Gerken G, Pontisso P, Roggendorf M, Grazia Rumi M, Simmonds P, Trepo C, Zeuzem S, Colucci G
Medizinische Klinik und Poliklinic, University of Mainz, Germany.
J Hepatol. 1996 Jan;24(1):33-7. doi: 10.1016/s0168-8278(96)80183-8.
BACKGROUND/AIMS: In the past few years the detection of HCV-RNA by polymerase chain reaction has become a well-established diagnostic tool for patients with chronic hepatitis C. However, the lack of reproducible results between laboratories and the relatively high proportion of false-positive results, has indicated the need for a standardized and reliable polymerase chain reaction assay. In the present study we have analyzed the performance of a commercial, HCV-RNA polymerase chain reaction assay based on a single, combined reverse transcription and amplification reaction and on the use of Uracil-N-glycosilase to prevent carry-over contamination (Amplicor HCV, Roche Molecular Systems).
In this assay the amplification products are detected in microwell plates using biotinylated primer and the HRP avidin colorimetric system. Serum samples collected from 446 patients, including 181 with chronic active hepatitis C, 50 with autoimmune chronic hepatitis, 117 in hemodialysis, 30 asymptomatic carriers of anti-HCV and 68 with indeterminate serology (RIBA indeterminate results), as well as from 121 controls were tested with the commercial, single-step assay and with nested polymerase chain reaction. Both techniques use primers located within the 5' non-coding region of the HCV genome.
In all cases a good concordance was observed between the commercial, single-step assay and nested polymerase chain reaction which, for patients with chronic active hepatitis, showed a sensitivity and specificity of 100% and 99.3% for the former and of 98.8% and 100% for the latter, when compared to clinical diagnosis taken as the gold standard. Most of the 11 discordant samples were seen in the group of RIBA-indeterminate cases and in patients with chronic active hepatitis C. Further analysis of these cases, based on repeat testing and clinical data showed that 64% and 36% of the discrepancies were due, respectively, to nested polymerase chain reaction and Amplicor inconsistent reactions. In hemodialyzed patients, patients with autoimmune hepatitis and asymptomatic carriers of anti-HCV, both assays produced results which were consistent with the clinical diagnosis. In the former group, polymerase chain reaction was able to identify the presence of active viral replication in some antibody negative samples.
Taken together, these results indicate that the commercial, single-step polymerase chain reaction assay has the same clinical sensitivity and specificity as nested polymerase chain reaction and that, because of its simplified procedures and fast turn-around time, it may be a valuable test for routine diagnostic applications.
背景/目的：在过去几年中，通过聚合酶链反应检测丙型肝炎病毒核糖核酸（HCV-RNA）已成为慢性丙型肝炎患者一种成熟的诊断工具。然而，各实验室之间缺乏可重复的结果以及相对较高的假阳性结果比例，表明需要一种标准化且可靠的聚合酶链反应检测方法。在本研究中，我们分析了一种基于单一、联合逆转录和扩增反应以及使用尿嘧啶-N-糖苷酶防止污染（Amplicor HCV，罗氏分子系统公司）的商用HCV-RNA聚合酶链反应检测方法的性能。
在该检测方法中，使用生物素化引物和辣根过氧化物酶抗生物素蛋白比色系统在微孔板中检测扩增产物。从446例患者中采集血清样本进行检测，这些患者包括181例慢性活动性丙型肝炎患者、50例自身免疫性慢性肝炎患者、117例血液透析患者、30例抗HCV无症状携带者以及68例血清学结果不确定（重组免疫印迹法结果不确定）的患者，同时还包括121例对照，采用商用单步检测方法和巢式聚合酶链反应进行检测。两种技术均使用位于HCV基因组5'非编码区内的引物。
在所有病例中，商用单步检测方法与巢式聚合酶链反应之间观察到良好的一致性。以临床诊断作为金标准，对于慢性活动性肝炎患者，前者的敏感性和特异性分别为100%和99.3%，后者为98.8%和100%。11例不一致的样本大多出现在重组免疫印迹法结果不确定的病例组和慢性活动性丙型肝炎患者中。基于重复检测和临床数据对这些病例进行进一步分析表明，分别有64%和36%的差异是由于巢式聚合酶链反应和Amplicor检测反应不一致所致。在血液透析患者、自身免疫性肝炎患者和抗HCV无症状携带者中，两种检测方法的结果均与临床诊断一致。在前者组中，聚合酶链反应能够在一些抗体阴性样本中识别出活跃的病毒复制。
总体而言，这些结果表明商用单步聚合酶链反应检测方法与巢式聚合酶链反应具有相同的临床敏感性和特异性，并且由于其操作程序简化和周转时间快，它可能是常规诊断应用中有价值的检测方法。
