CAJ | 학술논문

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。
채용역전록배개도등온확증기술（RT-LAMP），건립일충편첩、령민적대산X병독（Garlic virus X， GarVX）적쾌속검측방법。근거GarVX ORF4서렬적보수구설계6조특이성인물，분별식별해보수구적8개위점，재반전록매화Bst DNA취합매적작용하대파서렬진행확증반응。통과조건우화，재65℃항온조건하온욕60 min가성공검측GarVX。특이성검측결과표명，해방법가특이성검출GarVX，대대산A병독（ Garlic virus A， GarVA）、대산D병독（ Garlic virus D， GarVD）、대산E병독（ Garlic virus E， GarVE）등적검측균위음성，차검측령민도고，최저검출한위5 pg·μL-1，시RT-PCR방법적10배。시험결과표명，RT-LAMP검측방법가쾌속、특이、령민지검측GarVX，병괄합현장쾌속검측。
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 .