Soguero C, Campo E, Ribalta T, Sánchez-Tapias J M, Sáiz J C, Bruguera M
Liver Unit, Hospital Clínic Institut d'Investigacions Biomèdiques August Pí i Sunyer, Universidad de Barcelona, Spain.
Lab Invest. 2000 Jun;80(6):851-6. doi: 10.1038/labinvest.3780089.
Drawbacks of hepatitis C virus (HCV) RNA detection in paraffin-embedded liver tissue have satisfactorily been solved by RT-PCR amplification of the 5'non-coding region (5'NCR). However, detection of this highly conserved region does not provide information on epidemiological or pathogenetic aspects of HCV infection. This study explores whether other functionally important genetic regions of HCV, such as the hypervariable region 1 (HVR-1) and the interferon sensitivity-determining region (ISDR), can be retrieved from paraffin-embedded liver specimens by RT-PCR, and whether the amplified material is suitable for further molecular analyses. RT-PCR amplification of 5'NCR, HVR-1, and ISDR was assessed in RNA extracted from 50 formalin-fixed, paraffin-embedded liver specimens, including 23 needle liver biopsies (11 from patients with non-A, non-B chronic hepatitis diagnosed between 1971 and 1985, 8 from subjects with normal liver histology and 4 from sequential biopsies from a patient with HCV recurrence after liver transplantation), and 27 liver explants from patients undergoing transplantation between 1988 and 1996 (16 with HCV-related cirrhosis and 11 with other disorders). The 5'NCR was successfully amplified in 8 of 11 (73%) non-A, non-B chronic hepatitis biopsies and in all of the specimens from patients with serological documentation of HCV infection. There were no false-positive results. HCV genotype was identified by RFLP analysis of the 5'NCR in the 13 cases analyzed. HVR-1 and ISDR were amplified in 24 of 28 (86%) samples, which were positive for the 5'NCR. Efficient amplification was inversely related to the time of storage. The evolutionary changes of HVR-1 and ISDR were successfully analyzed by direct sequencing of amplificates from the explanted liver and from the sequential liver biopsies in a patient with HCV infection recurrence after transplantation. These observations indicate that paraffin-embedded liver tissue, even when stored for more than 20 years, is appropriate for advanced studies on the molecular biology of HCV.
通过对5'非编码区（5'NCR）进行逆转录聚合酶链反应（RT-PCR）扩增，石蜡包埋肝组织中丙型肝炎病毒（HCV）RNA检测的缺点已得到令人满意的解决。然而，检测这个高度保守的区域并不能提供关于HCV感染的流行病学或发病机制方面的信息。本研究探讨HCV其他功能重要的基因区域，如高变区1（HVR-1）和干扰素敏感性决定区（ISDR），是否可通过RT-PCR从石蜡包埋的肝标本中获取，以及扩增产物是否适合进一步的分子分析。对从50份福尔马林固定、石蜡包埋的肝标本中提取的RNA进行5'NCR、HVR-1和ISDR的RT-PCR扩增评估，这些标本包括23份肝穿刺活检标本（11份来自1971年至1985年期间诊断为非甲非乙型慢性肝炎的患者，8份来自肝组织学正常的受试者，4份来自肝移植后HCV复发患者的连续活检标本），以及27份1988年至1996年期间接受移植患者的肝外植体标本（16份患有HCV相关肝硬化，11份患有其他疾病）。11份非甲非乙型慢性肝炎活检标本中的8份（73%）以及所有有HCV感染血清学证据患者的标本中5'NCR成功扩增。没有假阳性结果。在所分析的13例病例中通过对5'NCR进行限制性片段长度多态性（RFLP）分析鉴定了HCV基因型。28份5'NCR呈阳性的样本中有24份（86%）扩增出了HVR-1和ISDR。有效扩增与储存时间呈负相关。通过对移植后HCV感染复发患者的肝外植体和连续肝活检标本的扩增产物进行直接测序，成功分析了HVR-1和ISDR的进化变化。这些观察结果表明，石蜡包埋的肝组织即使储存超过20年，也适合进行HCV分子生物学的深入研究。
