向日葵黑茎病菌TaqMan-MGB探针实时荧光快速检测方法  

Development of Taq Man-MGB fluorescent real-time PCR assay for the detection of Plenodomus lindquistii

作　　者：段维军 段丽君 吕燕 陈倩[3] 吴品珊[4] 蔡磊[3] DUAN Wei-jun;DUAN Li-jun;Lü Yan;CHEN Qian;WU Pin-shan;CAI Lei(Ningbo Academy of Inspection and Quarantine,Ningbo 315012,China;Ningbo Customs District P.R.China,Ningbo 315012,China;State Key Laboratory of Mycology,Institute of Microbiology,Chinese Academy of Sciences,Beijing 100101,China;Chinese Academy of Inspection and Quarantine Science,Beijing 100176,China)

机构地区：[1]宁波检验检疫科学技术研究院,宁波315012 [2]宁波海关,宁波315012 [3]中国科学院微生物研究所,北京100101 [4]中国检验检疫科学研究院,北京100176

出　　处：《植物病理学报》2021年第6期975-986,共12页Acta Phytopathologica Sinica

基　　金：浙江省公益技术研究项目(LGF20C140001);海关总署科研项目(2020HK162);中国检科院科研项目(2020JK048)。

摘　　要：向日葵黑茎病菌是我国进境检疫性有害生物名录中的一种检疫性真菌。根据向日葵黑茎病菌及其近似种的ITS序列差异,设计并合成特异性引物和探针,建立了向日葵黑茎病菌的实时荧光PCR检测方法。特异性试验结果表明,该检测方法能特异性检测向日葵黑茎病菌;灵敏度试验结果表明,最低检测限量为20μL反应体系中总DNA含量0.1 pg;实时荧光PCR优化反应条件为引物终浓度0.6μmol·L^(-1),探针终浓度0.3μmol·L^(-1)。实际样品检测结果表明,该方法可用于疑似携带向日葵黑茎病菌样品的检测与初筛。此方法快速、灵敏,整个反应过程约1 h,检测过程完全闭管,无需PCR后续处理,为早期快速检测向日葵黑茎病菌提供了重要参考。Plenodomus lindquistii is a quarantine fungus in the list of quarantine pests in China.A species-specific real-time polymerase chain reaction(PCR)assay was developed for the detection of P.lindquistii.A pair of specific primers and a TaqMan-MGB probe was designed and synthesized according to the difference of ITS sequence between P.lindquistii and related species.A novel real-time fluorescent PCR was established to detect P.lindquistii.The minimal detectable concentration of targeted DNA was 0.1 pg in 20μL reaction mixture.Optimal primer concentration and probe concentration were 0.6μmol·L^(-1)and 0.3μmol·L^(-1),respectively.The method could be used for the detection and preliminary screening of the samples suspected of carrying P.lindquistii.The method was specific,rapid,sensitive and completed within a single tube,the whole reaction took about one hour,without post-PCR handling,which provided a valuable tool for early rapid detection and identification of P.lindquistii.

关 键 词：向日葵黑茎病菌 实时荧光PCR TAQMAN-MGB探针 检测 

分 类 号：S435.655[农业科学—农业昆虫与害虫防治]