Furuwatari C, Hidaka E, Akahane M, Ishikawa M, Ueno I, Furihata K, Ogiso Y, Katsuyama T
Central Laboratories, Shinshu University Hospital, Matsumoto.
Rinsho Byori. 1998 Feb;46(2):151-7.
The COBAS Amplicor system is an automated diagnostic PCR system which contains a PCR internal control (P. I. C.) template to monitor the amplification. The applicability of COBAS Amplicor HCV was examined using sera of patients with hepatitis. Furthermore, the effects of possible interfering substance (total protein, triglyceride, hemoglobin, glucose, total bilirubin, heparin, lysis reagent including guanidium) on HCV-RNA detection were investigated. The sensitivity of COBAS Amplicor HCV was equivalent to the manual method of Amplicor HCV, moreover all of the results in 54 clinical samples analyzed on both COBAS Amplicor HCV and Amplicor HCV were in agreement. Detection sensitivity of HCV-RNA decreased in the presence of total bilirubin and heparin. Ten and 25mg/dl of total bilirubin affected HCV-RNA detection but did not affect P. I. C. This result suggested that total bilirubin interfered with the protein denature caused by the lysis reagent. Fifteen U/ml of heparin in the sample completely inhibited amplification both of the HCV-RNA and P. I. C. One U/ml of heparin did not affect amplification, but heparinized blood samples should not be used for the detection of HCV-RNA. To examine the effect of possible carry over contamination on the lysis reagent which contains guanidium, various concentrations of lysis reagent in P. I. C. were tested. RT-PCR was inhibited by 1/500 volume contamination of lysis reagent in specimen diluent. Other substances did not affect the sensitivity. Our results indicate that the carryover contamination of lysis reagent cause more "false negative" results than interfering substances in sera. In conclusion, HCV-RNA detection system containing P. I. C., such as COBAS Amplicor HCV, will become a very useful to differentiate "false negative" and "true negative" result.
COBAS Amplicor系统是一种自动化诊断PCR系统，它包含一个PCR内部对照（P.I.C.）模板来监测扩增情况。使用肝炎患者的血清检测了COBAS Amplicor HCV的适用性。此外，还研究了可能的干扰物质（总蛋白、甘油三酯、血红蛋白、葡萄糖、总胆红素、肝素、包括胍盐的裂解试剂）对HCV-RNA检测的影响。COBAS Amplicor HCV的灵敏度与Amplicor HCV的手工方法相当，而且在COBAS Amplicor HCV和Amplicor HCV上分析的54份临床样本的所有结果均一致。在总胆红素和肝素存在的情况下，HCV-RNA的检测灵敏度降低。10mg/dl和25mg/dl的总胆红素会影响HCV-RNA检测，但不影响P.I.C.。该结果表明总胆红素干扰了裂解试剂引起的蛋白质变性。样本中15U/ml的肝素完全抑制了HCV-RNA和P.I.C.的扩增。1U/ml的肝素不影响扩增，但肝素化血样不应用于检测HCV-RNA。为了检测含有胍盐的裂解试剂可能的残留污染的影响，对P.I.C.中不同浓度的裂解试剂进行了测试。标本稀释液中1/500体积的裂解试剂污染会抑制RT-PCR。其他物质不影响灵敏度。我们的结果表明，裂解试剂的残留污染比血清中的干扰物质导致更多的“假阴性”结果。总之，含有P.I.C.的HCV-RNA检测系统，如COBAS Amplicor HCV，对于区分“假阴性”和“真阴性”结果将非常有用。
