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为了能在植物发病早期快速、准确地鉴定出Phytophthora tentaculata引起的疫病,控制疫病的传播和流行,减少其造成的经济损失,笔者以Ypt1基因为靶序列应用环介导等温核酸扩增技术(loop-mediated isothermal am-plification,LAMP),建立了一种基于颜色判定的简单、快速和灵敏的P.tentaculata检测方法。结果表明:在等温条件下(64℃)进行核酸扩增反应80 min,反应后经2%琼脂糖凝胶电泳和扩增前加入染料HNB(羟基萘酚蓝)作为反应指示剂验证扩增结果,在特异性实验中,P.tentaculata菌株能够扩增到梯形状的条带,同时HNB显色观察到天蓝色的阳性反应,而从其他疫霉、腐霉和真菌供试菌株中均没有观察到这些现象。在灵敏度试验中,PtYpt1-LAMP技术最低检测限为1 ng/μL。
In order to rapidly and accurately identify the Phytophthora tentaculata-induced disease in the early stage of plant disease, to control the spread and spread of the disease and reduce the economic losses caused by Phytophthora tentaculata, we used the loop-mediated isothermal nucleic acid amplification -mediated isothermal am-plification (LAMP), a simple, rapid and sensitive P.tentaculata detection method based on color judgment was established. The results showed that the amplification reaction was carried out under isothermal conditions (64 ℃) for 80 min. The reaction was followed by 2% agarose gel electrophoresis and dye HNB (hydroxynaphthol blue) as the reaction indicator before amplification In specific experiments, P.teaculata strains were able to amplify trapezoidal bands with a sky-blue positive reaction when HNB was developed, but not from other Phytophthora, Pythium and fungi tested strains To these phenomena. The minimum limit of detection for PtYpt1-LAMP technology is 1 ng / μL in sensitivity tests.