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

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.