猪圆环病毒3型TaqMan实时荧光定量PCR检测方法的建立

A TaqMan RT-PCR Method for Detecting Porcine Circovirus 3

【目的】建立检测猪圆环病毒3型(Porcine circovirus 3,PCV3)感染的TaqMan实时荧光定量PCR方法。【方法】通过分析明确PCV3复制相关蛋白Rep基因特征,设计针对Rep基因的特异性引物和探针,经条件优化后建立检测PCV3感染的TaqMan实时荧光定量PCR方法。【结果】建立的TaqMan实时荧光定量PCR方法敏感性好,最低检测限为42.2拷贝·μL-1;特异性强,对常见猪群传染病均无交叉反应;重复性好,组内变异系数和组间变异系数均在1.48%以内。对福建省2014年至2018年保存的193份组织样品进行检测发现,PCV3在福建省猪群中存在较高的阳性感染率(65.80%),且和PCV2混合感染率较高(52.85%)。【结论】本方法的建立为开展Rep基因在PCV3复制和感染过程中的作用机制提供检测方法。

【Objective】A TaqMan RT-PCR method was established for detecting porcine circovirus 3(PCV3)infection in swine.【Method】Specific primers and probe were designed by Oligo 7 software targeting the Rep gene after genetic comparison.【Result】The established TaqMan RT-PCR method could detect 42.2 copies·uL-1 with no positive signal on common porcine infectious diseases indicating high specificity of the methodology.On the constructed positive plasmids,the coefficients of variation for the intra-and inter-assays were less than 1.48%showing a high detection reproducibility.From the193 clinical specimens collected in Fujian from 2014 to 2018,the newly developed method showed a high prevalence of PCV3 at 65.80%and high co-infection with PCV2 at 52.85%.【Conclusion】The established TaqMan RT-PCR method was made available for studying the function of Rep in PCV3.