【摘要】 本文将叠氮溴化丙锭(PMA)与荧光实时定量PCR技术相结合,建立了一种适于青枯菌不同小种菌株活细胞精准、快速检测的PMA-qPCR方法。通过单因素变化试验对PMA预处理反应体系中的各参数进行优化,确立了PMA终浓度为15 ng/μL,黑暗孵育时间为10 min,曝光时间为5 min的PMA预处理体系。试验结果表明,当活菌比例大于10%,PMA-qPCR的测定结果均在理论活菌数相对应的95%置信区间内。检测灵敏度测试结果显示,该方法适用于活菌数在5.0×10~2～5.0×10~8 cfu/mL范围内菌悬液的检测。本文建立的PMA-qPCR方法可在一定范围内有效去除青枯菌死菌的干扰,定量检测出活菌数量,研究结果可为植物细菌性青枯病的流行规律研究提供新的技术支撑。更多还原

【Abstract】 By combining propidium monoazide(PMA) with fluorogenic(TaqMan) PCR, we developed a novel method for specific detection of viable cells of Ralstonia solanacearum at species level. We optimized various parameters of PMA by single factor change test to establish the system of PMA pre-treatment. The optimal conditions for PMA-qPCR were: a final concentration of PMA of 15 ng/mL, a dark incubation time of 10 min, and an exposure time of 5 min. The test results showed that, within the range of 5.0×10~2-5.0×10~8 cfu/mL, when the proportion of viable bacteria was greater than 10%, the results of PMA-qPCR were almost identical with the theoretical values. The method could effectively remove the interference of the dead cells of R.solanacearum and quantitatively detect the number of viable cells, thereby providing a new technical support for the research of epidemiology of bacterial wilt caused by R.solanacearum.更多还原