玉米细菌性枯萎病菌荧光重组酶介导等温扩增检测方法的建立与应用  

Establishment and Application of Fluorescent Recombinase-aided Amplification Method for Pantoea stewartii subsp.stewarii Detection

作　　者：单长林 周圆 李孝军 SHAN Changlin;ZHOU Yuan;LI Xiaojun(Comprehensive Technology Service Center of Zhoushan Custom,Zhoushan 316021,China)

机构地区：[1]舟山海关综合技术服务中心,浙江舟山316021

出　　处：《广东农业科学》2021年第1期111-118,共8页Guangdong Agricultural Sciences

基　　金：浙江省基础公益研究项目(GN18C140001)。

摘　　要：【目的】建立一种快速、灵敏、特异的玉米细菌性枯萎病菌(Pantoea stewartii subsp.stewarii)重组酶介导等温扩增(recombinase-aided amplification,RAA)检测方法。【方法】以玉米细菌性枯萎病菌pstS-glmS基因序列作为靶标序列,设计、合成荧光RAA检测引物和探针,并进行灵敏性测定、特异性验证;使用建立的荧光RAA检测方法检测玉米模拟样本和玉米真实样本,参照标准SN/T 1375-2004方法同步复核,评估荧光RAA检测方法的可靠性。【结果】成功建立了玉米细菌性枯萎病菌荧光RAA检测方法。该方法反应快速,在39℃条件下20 min内即可完成检测反应,特异性好,且灵敏度为280 fg/μL;利用该方法检测6份模拟样本结果阳性,24份真实样本结果呈阴性,与参照标准SN/T 1375-2004检测结果一致。【结论】成功建立了玉米细菌性枯萎病菌快速、灵敏和特异的荧光RAA检测方法,可用于玉米细菌性枯萎病菌的现场检测。【Objective】The study was conducted to develop a rapid,sensitive and specific assay for detection of Pantoea stewartii subsp.stewarii based on fluorescent recombinase-aided amplification(RAA).【Method】The pstS-glmS sequence of P.stewartii subsp.stewarii was used as target sequence,and the specific primers and probes were designed and synthesized to establish a fluorescent RAA assay for rapid detection of P.stewartii subsp.stewarii.The sensitivity and specificity of the fluorescent method was detected.Simulated samples and actual samples were used to evaluate the reliability of fluorescent RAA,with the SN/T 1375-2004 as standard for verification.【Result】A fluorescent RAA for detection of P.stewartii subsp.stewarii was established,which could detect P.stewartii subsp.stewarii specifically at 39℃within 20 min.The sensitivity of the fluorescent RAA was 280 fg/μL.With the established method,6 simulated samples were positive and 24 real samples were negative with the same results as SN/T 1375-2004.【Conclusion】The fluorescent RAA method for detection of P.stewartii subsp.stewarii is rapid,sensitive and specific,which is proved to be a great choice for site monitoring of P.stewartii subsp.stewarii.

关 键 词：玉米细菌性枯萎病菌 pstS-glmS序列 重组酶介导等温扩增 荧光探针 快速检测 

分 类 号：S432.42[农业科学—植物病理学]