应用MGB探针检测人乙型肝炎病毒的基因型

Apply the MGB probe to detect HBV virus genotyping of the people

目的检测血浆或血清样本中人乙型肝炎病毒HBV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HBV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扩增的产物纯化后进行基因克隆,克隆成功后提取的质粒制备为阳性质控品,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HBV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50 IU/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HBV荧光定量PCR的基因分型检测方法,可以很好地定性的检测血浆或血清样本及血液制品中的人乙型肝炎病毒HBV的基因型及含量,对于该疾病的临床治疗具有重要的意义。

Objective A Real-Time PCR assay based on the MGB probe for detecting genotypes of hepatitis B virus existed in plasma or serum samples was built. Methods To ensure the specificity and high resolution, the primer and probe of Real-Time PCR of HBV was designed in the areas which genome type concentrated. To evaluate the methodology for the Real-Time PCR detection system of the MGB probe, we constructed plasmids as the positive quality products to build complete detection system. Results The detection system of HBV genotypcs of Real-Time PCR based on the MGB probe was established successfully. Methodological evaluation showed that the sensitivity was up to 50 IU/mL and repeatability is excellent. The tests of a varieties of plasma virus nucleic acid were negative to reveal high specificity. Conclusion A detection methods based on the MGB probe of HBV gcnotypes of Real-Time PCR was built to detect hepatitis B virus genotype and the content of blood, plasma or serum samples. The system is of great importance for the clinical treatment of HBV virus.