Park Y N, Abe K, Li H, Hsuih T, Thung S N, Zhang D Y
Lillian and Henry M. Stratton-Hans Popper Department of Pathology, Mount Sinai Medical Center, New York 10029, USA.
Am J Pathol. 1996 Nov;149(5):1485-91.
Reverse transcription polymerase chain reaction (RT-PCR) has been used to detect hepatitis C virus (HCV) sequences in liver tissue. However, RT-PCR has a variable detection sensitivity, especially on routinely processed formalin-fixed, paraffin-embedded (FFPE) specimens. RNA-RNA and RNA-protein cross-links formed during formalin fixation is the major limiting factor preventing reverse trans criptase from extending the primers. To overcome this problem, we applied the ligation-dependent PCR (LD-PCR) for the detection of HCV RNA in FFPE liver tissue. This method uses two capture probes for RNA isolation and two hemiprobes for the subsequent PCR. Despite cross-links, the capture probes and the hemiprobes are able to form hybrids with HCV RNAs released from the FFPE tissue. The hybrids are isolated through binding of the capture probes to paramagnetic beads. The hemiprobes are then ligated by a T4 DNA ligase to form a full probe that serves as a template for the Taq DNA polymerase. A total of 22 FFPE liver specimens, 21 with hepatocellular carcinoma (HCC) and 1 with biliary cirrhosis secondary to bile duct atresia were selected for this study, of which 13 patients were HCV seropositive and 9 seronegative. HCV RNA was detectable by ID-PCR from all 13 HCV-seropositive HCCs and from 5 of 8 HCV-seronegative HCCs but not from the HCV-seronegative liver with biliary atresia. By contrast, RT-PCR detected HCV sequences in only 5 of the HCV-sero-positive and in 1 of the HCV-seronegative HCCs. To resolve the discordance between the LD-PCR and RT-PCR results, RT-PCR was performed on frozen liver tissue of the discrepant specimens, which confirmed the LD-PCR positive results. In conclusion, LD-PCR is a more sensitive method than RT-PCR for the detection of HCV sequences in routinely processed liver tissues. A high rate of HCV infection (86%) is found in HCC specimens, indicating a previously underestimated role of HCV in HCC pathogenesis.
逆转录聚合酶链反应（RT-PCR）已用于检测肝组织中的丙型肝炎病毒（HCV）序列。然而，RT-PCR的检测灵敏度存在差异，尤其是对常规处理的福尔马林固定、石蜡包埋（FFPE）标本。福尔马林固定过程中形成的RNA-RNA和RNA-蛋白质交联是阻止逆转录酶延伸引物的主要限制因素。为克服这一问题，我们应用连接依赖PCR（LD-PCR）检测FFPE肝组织中的HCV RNA。该方法使用两个捕获探针进行RNA分离，两个半探针用于后续PCR。尽管存在交联，捕获探针和半探针仍能够与从FFPE组织中释放的HCV RNA形成杂交体。通过捕获探针与顺磁珠的结合分离杂交体。然后用T4 DNA连接酶连接半探针以形成完整探针，作为Taq DNA聚合酶的模板。本研究共选择了22个FFPE肝标本，其中21个为肝细胞癌（HCC），1个为胆管闭锁继发的胆汁性肝硬化，其中13例患者HCV血清学阳性，9例血清学阴性。通过ID-PCR可从所有13例HCV血清学阳性的HCC以及8例HCV血清学阴性的HCC中的5例检测到HCV RNA，但未从胆汁性闭锁的HCV血清学阴性肝脏中检测到。相比之下，RT-PCR仅在5例HCV血清学阳性和1例HCV血清学阴性的HCC中检测到HCV序列。为解决LD-PCR和RT-PCR结果之间的不一致，对差异标本的冷冻肝组织进行RT-PCR，证实了LD-PCR的阳性结果。总之，对于常规处理的肝组织中HCV序列的检测，LD-PCR是一种比RT-PCR更灵敏的方法。在HCC标本中发现HCV感染率很高（86%），表明HCV在HCC发病机制中的作用此前被低估。
