基于环介导等温扩增技术检测雪松疫霉根腐病菌

雪松疫霉根腐病菌(Phytophthora lateralis Tucker et Milbrath)是一种重要的毁灭性植物病原菌,也是我国进境植物检疫性有害生物。目前,我国未有该病害发生的报道,为了防止P.lateralis的传入和扩散,需对其进行快速、准确的检测。本文利用环介导等温扩增技术(loop-mediated isothermal amplification,LAMP),以Ypt1基因为靶标序列,设计LAMP特异性引物,建立LAMP反应体系,并对灵敏度和特异性进行检测。结果表明:整个检测过程仅需80 min,即可通过肉眼观察直接判定检测结果。在特异性检测中,P.lateralis菌株都能观察到天蓝色的阳性反应,而其他疫霉菌和真菌供试菌株均呈阴性反应。在灵敏度检测中,最低检测限为100 pg/μL。该方法的建立为P.lateralis的检疫鉴定及其所致病害的快速诊断提供了新技术。

A rapid detection method for Phytophthora lateralis based on loop-mediated isothermal amplification (LAMP)

Phytophthora lateralis is a devastating pathogen that usually causes severe root rot to the host plants and it is an entry plant quarantine pest of China.There were no reports of the disease in our country now.Therefore,there is a demand for rapid and specific detection method to prevent the pathogen entering and spreading.This study reports the development of a loop-mediated isothermal amplication (LAMP) assay targeting Ypt1 gene for visual detection of P.lateralis.The Ypt1-LAMP assay efficiently amplified the target gene within 80 min at 62 ℃ and was evaluated for specificity and sensitivity.The specificity of the assay was evaluated against isolates of P.lateralis,Phytophthora spp.,and fungi isolates.A positive color (sky blue) was only observed in the presence of P.lateralis by addition of hydroxynaphthol blue (HNB) prior to the amplification,whereas none of other isolates showed a color reaction.The detection limit of Ypt1-LAMP assay was 100 pg/μL of genomic DNA per reaction.These results suggest that the Ypt1-LAMP assay reported here provides a new alternative method for the rapid detection of P.lateralis.