人冠状病毒HCoV-HKU1和HCoV-NL63双重实时荧光RT-PCR检测方法的研究

    刘胜牙，朱玉兰，张树平，谢聪贤，甄胜西，李微

　　深圳国际旅行卫生保健中心，广东 深圳 518033

　　摘要：目的 建立一种快速、灵敏、高效的人冠状病毒HKU1（HCoV-HKU1）和NL63（HCoV-NL63）双重实时荧光RT-PCR检测方法。方法 根据GENBANK数据库中HCoV-HKU1和HCoV-NL63的基因序列，利用BIOEDIT5.0软件进行序列比对分析，选择基因组的保守序列，并借助引物设计生物信息学软件，设计筛选出一套针对HCoV-HKU1和HCoV-NL63双重实时荧光PCR扩增的引物。从特异性、最低检测限、重复性等方面进行评估，建立了HCoV-HKU1和HCoV-NL63双重实时荧光PCR检测方法。结果 分别以HCoV-HKU1和HCoV-NL63质粒为模板，HCoV-HKU1和HCoV-NL63双重实时荧光PCR方法的最低检测量分别为4.96×102copies/ml和4.96×102copies/ml。该方法针对其他病原微生物及人类基因组无反应信号，特异性好。4个梯度稀释质粒样本的4次重复检测Ct值变异系数均<5%，重复性好。结论 HCoV-HKU1和HCoV-NL63双重实时荧光RT-PCR方法准确性好、灵敏度高、稳定性好，在临床鉴别诊断和口岸冠状病毒的监测中具有很好的应用前景。

　　关键词：人冠状病毒HKU1；人冠状病毒NL63；双重实时荧光PCR；检测；研究

　　中图分类号：R446.6   文献标识码：B

　　Study of multiplex real-time RT-PCR method for the detection ofHCoV-HKU1 and HCoV-NL63

　　LIU Sheng-ya， ZHU Yu-lan， ZHANG Shu-ping， XIE Cong-xian， ZHENSheng-xi， LI Wei

　　Shenzhen International Travel Health Care Center， Shenzhen，Guangdong 518033， China

　　Abstract： Objective  To establish a rapid， sensitive，efficient method for the detection of HCoV-HKU1 and HCoV-NL63 bymultiplex real-time fluorescence RT-PCR.  Methods Specific primers and probes were designed based on highlyconserved region of HCoV-HKU1 and HCoV-NL63 depending onbioinformatics software． Specificity， minimum detection limits andrepeatability of the method were assessed. Results  Theminimum detection limits of the multiplex real-time RT-PCR methodfor HCoV-HKU1and HCoV-NL63 were 4.96×102 copies/ml and 4.96×102copies/ml. Other pathogenic organisms and human genome in themethod have not shown any response signal， displaying goodspecificity of the method. The four different gradient dilutionplasmid samples were repeated four times， the variationcoefficients of CT value were less than 5%， showing goodrepeatability.  Conclusion  HCoV-HKU1 and HCoV-NL63Multiplex real-time RT-PCR method with good specificity， highsensitivity and good stability， has a good application prospect inclinical differential diagnosis and coronavirus monitoring ofport.

　　Key words： HCoV-HKU1； HCoV-NL63； Multiplex Real-time PCR；Detection； Study

　　《中国国境卫生检疫杂志》2014年3期