应用多重SYBR Green-Ⅰ实时荧光定量PCR检测甘蔗黄叶病毒和高粱花叶病毒

Detection of Sugarcane Yellow Leaf Virus and Sorghum Mosaic Virus by Multiplex SYBR Green-Ⅰ Real Time PCR

基于已报道的甘蔗黄叶病毒(Sugarcane Yellow Leaf Virus,ScYLV)cp基因和高粱花叶病毒(Sorghummosaic virus,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(real time-quantitative,RT-qPCR)检测方法。在此基础上,利用扩增产物Tm值的不同,建立了可区分ScYLV与SrMV的多重SYBR Green-Ⅰ实时荧光定量PCR方法,两种病毒扩增产物的Tm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250 copy/μL;两种病毒Tm值的批内变异系数分别为0.03%和0.02%,批间变异系数分别为2.06%和3.12%。综上所述,所建立的多重SYBR Green-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。

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.