基于PMA-qPCR检测青枯菌5号生理小种活菌的方法  

Detection of Living Cells of Ralstonia solanacearum Race 5 by PMAqPCR Method

作　　者：曹梦琪[1] 王旭东[1] 王俊[1] 盛晟[1,2] 吴福安[1,2] 

机构地区：[1]江苏科技大学,江苏镇江212003 [2]中国农业科学院蚕业研究所,江苏镇江212018

出　　处：《蚕业科学》2015年第6期1004-1010,共7页ACTA SERICOLOGICA SINICA

基　　金：现代农业产业技术体系建设专项(No.CARS-22);江苏省普通高校研究生科研创新计划项目(No.CXZZ14_299)

摘　　要：青枯菌5号生理小种(Ralstonia solanacearum race 5)是引发桑青枯病的病原菌。为了改进常规PCR方法不能对活体病原菌进行准确鉴别和定量分析的不足,建立叠氮溴化丙锭(PMA)预处理结合荧光定量PCR(q PCR)检测青枯菌5号生理小种活菌的方法。该方法首先将待检测样品的病原菌细胞用质量浓度为15 ng/μL的PMA暗孵育10 min后,在卤钨灯下曝光5min,以消除死菌细胞DNA的PCR扩增信号。用PMA-q PCR方法对含青枯菌5号生理小种活菌和死菌的混合样本的检测结果表明,死菌的存在不会对活菌细胞DNA的扩增产生影响。建立的PMA-q PCR标准曲线显示,在菌液浓度为103~107CFU/m L的范围内,qPCR循环阈值(Ct)与菌液浓度的对数值之间呈良好的线性关系,线性方程为y=-3.477x+47.489,R^2=0.997。建立的PMA-q PCR方法能更为准确地检测复杂样本中的青枯菌5号生理小种活菌数量,有助于对桑树青枯病的早期诊断和及时制定病害防控措施。Ralstonia solanacearum race 5 is the pathogen causing mulberry bacterial wilt. Conventional PCR cannot be used for accurate identification and quantitative analysis of living pathogen, so pre-treatment with propidium monoazide （PMA） and real-time fluorescent quantitative PCR （qPCR） were employed to detect the living cells of R. solanacearum race 5. Pathogenic cells were first treated with 15 ng/μL PMA for 10 min in the dark followed by 5 min halogen light expo- sure to eliminate PCR amplified signal of dead cell DNA. PMA-qPCR results of mixed samples of living cells and dead cells indicated that dead cells did not influence DNA amplification of living cells. Analysis of the constructed PMA-qPCR standard curve showed that there was a good linear relationship between qPCR cycle threshold （Ct） and the logarithm value of bacterial solution concentration within the range of 10^3-10^7 CFU/mL. The linear equation of standard curve was y=-3. 477x＋47. 489, R^2 =0. 997. The established PMA-qPCR method can be used to accurately quantify the number of living cells of R. solanacearum race 5 in complex samples, and it is conducive to early diagnosis of mulberry bacterial wilt and prompt formulation of prevention and control meas- ures.

关 键 词：桑树青枯病 青枯菌5号生理小种 叠氮溴化丙锭 荧光定量PCR 活细胞 

分 类 号：S888.71[农业科学—特种经济动物饲养]