应用MGB探针检测人丙型肝炎病毒的基因型

目的检测血浆或血清样本中人丙型肝炎病毒（HCV）的基因型,建立了基于MGB探针的实时荧光定量PCR检测体系。方法在HCV基因组型特异性碱基的聚集区域设计荧光定量PCR引物及探针,为保证特征特异性及高分辨率,选择设计MGB探针,然后将PCR扩增的产物先进行基因克隆,而后制备病毒样颗粒做为阳性质控品,从而建立完整的MGB探针法的荧光定量PCR检测体系,最后对该检测体系进行方法学评价。结果成功建立了基于MGB探针的HCV荧光定量PCR的基因分型检测体系。方法学评价显示,该检测体系的灵敏度达100IU/mL；重复性好；特异性良好,以多种血浆病毒的核酸样品为模板进行检测时结果全为阴性。结论本研究建立基于MGB探针的HCV荧光定量PCR的基因分型检测方法,可定性检测血浆或血清样本及血液制品中的人丙型肝炎病毒HCV的基因型及含量,对于该疾病的研究及临床治疗都具有重要的意义。

Genotyping of Hepatitis C Virus Detected by the MGB Probe

Objective To establish the real-time fluorescence quantitative PCR detection system based on MGB probe by detecting the Genotype of HCV in the plasma or serum samples.Methods The fluorescence quantitative PCR primer and probe will be designed in specific base aggregation area of the HCV genome.In order to ensure the characteristics of specificity and high resolution,the MGB probe was chosen.The PCR product underwent the gene cloning,then the virus like particles were prepared as positive control to establish the real-time fluorescence quantitative PCR detection system which was carried out by methodological evaluation.Results The real-time fluorescence quantitative PCR detection system based on MGB probe has been established.The methodological evaluation showed the sensitivity of the detection system was 100 IU/mL.The repeatability and characteristics of specificity were good.The results were negative when many plasma viral nucleic acid samples as templates were detected.Conclusions This study established the real-time fluorescence quantitative PCR detection system based on MGB probe and can detect the Genotype of HCV in the plasma or serum samples,which has important significance for the study of the disease and the clinical therapy.