绵羊肺腺瘤病毒SYBRGreenI实时荧光定量PCR检测方法的建立及应用

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-TVector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SYBRGreenI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SYBR—GreenI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。

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

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.