猪圆环病毒Ⅰ型TaqMan荧光定量PCR检测方法的建立  

Development of a taqman-based real-time quantitative PCR assay for detection of porcine circovirus type 1

作　　者：原霖[1,2] 陈亚娜[1] 翟新验[1] 

机构地区：[1]中国动物疫病预防控制中心OIE猪繁殖与呼吸综合征参考实验室,北京102618 [2]中国农业大学动物医学院,北京100193

出　　处：《黑龙江畜牧兽医》2017年第5期168-171,共4页Heilongjiang Animal Science And veterinary Medicine

基　　金：科技部科技基础性工作专项(2013FY113300-6)

摘　　要：为了建立一种用于检测猪圆环病毒Ⅰ型(PCV-1)的Taq Man荧光定量PCR检测方法,试验以PCV-1 ORF1为靶基因,以PCV-1全基因质粒为标准品,同时还对该方法进行了特异性、敏感性、重复性试验以及临床样品检测。结果表明:该方法检测灵敏度可达5.12拷贝/μL,比常规PCR方法高10倍;线性相关系数(R2)为0.997,扩增效率为104.92%,显示出良好的线性关系和扩增效率;重复性试验的变异系数小于2%,与其他病毒无交叉反应;临床样品检测显示,该方法的检测灵敏度高于普通PCR。说明新建立的方法敏感性高、特异性强,可用于PCV-1检测。In the current study, a taqman based real - time quantitative PCR（qPCR） assay was developed for detection of porcine circovirus type 1 （ PCV - 1 ）. The primers and probe were designed according to the conserved nucleotide sequences of ORF1 gene of PCV - 1. The recom-binant plasmid containing the complete PCV - 1 genome sequence was constructed as a standard control. The sensitivity, specificity and repeat- ability of the qPCR were evaluated, respectively. Results indicated that the Limit of detection was 5.12 copies/μL ,which is higher with 10 fold than that of the conventional PCR. The R2 value of the qPCR standard curve was 0. 997; amplification efficiency of the qPCR was 104.92% ; the qPCR had a good linear response and high qpcr amplification efficiency. The coefficient of variation was less than 2 % ; the qPCR was specific for detection of PCV - 1. The results indicated that the developed qPCR assay had a potential use for rapid detection of PCV-1.

关 键 词：猪 猪圆环病毒I型(PCV-1) TAQMAN探针 荧光定量PCR 检测 

分 类 号：S828[农业科学—畜牧学] S852.651[农业科学—畜牧兽医]