【摘要】 采用PCR方法克隆了猪圆环病毒1型(PCV1)ORF2基因,构建了重组质粒pMD-ORF2并将其作为标准阳性模板。通过对反应条件进行优化,以定量的10倍系列稀释的质粒pMD-ORF2为标准品进行荧光定量PCR扩增,建立了检测PCV1的荧光定量PCR方法。结果显示,该方法的检测灵敏度可达1.0×101拷贝/μL;对起始浓度为1.0×107、1.0×106、1.0×105拷贝/μL标准品的最终实际测得值分别为1.001×107、0.869×106和0.988×105拷贝/μL,变异系数分别为4.758%、1.865%和3.836%。表明,此方法具有良好的准确性和重现性。更多还原

【Abstract】 An open reading frame(ORF) 2 gene of porcine circovirus(PCV) serotype 1 was amplified by PCR and the amplicon was cloned into pMD18-T vector.The recombinant plasmid pMD-ORF2 was constructed and used as a standard positive template.A real-time fluorescent quantitative PCR(FQ-PCR) assay was carried out at quantitative concentrations of serial 10 fold dilutions of pMD-ORF2 DNA by optimizing circulation parameters.The sensitivity of this method was 1.0×101copies/μL.The standardized pMD-ORF2 DNAs at begining concentrations of 1.0×107,1.0×106 and 1.0×105copies/μL were detected finally to be 1.001×107,0.869×106and 0.988×105copies/μL in concentration and 4.758%,1.865%and 3.836% in variation coefficient,respectively.The results demonstrated that the FQ-PCR was accurate and reproducible.更多还原