SYBR GreenⅠ实时荧光定量PCR检测猪细小病毒方法的建立及初步应用  

Development and application of SYBR-Green Ⅰ real-time quantitative PCR technique for detecting porcine parvovirus virus

作　　者：沈志强[1,2] 王金良[1] 郭显坡 王晓虎 汪明[3] 赵德明[3] 

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 [3]中国农业大学动物医学院,北京100094

出　　处：《中国兽医学报》2011年第1期11-15,共5页Chinese Journal of Veterinary Science

摘　　要：根据GenBank登录的猪细小病毒株NADL-2(NC001718)的VP2基因序列设计1对特异性引物,经PCR扩增出431bp的目的片段。回收产物与pMD18-T vector连接后转化到基因工程菌DH5α中,提取重组质粒,经PCR及测序鉴定后,作为标准模板建立SYBR GreenⅠ荧光定量PCR标准曲线,并进行敏感性试验、特异性试验和重复性试验。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,R2=0.997 6,产物Tm为82.3～82.9℃,检测灵敏度为72.1拷贝/μL,特异性和重复性良好。本试验所建立的检测PPV VP2基因的SYBR GreenⅠ实时定量PCR方法,为该病毒的致病机制研究、临床早期诊断及定量分析PPV感染程度奠定了基础。According to the porcine parvovirus virus strain NADL-2（NC001718） VP2 gene sequence available in GenBank,a pair of specificity which can amplify about 431bp fragment was designed.The purified PCR products were inserted into pMD18-T vector,and then transformed to E.coli DH5α.After PCR identifying and sequencing,recombinant plasmid was extracted as a positive template to establish SYBR-GreenⅠfluorescence quantitative PCR standard curve.The method of sensitivity,reproducibility and specificity were determined.The results indicated that standard curve established by recombinant plasmid showed a good linear relationship between threshold cycle and template concentration,R2=0.997 6,the Tm was between 82.3-82.9℃ and the sensitive degree is 72.1 copies per μL,and the quantitative PCR was high reproducibility and more specific than that of traditional PCR.A SYBR Green Ⅰ fluorescent quantitative PCR assay for detecting VP2 gene of PPV was developed and provided a basis of the early,rapid detection and quantitative analyzation for the infect degree of PEDV.

关 键 词：猪细小病毒 实时荧光定量PCR 检测 

分 类 号：S852.65[农业科学—基础兽医学]