实时荧光定量PCR检测乙型肝炎病毒核酸载量的测量不确定度构成分析  

Uncertainty in measuring HBV DNA load by quantitative real-time PCR

作　　者：余南 毕晓云 崔进 陈小坚 李汛 

机构地区：[1]广州经济技术开发区医院生物中心实验室,510730

出　　处：《中国医药生物技术》2006年第1期37-41,共5页Chinese Medicinal Biotechnology

基　　金：广东省社会发展领域科技计划项目(63104);广州市医药卫生科技基金资助项目(2006-YB-196)

摘　　要：目的研究实时荧光定量PCR用于乙型肝炎病毒(HBV)核酸载量检测时的测量不确定度构成。方法采用实时荧光定量PCR检测乙型肝炎患者血清标本中HBV核酸载量,收集检测过程中的各类数据,计算以下6种来源的测量不确定度,对该方法的测量不确定度构成进行评估。⑴标本浓缩的不确定度(uenrich);⑵核酸提取的不确定度(uex);⑶扩增反应体系的不确定度(upcr);⑷热循环仪的不确定度(uins);⑸数据处理的不确定度(uana);⑹加样枪的不确定度(upip)。其中热循环仪的不确定度检测样本数为7份,其他各种不确定度检测的样本数为10份。结果核酸浓度为5.610E+07拷贝/ml的标本,其浓缩过程来源的相对不确定度达0.437;核酸提取来源的浓度真值无偏估计在6.585E+03、9.067E+04、7.223E+06拷贝/ml样本的相对不确定度分别为0.249、0.173、0.140;热循环仪和数据分析来源的相对不确定度分别为0.020和不大于0.050。结论标本制备过程中的浓缩和DNA提取步骤所带来的不确定度是实时荧光定量PCR检测HBV核酸载量测量不确定度的主要分量,因此标本制备处理的方法与标准操作程序能否有效地提取核酸并去除PCR反应的抑制物对于该项检测十分关键;起始模板浓度与测量不确定度相关,低浓度的样本显示出更大的相对不确定度。Objective To investigate the uncertainty of quantitative real-time PCR in measuring HBV DNA load. Methods The uncertainty and its composition of quantitative real-time PCR measurement were analyzed by means of processing data mainly from Hepatitis B virus DNA loading test of patients’ serum samples. Six sources of uncertainties were caculated including: (1)Uncertainty from sample enrichment step (uenrich); (2)Uncertainty from DNA extraction step (uex); (3)Uncertainty from PCR mix (uper); (4)Uncertainty from thermocycle instrument (uins); (5)Uncertainty from data analysis step (uana); (6)Uncertainty from pipet (upip). Seven samples were caculated in group (4), while ten samples in each group of the rest. Results In samples of 5.610×107 copies/ml, the relative Uex caused by enrichment of DNA reached 0.437. In those with an estimated HBV-DNA level of 6.585×103 copies/ml, 9.067×104 copies/ml and 7.223×106 copies/ml, the Uex was 0.249, 0.173, and 0.140, respectively. The Uins and Uana were 0.020 and ≤0.050, respectively. Conclusions The enrichment and extraction of DNA may be the main cause of uncertainty in the whole measurement. Therefore, the pre-treatment of samples and its standard operating protocol are critical points in extracting DNA and diminishing PCR inhibitors. In addition, the initial DNA concentration is related to the uncertainty, and the samples of lower DNA concentration showed higher relative uncertainty.

关 键 词：聚合酶链反应 肝炎病毒 乙型 核酸类 不确定度 

分 类 号：R450[医药卫生—治疗学]