乙型肝炎病毒核酸定量检测中不确定度的研究

Uncertainties of Measurement in HBV-DNA Quantification by FQ-PCR

目的研究应用实时荧光定量PCR(Fluorescence Quantitative PCR,FQ-PCR)开展乙型肝炎病毒(Hepatitis Bvirus,HBV)核酸定量检测中的测量不确定度。方法通过实时荧光定量PCR进行乙型肝炎病毒核酸定量检测的实验分组设计与数据处理分析,计算分析该检测方法不同来源的测量不确定度值。结果对3组不同水平浓度值样本来源的数据采用两种方法进行统计分析,QDNA的组标准差远远大于LGQDNA的标准差,初始浓度数值经过对数转化后离散程度降低,统计平均值也发生了偏离。LGQDNA为3.465、4.927和6.018的样本,其浓缩过程来源的相对不确定度分别为0.020、0.019和0.009;LGQ无偏估计在3.706、6.750的样本的核酸提取来源相对不确定度分别为0.016和0.009;数据分析来源的LGQ不确定度为0.000～0.008;热循环仪来源的LGQ不确定度为0.002～0.008。结论使用核酸浓度值QDNA与使用其对数值LGQDNA进行数据统计分析的结果存在差异;标本制备过程中的浓缩和DNA提取是实时荧光定量PCR进行HBV核酸定量检测时测量不确定度的重要来源,标本制备环节是影响该项检测的关键因素,包括试剂、方法和操作者的准确操作能力。

Objective To investigate sources and composition of uncertainties accompanying with the HBV-DNA quantitative detection by FQ-PCR technique. Method Through different approaches of experimental design and data processing on hepatitis B virus DNA quantitative detection using FQ-PCR, the uncertainties and its composition were analyzed on the data obtained from hepatitis B virus DNA quantitative detection of clinical serum samples. Result The standard deviations of QDNA far exceeded those of LGQDNA of the data obtained from 3 groups derived from 3 samples with different levels of HBV-DNA concentration. Dispersion degree decreased greatly when QDNA value was converted to LGQDNA value at the initial concentration of DNA. Those with higher standard deviation accompanied with greater departure after logarithm-convertion. As for samples with estimated LGQDNA of 3.465, 4.927 and 6.018, the relative uncertainties from enriching step reached 0.020, 0.019 and 0.009, respectively. Samples with estimated LGQDNA of 3.706 and 6.750, accompanied with relative uncertainties of 0.016 and 0.009, respectively. The LGQ uncertainties generated from data analysis is ranged 0.000-0.008, and that caused by PCR instruments is 0.002 to 0.008. Conclusion Statistic analysis resulted differently between using the DNA concentration QDNA directly and using the corresponding logarithm, LGQDNA. The enriching and DNA extraction in pre-treat step might be the crucial steps caused the major uncertainties of whole measurement. Sample pre-treat method, reagents and operation were important factors in hepatitis B virus DNA quantitative detection since they influenced both effective extraction of nucleic acid and exhaustively elimination of components which may inhibit PCR amplification.