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

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RTPCR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4.96×102 copies/ml和4.96×102 copies/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。

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

Objective To establish a rapid, sensitive, efficient method for the detection of HCoV-HKU1 and HCoV- NL63 by multiplex real-time fluorescence RT-PCR. Methods Specific primers and probes were designed based on highly conserved region of HCoV-HKU1 and HCoV-NL63 depending on bioinformatics software. Specificity, minimum detection limits and repeatability of the method were assessed. Results The minimum detection limits of the multiplex real-time RT-PCR method for HCoV-HKUland HCoV-NL63 were 4.96×10^2 copies/ml and 4.96×10^5 copies/ml. Other pathogenic organisms and human genome in the method have not shown any response signal, displaying good specificity of the method. The four different gradient dilution plasmid samples were repeated four times, the variation coefficients of CT value were less than 5%, showing good repeatability. Conclusion HCoV- HKU1 and HCoV-NL63 Multiplex real-time RT-PCR method with good specificity, high sensitivity and good stability, has a good application prospect in clinical differential diagnosis and coronavirus monitoring of port.