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. 39 ›› 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, 39(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, 39(06): 1-6.DOI: 10.3969/j.issn.1000-2006.2015.06.001.

参考文献

[1] Kröber H, Marwitz R. Phytophthora tentaculata sp. nov. and Phytophthora cinnamomi var. parvispora var. nov., zwei neue Pilze von Zierpflanzen in Deutschland/Phytophthora tentaculata sp. nov. and Phytophthora cinnamoml var. parvispora var. nov., two new fungi from ornamental plants in Germany[J]. Zeitschrift für Pflanzenkrankheiten und Pflanzenschutz/Journal of Plant Diseases and Protection, 1993, 100(3): 250-258.
[2] Moralejo E, Puig M, Man in't Veld W A. First report of Phytophthora tentaculata on Verbena sp. in Spain [J]. Plant Pathology, 2004, 53(6), 806.
[3] Meng J, Wang Y C. First report of stalk rot caused by Phytophthora tentaculata on Aucklandia lappa in China[J]. Plant Disease, 2008, 92(9): 1365.
[4] 郑小波.疫霉菌及其研究技术[M].北京:中国农业出版社,1997:81-82.
[5] 孟军,刘燕,方志翔.基于Ypt1基因为靶标的木香疫病病组织及病田土壤分子检测技术[J].植物病理学报,2013,43(1): 95-99. Meng J, Liu Y, Fang Z X. Molecular detection of Phytophthora tentaculata in infected Aucklandia lappa tissues and soil on the basis of Ypt1 Gene[J].Acta Phytopathological Sinica, 1993, 43(1):95-99.
[6] Notomi T, Okayama H, Masubuchi H, et al. Loop-mediated isothermal amplification of DNA[J]. Nucleic Acids Research, 2000, 28(12):63.
[7] Pan W, Wang J Y, Shen H Y, et al. Development and application of the novel visual loop-mediated isothermal amplification of Omp25 sequence for rapid detection of Brucella sp[J]. Journal of Animal and Veterinary Advances, 2011, 10(16):2120-2126.
[8] Parida M, Horioke K, Ishida H, et al. Rapid detection and differentiation of dengue virus serotypes by a real-time reverse transcription-loop-mediated isothermal amplification assay[J]. Journal of Clinical Microbiology, 2005, 43(6):2895-2903.
[9] Niessen L, Vogel R F. Detection of Fusarium graminearum DNA using a loop-mediated isothermal amplification(LAMP)assay[J]. International Journal of Food Microbiology, 2010, 140(2): 183-191.
[10] Imai M, Ninomiya A, Minekawa H, et al. Development of H5-RT-LAMP(loop-mediated isothermal amplification)system for rapid diagnosis of H5 avian influenza virus infection[J]. Vaccine, 2006, 24(44):6679-6682.
[11] 张宏伟,叶露萌,彭杨思,等. 利用环介导等温扩增技术对沙门氏菌进行检测[J]. 食品研究与开发,2009.30(5): 115-118. Zhang H W, Ye L M, Peng Y S, et al. Development of LAMP detection of salmonella in food[J]. Food Research and Development, 2009, 30(5):115-118.
[12] Abd-Elsalam K, Bahkali A, Moslem M, et al. An optimized protocol for DNA extraction from wheat seeds and Loop-Mediated Isothermal Amplication(LAMP)to detect Fusarium graminearum contamination of wheat grain[J]. International Journal of Molecular Sciences, 2011, 12(6):3459-3472.
[13] Dai T T, Lu C C, Dong S M, et al. Development of a loop-mediated isothermal amplification assay for detection of Phytophthora sojae [J]. FEMS Microbiology Letters, 2013, 334(1):27-34.
[14] Wang Y, Zhang W, Zheng X. Rapid and sensitive detection of Phytophthora sojae in soil and infected soybeans by species-specific polymerase chain reaction assays[J]. Phytopathology,2006,96(12):1315-1321.
[15] Goto M, Honda E, Ogura A, et al. Short technical reports:colorimetric detection of loop-mediated isothermal amplifcation reaction by using hydroxynaphthol blue[J]. Biotechniques, 2009, 46(3):167-172
[16] Schena L, Cooke D E L. Assessing the potential of regions of the nuclear and mitochondrial genome to develop a ‘molecular tool box' for the detection and characterization of Phytophthora species[J]. Journal of Microbiological Methods, 2006, 67(1):70-85.
[17] Schena L, Hughes K J D, Cooke D E L. Detection and quantification of Phytophthora ramorum, P. kernoviae, P. citricola and P. quercina in symptomatic leaves by multiplex real-time PCR Mole[J]. Plant Pathology, 2006, 7(5):365-369.
[18] Uemura N, Makimura K, Onozaki M, et al. Development of a loop-mediated isothermal amplification method for diagnosing Pneumocystis pneumonia[J]. Journal of Medical Microbiology, 2008, 57(1):50-57.
[19] Martini P, Pane A, Raudino F, et al. First report of Phytophthora tentaculata causing root and stem rot of Oregano in Italy[J]. Plant Disease, 2009, 93(8):843-843.
[20] Rooney-Latham S, Blomquist C L. First report of root and stem rot caused by Phytophthora tentaculata on Mimulus aurantiacus in North America[J]. Plant Disease, 2014, 98(7):996-996.
[21] 邱秀文,叶建仁,张林平.环介导等温基因扩增技术在林木检测中的应用展望[J].南京林业大学学报:自然科学版, 2012,36(6): 135-139. Qiu X W, Ye J R, Zhang L P.The application prospect of loop-mediated isothermal-amplification method in forest disease detection[J]. Journal of Nanjing Forestry University:Natural Sciences Edition, 2012, 36(6):135-139.

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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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