圆圈病毒3型TaqMan荧光定量PCR检测方法的建立及应用

A TaqMan RT-qPCR Assay for Gyrovirus 3 Detection

【目的】建立一种检测圆圈病毒3型(Gyrovirus 3,GyV3)的TaqMan荧光定量PCR方法,为GyV3提供快速便捷的检测技术手段。【方法】根据NCBI数据库中GyV3 VP2基因保守区序列,设计一对特异性检测引物和一条标记FAM荧光基团的探针,经过条件优化,建立用于检测GyV3的TaqMan实时荧光定量PCR快速检测方法,并进行特异性、敏感性、重复性以及模拟样品的检测试验。【结果】该方法灵敏性高,最低检测限度为1.585 copies·μL^(−1);特异性强,对其他常见禽病原不发生非特异性反应;重复性好,批内和批间变异系数均小于1%;对鸡肝脏组织DNA加入重组质粒制备的模拟样品同时进行TaqMan荧光定量PCR与SYBR GreenⅠ实时荧光定量PCR检测,均扩增出与预期相符的扩增曲线。【结论】首次建立用于检测GyV3的TaqMan实时荧光定量PCR快速检测方法,具有灵敏性高、特异性强、重复性好等优点,为GyV3的诊断和流行病学调查提供了技术支持。

【Objective】An RT-qPCR method to accurately detect gyrovirus 3(GyV3)was established.【Methods】A pair of specific primers and an FAM-labeled fluorophobe were designed according to the conserved region of GyV3 VP2 in Genbank database.Reaction conditions of a rapid TaqMan RT-qPCR assay were optimized,and specificity,sensitivity,repeatability,and simulation tests conducted to verify the applicability of the methodology.【Results】The assay exhibited a high sensitivity with a minimum detection limit of 1.585 copies·μL^(−1),a specificity free of cross-reactivity with other prevalent avian pathogens,and a reproducibility with less than 1%coefficients of variation on intra-and inter-batch determinations.On a simulated sample with an added recombinant plasmid to the chicken liver tissue DNA,the assay positively identified GyV3 with same result as did the SYBR GreenⅠPCR.【Conclusion】The newly developed TaqMan RT-qPCR assay showed high sensitivity,specificity,repeatability,and rapid turn-around time in detecting GyV3.It was considered appropriate for clinical diagnosis and epidemiological investigation on the virus.