中东呼吸综合征冠状病毒实时荧光定量PCR检测方法的建立

目的建立一种快速、敏感、特异的实时荧光定量PCR(Real-time PCR)方法,用于中东呼吸综合征冠状病毒(MERS-CoV)的检测。方法根据中东呼吸综合征冠状病毒S蛋白基因的保守序列设计并合成一对引物及一条特异性TaqMan探针。通过条件优化,以10倍系列稀释重组质粒为标准品,进行Real-time PCR扩增,绘制标准曲线,并进行重复性、准确性、特异性及敏感性检测。结果建立的Real-time PCR方法检测中东呼吸综合征冠状病毒所绘制标准曲线的相关系数0.99,灵敏度为1.00×101拷贝,高于常规PCR方法(1.00×102拷贝);用该方法检测中东呼吸综合征冠状病毒基因为阳性,其他6种对照呼吸道病原体及冠状病毒基因检测均呈阴性;批内、批间重复试验的变异系数均1%。结论建立的中东呼吸综合征冠状病毒Real-time PCR检测方法灵敏、特异、重复性好,可用于中东呼吸综合征冠状病毒感染的快速诊断和流行病学调查。

Development of a real-time PCR assay to detect Middle East respiratory syndrome coronavirus

Objective To establish a rapid,sensitive,and specific assay to detect Middle East respiratory syndrome coronavirus（MERS-CoV）using real-time fluorescence quantitative PCR（real-time PCR）. Methods A pair of primers and a TaqMan probe were designed in accordance with the conserved sequence of MERS-CoV spike protein gene.Realtime PCR was carried out using optimized parameters and positive DNA prepared by serial（10-fold）dilution of a recombinant plasmid and plotting a standard curve.In addition,the reproducibility,accuracy,specificity,and sensitivity of the assay were tested. Results Real-time PCR results and the MERS-CoV standard curve had a correlation coefficient greater than 0.99;the assay was sensitive enough to detect as few as 1.00×101 copies/reaction,which was more sensitive than conventional PCR（1.00×102 copies/reaction）.Positive signals were observed in MERS-CoV recombinant plasmid samples while no signals were observed when six control respiratory disease pathogens and coronavirus pathogens were detected using this assay.The intra-batch and inter-batch coefficients of variation for the same DNA sample were both below 1% Conclusion A real-time PCR detection assay was successfully created.This assay is highly sensitive,specific,and stable,so it lays the foundation for rapid diagnosis and epidemiological study of MERS-CoV infection.