基于丙型肝炎病毒NS5B基因序列分析的基因分型

目的：建立基于丙型肝炎病毒(HCV)NS5B基因序列分析的基因分型方法，对上海地区慢性丙型肝炎进行基因分型。方法：用HCVRNA实时荧光定量检测试剂盒对慢性丙型肝炎患血清标本的HCV RNA水平进行定量分析。用逆转录-聚合酶链反应(RT-PCR)和套式-PCR扩增HCV RNA阳性的41份标本的部分HCV NSSB区基因，PCR产物经回收、纯化后直接进行测序分析。从GenBank中下载25份不同基因型的HCV原型序列，用NSSB区222bp的基因片段构建系统进化树，对临床标本相应的HCVNS5B区序列进行分析。结果：基于HCVNS5B基因序列的系统进化树分析，可成功地对本组41份标本进行基因分型。分型结果显示，1b型占85．4％(35／41)，2a型占12．20％(5／41)，3a型占2．44％(1／41)。结论：基于HCVNSSB区基因序列的系统进化树分析是一种可靠和方便的HCV基因分型方法。上海地区的HCV基因型以1b型为主。

Genotyping of hepatitis C virus based on NS5B region sequence analysis

Objective: To establish a method of genotyping for HCV based on NS5B region sequence analysis. Methods:Serum HCV RNA levels in 41 patients with chronic hepatit is C in Shanghai area were determined using the real time fluorescent quantitat ive polymerase chain reaction (PCR) assay. Part of HCV NS5B region was amplified by RT PCR and nested PCR, the PCR products were subjected to direct sequencing. A 222 bp segment of the HCV NS5B region was used to construct phylogenetic trees for analyzing the relationships between the amplified NS5B region sequences and 25 published proto type sequences retrieved from the GenBank data base.Results:Genotype classification based on phylogenetic analysis o f the commonly used 222 bp long NS5B region was possible for all serum samples tested in this study. Among the 41 samples tested 35(85.4%) were 1b subtype, 5( 12.2%)and 1(2.4%) were 2a subtype and 3a subtype respectively. Other genotypes a nd subtypes were not detected. Conclusions:Phylogenetic analysis based on the NS5B region was a reliable and convenient method for HCV genotyping. 1b subtype seemed to be the dominant genotype of HCV in Shanghai area.