丙型肝炎病毒核酸荧光定量检测法的性能验证

Performance verification analysis of HCV-RNA fluorescence quantitative assay in nucleic acid treatment platform of Molecular Biology Laboratory

目的对基于QIGEN cube全自动核酸提取仪与ABI 7500核酸扩增仪的丙型肝炎病毒(HCV)-RNA定量检测系统进行性能评估。方法参照《临床实验室对商品定量试剂盒》WS/T 420-2013的性能验证方案,采用质量控制(质控)血清与临床标本评价所用商品试剂盒的精密度、正确度、线性范围与检测下限;同时参照《医学实验室质量和能力认可准则在分子诊断领域的应用说明》标准,统计分析实验偏差并与厂家声明的性能指标进行比较。结果精密度验证结果显示,2个浓度水平(level 1、level 2)质控品的批内变异系数(CV_(批内))分别为0.3%、0.9%,批间变异系数(CV_(批间))分别为0.4%、1.11%,均小于厂家声明值(5%)。线性范围试验显示,在1.0×10~2～1.0×10~8 kU/L范围内呈良好线性(R^2=0.999,P<0.001),高于厂家声明区间(2.0×10~1～1.0×10~9 kU/L)。正确度验证结果显示,室间质评中的5个浓度水平质控品的实测值与质控样本理论值间偏倚分别为0.10、0、0、0.26和0.26,均小于0.4对数值(log值)。最低定量检测下限为1.0×10~2 kU/L,高于厂家声明的最低检测限(0.5×10~2 kU/L),但与定量限(1.0×10~2 kU/L)一致。结论本实验室建立的HCV-RNA定量检测系统在精密度、正确度、线性范围与检测下限方面的性能指标均满足卫生行业标准与ISO 15189医学实验室质量认可准则的要求,能为临床提供可靠的检验报告。

Objective To evaluate the performance of the hepatitis C（HCV）-RNA quantitative detection based on the QIGEN cube automatic nucleic acid extraction system and the ABI 7500 nucleic acid amplification system. Methods The precision, accuracy, linear range and lower limit detection of the used commodity kit were evaluated through testing quality controlled serum and clinical specimen by referring to verification of analytical performance of commodity quantitative kits in clinical laboratory WS/T 420-2013; in the mean time, referring to the guidance on the application of accreditation criteria for the medical laboratory quality and competence in the field of molecular diagnosis, we analyzed the test bias statistically and compared with the declaration of manufacturer＇s performance index. Results The results of precision degree verification showed that the intra-batch coefficient of variation（CVintra） of quality controlled material at 2 different concentration levels（level 1, level 2） were 0.3%, 0.9%, and that the inter-batch（CVinter） were 0.4%, 1.11%, demonstrating that they all lower than the statement declared by the manufacturer（5%）. The linear range experiment showed that in this range 1.0×10^2-1.0×10^8 kU/L, the linearity presented excellent result（R^2 = 0.999, P〈0.001）, that was a little higher than the manufacturer＇s claimed value（2.0×10^1-1.0×10^9 kU/L）. The results of accuracy verification showed that in the inter-laboratory quality assessments on the 5 quality controlled material with different concentration levels, the bias values between practical measured values and the theoretical values were 0.10, 0, 0, 0.26 and 0.26 respectively, which were all lowered than 0.4 log value（standard requirements）. The minimum quantitative limit was determined to be 1.0×10^2 kU/L, which was higher than manufacturer＇s claimed minimum detection limit（0.5×10^2 kU/L）, but consistent with manufacturer＇s claimed quantitative limit（1.0×10^2 kU/L）. Conclusions The precision, accuracy, linearity assessment andminimum quantitative limit of performance of the HCV-RNA quantitative detection system established in our laboratory are in accord to the requirements of health industry standard and ISO 15189＇s medical laboratory quality accreditation standard. This detection system can provide reliable test reports for clinical practice.