摘要
基于叠氮溴化丙锭(PMA)结合荧光定量PCR技术能利用活细胞的膜完整性区分活菌和死菌的原理,以玉米细菌性枯萎病菌为研究对象,探索PMA-qPCR方法中各项因素的优化,并建立该病菌的活菌检测方法。试验结果表明,当添加PMA质量浓度为10μg/mL,黑暗孵育20 min,再进行强光照射10 min后,死细胞的DNA扩增被有效抑制。通过对比发现qPCR和PMA-qPCR的灵敏度相近,最低检出限均为10^(3)cfu/mL。应用PMA-qPCR、qPCR、平板分离法对不同浓度的死活菌悬液和实际样品进行检测,结果显示PMA-qPCR能准确反映实际样品带活菌的情况,并能有效避免qPCR检测实际样品可能出现的假阳性结果,为后续进一步开展检测技术研究提供了参考。
This study sets out to establish a methodology for the detection of live bacteria of the Pantoea stewartii subsp stewartii strain,which is based upon the principle that propidium monoazide quantitative PCR(PMA-qPCR)can differentiate between live bacteria and dead bacteria by assessing the membrane integrity of living cells.To this end,the study explores the optimization of various factors in the PMA-qPCR method.The results showed that when the PMA concentration was set at 10μg/mL,theDNAamplification of dead cells was effectively inhibited following a 20-minute dark incubation,and subsequently,a 10-minute stronglight irradiation.A comparison of the real-time qPCR and PMAqPCR revealed no significant difference in sensitivity between the two methods,with a minimum detection limit of 10^(3) cfu/mL observed for both.The detection of different ratios of dead and live bacterial suspensions and actual samples was accomplished by utilising PMA-qPCR,qPCR,and plate count methodologies.The findings indicated that PMA-qPCR could accurately reflect the presence of live bacteria in actual samples and effectively avoid the false positive results that might occur in qPCR detection of samples.This provides an effective technical means for further research.
作者
于璇
冯黎霞
黄娇芬
魏霜
李献锋
Yu Xuan;Feng Lixia;Huang Jiaofen;Wei Shuang;Li Xianfeng(Guangzhou Customs Technology Center,Gaungzhou 510623,Guangdong Province;Foshan Customs Comprehensive Technology Center,Foshan Guangdong 528000,China)
出处
《中国植保导刊》
北大核心
2025年第10期109-114,共6页
China Plant Protection
基金
国家重点研发计划(2021YFD1400104)。
关键词
玉米细菌性枯萎病菌
活菌
检测
Pantoea stewartii subsp.stewartii
live bacteria
detection
