樱桃灰霉病菌LFD-RPA快速检测方法的建立  

Development of LFD-RPA assay for rapid detection of Botrytis cinerea in cherry

作　　者：王燕[1] 王春伟[1] 王琳 荆琦 高海馨 余廷濠 王美琴[1] 张作刚[1] 王建明[1] WANG Yan;WANG Chun-wei;WANG Lin;JING Qi;GAO Hai-xing;YU Ting-hao;WANG Mei-qin;ZHANG Zuo-gang;WANG Jjan-ming(College of Agriculure,Shanxi Agiculural Univrsity,Taigu 030801,China;Yuncheng sub-center,Shanxi Entry-Exit Inspection and Quarantine Bureau Technology Center,Yuncheng 044600,China)

机构地区：[1]山西农业大学农学院,太谷030801 [2]山西出入境检验检疫局检验检疫技术中心运城分中心,运城044600

出　　处：《植物病理学报》2020年第2期238-245,共8页Acta Phytopathologica Sinica

基　　金：山西省应用基础研究项目(201701D221191);山西农业大学科技创新基金项目(2016YJ11,2016YJ12)。

摘　　要：灰霉病是樱桃的主要病害,由灰葡萄孢(Botrytis cinerea)侵染所致。该病菌可为害樱桃的花、叶、果实等多个部位,尤其在樱桃生长中后期及贮藏期发病率极高,危害严重。依据樱桃灰霉病菌内转录间隔区(ITS)序列设计引物,建立了樱桃灰霉病菌的重组酶聚合酶扩增结合侧流层析试纸条(LFD-RPA)检测方法。结果表明,该方法检测条件为37℃扩增30 min,能特异性地检测灰葡萄孢,灵敏度为100 fg·μL-1,略低于常规PCR(10 fg·μL-1)和荧光定量PCR(7.43 fg·μL-1);检测方法用时短,PRA扩增仅需30 min,LFD检测仅需10 min。本研究建立的快速检测方法可用于樱桃灰霉病菌的实时监测及病害的田间快速诊断。Gray mold, caused by Botrytis cinerea, is one of the main diseases on cherry flowers, leaves, and fruits in the middle and late growth period and the storage period with high incidence rate. Based on the internal transcribed spacer(ITS) sequence, a pair of primers were designed and used to develop recombinase polymerase amplification combined with a lateral flow dipstick(LFD-RPA) assay. The results showed that the optimal reaction temperature and time were 37℃ for 30 min for specific detection of B. cinerea with the sensitivity at 100 fg·μL-1, which was slightly lower than conventional PCR at 10 fg·μL-1 and real-time quantitative PCR at 7.43 fg·μL-1. However, the detection process was relatively simple and rapid, just including RPA reaction in 30 min and LFD detection in 10 min. This method can be of great value for the surveillance of pathogen and early diagnosis of the disease.

关 键 词：樱桃 灰葡萄孢 重组酶聚合酶扩增 侧流层析试纸条 ITS 快速检测 

分 类 号：S432.44[农业科学—植物病理学]