基于环介导等温扩增技术检测P-南京林业大学.DOCVIP

可视化环介导等温扩增技术检测Phytophthora tentaculata的研究 戴婷婷1,2，康振烨1,2，吴小芹1,2 （1.南京林业大学南方现代林业协同创新中心,江苏 南京 210037;2.江苏省有害生物入侵预防与控制重点实验室,南京林业大学林学院,江苏 南京 210037Phytophthora tentaculata引起的疫病，控制疫病的传播和流行、减少其造成经济损失，本研究以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的检测方法。结果表明在等温条件下（64°C）进行核酸扩增反应80min，反应后经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菌株中扩增到的条带而从其它疫霉腐霉和真菌供试菌株中P. tentaculata引起的疫病的检疫和所致病害的快速诊断提供了新的技术。 关键词：Phytophthora tentaculata；环介导等温扩增；PtYpt1；羟基萘酚蓝（HNB） 中图分类号： 文献标志码：A 文章编号： Rapid diagnostic methods for Phytophthora tentaculata using LAMP DAI Ting-ting1,2*, KangzhenYe1,2, Xiaoqin Wu1,2 （1. Co-Innovation Center for the Sustainable Forestry in Southern China, Nanjing Forestry University, Nanjing 210037, China;2. Jiangsu Key Laboratory for Prevention and Control of Invasive Species,College of Forestry, Nanjing Forestry University,Nanjing 210037,China） Abstract: In order to quickly and accurately identify the disease which was caused by Phytophthora tentaculata, keep the spread of the disease under control and reduce the economical losses, Loop-mediated isothermal amplification （LAMP） assay targeting the Phytophthora tentaculata. The PtYpt1-LAMP assay efficiently amplified the target element in less than 80 min at 64°C and was evaluated for specificity and sensitivity. 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 on 2% gel electrophoresis. A positive colour （sky blue） was only observed in the presence of P 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/礚t of genomic DNA per reaction. Establishment of LAMP provides a new alternative method for the rapid detection of P. tentaculata. Keywords: Ph