聚合酶链反应系统检测人巨细胞病毒DNA的性能验证及评价

Performance verification and evaluation of PCR system in detecting HCMV DNA

目的:利用实时荧光定量-聚合酶链式反应(FQ-PCR)系统检测人巨细胞病毒脱氧核糖核酸(HCMV DNA)的性能验证及评价,以满足临床检测需要。方法:收集医院临床样本及第三方提供的标准品,采用实时荧光定量PCR系统检测HCMV DNA试剂的性能及进行评价。依据中国合格评定国家认可委员会发布的《分子诊断检验程序性能验证指南(CNAS-CL039)》《临床化学定量检验程序性能验证指南(CNAS-GL037)》及美国临床和实验室标准化协会(CLSI)扩展(EP)系列文件相关要求对实验室HCMV DNAFQ-PCR系统的检测方法的正确度、测量精密度(含测量批内重复性和测量批间精密度)、线性区间、检出限、抗干扰能力、交叉反应等性能进行验证及评价。结果:荧光定量PCR系统检测HCMV DNA的正确度在允许范围内;低浓度(1.72E+04 copies/ml)、高浓度(2.74E+09 copies/ml)样本批内重复性测量精密度变异系数(CV)分别为3.90%和0.11%,批间测量精密度CV分别为4.05%和0.94%;在4.00E+02~4.00E+09 copies/ml范围内线性关系良好(R^(2)=0.999),符合要求;检测下限可以达到4.00E+02 copies/ml;干扰物(总胆红素、甘油三脂、血红蛋白)样本检测结果与对照样本绝对偏差≤±log0.4;交叉反应病原体[乙型肝炎病毒、丙型肝炎病毒、单纯疱疹病毒、人类疱疹病毒4型、人乳头瘤病毒6/11型]检测结果均为阴性。结论:HCMV DNA荧光定量PCR检测方法的各项性能指标与厂家声明相符,可用于临床检测。

Objective:To conduct performance verification and evaluation of real-time fluorescent quantitativepolymerase chain reaction(FQ-PCR)system in detecting human cytomegalovirus deoxyribonucleic acid(HCMV DNA)so as to satisfy the demands of clinical detection.Methods:The clinical samples of hospital and the standard samples provided by third party were collected,and the real-time FQ-PCR system was adopted to detect the performance of HCMV DNA reagent and conduct evaluation.The accuracy,measurement precision(intra batch repeatability and inter batch precision),linear interval,detection limit,anti-interference ability,cross reaction and other performance of the detection method of HCMV DNA FQ-PCR system were verified and evaluated according to the corresponding requirements of the series of documents of<Guidance on the Performance Verification for Molecular Diagnostic Procedures(CNAS-CL039)>and<Guidance on the Verification of Quantitative Measurement Procedures used in the Clinical Chemistry(CNASGL037)>of China National Accreditation Service for Conformity Assessment,and the EP series files of Clinical and Laboratory Standards Institute(CLSI)of American.Results:The accuracy of FQ-PCR system was within the permissible range in detecting HCMV DNA,and the coefficients of variation(CVs)of precision of intra batch repeatability of low and high concentrations samples(1.72E+04copies/ml and 2.74E+09 copies/ml)were respectively 3.90% and 0.11%,and the inter batch precision CVs of them were respectively 4.05% and 0.94%.The linear relationship was favorable within the range between 4.00E+02 and 4.00E+09 copies/ml(R^(2)=0.999),which met the requirements.The lower limit of detection could reach to 4.00E+02 copies/ml,and the absolute deviations between the detective results of the interferences(total bilirubin,triglycerides and hemoglobin)and the control sample were≤±log0.4.The detective results of cross reactions of pathogens[hepatitis B virus,hepatitis C virus,herpes simplex virus,human herpesvirus type 4(EB virus),human papillomavirus type (6/11)]were negative.Conclusion:The each performance indicator of the FQ-PCR detection method of HCMV DNA is consistent with the statement of the manufacturer,which can be used in clinical detection.