[1]吴旭东,陆辰晨,沈浩,等.大豆茎褐腐病菌环介导等温扩增检测技术的建立[J].南京农业大学学报,2015,38(4):568-574.[doi:10.7685/j.issn.1000-2030.2015.04.007]
 WU Xudong,LU Chenchen,SHEN Hao,et al.A rapid detection method for the plant pathogen Phialophora gregata f.sp.sojae based on loop-mediated isothermal amplification(LAMP)[J].Journal of Nanjing Agricultural University,2015,38(4):568-574.[doi:10.7685/j.issn.1000-2030.2015.04.007]
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大豆茎褐腐病菌环介导等温扩增检测技术的建立()
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《南京农业大学学报》[ISSN:1000-2030/CN:32-1148/S]

卷:
38卷
期数:
2015年4期
页码:
568-574
栏目:
出版日期:
2015-06-28

文章信息/Info

Title:
A rapid detection method for the plant pathogen Phialophora gregata f.sp.sojae based on loop-mediated isothermal amplification(LAMP)
作者:
吴旭东1 陆辰晨1 沈浩1 吴翠萍2 张海峰1 王源超1 郑小波1
1. 南京农业大学植物保护学院卵菌与真菌分子生物学实验室, 江苏 南京 210095;
2. 江苏出入境检验检疫局, 江苏 南京 210001
Author(s):
WU Xudong1 LU Chenchen1 SHEN Hao1 WU Cuiping2 ZHANG Haifeng1 WANG Yuanchao1 ZHENG Xiaobo1
1. Laboratory of Oomycetes and Fungi Molecular Biology, College of Plant Protection, Nanjing Agricultural University, Nanjing 210095, China;
2. Jiangsu Entry-Exit Inspection and Quarantine Bureau, Nanjing 210001, China
关键词:
大豆茎褐腐病菌环介导等温扩增ITS羟基萘酚蓝(HNB)
Keywords:
Phialophora gregata f.sp.sojaeloop-mediated isothermal amplificationITShydroxynaphthol blue(HNB)
分类号:
Q93-331
DOI:
10.7685/j.issn.1000-2030.2015.04.007
摘要:
[目的]应用环介导等温核酸扩增技术(loop-mediated isothermal amplification,LAMP)建立基于颜色判定的简便、快速和灵敏的大豆茎褐腐病菌[Phialophora gregata f.sp.sojae(PGS)]检测方法。[方法]利用LAMP技术,以ITS为靶序列,设计了4条特异性的LAMP引物和2条环引物,在此基础上建立了检测大豆茎褐腐病菌的LAMP体系。[结果]整个检测过程仅需1 h就可通过肉眼直接目测试验结果。在等温条件下(64 ℃)进行核酸扩增反应1 h,在扩增前加入染料HNB(羟基萘酚蓝)作为反应指示剂,根据HNB的颜色变化可以判断PGS的存在与否。特异性试验中,大豆茎褐腐病菌菌株DNA扩增后HNB呈天蓝色的阳性反应,而在其他大豆常见病害的供试菌株中均呈紫色的阴性反应。在灵敏度试验中,ITS-LAMP技术最低检测限为10 pg·μL-1,对于进口大豆残体样本中PGS的检测灵敏度为每10 g大豆植株残体50个孢子。[结论]该方法的建立为检疫性病害大豆茎褐腐病菌的检测提供了新的技术平台,符合我国口岸对大豆茎褐腐病菌快速检测的迫切需要。
Abstract:
[Objectives]rDNA sequences between closely related species are highly conserved,limiting the development of species-specific detection among Phialophora gregata f.sp.sojae(PGS). In this paper,we report the development of loop-mediated isothermal amplification(LAMP)assays targeting ITS element for visual detection of PGS. [Methods]Via utilizing the technology of LAMP,and setting the ITS as target sequence,we designed four special LAMP primers and two loop primers and therefore based on this we set up the LAMP system of testing PGS. The ITS-LAMP assay was evaluated for specificity,sensitivity,detections of artificial incubation and diseased soybean tissue. [Results]The ITS-LAMP assay efficiently amplified the target element in less than 60 min at 64 ℃ and was evaluated for specificity and sensitivity. The specificity was evaluated against PGS and other fungi isolates. A positive colour(sky blue)was only observed in the presence of PGS by addition of hydroxynaphthol blue(HNB)prior to amplification,whereas none of other isolates showed a colour change. The detection limit of the ITS-specific LAMP assay for PGS was 10 pg·μL-1 of genomic DNA per reaction,and its sensitivity of imported beans residue samples was 50 spores per 10 g beans residue. [Conclusions]Establishment of LAMP provides a new alternative method for the rapid detection of PGS.

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备注/Memo

备注/Memo:
收稿日期:2014-09-22。
基金项目:国家863计划项目(2012AA101501);国家质检总局项目(2012IK278)
作者简介:吴旭东,硕士研究生。
通讯作者:郑小波,教授,主要从事卵菌与真菌分子遗传的研究,E-mail:xbzheng@njau.edu.cn。
更新日期/Last Update: 1900-01-01