可视化环介导等温扩增技术检测 Phytophthora tentaculata

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 amplification,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 identify the disease caused by Phytophthora tentaculata, and keep the spread of the disease under control and reduce the economic losses quickly and accurately, loop-mediated isothermal amplification(LAMP)assay targeting the PtYpt1 element was used to detect Phytophthora tentaculata. The PtYpt1-LAMP assay amplified the target element in less than 80 min at 64 ℃ efficiently and was evaluated specificitily and sensitivitily. The specificity was evaluated against Phytophthora tentaculata, Phytophthora spp., Pythium spp., and true fungi isolates. The P. tentaculata DNA products were visualised as a ladder-like banding pattern by 2% gel electrophoresis. A positive colour(sky blue)was only observed in the presence of P. tentaculata by addition of hydroxynaphthol blue prior to amplification, whereas none of other isolates showed a colour change. The detection limit of the PtYpt1-specific LAMP assay for P. tentaculata was 1 ng/μL of genomic DNA per reaction