1,2];Li, Yuanguang[1,2]

机构：[1]East China Univ Sci & Technol, State Key Lab Bioreactor Engn, Shanghai 200237, Peoples R China;[2]Mail Box 301,Meilong Rd 130, Shanghai 200237, Peoples R China

年份：2022

卷号：199

外文期刊名：JOURNAL OF MICROBIOLOGICAL METHODS

收录：;WOS:【SCI-EXPANDED(收录号:WOS:000828687500002)】；

基金：This work was financially supported by the National Key Research and Development Program of China (2017YFD0201107-2-3) . We are furthermore thankful to Shanghai Zeyuan Marine Biotechnology Co., Ltd. for providing marine Bacillus WP with one billion CFU/g for this study.

语种：英文

外文关键词：BacillusvelezensisB-9987; Propidiummonoazide; qPCR; SporegenomicDNA; Viablebacterianumber

摘要：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.