大蒜X病毒逆转录环介导等温扩增检测方法的建立

›› 2013, Vol. 25 ›› Issue (6): 0-1331.

• 植物保护 •

大蒜X病毒逆转录环介导等温扩增检测方法的建立

赵桐1，李长红1，周前进1，*，陈先锋1，2，陈炯1，3，陈剑平3

1 宁波大学生物化学与分子生物学实验室，浙江宁波 315211；2 宁波检验检疫科学技术研究院，浙江宁波 315012；3浙江省农业科学院农业部植物保护和生物技术重点实验室，浙江杭州 310021

收稿日期:1900-01-01 修回日期:1900-01-01 出版日期:2013-11-25 发布日期:2013-11-25

Rapid detection of Garlic virus X by reverse transcription loop\|mediated isothermal amplification

ZHAO Tong;LI Chang\|hong;ZHOU Qian\|jin;*;CHEN Xian\|feng;CHEN Jiong;CHEN Jian\|ping

1 Laboratory of Biochemistry and Molecular Biology， Ningbo University， Ningbo 315211， China;2 Ningbo Academy of Science & Technology for Inspection & Quarantine， Ningbo 315012， China;3 MOA Key Laboratory for Plant Protection and Biotechnology， Zhejiang Academy of Agricultural Sciences， Hangzhou 310021， China

Received:1900-01-01 Revised:1900-01-01 Online:2013-11-25 Published:2013-11-25

摘要/Abstract

摘要： 采用逆转录环介导等温扩增技术（RT\|LAMP），建立一种便捷、灵敏的大蒜X病毒（Garlic virus X， GarVX）的快速检测方法。根据GarVX ORF4序列的保守区设计6条特异性引物，分别识别该保守区的8个位点，在反转录酶和Bst DNA聚合酶的作用下对靶序列进行扩增反应。通过条件优化，在65℃恒温条件下温浴60 min可成功检测GarVX。特异性检测结果表明，该方法可特异性检出GarVX，对大蒜A病毒（Garlic virus A， GarVA）、大蒜D病毒（Garlic virus D， GarVD）、大蒜E病毒（Garlic virus E， GarVE）等的检测均为阴性，且检测灵敏度高，最低检出限为5 pg·μL-1，是RT\|PCR方法的10倍。试验结果表明，RT\|LAMP检测方法可快速、特异、灵敏地检测GarVX，并适合现场快速检测。

关键词: 大蒜X病毒, ORF4, 逆转录环介导等温扩增

Abstract: A one step reverse transcription loop\|mediated isothermal amplification （RT\|LAMP） assay was developed for detection of Garlic virus X（GarVX）. A set of six primers that recognized eight distinct regions of GarVX were designed based on the ORF4 sequence of the GarVX. Specificity amplification was carried out with the addition of reverse transcriptase and Bst DNA polymerase. The assay was optimized， and could detect GarVX by incubation at 65℃ for only 60 min. Specificity detection showed that GarVX could be specifically detected， and no amplification was observed when the RNA template from Garlic virus A （GarVA）， Garlic virus D （GarVD）， or Garlic virus E（GarVE） was used. The detection limit of the RT\|LAMP method was 5 pg·μL-1， which is 10 times higher than that of RT\|PCR assay. Results demonstrated that this RT\|LAMP method can detect GarVX quickly with high specificity and sensitivity， and is suitable for field testing.

Key words: Garlic virus X, ORF4 sequence, reverse transcription loop\|mediated isothermal amplification

赵桐;李长红;周前进;*;陈先锋;陈炯;陈剑平. 大蒜X病毒逆转录环介导等温扩增检测方法的建立[J]. , 2013, 25(6): 0-1331.

大蒜X病毒逆转录环介导等温扩增检测方法的建立

Rapid detection of Garlic virus X by reverse transcription loop\|mediated isothermal amplification

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