Shieh Y S, Shim K S, Lampertico P, Balart L A, Jeffers L J, Thung S N, Regenstein F, Reddy K R, Farr G, Schiff E R
Department of Pathology, Tulane University School of Medicine, New Orleans, Louisiana.
Lab Invest. 1991 Oct;65(4):408-11.
Although sensitive assays for serum antibodies to hepatitis C virus (HCV/anti-HCV) have been developed recently, the relation of anti-HCV to HCV infection of the liver has not been clarified. Therefore, we determined the presence of HCV RNA by the reverse transcription-polymerase chain reaction (PCR) in liver biopsy specimens of 21 patients with chronic liver disease and 5 control patients. RNA was extracted from frozen liver tissues by the guanidinium method, HCV cDNA was synthesized by reverse transcription, and core region and NS3 region sequences were amplified by PCR. The sensitivity and specificity of the reaction was significantly enhanced by double PCR with nested primers followed by Southern blotting with an HCV specific oligomer probe. NS3 region sequences were detected in the liver specimens of 12 out of 15 anti-HCV positive patients. Core region sequences were detected in 9 patients, all of whom were also positive for NS3 region sequences. HCV sequences were not detected in 11 anti-HCV negative patients. In all cases, the integrity of the extracted RNA was demonstrated by successful amplification of albumin mRNA as internal control. Our findings demonstrate the feasibility of the reverse transcription-double PCR method followed by Southern blotting for the detection of HCV sequences in liver tissues. In this system, the detection rate of NS3 region sequences is higher than that of core region sequences. There is a statistically significant correlation between high titer anti-HCV antibodies in serum and NS3 region sequences in liver tissue. However, not all anti-HCV positive patients had HCV positive hepatitis. The reverse transcription-polymerase chain reaction for HCV sequences on liver tissue extracts may reveal valuable information on the diagnosis of HCV infection and the pathogenesis of chronic hepatitis C.
尽管最近已开发出检测丙型肝炎病毒血清抗体（HCV/抗-HCV）的灵敏检测方法，但抗-HCV与肝脏HCV感染之间的关系尚未阐明。因此，我们采用逆转录聚合酶链反应（PCR）检测了21例慢性肝病患者和5例对照患者肝活检标本中HCV RNA的存在情况。通过胍盐法从冷冻肝组织中提取RNA，经逆转录合成HCV cDNA，并用PCR扩增核心区和NS3区序列。采用巢式引物双重PCR，随后用HCV特异性寡聚体探针进行Southern印迹，可显著提高反应的灵敏度和特异性。在15例抗-HCV阳性患者的肝标本中，有12例检测到NS3区序列。9例患者检测到核心区序列，所有这些患者的NS3区序列也呈阳性。11例抗-HCV阴性患者未检测到HCV序列。在所有病例中，通过成功扩增白蛋白mRNA作为内对照，证明了提取RNA的完整性。我们的研究结果证明了逆转录双重PCR法结合Southern印迹检测肝组织中HCV序列的可行性。在该系统中，NS3区序列的检测率高于核心区序列。血清中高滴度抗-HCV抗体与肝组织中NS3区序列之间存在统计学显著相关性。然而，并非所有抗-HCV阳性患者都患有HCV阳性肝炎。对肝组织提取物进行HCV序列的逆转录聚合酶链反应可能会揭示有关HCV感染诊断和慢性丙型肝炎发病机制的有价值信息。
