Chou W H, Yoneyama T, Takeuchi K, Harada H, Saito I, Miyamura T
Department of Enteroviruses, National Institute of Health, Tokyo, Japan.
J Clin Microbiol. 1991 Dec;29(12):2860-4. doi: 10.1128/jcm.29.12.2860-2864.1991.
We investigated the presence of the hepatitis C virus (HCV) genome in liver tissues of eight different patients with hepatocellular carcinoma by using the reverse transcriptase polymerase chain reaction (PCR) method. RNA was extracted separately from cancerous and peripheral noncancerous portions of the liver tissues of each patient. For reverse transcriptase PCR, we used sets of primers derived either from nonstructural region 3 (the NS3 region) or from the nucleocapsid-envelope (C/E) region of the HCV genome. The nucleotide sequences of the amplimers were directly determined without subcloning. Of 16 samples tested, cDNA of the HCV genome was detected in 2 cancerous tissues and in 4 noncancerous tissues by either pair of primers. Nucleotide sequences of HCV cDNA fragments amplified from cancerous and peripheral noncancerous tissues from the same patients were identical. However, 4.4 to 6.3% and 7.5 to 11.3% sequence variation was observed in NS3 and C/E regions, respectively, among cDNA fragments from different patients. The result indicated that the HCV genome detected in a given patient is distinguishable from that in others by a simple direct nucleotide sequencing of the reverse transcriptase PCR products.
我们采用逆转录聚合酶链反应（PCR）方法，对8例不同的肝细胞癌患者肝组织中的丙型肝炎病毒（HCV）基因组进行了研究。分别从每位患者肝组织的癌组织和外周非癌组织中提取RNA。对于逆转录PCR，我们使用了源自HCV基因组非结构区3（NS3区）或核衣壳-包膜（C/E）区的引物组。扩增产物的核苷酸序列直接测定，无需亚克隆。在检测的16个样本中，两组引物均在2个癌组织和4个非癌组织中检测到HCV基因组的cDNA。从同一患者的癌组织和外周非癌组织中扩增得到的HCV cDNA片段的核苷酸序列相同。然而，不同患者的cDNA片段在NS3区和C/E区分别观察到4.4%至6.3%和7.5%至11.3%的序列变异。结果表明，通过对逆转录PCR产物进行简单的直接核苷酸测序，可区分特定患者中检测到的HCV基因组与其他患者的HCV基因组。
