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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。
Phytophthora lateralis Tucker et Milbrath is an important pathogen of destructive plant pathogen and a quarantine pest of entry plants in China. At present, there is no report on the occurrence of this disease in our country. In order to prevent the introduction and spread of P. lateralis, it is necessary to detect it rapidly and accurately. In this study, LAMP-specific primers were designed by using loop-mediated isothermal amplification (LAMP). The LAMP reaction system was designed and the sensitivity and specificity were determined. The results show that the entire detection process takes only 80 min, which can directly determine the test results by naked eye observation. In the specificity test, azure positive reaction was observed in both P. lateralis strains, while other Phytophthora and fungal tested strains were negative. In sensitivity testing, the minimum detection limit is 100 pg / μL. The establishment of this method provides a new technique for the quarantine and identification of P. lateralis and the rapid diagnosis of the disease caused by it.