[1]沈浩,戴婷婷,吴翠萍,等.基于环介导等温扩增技术检测大豆北方茎溃疡病菌[J].南京农业大学学报,2015,38(2):255-260.[doi:10.7685/j.issn.1000-2030.2015.02.012]
 SHEN Hao,DAI Tingting,WU Cuiping,et al.The tef1α-LAMP method for rapid detection of Diaporthe phaseolorum var.caulivora[J].Journal of Nanjing Agricultural University,2015,38(2):255-260.[doi:10.7685/j.issn.1000-2030.2015.02.012]
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基于环介导等温扩增技术检测大豆北方茎溃疡病菌()
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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2期
页码:
255-260
栏目:
出版日期:
2015-03-23

文章信息/Info

Title:
The tef1α-LAMP method for rapid detection of Diaporthe phaseolorum var.caulivora
作者:
沈浩1 戴婷婷2 吴翠萍3 杨辉耀1 宋必1 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, 江苏 南京 210095;
2. 南京林业大学林学院, 江苏 南京 210037;
3. 江苏省出入境检验检疫局, 江苏 南京 210001
Author(s):
SHEN Hao1 DAI Tingting2 WU Cuiping3 YANG Huiyao1 SONG Bi1 ZHANG Haifeng1 WANG Yuanchao1 ZHENG Xiaobo1
1. College of Plant Protection, Nanjing Agricultural University, Nanjing 210095, China;
2. College of Forestry, Nanjing Forestry University, Nanjing 210037, China;
3. Jiangsu Entry-Exit Inspection and Quarantine Bureau, Nanjing 210001, China
关键词:
大豆北方茎溃疡病菌环介导等温扩增翻译延伸因子SYBR greenⅠ
Keywords:
Diaporthe phaseolorum var.caulivora(DPC)loop-mediated isothermal amplification(LAMP)translation elongation factor 1α(tef1α)SYBR greenⅠ
分类号:
Q93-331
DOI:
10.7685/j.issn.1000-2030.2015.02.012
摘要:
[目的]传统的以ITS(内转录间隔区)为靶序列对大豆北方茎溃疡病菌(Diaporthe phaseolorum var.caulivora,DPC)的分子检测无法区分大豆南方茎溃疡病菌(D.phaseolorum var.meridionalis)和大豆拟茎点种腐病菌(Phomopsis longicolla)等近似种。笔者在大豆北方茎溃疡病菌靶标序列筛选中,发现了翻译延伸因子(translation elongation factor 1α,tef1α)序列。通过目标病原菌与其相似种比对发现,大豆北方茎溃疡病菌在tef1α序列上有很好的特异性,适合作为分子检测的靶标。[方法]基于环介导等温扩增技术(loop-mediated isothermal amplification,LAMP),以tef1α为靶序列,设计LAMP特异性引物,建立了一种基于颜色判定的简单、快速和灵敏的DPC检测方法,并进行特异性、灵敏度、植株接种试验和进口大豆夹带大豆病残体检测。[结果]该方法仅需60 min,即可通过肉眼直接目测试验结果。64 ℃等温条件下进行核酸扩增反应60 min,反应结束后,加入SYBR greenⅠ染料,根据染料颜色变化判定扩增结果。特异性试验中,在DPC菌株中扩增到梯形条带,同时加入SYBR greenⅠ染料后可观察到绿色荧光的阳性反应;而在其他供试菌株中均没有出现梯形条带,加入SYBR greenⅠ染料后则保持橙色的阴性反应。该技术最低检测限为1 pg·μL-1目标菌纯DNA。tef1α-LAMP技术能够检测出所有人工接种发病豆苗中的目标菌。在口岸截获的大豆病残体检测中,tef1α-LAMP技术能够在10 g进口大豆中夹带的大豆植株残体中最低检测出10个子囊孢子。[结论]该方法的建立为大豆北方茎溃疡病菌的检疫以及其所致病害的快速诊断提供了新的技术。
Abstract:
[Objective]rDNA sequences between closely related species are highly conserved, limiting the development of species-specific detection among Diaporthe phaseolorum var.caulivora, D.phaseolorum var.meridionalis and Phomopsis longicolla. [Methods]In this research, translation elongation factor 1α(tef1α)was identified as a suitable target for detecting D.phaseolorum var.caulivora using loop-mediated isothermal amplification(LAMP) method.The tef1α-LAMP assay efficiently amplified the target gene in 60 min at 64 ℃ and was evaluated for specificity, sensitivity, detections of artificial incubation and diseased soybean tissue. [Results]The specificity was evaluated against D.phaseolorum var.meridionalis, Phomopsis longicolla and other fungi isolates. The DNA products of D.phaseolorum var.caulivora were visualized as a ladder-like banding pattern on 20 g·L-1 gel electrophoresis and a positive color(yellow-green) was only observed in the presence of D.phaseolorum var.caulivora by adding SYBR Green Ⅰ after the amplification, whereas all other isolates showed orange color. The detection limit of the tef1α-LAMP assay for D.phaseolorum var.caulivora was 1 pg·μL-1 of genomic DNA per reaction. The assay also detected D.phaseolorum var.caulivora from inoculated soybean tissues and the tef1α-LAMP assay can only detect 10 ascospore in 10 g soybean residues which are carried by the soybean trading between China and other countries. [Conclusions]These results suggest that this tef1α-LAMP provides a rapid and sensitive tool for detecting D.phaseolorum var.caulivora in plants and production fields.

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备注/Memo

备注/Memo:
收稿日期:2014-7-9。
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:沈浩,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