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。
Abstract
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.
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基金
收稿日期:2014-11-04 修回日期:2015-05-20
基金项目:国家自然科学基金项目(31500526); 中国博士后科学基金项目(2014M561657); 江苏省博士后科学基金项目(1402159C); 江苏高校优势学科建设工程资助项目(PAPD); 南京林业大学高层次人才启动基金(G2014003)
第一作者:戴婷婷,讲师。 E-mai: 13770647123@163.com。
引文格式:戴婷婷,康振烨,吴小芹. 可视化环介导等温扩增技术检测Phytophthora tentaculata[J]. 南京林业大学学报:自然科学版,2015,39(6):1-6.
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