乙型肝炎病毒核酸两种PCR定量方法的比较

Comparison of two quantitative PCR assaies for the determination of serum HBV DNA

目的 选用PCR 杂交酶免疫法和荧光能量转换PCR法 ,对它们在乙型肝炎病毒 (HBV)DNA定量分析中的灵敏度、特异性及稳定性进行比较研究 ,以便寻求一种较为精确的定量PCR法。方法 分别用上述两种方法测定 2 0份正常人和 30份慢性乙型肝炎病人血清中HBVDNA的含量 ,在检测过程中 ,用罗氏Amplicor法作为阳性参照 ,衡量实验体系的稳定性。结果 1.灵敏度 :两种方法的灵敏度很接近 ,PCR 杂交酶免疫法为 4× 10 3 拷贝 /ml,而荧光能量转换PCR法为 3× 10 3 拷贝 /ml。 2 .特异性 :所有HBsAg、HBeAg、抗 HBs和抗 HBc均为阴性的血清 ,用两种PCR方法比较 ,结果相同 ,血清中HBVDNA均为阴性。 3 .稳定性 :重复实验结果表明 ,PCR 杂交酶免疫法优于荧光能量转换PCR法 ,它们的变异系数分别为 31.33 %和 5 2 .0 6 % ,两种PCR方法均显示出良好的线性关系。结论 PCR 杂交酶免疫法是一种较为精确的定量PCR方法 ,可用于检测病毒DNA的复制情况 ,并对评价药物的疗效和疾病的转归 。

Objective To compare with the accuracy sensitiveness of PCR hybridized enzyme linked immune assay and fluorescence amplisensor energy transfer assay, for the detection of serum HBV DNA levels. Methods Serum samples from 20 normal person and 30 chronic hepatitis B patients were quantitated for HBV DNA by PCR hybridized enzyme linked immune assay and amplisensor energy transfer assay. Amplicor assay is used as standard control. Results 1. Sensitivity: sensitivity of two quantitative PCR assaies were 4×10 3 and 3×10 3 copies/ml. 2. Specificity: the results are identical in serun samples correspond to serum of HBsAg、HBeAg、Anti HBs and Anti HBc(-). Serum HBV DNA did not be detected in normal controls. 3. Reproducibility: the average variant coefficient of PCR hybridized enzyme linked immune assay is 31.33%, and amplisensor energy transfer essay is 52.06% by duplicate tests. 4. Regression: The results of tests showsed that two qualtitative PCR assaies display linear relationship. Conclusion The two PCR based quantitation assaies were useful method for determination of serum HBV DNA levels.