HBV DNA超敏定量检测试剂盒的性能验证

Performance verification of a hepatitis B virus DNA ultra-sensitive quantification kit

目的:验证天隆HBV DNA超敏定量检测试剂盒在cobas?Z480全自动荧光定量PCR分析仪上的检测性能,以评估其是否满足临床应用要求。方法:参考CNAS-GL039《分子诊断检验程序性能验证指南》和CNAS-CL02-A009《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》等相关文件,应用临床血清样本和HBV DNA国家标准物质以及WHO国际标准物质,对精密度、正确度、线性范围、定量限、检出限、抗干扰能力、交叉污染、试剂批间差异、核酸室温放置时间等性能进行验证。结果:该系统检测102、103、106 IU/mL混合血清样本的重复性精密度分别为2.60%、2.43%、0.91%,中间精密度分别为4.41%、3.36%、2.48%;HBV DNA国家标准物质GBW(E)090586(200 IU/mL)、GBW(E)090137(1.41×10^(3) IU/mL)和GBW(E)090139(4.60×10^(6) IU/mL)的实测值与靶值差异均在±0.4 Log10IU/mL范围内;该系统在2.32×10^(1)~2.32×10^(9)IU/mL范围内呈线性(线性回归方程为Y=0.993 2X+0.161 2,R2=0.998 9);该系统的定量限为30 IU/mL,检出限为10 IU/mL;干扰物质总胆红素(Tb)浓度≤512μmol/L、血红蛋白(Hb)浓度≤10 g/L、甘油三酯(TG)浓度≤18 mmol/L时,均对检测结果没有明显影响(偏倚<±7.5%);交叉防污染实验中阳性标本检出率为100%,阴性标本检出率为0,全部阴性样本未受污染;不同批次试剂间检测结果差异符合要求(偏倚<±7.5%);磁珠法提取的核酸室温放置2 h,再进行加样扩增,对检测结果没有明显影响(偏倚<±7.5%)。结论:天隆HBV DNA超敏定量检测试剂盒在全自动荧光定量PCR仪cobas?Z480上的检测性能基本符合临床应用要求。

Objective: To verify the detection performance of Tianlong HBV nucleic acid ultra-sensitive quantification kit on cobas? Z480 automated quantitative PCR thermocycler and assess whether it meets clinical application requirements. Methods: According to CNAS-GL039 "Guidance on the Performance Verification for Molecular Diagnostic Procedures" and CNAS-CL02-A009 "Guidance on the Application of Accreditation Criteria for the Medical Laboratory Quality and Competence in the Field of Molecular Diagnostics", clinical serum samples, HBV DNA national serum standard materials and WHO international standard were used to verify the precision, accuracy, linear range, quantitative limit, detection limit, anti-interference ability, cross-contamination, reagent batch difference and nucleic acid room temperature placement time. Results: The repeatability precision of the system at 102, 103and 106IU/mL was 2.60%, 2.43%, and 0.91%, respectively, and the intermediate precision was 4.41%, 3.36%, and 2.48%, respectively. The differences between the expected and tested concentrations of the HBV DNA national standards including GBW(E) 090586(200 IU/mL), GBW(E) 090137(1.41×10^(3)IU/mL), and GBW(E) 090139(4.60×10^(6)IU/mL) were all within 0.4 Log10IU/mL. The linearity range was 2.32×10^(1)-2.32×10^(9)IU/mL, and the linear regression equation was Y=0.9932 X+0.161 2, R^(2)=0.998 9. The detection limit of the system was 10 IU/mL, and its limit of quantification was 30 IU/mL. When the concentration of hemoglobin(Hb) was less than 10 g/L, total bilirubin(Tb) was less than 512 μmol/L, and triglyceride(TG) was less than 18 mmol/L, there was no effect on the detection results(bias<±7.5%). There was no cross-contamination during the"checkerboard"test. The detection differences between different batches of the kits met the requirements(bias<±7.5%). When the nucleic acid exacted by the magnetic bead method was placed at room temperature for 2 hours, there was no effect on the detection results(bias<±7.5%). Conclusion: The detection performance of the Tianlong HBV nucleic acid quantification kit on the cobas? Z480 automated quantitative PCR thermocycler basically can meet clinical application requirements.