【摘要】 采用PCR方法克隆了猪圆环病毒2型(PCV2)ORF2基因,将构建的重组质粒pMD-ORF2作为PCV2实时荧光定量PCR(FQ-PCR)检测的标准阳性模板,并对该模板进行了酶切及测序鉴定。结果显示,此模板特异性强,稳定性好,-20℃保存20 d无显著变化;对起始浓度为1.0×106、1.0×105、1.0×104拷贝/μL的pMD-ORF2标准品的最终实际测得值分别为1.000×106、0.935×105和0.987×104,其变异系数分别为2.527%、2.067%和2.092%。表明,以此模板进行FQ-PCR具有良好的准确性和重现性。该模板的检测线性范围宽,当稀释度为1.0×109~1.0×105拷贝/μL时,相关系数r=-0.989。更多还原

【Abstract】 An open reading frame（ORF） 2 gene of porcine circovirus（PCV） serotype 2 was amplified by PCR and cloned into pMD-18 T vector to construct a recombinant plasmid pMD-ORF2 to be used as standard positive template for the real-time fluorescent quantitative PCR（FQ-PCR） for detection of PCV2.The pMD-ORF2 was identified by digestion with KpnⅠ+Hind Ⅲ and sequencing.The pMD-ORF2 was high specific and stable for the FQ-PCR.The final values on initiative concentrations of 1.0×10⁶,(1.0×10⁵,)1.0×10⁴ （copies/μL） of DNA were 1.000×10⁶,0.935×10⁵ and 0.987×10⁴ and their coefficients of variation were 2.527%,2.067% and 2.092%,respectively.The results demonstrated that the plasmid for FQ-PCR was favourable accurate and repeatable,with wide linear range.The correlation coefficient was-0.989 when dilution was among 1.0×10⁹ to 1.0×10⁵ copies/μL.更多还原