Miyachi Hayato, Masukawa Atsuko, Asai Satomi, Miura Toshiaki, Tamatsukuri Shigeru, Hirose Toru, Ando Yasuhiko
Department of Laboratory Medicine, Tokai University School of Medicine,Tokyo, Japan.
J Clin Microbiol. 2003 Feb;41(2):572-5. doi: 10.1128/JCM.41.2.572-575.2003.
A commercially available automated specimen preparation instrument for specific probe capture and paramagnetic separation has been developed (AmpliCap/GT-12; Roche Molecular Systems). We evaluated assay performance of the AmpliCap/GT-12 in the quantitative assay for hepatitis C virus (HCV) RNA with the AMPLICOR HCV MONITOR Test (version 2.0). Assay linearity using serial dilutions from a serum panel was observed in the range of 500 to 850000 IU/ml, with a slightly compromised slope in the higher viral titers. The overall within-run and between-run reproducibility of the entire detection process for 3 and 5 log(10) (IU/ml) of HCV RNA in samples had a standard deviation of <0.2, which was comparable to a manual method based on organic extraction and isopropanol precipitation (Roche Molecular Systems). Comparison of the test results with those obtained by the manual method showed a good correlation (R(2) = 0.972, n = 86). Using heparin (3, 6.5, and 13 U/ml), dextran sulfate (0.1, 1, and 5 mM), hemoglobin (1.13, 2.25, and 4.5 g/liter), conjugated or unconjugated bilirubin (7.5, 15, and 30 mg/dl), and ATP (1.25, 2.5, and 5.0 mM) as known inhibitors, inhibition was only detected at a dextran sulfate concentration of 1 mM with the manual method but not with the AmpliCap/GT-12 extraction. In summary, the AmpliCap/GT-12 system was shown to permit a stable extraction process and accurate results for the quantitative assay of HCV RNA, successfully eliminating the inhibitory effect of dextran sulfate. This automated extraction system provides reliable and reproducible test results and saves labor; thus, it is suitable for routine diagnostic PCR.
已开发出一种用于特定探针捕获和顺磁分离的市售自动化样本制备仪器（AmpliCap/GT-12；罗氏分子系统公司）。我们使用AMPLICOR HCV MONITOR检测法（2.0版）评估了AmpliCap/GT-12在丙型肝炎病毒（HCV）RNA定量检测中的分析性能。使用血清样本系列稀释液观察到分析线性范围为500至850000 IU/ml，在较高病毒滴度时斜率略有下降。样本中3和5 log(10)（IU/ml）的HCV RNA整个检测过程的批内和批间总体重复性标准差<0.2，这与基于有机萃取和异丙醇沉淀的手工方法（罗氏分子系统公司）相当。将检测结果与手工方法获得的结果进行比较，显示出良好的相关性（R(2)=0.972，n=86）。使用肝素（3、6.5和13 U/ml）、硫酸葡聚糖（0.1、1和5 mM）、血红蛋白（1.13、2.25和4.5 g/升）、结合或未结合胆红素（7.5、15和30 mg/dl）以及ATP（1.25、2.5和5.0 mM）作为已知抑制剂，手工方法仅在硫酸葡聚糖浓度为1 mM时检测到抑制作用，而AmpliCap/GT-12萃取法未检测到抑制作用。总之，AmpliCap/GT-12系统被证明能够实现稳定的萃取过程以及HCV RNA定量检测的准确结果，成功消除了硫酸葡聚糖的抑制作用。这种自动化萃取系统提供可靠且可重复的检测结果并节省人力；因此，它适用于常规诊断性PCR。
