猪圆环病毒3型TaqMan实时荧光定量PCR检测方法的建立

陈如敬; 吴学敏; 陈秋勇; 车勇良; 王隆柏; 严山; 周伦江

doi:10.19303/j.issn.1008-0384.2020.07.007

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

摘要

摘要:
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。

Abstract:
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 the 193 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.

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