PMA-qPCR检测十字花科黑斑病菌活菌方法的建立

PMA-qPCR assay for the detection of viable Pseudomonas syringae pv.maculicola

利用叠氮溴化丙锭(propidium monoazide,PMA)与实时荧光PCR技术相结合(PMA-qPCR),对PMA质量浓度、暗孵育和曝光时间等因素进行优化,建立了检测十字花科黑斑病菌活菌的方法。结果显示,当PMA质量浓度为10μg/mL,避光条件下孵育5 min、然后曝光10 min,此条件下能够抑制10-7CFU/mL十字花科黑斑病菌死菌的DNA扩增,对活菌扩增无影响,通过对比qPCR和PMA-qPCR的灵敏度可知,两者灵敏度差异不大,最低检出限均为10-3CFU/mL,将PMA-qPCR方法用于实际5批十字花科黑斑病菌阳性的油菜籽样品检测,结果显示该方法能准确反映实际样品的带活菌的情况。本研究为十字花科黑斑病菌活菌的检测提供了快速检测方法,可为实验室该病菌的快速筛检提供参考。

A rapid method of PMA-qPCR for the detection of viable bacteria Pseudomonas syringae pv.maculicola was established by optimizing the concentration,dark incubation and exposure time of propidium monoazide(PMA)through the combination of PMA and real-time fluorescent PCR.The results showed that when PMA concentration was 10μg/mL,incubated in dark condition for 5 min,then exposed for 10 min,the DNA amplification of 107 CFU/mL dead bacteria could be inhibited but has no effect on the amplification of viable bacteria of P.syringae pv.maculicola.By comparing the sensitivity of real-time PCR and PMA-qPCR,we could see that there was little difference between them and minimum detection limits were all 103 CFU/mL.PMA-qPCR method was used for actual 5 batches of positive rapeseed samples,the results displayed that this method could accurately reflect the actual samples with living bacteria.This study provides a rapid detection method for the detection of viable bacteria Pseudomonas syringae pv.maculicola,and provides a reference for rapid screening of the pathogen in laboratory.