Lo Y M, Mehal W Z, Fleming K A
University of Oxford, Nuffield Department of Pathology, John Radcliffe Hospital.
J Clin Pathol. 1989 Aug;42(8):840-6. doi: 10.1136/jcp.42.8.840.
A 185 base pair fragment from the core-polymerase overlap region of the hepatitis B virus (HBV) genome was amplified using the polymerase chain reaction (PCR). The results were compared with those of Southern blotting on extracted DNA from eight hepatocellular carcinomata. The data agreed with those of Southern blotting in six cases (two positive, four negative) but in two other positive cases PCR failed to amplify HBV sequences. This suggests deletion or mutation, or both, of this viral region in these cases. PCR was also used to amplify HBV sequences from formalin fixed, paraffin wax embedded tissue. Tissue inhibition of PCR occurred which increased with the number of tissue sections. It was present in tissues from different organs and species and fixed by different procedures, thus highlighting the need for a positive control during amplification. Use of formalin fixed Alexander cells, however, showed a sensitivity of one viral copy per 5000 cells. Confirmation of the identity of the PCR products was carried out using PCR-generated biotinylated probes, and suggested the insertion of extra nucleotide sequences or infection with an HBV variant in one case.
使用聚合酶链反应（PCR）扩增了来自乙型肝炎病毒（HBV）基因组核心 - 聚合酶重叠区域的一段185个碱基对的片段。将结果与对8例肝细胞癌提取的DNA进行Southern印迹分析的结果进行比较。在6例病例中（2例阳性，4例阴性）数据与Southern印迹分析结果一致，但在另外2例阳性病例中，PCR未能扩增出HBV序列。这表明在这些病例中该病毒区域发生了缺失或突变，或两者皆有。PCR还用于从福尔马林固定、石蜡包埋的组织中扩增HBV序列。出现了PCR的组织抑制现象，且随着组织切片数量的增加而增强。它存在于来自不同器官和物种且经不同程序固定的组织中，因此突出了扩增过程中设置阳性对照的必要性。然而，使用福尔马林固定的亚历山大细胞显示每5000个细胞中有一个病毒拷贝的灵敏度。使用PCR产生的生物素化探针进行PCR产物的身份确认，结果表明在一例病例中存在额外核苷酸序列的插入或感染了HBV变异体。
