CAJ | 학술논문

基于已报道的甘蔗黄叶病毒（SugarcaneYellowLeafVirus．SCYLV）cp基因和高粱花叶病毒（Sorghummosaicvirus．SrMV）NSP基因序列设计特异性引物．建立了针对这两种病毒的实时荧光定量PCR（re矗time-quantitative，RT-qPCR）检测方法。在此基础上，利用扩增产物‰值的不同，建立了可区分ScYLV与SrMV的多重SYBRGreen—I实时荧光定量PCR方法．两种病毒扩增产物的‰值分别为80．8～82．8℃和84．6～86．6℃。检测数据显示：SrMV和ScYLV均有各自的特异性峰值出现，而健康植株无特异性峰值；在单独检测中的检出水平分别可达500和250copy／ixL：两种病毒m值的批内变异系数分别为0．03％和0．0270．批间变异系数分别为2．0670和3．1270。综上所述．所建立的多重SYBRGreen—I实时荧光定量PCR方法在对以上两种病毒的检测中具有较好的特异性、敏感性和稳定性。
기우이보도적감자황협병독（SugarcaneYellowLeafVirus．SCYLV）cp기인화고량화협병독（Sorghummosaicvirus．SrMV）NSP기인서렬설계특이성인물．건립료침대저량충병독적실시형광정량PCR（re촉time-quantitative，RT-qPCR）검측방법。재차기출상，이용확증산물‰치적불동，건립료가구분ScYLV여SrMV적다중SYBRGreen—I실시형광정량PCR방법．량충병독확증산물적‰치분별위80．8～82．8℃화84．6～86．6℃。검측수거현시：SrMV화ScYLV균유각자적특이성봉치출현，이건강식주무특이성봉치；재단독검측중적검출수평분별가체500화250copy／ixL：량충병독m치적비내변이계수분별위0．03％화0．0270．비간변이계수분별위2．0670화3．1270。종상소술．소건립적다중SYBRGreen—I실시형광정량PCR방법재대이상량충병독적검측중구유교호적특이성、민감성화은정성。
In the present research, two pairs of primers used in the SYBR Green I real time PCR were designed based on the sequences of cp gene of Sugarcane Yellow Leaf Virus （ScYLV） and NSP gene of Sorghum mosaic virus（SrMV） respectively in genebank. On this basis, the multiplex SYBR Green-real time PCR was established successfully by the Tm value disparity of the two amplicons （ScYLV 80.8 -82.8℃; SrMV 84.6-86.6℃ ） which made them easy to be discriminated.. The result of real time PCR showed that the infection of ScYLV, SrMV could be specifically detected whereas healthy samples had no amplification. The sensitivity was 250 copies/p,L for scylv and 500 copies/p~L for SrMV. The reproducibility of intra-assay was that the cv% of Tm was 0.02% for scylv, 0.03% for srmv, the cv% of Tm was 3.12% for scylv, 2.06% for srmv. in inter-assay. In sum, this multiplex SYBR Green-I real time PCR system had qualified specificity, reproducibility and sensitivity.