3种常见金银花致病菌多重实时荧光定量PCR检测方法研究  

Study on Multiplex Real-Time Fluorescence Quantitative PCR Detection Method for Three Common Lonicera japonica Pathogens

作　　者：朱桐杉 刘艳艳[2] 谭晴晴[2] 刘国霞[2] 陈雪燕[2] 步迅[2] 王文博 于子涵 张全芳[2] 张永清[1] Zhu Tongshan;Liu Yanyan;Tan Qingqing;Liu Guoxia;Chen Xueyan;Bu Xun;Wang Wenbo;Yu Zihan;Zhang Quanfang;Zhang Yongqing(College of Pharmacy,Shandong University of Traditional Chinese Medicine,Jinan 250355,China;Institute of Crop Germplasm Resources,Shandong Academy of Agricultural Sciences,Jinan 250100,China;Institute of Agricultural Quality Standards and Testing Technology,Shandong Academy of Agricultural Sciences,Jinan 250100,China;Affiliated Hospital of Shandong University of Traditional Chinese Medicine,Jinan 250013,China)

机构地区：[1]山东中医药大学药学院,山东济南250355 [2]山东省农业科学院农作物种质资源研究所,山东济南250100 [3]山东省农业科学院农业质量标准与检测技术研究所,山东济南250100 [4]山东中医药大学附属医院,山东济南250013

出　　处：《山东农业科学》2023年第7期145-151,共7页Shandong Agricultural Sciences

基　　金：山东省高校中药质量控制与全产业链建设协同创新中心项目(CYLXTCX2021-06);山东省创新公共服务平台计划中药分子鉴定公共服务平台项目(2018JGX111);山东省农业科学院齐鲁农科英才工程(创新类)项目。

摘　　要：为了建立可同时检测炭疽病病原菌(Colletotrichum gloeosporioides)、白粉病病原菌(Erysiphe lonicera)和褐斑病病原菌(Cercospora rhamni)3种金银花重要叶部病原菌的多重实时荧光定量PCR快速检测方法,本试验基于3种病原菌ITS片段序列分别设计特异引物和TaqMan荧光探针,并制备重组质粒建立标准曲线;对建立的多重实时荧光定量PCR检测方法进行特异性、灵敏度、准确性验证。结果表明,本研究建立的多重实时荧光定量PCR快速检测方法仅针对金银花炭疽病、白粉病和褐斑病病原菌有特异性扩增;最低检测限为2.94×10^(2)copies/μL,比普通PCR法高100倍;利用本方法检测15份有叶部病害症状的金银花病叶,与DNA测序结果完全一致。综之,本研究建立的多重实时荧光PCR检测方法特异性强、灵敏度高,为金银花炭疽病、白粉病和褐斑病的早期诊断提供了技术支持。In order to establish a multiplex real-time fluorescence PCR method for rapid detection of an-thracnose(Colletotrichum gloeosporioides),powdery mildew(Erysiphe lonicera)and brown spot(Cercospora rhamni)of Lonicera japonica Thunb.,specific primers and TaqMan fluorescent probes were designed in this study based on the ITS fragment sequence of three pathogens,and the standard curve was established by pre-paring recombinant plasmids.The specificity,sensitivity and accuracy of the established multiplex real-time fluorescence quantitative PCR detection method for the three pathogens were verified.The results showed that the multiplex real-time fluorescent PCR rapid detection method established in this study only had specific am-plification for the pathogens of Colletotrichum gloeosporioides,Erysiphe lonicera and Cercospora rhamni.The lowest detection limit was 2.94×10^(2) copies/μL,which was 100 times higher than that of the ordinary PCR method.Fifteen Lonicera japonica samples with symptoms of leaf disease were detected by this method,which was consistent with the results of DNA sequencing.In conclusion,the multiplex real-time quantitative PCR de-tection method established in this study had high specificity and sensitivity,which provided technical supports for the early diagnosis of anthracnose,powdery mildew and brown spot of Lonicera japonica.

关 键 词：金银花 炭疽病 白粉病 褐斑病 多重实时荧光定量PCR 

分 类 号：S435.121.4[农业科学—农业昆虫与害虫防治]