Detection of hepatitis C virus RNA by sensitive PCR method

非甲非乙型肝炎病毒是日本慢性肝病的主要病原体。最近，M.Houghton et al.分子克隆了丙型肝炎病毒核酸，并报道了其基因组序列。建立了检测抗-丙型肝炎病毒抗体的检测体系。然而，目前还没有建立丙型肝炎病毒抗原或核酸的检测方法。其原因可能是由于病毒数量较少所致。本研究试图通过应用聚合酶链式反应(PCR)这一检测微量核酸的有效方法，建立一种灵敏的检测HCVRNA的方法。为了检测丙型肝炎病毒核糖核酸，我们从患者的血液和肝组织中提取核酸，然后将其逆转录成cDNA，然后用合成的寡核苷酸引物进行聚合酶链式反应扩增。所用的引物是根据丙型肝炎病毒序列和黄病毒序列合成的。用该RT-PCR方法检测了非甲、非乙型肝炎患者血清中的HCVRNA。在90例患者中，83例(92%)可检测到丙型肝炎病毒RNA。在40名患者中，有36名(90%)的肝组织中可检测到该病毒。提示非甲、非乙型肝炎患者MST感染丙型肝炎病毒。同时检测干扰素治疗后患者血清丙型肝炎病毒核糖核酸的变化。当患者得到有效治疗时，血清中的丙型肝炎病毒RNA持续消失，相反，在治疗成功时，血清中的丙型肝炎病毒核糖核酸保持阳性。丙型肝炎病毒核糖核酸的检测似乎预示着治疗是否有效。丙型肝炎病毒RNA检测方法的发展对丙型肝炎的诊断和治疗具有重要意义。

Non-A, non-B hepatitis virus is a major etiologic agent of chronic liver disease in Japan. Recently, M. Houghton et al. mleculary cloned the hepatitis C virus(HCV)nucleic acid and reported its genomic sequence. An assay system for detection of anti-HCV antibody was also established. However, detection method for HCV antigen or HCV nucleic acid have not been established. The reason might probably be because of small amount of the virus in spechmns. Here we tried to establish a sensitive method for detection of HCV RNA by applying polymerase chain reaction(PCR)which is a powerful method to detect small amount of nucleic acid. To detect the HCV RNA, we extracted the nucleic acid from patients samples(blood and liver tissue), then reversetranscribed the nucleic acid to cDNA, then amplified with PCR method using synthetic oligonucleotide primers. The primes used were synthesized based on the HCV sequence and flaviviral sequences. With this RT-PCR rmthod we detected the HCV RNA in the sera from patients with non-A, non-B hepatitis. HCV RNA was detectable in 83 of 90(92%)patents. It was detectable in liver tissue in 36 of 40(90%)patients. Thus, it was revealed that mst of patients with non-A, non-B hepatitis were infected with hepatitis C virus. We also detected the change of HCV RNA in patients treated by interferon. When patients were effectively treated, HCV RNA in serum persistently disappeared, in contrast it remained positive when the treatment was resuccessful. Detection of HCV RNA seems to indicate whether the treatment is effective or not. The developrant of detection method of HCV RNA seems to be important for the diagnosis and treatment of type C hepatitis.