基于Pre—NAT全自动核酸提取平台的高敏HBVDNA检测性能评价

Evaluation of a novel fully automated real-time PCR assay for hepatitis B virus DNA quantification

目的评价基于Pre—NAT全自动核酸提取系统的高敏HBV核酸定量试剂的检测性能。方法方法学性能验证。于2014年5月和7至9月分别在北京大学医学部病原生物学系实验室和上海交通大学医学院附属仁济医院检验科进行实验，采用HBVWHO标准品验证正确度与检测下限，A、B、c、D型标准血浆（6个浓度）重复检测验证精密度，高浓度HBV假病毒颗粒梯度稀释验证线性范围，以及144份临床血清评价与罗氏系统的相关性。结果正确度方面，该试剂在5个浓度水平的实测值与标准品理论值偏差均小于±0．35lgIU／ml（-0．17～0．32lgIU／ml）。精密度方面，A、B、C、D型标准血浆的检测重复性（CV）分别为：3．87％～6．32％、0．45％-14．68％、0．16％～8．36％、0．64％-13．01％；中间精密度（CV）分别为：5．67％-9．69％、1．28％～15．68％、0．36％～9．05％、1．69％-13．65％。线性范围评价显示在20～1×10^10IU／ml范围内呈良好的线性关系（r=0．998，P〈0．001）。检测下限验证结果，20IU／ml浓度水平检出率5／5。临床标本检测中，与国际通用Roche试剂符合率为100％（144／144）；104份阳性标本两者数据显著相关（r=0．984，P〈0．0001）。结论该高敏HBVDNA检测的正确度、精密度、检测下限和线性范围均表现良好，并与Roche试剂有良好相关性，有一定临床应用价值。

Objective To evaluate the analytical performance of a novel HBV DNA assay based on automated DNA extraction and real-time fluorescence quantitative PCR. Methods Analytic verification studies. Accuracy and lower limit of detection were assessed by determining a panel of HBV standard plasma of WHO. HBV standard plasma （genotype A, B, C and D） at 6 different concentrations were measured 18 times to evaluate precision and reproducibility. Pseudo-viral particles at high HBV DNA concentration were serially diluted to assess linear range. One hundred and forty-four clinical specimens were quantified for HBV DNA so as to evaluate the correlation between the new test and COBAS system. Results Quantification of HBV standard plasma showed acceptable accuracy, with each deviation between observed and expected values within ± 0. 35 lg IU/ml （ -0. 17 -0. 32 lg IU/ml）. Intra-assay coefficients of variation （CV） for genotype A, B, C and D were 3.87% -6.32%, 0.45% - 14. 68%, 0.16% -8.36% and 0. 64% - 13.01% respectively, and the inter-assay CV were 5.67% -9. 69% , 1.28% - 15.68% , 0. 36% -9. 05% and 1.69% -13.65% , separately. Linearity assessment exhibited an excellent dynamic range of linear quantification from 20 to 1.0 ×10^10IU/ml （ r = 0. 998, P 〈 0. 001 ）. And the satisfactory results obtained at 3 levels of HBV DNA concentration （ 10, 20, 50 IU/ml, respectively） confirmed the claimedlower limit of detection with 5/5 detectable rate at 20 IU/ml. Furthermore, good correspondence was observed between the new HBV DNA assay and the COBAS system with 100% （ 144/144 ） qualitative coincidence and significant correlation based on 104 positive data （ r = 0. 984, P 〈 0. 000 1 ）. Conclusions The novel fully-automated real-time PCR assay displayed good analytical and clinical performance for highly sensitive detection of HBV DNA. It was well suited for monitoring antiviral responses as well as drug resistance according to current clinical practice guidelines for the management of chronic HBV infection.