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

doi:10.3969/j.issn.1000-2006.2015.06.001

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南京林业大学学报（自然科学版） ›› 2015, Vol. 58 ›› Issue (06): 1-6.doi: 10.3969/j.issn.1000-2006.2015.06.001

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

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 210037

出版日期:2015-11-30 发布日期:2015-11-30
基金资助:
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.

Rapid diagnostic methods for Phytophthora tentaculata using LAMP

DAI Tingting^1,2, KANG Zhenye^1,2, WU Xiaoqin^1,2

1. Co-Innovation Center for the Sustainable Forestry in Southern China, Nanjing 210037, China;
2. Jiangsu Key Laboratory for Prevention and Control of Invasive Species, College of Forestry, Nanjing Forestry University, Nanjing 210037,China

Online:2015-11-30 Published:2015-11-30

摘要/Abstract

摘要： 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.

中图分类号:

S763

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, 58(06): 1-6.

DAI Tingting, KANG Zhenye, WU Xiaoqin. Rapid diagnostic methods for Phytophthora tentaculata using LAMP[J].Journal of Nanjing Forestry University (Natural Science Edition), 2015, 58(06): 1-6.DOI: 10.3969/j.issn.1000-2006.2015.06.001.

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

Rapid diagnostic methods for Phytophthora tentaculata using LAMP

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