锁核酸捕获TaqMan探针实时聚合酶链反应构建及检测乙型肝炎病毒DNA

Construction of real-time RT-PCR using locked nucleic acid capture TaqMan probes and its detection of hepatitis B virus DNA

目的建立和检测新型乙型肝炎病毒（HBV）DNA检测技术锁核酸（LNA）捕获TaqMan探针实时聚合酶链反应（PCR）。方法2009年8月至2010年3月选取本院20例健康献血者和肝病专科住院的75例经COBAS Amplicor检测HBVDNA阳性慢性乙型肝炎（CHB）患者，分别采集血液样本进行LNA捕获TaqMan探针实时PCR特异性试验。同时对LNA一实时PCR方法的重复性、特异性和线性范围等进行分析。结果其线性范围为10～2．3×10^9IU／ml。将10IU／ml作为检测下限，具有95％可信区间。线性回归分析显示其斜率接近1．0（Rz=0．999）。批内变异值为3．88％～4．36％，批间变异值为8．5％～11．7％。20例健康献血者的阴性对照血清HBV．DNA检测均为阴性，而75例经COBAS Amplicor检测HBVDNA阳性患者的血清样本除1例阴性外，其余都为阳性。结论LNA捕获TaqMan探针实时PCR方法为HBV—DNA检测的一种快捷灵敏、准确度高的新方法，值得临床实验室推广应用。

Objective To establish a novel system of real-time RT-PCR using locked nucleic acid （LNA） capture TaqMan probes for detection of hepatitis B virus （HBV） DNA. Methods Between August 2009 and March 2010, 20 healthy blood donors and 75 patients with COBAS Amplicor-identified HBV DNA- positive chronic hepatitis B （CHB） in our Department of Hepatology received phlebotomy for specific assay with RT-PCR using LNA capture TaqMan probes. Meanwhile, the reproducibility, specificity and range of linearity of LNA RT-PCR were analyzed. Results The outcomes showed good linearity of LNA RT-PCR within the range of 10- 2.3× 10^9 IU/ml. A 95% confidence interval （CI） was identified with 10 IU/ml as the low limit. Linear regression analysis demonstrated a slope of 1.0 （R^2=0.999）, an intra-assay coefficient of variance ranging from 3.88% to 4.36%, and an inter-assay coefficient of variance ranging from 8.5% to 11.7%. The samples of control serum from 20 healthy blood donors were negative for HBV DNA, and all the samples without one from 75 patients were HBV DNA positive as detected by COBAS Amplicor. Conclusion RT-PCR using LNA capture TaqMan probes is a novel and quick test with high sensitivity and accuracy, which warrants its widespread use in clinical laboratory.