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当前位置: 首页 > 文章 > 基于qPCR技术快速检测青枯劳尔氏菌5号生理小种的方法 蚕业科学 2015,41 (5) 807-814
Position: Home > Articles > Establishment of a qPCR Method for Rapid Detection of Ralstonia solanacearum Race 5 Science of Sericulture 2015,41 (5) 807-814

基于qPCR技术快速检测青枯劳尔氏菌5号生理小种的方法

作  者：
曹梦琪;包奇;杨采风;王俊;盛晟
关键词：
青枯劳尔氏菌;荧光定量PCR;桑青枯病;早期检测
摘  要：
由青枯劳尔氏菌(以下简称青枯菌)5号生理小种(Ralstonia solanacearum race 5)引起的桑青枯病是桑树的重大病害。采用荧光定量PCR(qPCR)技术对病原菌进行早期检测,为病害的预测预报及有效防控提供依据。从青枯菌差减基因文库筛选一对引物RS72F/RS312R,经PCR扩增试验及qPCR产物的熔解曲线分析验证其可特异性地扩增出青枯菌5号生理小种241 bp的基因片段。建立的qPCR标准曲线显示:以青枯菌5号生理小种基因组DNA为模板,在10-4~102 ng/μL范围内,DNA质量浓度的对数值与扩增Ct值呈现良好的线性关系,线性方程为y=-2.747x+21.834,R2=0.993;以青枯菌5号生理小种菌液为模板,在103~107 CFU/mL范围内,菌液浓度的对数值与扩增Ct值呈现良好的线性关系,线性方程为y=-3.418x+45.447,R2=0.998。应用建立的qPCR检测方法对经青枯菌5号生理小种菌液处理的土壤样品进行检测,结果显示可检测出自然环境中存在的低于致病浓度的病原青枯菌,且用菌液处理灭菌土壤和不灭菌土壤的样品间青枯菌检测的Ct值无显著差异。建立的检测方法具有特异性强、灵敏度高、可定量的特点,适用于由青枯菌5号生理小种引起的桑青枯病的早期检测诊断
译  名：
Establishment of a qPCR Method for Rapid Detection of Ralstonia solanacearum Race 5
作  者：
Cao Mengqi;Bao Qi;Yang Caifeng;Wang Jun;Sheng Sheng;Wu Fuan;Jiangsu University of Science and Technology;The Sericultural Research Institute,Chinese Academy of Agricultural Sciences;
关键词：
Ralstonia solanacearum;;Real-time fluorescent quantitative PCR;;Mulberry bacterial wilt;;Early detection
摘  要：
Mulberry bacterial wilt,caused by Ralstonia solanacearum race 5,is one of the most serious bacterial diseases of mulberry. In this study,real-time fluorescent quantitative PCR( qPCR) was employed for the early detection of pathogenic bacteria to provide evidence for forecast and control of mulberry bacterial wilt. PCR verification and melting curve analysis of qPCR products indicated that a 241 bp gene fragment was specially amplified from Ralstonia solanacearum race 5with the primer pair RS72F/RS312 R designed according to R. solanacearum subtracted gene library. The established standard curve of qPCR showed that using genomic DNA of R. solanacearum race 5 as template,the logarithm of DNA concentration had good linear relationship with amplified Ct value within the range of 10-4 to 102 ng /μL. The linear regression equation was y=-2. 747x+21. 834( R2 =0. 993). Using bacterial solution of R. solanacearum race 5 as template,the logarithm of bacterial solution concentration showed good linear relationship with amplified Ct value within the range of 103to 107 CFU/mL. The linear regression equation was y =-3. 418 x + 45. 447( R2 = 0. 998). Using the established qPCR detection method to detect the soil samples treated with bacterial solution of R. solanacearum race,the results showed that R. solanacearum in natural environment,of which concentration was low and cannot cause mulberry bacterial wilt,could be detected. There was no significant difference in Ct value between sterilized and non-sterilizedsoil samples. The established qPCR method for R. solanacearum detection is highly specific,sensitive,quantitative,being suitable for early detection of mulberry bacterial wilt caused by R. solanacearum race 5.

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