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

作　　者：赵桐[1] 李长红[1] 周前进[1] 陈先锋[1,2] 陈炯[1,3] 陈剑平[3] 

机构地区：[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

出　　处：《浙江农业学报》2013年第6期1326-1331,共6页Acta Agriculturae Zhejiangensis

基　　金：国家自然科学基金面上项目(30771402);浙江省大学生科技创新活动计划(新苗人才计划) 项目( 2013R405026)

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。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 de-signed based on the ORF 4 sequence of the GarVX .Specificity amplification was carried out with the addition of re-verse 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 .

关 键 词：大蒜X病毒 ORF4 逆转录环介导等温扩增 

分 类 号：S917.1[农业科学—水产科学]