Hu K Q, Yu C H, Vierling J M
Department of Medicine, University of California, Los Angeles.
J Clin Invest. 1992 Jun;89(6):2040-5. doi: 10.1172/JCI115815.
Diagnostic testing for hepatitis C virus (HCV) infection currently is based on the presence of anti-HCV antibodies or a positive HCV RNA polymerase chain reaction (PCR) test. Although HCV RNA PCR is a sensitive and specific technique, widespread application is limited. Moreover, HCV RNA PCR is subject to false-positive reactions through contamination and is inherently difficult to standardize and quantitate. To overcome limitations of HCV RNA PCR, we produced both cDNA and riboprobes from a 241 nucleotide sequence of the 5' untranslated region of the HCV genome for slot hybridization. Hybridization was absent using normal human serum, horse serum, or hepatic cellular RNA from noninfected liver. Hybridization occurred predominantly with positive-stranded HCV RNA and was abolished by pretreatment with RNase A. Slot hybridization was performed on serum samples from 60 patients with chronic HCV infection and a positive HCV RNA PCR and 20 patients with liver diseases unrelated to HCV who had a negative HCV RNA PCR. Slot hybridization with cDNA and riboprobes showed concordance with HCV RNA PCR of 95 and 98.3%, respectively. There were no false-positive reactions in controls. The sensitivity of riboprobe hybridization was comparable to that of one stage HCV RNA PCR using 5' untranslated region primers. Riboprobe hybridization with the HCV H strain standard was positive in the dilution corresponding to 10(-6) chimpanzee infectious doses50/ml. The density of the hybridization signals correlated significantly with the mass of an RNA standard extracted from the liver of a patient with HCV infection. The relative quantities of HCV RNA in the sera of selected patients varied and were not correlated with the duration of disease or the histopathological stage. The highest relative quantities were associated with concurrent immunosuppression. We conclude that slot hybridization is a sensitive, specific alternative to HCV RNA PCR that can be directly quantitated using appropriate HCV RNA standards.
目前，丙型肝炎病毒（HCV）感染的诊断检测基于抗HCV抗体的存在或HCV RNA聚合酶链反应（PCR）检测呈阳性。尽管HCV RNA PCR是一种灵敏且特异的技术，但其广泛应用受到限制。此外，HCV RNA PCR会因污染而出现假阳性反应，并且本身难以标准化和定量。为克服HCV RNA PCR的局限性，我们从HCV基因组5'非翻译区的241个核苷酸序列制备了cDNA和核糖探针用于狭缝杂交。使用正常人血清、马血清或未感染肝脏的肝细胞RNA时未出现杂交。杂交主要发生于正链HCV RNA，且经核糖核酸酶A预处理后杂交被消除。对60例慢性HCV感染且HCV RNA PCR呈阳性的患者以及20例HCV RNA PCR呈阴性的非HCV相关性肝病患者的血清样本进行狭缝杂交。与cDNA和核糖探针的狭缝杂交分别显示与HCV RNA PCR的一致性为95%和·98.3%。对照中未出现假阳性反应。核糖探针杂交的灵敏度与使用5'非翻译区引物的一步法HCV RNA PCR相当。与HCV H株标准品的核糖探针杂交在相当于10^(-6)黑猩猩感染剂量50/ml的稀释度下呈阳性。杂交信号的密度与从HCV感染患者肝脏中提取的RNA标准品的量显著相关。所选患者血清中HCV RNA 的相对量各不相同，且与疾病持续时间或组织病理学分期无关。相对量最高的与同时存在的免疫抑制相关。我们得出结论，狭缝杂交是HCV RNA PCR的一种灵敏、特异的替代方法，可使用适当的HCV RNA标准品直接定量。
