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 .
出处
《浙江农业学报》
CSCD
北大核心
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)
关键词
大蒜X病毒
ORF4
逆转录环介导等温扩增
Garlic virus X
ORF4 sequence
reverse transcription loop-mediated isothermal amplification
作者简介
赵桐（1992-），女，河南洛阳人，研究方向为生物技术，Tel：0574-87609571；E-mail：m18815282820@163．com
通讯作者，周前进，Tel：0574-87609571；E-mail：mumu2325@163．com
