Sergi C, Goeser T, Otto G, Otto H F, Hofmann W J
Department of Pathology, University of Heidelberg, Germany.
J Clin Pathol. 1996 May;49(5):369-72. doi: 10.1136/jcp.49.5.369.
To develop a polymerase chain reaction (PCR) based technique that would permit the rapid and highly specific detection of hepatitis C virus (HCV) RNA extracted from frozen liver tissue.
Samples of liver tissue from 18 patients undergoing orthotopic liver transplantation were studied. Nine patients were HCV positive. Total RNA was extracted from between one and 10 sections, 10 microns thick, from each tissue sample. HCV RNA was amplified by (1) conventional, multistep reverse transcription PCR (RT-PCR) and by (2) combined, single step RT-PCR using coupled oligonucleotide primers based on the sequence of the 5' untranslated region of the viral genome. Positive results were confirmed by dot blot analysis using a digoxigenin labelled oligoprobe (Alx 89).
HCV RNA was detected in the nine HCV positive patients by both conventional and combined RT-PCR. HCV RNA was not detected in the HCV negative patients. As little as 500 ng total RNA was needed as the template to yield detectable amounts of amplified cDNA. The digoxigenin labelled oligoprobe hybridised with HCV RNA positive specimens only.
The combined, single step RT-PCR is a rapid and sensitive technique for detecting HCV RNA in frozen liver tissue.
开发一种基于聚合酶链反应（PCR）的技术，用于快速、高度特异性地检测从冷冻肝组织中提取的丙型肝炎病毒（HCV）RNA。
研究了18例接受原位肝移植患者的肝组织样本。9例患者HCV呈阳性。从每个组织样本的1至10个10微米厚的切片中提取总RNA。HCV RNA通过以下方法进行扩增：（1）传统的多步逆转录PCR（RT-PCR）；（2）使用基于病毒基因组5'非翻译区序列的偶联寡核苷酸引物进行联合单步RT-PCR。通过使用地高辛标记的寡核苷酸探针（Alx 89）的斑点印迹分析来确认阳性结果。
通过传统RT-PCR和联合RT-PCR在9例HCV阳性患者中均检测到HCV RNA。在HCV阴性患者中未检测到HCV RNA。仅需500 ng总RNA作为模板即可产生可检测量的扩增cDNA。地高辛标记的寡核苷酸探针仅与HCV RNA阳性标本杂交。
联合单步RT-PCR是一种用于检测冷冻肝组织中HCV RNA的快速、灵敏技术。
