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 as2 say 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 infected2porcine serum, then cloned into pMD218 T vector.The recombinant plasmid was sequenced and analyzed by BLAST, and the SYBR Green I fluorescent quantitative PCR assaywas established based on positive plas2 mid template. The sensitivity and specificity of the assay were performed.The recombinant plasmid was confirmed by se2 quencing and the fragment belonged to P1. The P1 real2time PCR assay had a dynamic range of detection between 101 and 108 copies/ LL,with a sensitivity of 10 copies/ LL, while the detecting results of porcine pseudorabies virus, porcine par2 vovirus 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.
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