[1]张雯娜,李晋玉,田金艳,等.逆转录环介导等温扩增技术快速检测大豆花叶病毒[J].大豆科学,2014,33(03):422-428.[doi:10.11861/j.issn.10009841.2014.03.0422]
　ZHANG Wen-na,LI Jin-yu,TIAN Jin-yan,et al.Rapid Detection of Soybean Mosaic Virus by Reverse Transcription Loop Mediated Isothermal Amplification[J].Soybean Science,2014,33(03):422-428.[doi:10.11861/j.issn.10009841.2014.03.0422]
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逆转录环介导等温扩增技术快速检测大豆花叶病毒

《大豆科学》[ISSN:1000-9841/CN:23-1227/S] 卷: 第33卷 期数: 2014年03期 页码: 422-428 栏目: 出版日期: 2014-06-25
Title:
Rapid Detection of Soybean Mosaic Virus by Reverse Transcription Loop Mediated Isothermal Amplification
作者:
张雯娜1; 李晋玉1; 田金艳1; 卫其巍1; 智海剑2; 陶小荣1
1.南京农业大学 植物保护学院，江苏 南京 210095； 2.南京农业大学 大豆研究所/国家大豆改良中心，作物遗传与种质创新国家重点实验室，江苏 南京 210095
Author(s):
ZHANG Wen-na1; LI Jin-yu1; TIAN Jin-yan1; WEI Qi-wei1; ZHI Hai-jian2; TAO Xiao-rong1
1.Plant Protection College of Nanjing Agricultural University,Nanjing 210095,China;2.Soybean Research Institute,Nanjing Agricultural University/ National Key Laboratory for Crop Genetics and Germplasm Enhancement,National Center for Soybean Improvement,Nanjing 210095,China
关键词:
大豆花叶病毒; 快速检测; RT-LAMP; 大豆; 种子带毒检测
Keywords:
Rapid detection; Soybean mosaic virus; RTLAMP; Soybean; Detection of infected seeds
分类号:
S435.651
DOI:
10.11861/j.issn.10009841.2014.03.0422
文献标志码:
A
摘要:
大豆花叶病毒（Soybean mosaic virus，SMV）是一种在世界很多国家广泛分布并极具破坏性的植物病毒，导致大豆严重减产和种质衰退，每年都会给很多国家带来巨大的农业经济损失，为此，建立了一种逆转录环介导等温扩增(Reverse transcription loop mediated isothermal amplification,RT LAMP)全新的SMV检测技术，利用该技术可以快速、准确与有效地检测大豆植株和种子中携带的SMV。根据GenBank数据库中大豆花叶病毒的外壳蛋白基因序列，设计并筛选到了SMV RT-LAMP特异性引物组，应用该引物组优化了RT-LAMP反应参数，灵敏度实验结果表明，RT-LAMP检测SMV比普通RT-PCR灵敏度高10～100倍。结合RNA粗提法，建立了一步法RT-LAMP快速、灵敏地检测到植物叶片中的SMV。加入荧光染料SYBR Green I则可在反应管中直接观察检测结果，实现了检测的可视化。该RT-LAMP技术还可灵敏地检测到大豆种子中携带的SMV。RT-LAMP检测方法第一次应用于SMV的检测，具有灵敏度高、速度快和特异性强的优点,可应用于实验室和田间大豆样品SMV的快速检测。
Abstract:
Soybean mosaic virus(SMV)is a widely distributed and highly destructive plant virus worldwide.Infection by SMV usually results in severe yield losses and seed quality reduction.SMV infected disease has caused a huge economic losses in many countries.There fore,reversetranscription loopmediated isothermal amplification(RTLAMP),a method to achieve rapid,sensitive and accurate detection of SMV in soybean leaves and seeds was established.Using SMV coat protein gene sequences from GenBank database,the RTLAMP primers sets were designed and the best primers set was selected.Then they were used for the optimization of the RTLAMP parameters.The sensitivity of the RTLAMP was 10100 times higher than the conventional RTPCR.When crude RNA was integrated in this assay,the developed onestep RTLAMP could rapidly and sensitively detect SMV in soybean leaves.The reaction product could be directly visualized in tube by adding the fluorescent dye SYBR I.The developed RTLAMP could also successfully detect SMV in soybean seeds.This is the first report of the application of the RTLAMP assay to detect SMV in soybean leaves and seeds.The rapid,sensitive and specific RTLAMP assay developed in this study can be applied widely in laboratories and the field detection of SMV.

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备注/Memo

收稿日期：2013-12-05
基金项目：国家高技术研究发展计划“863计划” (2012AA101501)；公益性行业（农业）科研专项(201303028)；教育部高校博士点基金（20130097110004）；江苏省基础研究计划(自然科学基金)（BK2012769）。
第一作者简介：张雯娜(1988-)，女，硕士，主要从事植物病毒学研究。E-mail:wenna_521@163.com。通讯作者：陶小荣（1977-），男，教授，主要从事植物病毒学研究。E-mail:taoxiaorong@njau.edu.cn。

更新日期/Last Update: 2014-08-01