摘要
目的了解长春地区乙型肝炎(乙肝)患者基因型特点及与肝炎严重程度、疾病转归的关系,以指导临床对疾病进展的判断和指导临床用药。方法选择216例乙肝病毒(HBV)阳性肝炎患者,年龄33-87岁,平均(60．5±11．4)岁。病变程度的判断根据2000年《病毒性肝炎防治方案》中的诊断标准。应用巢式聚合酶链反应方法(nPCR)扩增HBV S基因组序列,用第二轮PCR产物直接进行限制性片段长度多态性(RFLP)分析;然后用HBV S基因扩增产物采用Sanger双脱氧链终止法进行DNA测序,将所得到的核苷酸序列及推测的相应氨基酸序列通过DNASIS软件处理,并与GenBank中已发表的HBV中国株及国外株进行同源性比较以确定HBV的基因型,证明用S基因序列的RFLP法HBV分型结果与用全基因组序列的基因分析是一致的。表明S基因序列RFLP法的基因分型结果与S基因测序分型结果是一致的。结果216例HBV DNA阳性标本中单基因型分别检测出A、B、C、D四种,其中单基因型中A型有1例(0．46％),B型有19例(8．8％),C型有175例(81．02％),D型有21例(9．72％)。86例HBV DNA阳性住院患者标本中,C型为感染的主要基因型,69例(80．23％)。B型次之,9例(10．47％)。6例原发性肝癌均为C型;20例肝炎后肝硬化的患者中有17例(85．0％)为C型。结论长春地区HBV基因型仍以C型为主(81．02％),B、D型次之(分别是8．8％和9．72％)。同时结果显示长春地区HBV感染有基因多样性趋势,表现在检测出了A单基因型。肝癌、肝硬化与C基因型HBV的相关性最大。C基因型HBV DNA拷贝数较非C基因型高,提示与HBV DNA在C型患者体内长期存在有关。HBV病毒载量与肝脏病变严重程度的关系无统计学意义。
Objective To study the hepatitis B virus （HBV） genotype and its relation to clinical degree and responsiveness to antiviral therapy on hepatitis in order to guide the clinical therapy. Methods We amplified HBV S gene by polymerase chain reaction （PCR）, using the second-round PCR product, which was digested by restriction fragment length polymorphism （RFLP）. This genotype method was designed under the analysis of the restriction fragment length polymorphism and using the restriction enzymes that identified the genotype-specific sequences. Five restriction enzymes, Hph Ⅰ , Nci Ⅰ , Alw Ⅰ , Ear Ⅰ and Nla Ⅳ, were identified in genotype-speclfic RFLP from the S gene region. Representative sequences from the S genome region of each HBV genotype were aligned to show the restriction sites by the five restriction enzymes. The amplified S gene nucleotide sequences were sequenced by dideoxy-chaintermination method and the corresponding amino acid sequence was deduced using DNASIS software. Later, they were genotyped by comparing to representative S gene sequences obtained from GenBank. This confirmed the results of RFLP HBV genotyping methods,coincident with that of S gene sequence. Results Genotypes A, B, C, D were classified in 216 patients with HBV and DNA positive. The results showed that： 1 case （0.46%） of genotype A,19 cases genotype B（8.8% ） ,175 genotype C（81.02% ） and 21 genotype D（9.72% ）. A total of 86 patients in the hospital were divided into either genotype C cases （69） or nongenotype C cases （17）. Conclusion Genotype C was the major genotype in Changchun. Among HBV patients,type C was 80.95 %, followed by genotypes B and D. Both hepatocellular carcinoma and liver cirrhosis showed relations with genotype C.
出处
《中华流行病学杂志》
CAS
CSCD
北大核心
2006年第12期1057-1060,共4页
Chinese Journal of Epidemiology
