多重引物聚合酶链反应扩增丙型肝炎病毒基因及基因分型

Hepatitis C virus gene amplification and gene typing by multiplex polymerase chain reaction

利用聚合酶链反应(PCR)技术对丙型肝炎病毒(HCV)的5’-非编码区(5’-NCR)、C及NS4基因区的3对引物分别及同时扩增,检测80例抗-HCV阳性患者的血清HCV RNA,并进行了HCV基因分型研究。各不同引物所介导的PCR检出HCV RNA的结果为:5’-NCR基因区60%(48/80),C基因区37%(30/80),NS4基因区30%(24/80)。以上3对引物同时扩增仅42%(34/80),5’-NCR与NS4区同时扩增为40%(32/80),C与NS4区同时扩增为38%(31/80)。经5’-NCR检测HCVRNA阳性的48份血清标本中HCVⅡ型占68%(33/48);Ⅲ型占20%(10/48);混合型(包括Ⅱ/Ⅲ、Ⅰ/Ⅲ各1例)占4%(2/48);没有发现Ⅳ型;未分型3例,占6%。说明高度保守的HCV 5’-NCR区引物扩增效果较好。

Hepatitis C virus (HCV)-RNA extracted from 80 anti-HCV positive patients were amplified with three pairs of primers corresponding to the genes of 5'-noncoding region (5'-NCR), C region and NS4 region by multiplex polymerase chain reaction (multiplex PCR), and the genotypes of HCV were analyzed. The results showed that ① HCV RNA positive rates were 60% by the primer of 5'-NCR gene, 37% by the primer of C gene and 30% by the primer of NS4 gene, ② 42 per cent (34/80) was HCV-RNA positive when the PCR was perforemed by using these three primers at the same time, ③ 40 percent (32/80) was positive when using two pairs of the primers: 5'-NCR and NS4 gene, ④ 38 percent. (13/80) was positive when using two pairs of the primers: C and NS4 genes, ⑤ genotypes of HCV samples from 48 HCV-RNA positive patients were tested and 68 per cent (33/48) of them were genotype Ⅱ, 20 per cent (10/48) were Ⅲ, 4 percents (2/48) were Ⅱ/ Ⅲ, and Ⅰ/Ⅲ, and 6 per cents (3/48) were undetermined but type Ⅳ was not found.