燕麦镰孢菌环介导等温扩增(LAMP)检测方法的建立与应用

doi:10.11733/j.issn.1007-0435.2018.04.028

草地学报 ›› 2018, Vol. 26 ›› Issue (4): 1004-1010.DOI: 10.11733/j.issn.1007-0435.2018.04.028

燕麦镰孢菌环介导等温扩增(LAMP)检测方法的建立与应用

漆永红^1,3, 曹素芳², 李雪萍¹, 李敏权¹

1. 甘肃省农业科学院植物保护研究所, 甘肃兰州 730070;
2. 甘肃省农业科学院林果花卉研究所, 甘肃兰州 730070;
3. 甘肃农业大学草业学院, 甘肃兰州 730070

收稿日期:2017-12-18 修回日期:2018-07-29 出版日期:2018-08-15 发布日期:2018-09-20
通讯作者: 曹素芳, 李敏权
作者简介:漆永红(1978-),男,甘肃天水人,副研究员,主要从事经济作物病害研究,E-mail:qiyonghong920@gsagr.ac.cn
基金资助:
国家公益性行业专项“作物根腐病综合治理技术方案”（201503112）；国家重点研发计划“特色经济作物化肥农药减施技术集成研究与示范”（2018YFD0201100）；甘肃省农业科学院科研条件建设及成果转化项目（2017GAAS73）资助

Development of a Loop-mediated Isothermal Amplification Assay for Detection of Fusarium avenaceum

QI Yong-hong^1,3, CAO Su-fang², LI Xue-ping¹, LI Min-quan¹

1. Institute of Plant Protection, Gansu Academy of Agricultural Sciences, Lanzhou, Gansu Province 730070, China;
2. Institute of Fruit and Floriculture Research, Gansu Academy of Agricultural Sciences, Lanzhou, Gansu Province 730070, China;
3. College of Pratacultural, Gansu Agrcultural University, Lanzhou, Gansu Province 730070, China

Received:2017-12-18 Revised:2018-07-29 Online:2018-08-15 Published:2018-09-20

摘要/Abstract

摘要： 为了明确燕麦镰孢菌（Fusarium avenaceum）快速检测方法，以荧光染料SYBR Green I为指示剂，以燕麦镰孢菌（F.avenaceum）的ITS序列为目的DNA片段，应用LAMP设计软件设计4条引物，优化LAMP反应体系和反应条件，用2%琼脂糖凝胶电泳和LAMP反应液颜色变化进行特异性和灵敏度验证，同时进行田间发病组织检测和土壤燕麦镰孢菌灵敏度验证。结果表明：优化的LAMP反应体系，最佳反应温度为65℃，反应程序为65℃下1 h，80℃下20 min。特异性检测结果表明，燕麦镰孢菌LAMP检测均呈黄绿色（阳性），对照和其他病原菌均呈橘色（阴性）。灵敏度验证结果表明，LAMP反应液检测灵敏度在DNA水平达到10 pg·μL^-1，每克土壤中检测的灵敏度为40个燕麦镰孢菌孢子。对采自甘肃省甘南州卓尼县和临潭县的20份疑似病害样本提取的DNA进行检测，其中13份为阳性，表明该技术能够有效的检测出青稞发病组织中的燕麦镰孢菌。本方法的建立与应用能为燕麦镰孢菌的检测及其青稞茎基腐病的诊断提供一种新技术。

关键词: 青稞, 茎基腐病, 燕麦镰孢菌, 环介导等温扩增(LAMP)

Abstract: In order to determine Fusarium avenaceum with rapid method,a loop-mediated isothermal amplification (LAMP) was first developed using SYBR Green I as a fluorescence indicator in this study. Based on the internal transcribed spacer (ITS) sequence of DNA genes in F. avenaceum,a set of four LAMP primers were designed using software. The reaction systems and conditions were optimized. The specificity and sensitivity of LAMP were assayed by electrophoresis on 2.0% agarose gel and SYBR Green I stain. Diseased naked barley tissue and F. avenaceum spores in soil were evaluated. The results showed that the LAMP assay efficiently amplified the target DNA in 1 h at 65℃ and 20 min at 80℃ with the optimum reaction temperature at 65℃. A positive yellow green color was only observed in the presence of F. avenaceum,whereas all other isolates showed orange color as negative control. The detection limit of the LAMP assay for F. avenaceum was 10 pg·μL^-1 of target DNA or 40 spores in 1 g soil in the high sensitivity. The diagnostic validation was performed to detect F. avenaceum in 13 out of 20 suspect diseased naked barley samples collected from Lintan and Zhuoni County in Gannan state,Gansu province. This study provided a new approach for the detection of F. avenaceum and naked barley crown rot disease.

Key words: Naked barley, Crown rot disease, Fusarium avenaceum, Loop-mediated isothermal amplification (LAMP)

中图分类号:

S543

漆永红, 曹素芳, 李雪萍, 李敏权. 燕麦镰孢菌环介导等温扩增(LAMP)检测方法的建立与应用[J]. 草地学报, 2018, 26(4): 1004-1010.

QI Yong-hong, CAO Su-fang, LI Xue-ping, LI Min-quan. Development of a Loop-mediated Isothermal Amplification Assay for Detection of Fusarium avenaceum[J]. Acta Agrestia Sinica, 2018, 26(4): 1004-1010.

0
/ / 推荐

导出引用管理器 EndNote|Ris|BibTeX

链接本文: https://manu40.magtech.com.cn/Jweb_cdxb/CN/10.11733/j.issn.1007-0435.2018.04.028

https://manu40.magtech.com.cn/Jweb_cdxb/CN/Y2018/V26/I4/1004

参考文献

[1] 李雪萍. 青藏高原青稞根腐类病害及其对根际土壤微生态的影响[D]. 兰州:甘肃农业大学,2017:32-43
[2] Mori Y,Notomi T. Loop-mediated isothermal amplification (LAMP):A rapid,accurate,and cost-effective diagnostic method for infectious disease[J]. Journal of Infection and Chemotherapy,2009,15(2):62-69
[3] Notomi T,Okayama H,Masubuchi H,et al. Loop-mediated isothermal amplification of DNA[J]. Nucleic Acids Research,2000,28(12):63
[4] Tomita N,Mori Y,Kanda H,et al. Loop-mediated isothermal amplification(LAMP) of gene sequences and simple visual detection of products[J]. Nature Protocols,2008,3(5):877-882
[5] Goto M,Honda E,Ogura A,et al. Colorimetric detection of loop-mediated isothermal amplification reaction by using hydroxyl naphthol blue[J]. Biotechniques,2009,46(3):167-172
[6] Wang X D,Lin X J,Hu H Q,et al. Establishment and application of visual loop-mediated amplification assay on citrus Huanglongbing[J]. Chinese Journal of Tropical Crops,2014,35(5):918-924
[7] Hayashi N,Arai R,Tada S,et al. Detection and identification of Brettanomyces/Dekkera sp. Yeasts with a loop-mediated isothermal amplification method[J]. Food Microbiology,2007,24(7):778-785
[8] Dai T T,Lu C C,Lu J,et al. Development of a loop-mediated isothermal amplification assay for detection of Phytophthora sojae[J]. FEMS Microbiology Letters,2012,334(1):27-34
[9] Qiao Y M,Guo Y C,Zhang X E,et al. Loop-mediated isothermal amplification for rapid detection of Bacillus anthracis spores[J]. Biotechnology Letters,2007,29(12):1939-1946
[10] Liu Y,Zhang J F,Zhang H Y,et al. Development of a loop-mediated isothermal amplification assay for rapid detection of transgenic soybean[J]. Soybean Science,2009(04):32
[11] Boubourakas I N,Fukuta S,Kyriakopoulou P E. Sensitive and rapid detection loop-mediated isothermal amplification[J]. Journal of Virological Methods,2009,160(1):63-68
[12] 王旭哲,张凡凡,马春晖.青贮饲料中真菌、真菌毒素及其检测方法的研究进展[J]. 草地学报,2016,24(3):509-517
[13] 史亚娟,王英志,王振跃,等. 假禾谷镰孢菌LAMP快速检测方法的建立[J]. 植物病理学报,2016,46(4):566-568
[14] 田擎,张海峰,曾丹丹,等. 环介导等温扩增技术检测大丽轮枝菌[J]. 植物病理学报,2016,46(4):721-729
[15] 吴敏亮,张溢溪,兰成忠,等. 潘石榴焦腐病菌LAMP检测方法的建立及应用[J]. 植物病理学报,2013,43(6):574-580
[16] Klimes A,Dobinsin K F. A hydrophobin gene,VDH1,is involved in microsclerotial development and spore viability in the plant pathogen Verticillium dahliae[J]. Fungal Genetics and Biology,2006,43(4):283-294
[17] Pérez-Artés E,García-Pedrajas M D,Bejarano-Alcázar J,et al. Differentiation of cotton-defoliating and nondefoliating pathotypes of Verticillium dahliae by RAPD and specific PCR analyses[J]. European Journal of Plant Pathology,2000,106(6):507-517
[18] Mercado-Blanco J,Rodríguez-Jurado D,Pérez-Artés E,et al. Detection of the nondefoliating pathotype of Verticillium dahliiae in infected olive plants by nested PCR[J]. Plant Pathology,2001,50(5):609-619
[19] Mercado-Blanco J,Rodríguez-Jurado D,Pérez-Artés E,et al. Detection of the defoliating pathotype of Verticillium dahliiae in infected olive plants by nested PCR[J]. European Journal of Plant Pathology,2003,118(1):1-13
[20] 任海英,戚行江,梁森苗,等. 利用常规PCR和实时荧光定量PCR检测杨梅凋萎病菌[J]. 植物病理学报,2016,46(1):1-10
[21] Noris E,Miozzi L. Real-time PCR protocols for the quantification of the begomovirus tomato yellow leaf curl Sardinia virus in tomato plants and in its insect vector[J]. Methods in Molecular Biology,2015(01236):61-72
[22] 伍文宪,刘勇,黄小琴,等. 尖孢镰孢菌分子检测技术的建立与应用[J]. 草业学报,2016,25(5):109-115
[23] Lu C C,Dai T T,Zhang H F,et al. Development of a loop-mediated isothermal amplification assay to detect Fusarium oxysporum[J]. Journal of Phytopathology,2015,163(1):63-66
[24] Lu C C,Zhang H F,Wang Y C,et al. Rapid diagnosis of Fusarium root rot in soybean caused by Fusarium equiseti or Fusarium graminearum using loop-mediated isothermal amplification (LAMP) assays[J]. Australasian Plant Pathology,2015,44(4):437-443
[25] 纪睿,曾丹丹,廖太林,等. 基于环介导等温扩增技术检测雪松疫霉根腐病菌[J]. 植物检疫,2017,30(1):42-47
[26] 张吉红,陈先锋,孙辉,等. 逆转录环介导等温扩增技术检测烟草环斑病毒的研究[J]. 植物病理学报,2014,44(6):629-633

燕麦镰孢菌环介导等温扩增(LAMP)检测方法的建立与应用

Development of a Loop-mediated Isothermal Amplification Assay for Detection of Fusarium avenaceum

PDF

可视化

摘要/Abstract

引用本文

使用本文

参考文献

相关文章 5

编辑推荐

Metrics

本文评价

[1]	漆永红, 曹素芳, 李雪萍, 李敏权. 甘南州临潭县青稞根际土壤养分含量、酶活性和微生物数量与根腐病的关系研究[J]. 草地学报, 2018, 26(4): 877-884.
[2]	辛鹏程, 原现军, 郭刚, 闻爱友, 肖慎华, 参木有, 陈裕祥, 邵涛. 添加丙酸对青稞秸秆和多年生黑麦草混合青贮发酵品质及有氧稳定性的影响[J]. 草地学报, 2015, 23(3): 594-600.
[3]	崔棹茗, 郭刚, 原现军, 李君风, 杨晓丹, 丁良, 余成群, 邵涛. 青稞秸秆青贮饲料中优良乳酸菌的筛选及鉴定[J]. 草地学报, 2015, 23(3): 607-615.
[4]	李龙兴, 郭刚, 原现军, 闻爱友, 王坚, 参木有, 陈裕祥, 邵涛. 添加剂对西藏青饲玉米和青稞秸秆混合青贮发酵品质的影响[J]. 草地学报, 2015, 23(2): 401-406.
[5]	张堃, 姚拓, 张德罡, 辛国荣, 师尚礼. 不同剂型联合固氮菌肥对青稞促生效应和固氮能力研究[J]. , 2010, 18(3): 426-430.