中国HCV5′-非编码区复合酶切分型及其3b基因型序列分析

Hepatitis C virus (HCV) 5′NCR genotype research and 3b sequencing analysis in China

目的 研究中国丙型肝炎病毒 (HCV)不同基因型感染分布情况 ,并对 3b序列进行分析。方法 采用逆转录 聚合酶链反应 (RT PCR)技术扩增HCV5’NCR 1a、1b、2a、2b、3a、3b、4a、6a不同基因型的cDNA及 187份HCVRNA阳性样品中cDNA。A应用BHH (BsrBI、HaeⅡ、HinfI)复合内切酶消化 5′ NCRcDNA ,B应用BstUⅠ消化 ,C应用HaeⅢ消化 ,电泳检测片段大小。结果 187例HCVRNA阳性患者ABC分型结果表明 ,1b型感染率占 6 7 38% ,2a型 12 30 % ,1b/ 2a型为5 88% ,3b型 5 35 % ,2b型 3 2 1% ,2a/ 2b、1b/ 2b型各为 2 14 % ,1a型 1 0 7,6a型 0 5 4 %。 3份 3b基因型 5’ NCRA T克隆测序结果表明 ,3株 3b型之间同源性为 99 5 4 %～ 10 0 %。其中chiKQ5 0与GI5 14 395 3a株为 96 2 8%与GI6 76 8773b株同源性为 98 6 % ,与 1a型为 93 4 9% ,与 1b型为94 88% ,与 2a型为 91 6 3% ,与 2b型为 89 30 % ,与 4a型为 95 35 % ,与 6a型为 93 4 % ,结论 研究结果表明该项分型技术既保证了RT PCR检测灵敏度 ,又具有良好的分型效果。提示 :该分型方法不仅适合中国HCV基因型检测 。

Objective To study the distribution of HCV genotype 3b in China.Methods HCV 5’noncoding region (5’-NCR) of cDNA of regular samples (HCV genotype 1a,1b,2a,2b,3a,3b,4a,6a) and 187 HCV-RNA positive samples was amplified by RT-PCR. A. Using a combination of three restriction endonuclease BHH′(BsrB I、Hae II、Hinf I) digestions at the same time. B. using restriction endonuclease BstU I digestion. C.Using restriction endonuclease Hae III digestion.Results In 187 HCV-RNA positive samples , HCV genotype distribution in China were 67.38% for 1b, 12.30% for 2a,5.88% for 1b/2a, 5.35% for 3b, 3.21% for 2b, 2.14% for 2a/2b,2.14% for 1b/2b 1.07% for 1a, 0.54% for 6a.3 strains of genotype 3b sequencing suggest that the nucleotide identities of 5’-NCR are in the range of 99.54%～100%.The nucleotide identity is 96.28% between chiKQ50 and the isolate GI514395,(genotype 3a ) 98.6% between the isolate chiKQ50 and the isolate GI676877,(genotype 3b),93.49% between chiKQ50 and 1a,94.88% 2a,89.30% 2b,95.35% 4a, 93.4%6a.Conclusion The study indicates that this typing method is sensitive to detect HCV-RNA and is efficient to discriminate HCV genotype. This typing method can be applied not only to detect HCV genotypes in China, but also of great value in Asia, Europe and Africa.