SYBR Green Ⅰ荧光定量PCR检测类猪圆环病毒因子P1  

Detection of Porcine Circovirus-like Agent P1 Using SYBR Green Ⅰ Fluorescent Quantitative PCR Assay

作　　者：温立斌[1] 何孔旺[1] 杨汉春[2] 郭容利[1] 周俊明[1] 钟书霖[1] 

机构地区：[1]江苏省农业科学院兽医研究所,农业部动物疫病诊断与免疫重点开放实验室,国家兽用生物制品工程技术研究中心,江苏南京210014 [2]中国农业大学动物医学院,农业部预防兽医学重点开放实验室,北京100094

出　　处：《华北农学报》2009年第4期31-35,共5页Acta Agriculturae Boreali-Sinica

基　　金：国家“973”计划前期研究专项(2007CB116308);江苏省自然科学基金(BK2008351)

摘　　要：该研究旨在建立快速、敏感和特异的检测类猪圆环病毒因子P1的SYBR Green Ⅰ荧光定量PCR法,用于P1的早期诊断。以感染P1的猪血清DNA提取物为模板,采用PCR扩增P1 101 bp的基因片段,将其克隆至pMD18-T载体,重组质粒测序并进行同源性分析;以阳性质粒为模板,建立SYBR Green Ⅰ荧光定量PCR检测方法,并进行敏感性和特异性检测。经测序证实扩增片段属于P1,所建立的SYBR Green Ⅰ荧光定量PCR检测P1的反应在101-108拷贝/μL之间具有良好的线性关系,反应的检出下限为10拷贝/μL,而对猪伪狂犬病病毒、猪细小病毒、猪繁殖与呼吸综合征病毒等的检测为阴性,表明该方法敏感、特异。成功建立了SYBR Green Ⅰ荧光定量PCR检测P1载量的方法,为P1致病机制和机体免疫保护机制的研究提供了技术平台。This experiment was to establish a fast, sensitive, specific SYBR Green I fluorescent quantitative PCR assay for early diagnosis of P1 infection. A 101 bp fragment of P1 gene was amplified by PCR based on genomic DNA of P1 obtained from the P1 infected-porcine serum, then cloned into pMD-18 T vector. The recombinant plasmid was sequenced and analyzed by BLAST, and the SYBR Green I fluorescent quantitative PCR assay was established based on positive plasmid template. The sensitivity and specificity of the assay were performed. The recombinant plasmid was confirmed by sequencing and the fragment belonged to P1. The P1 real-time PCR assay had a dynamic range of detection between 101 and l0s copies/tLL, with a sensitivity of 10 copies/μL, while the detecting results of porcine pseudorabies virus, porcine parvovirus and porcine reproductive and respiratory syndrome virus were negative, which indicated this assay was sensitive and specific for P1 detection. We successfully established a SYBR Green I Fluorescent Quantitative PCR Assay for the quantification of P1 DNA, and it has potential application for investigating the pathogenesis of P1 and the protective me chanism of animal body.

关 键 词：类猪圆环病毒因子 P1 荧光定量PCR 熔解曲线 检测 

分 类 号：S858.285.3[农业科学—临床兽医学] TS207.3[农业科学—兽医学]