绵羊肺腺瘤病毒SYBR GreenⅠ实时荧光定量PCR检测方法的建立及应用  

Development of SYBR-GreenI real-time quantitative RT-PCR assay for detection of Jaagsiekte sheep retrovirus

作　　者：王金良[1] 于新友 沈志强[1,2] 孙翠平 唐娜[1] 

机构地区：[1]山东省滨州畜牧兽医研究院,山东滨州256600 [2]山东绿都生物科技有限公司,山东滨州256600

出　　处：《中国兽医学报》2013年第11期1657-1661,1678,共6页Chinese Journal of Veterinary Science

基　　金：山东省现代农业产业技术体系羊产业创新团队项目(SDNY201226)

摘　　要：根据GenBank登录的绵羊肺腺瘤病毒gag基因序列设计1对特异性引物,经RT-PCR扩增出了276bp的片段。产物经回收与pMD18-T Vector连接后转化到基因工程菌DH5a中,提取重组质粒,经PCR及测序鉴定后,作为阳性模板建立SYBR GreenⅠ荧光定量PCR标准曲线,并做敏感性试验、特异性试验、重复性试验和临床检测应用。结果表明,标准曲线循环阈值与模板浓度呈良好的线性关系,产物Tm在82～82.5℃之间,灵敏度为2.22个拷贝/μL,特异性和重复性较好,较常规RT-PCR方法提前1h出检测结果。本试验建立了检测JSRV的SYBRGreenⅠ荧光定量PCR方法,为该病的早期快速诊断,并定量分析JSRV感染程度奠定了基础。According to the Jaagsiekte sheep retrovirus strain gag gene sequence available in Gen- Bank,a pair of specificity which can amplify about 276 bp fragment was designed. The purified RT-PCR products were inserted into pMD18-T vector,and then transformed to E. coli DHSa. Af- ter PCR identifying and sequencing, recombinant plasmid was extracted as a positive template to establish SYBR Green I fluorescence quantitative RT-PCR standard curve. Sensitivity assay,re- producibility of the assay, specificity assay and clinical application were determined. The results indicated that standard curve established by recombinant plasmid showed a good linear relation- ship between threshold cycle and template concentration,the TM was between 82-82.5℃ and the sensitive degree is 2. 22copies/μL, and the quantitative RT-PCR was higher reproducibility and specificity than traditional RT-PCR,and shorten 1 h for test time compara ted with conventional RT-PCR methods. A SYBR Green I fluorescent quantitative RT-PCR assay for detecting JSRV developed the basis of the early and rapid detection and quantitatively analysis the infect degree of JSRV.

关 键 词：绵羊肺腺瘤病毒 SYBR Green I 实时荧光定量PCR 标准曲线 

分 类 号：S852.65[农业科学—基础兽医学]