成都地区HBV基因型分布研究及准种筛查

发布时间：2018-06-07 12:55

本文选题：乙型肝炎病毒 + 基因型　；参考：《四川大学》2007年硕士论文

【摘要】： 目的：乙型肝炎是一种流行范围广、危害程度大的世界性传染病。我国是此病的高发区，乙肝携带者占到国民总数1／10以上，正确认识乙肝病毒(HBV)高突变、多亚型的遗传异质性，对发病机制研究和临床病毒诊疗均有重大意义。基于其广泛存在的准种现象，本实验拟采用长片段高保真聚合酶链反应(LA-PCR)方法，对成都地区HBV基因型分布试作调查。方法：设计合成HBV基因组间同源性较高的表面抗原蛋白(HBsAg)编码区特异引物，对90例已确诊的HBV-DNA阳性感染者血清裂解物进行高保真PCR扩增。采用双脱氧链末端终止法(Sanger法)测序技术对所获得PCR产物进行测序；测序结果与GeneBank标准序列比对分型，并分析其准种特征。同时，采用文献中常用的型特异引物分型法作为标准对照，通过使用B、C、D三种基因型的特异引物组进行巢式PCR扩增检验，比较两种方法的分型结果以评估其可靠性和准确性。基因型和混合型；基因型特异引物分型法检测结果为：B型43例(47.8％)，C型21例(23.3％)，，D型3例(3.3％)，B／C混合型22例(24.4％)，B／D混合型1例(1.1％)。测序结果显示了HBV基因组中存在高度变异性的遗传特征，不仅同一基因型内广泛存在多态性和异质性，有些患者体内的HBV病毒株也以多克隆的准种形式存在。结论：基因分型结果表明成都地区HBV优势基因型以B型为主，C型次之，偶见D型。这与文献报道我国南方主要流行基因型为B型一致。对乙型肝炎患者血清病毒的测序图进一步分析表明，LA-PCR扩增产物并非序列完全一致的单一克隆，而是一个以优势株为主的相关突变株病毒群(准种)。S基因直接测序法能够准确地鉴定出相关突变株病毒群中的优势株基因型。因而与型特异引物分型法相比，排除了非优势突变株产生的假阳性干扰，更好地反映出了HBV基因的多克隆准种特征，此结果差异具有统计学显著性意义(P＜0.001)。实验结果证实了HBV高突变易重组及存在准种现象的遗传学特征，其遗传异质性，特别是一些重要多态位点的进化意义以及对于乙肝诊疗、预后的影响尚有待进一步研究。
[Abstract]:Objective: hepatitis B is a worldwide infectious disease with wide epidemic scope and great harm. China is a high incidence area of this disease, hepatitis B carriers account for more than 1 / 10 of the total number of people. It is of great significance to correctly understand the high mutation of hepatitis B virus (HBV) and the genetic heterogeneity of multiple subtypes, which is of great significance to the study of pathogenesis and clinical virus diagnosis and treatment. Based on the widespread quasispecies phenomenon, the long fragment high fidelity polymerase chain reaction (LPCR) method was used to investigate the genotype distribution of HBV in Chengdu. Methods: specific primers for the coding region of surface antigen protein (HBsAg) with high homology among genomes of HBV were designed and synthesized, and high fidelity PCR amplification was performed on 90 serum lysates of HBV-DNA positive patients. The PCR products were sequenced by dideoxy chain terminating method (Sanger method), and the results were compared with the standard GeneBank sequence, and the quasi species characteristics were analyzed. At the same time, the type-specific primer typing method commonly used in literature was used as the standard control, and the nested PCR amplification test was carried out by using the specific primer groups of three genotypes of BCU D, and the results of the two methods were compared to evaluate the reliability and accuracy of the two methods. The results of genotypic and mixed genotyping were as follows: 43 cases of type B, 47.8% and 21 cases of type C, 23. 3 and D, 3 cases of B / C mixed type, 22 cases of B / C mixed type 24. 4% B / D mixed type, 1 case of B / D mixed type. Sequencing results showed that there was a high degree of genetic variability in the HBV genome. Not only was there polymorphism and heterogeneity in the same genotype, but also in some patients, the HBV virus strains existed in the form of polyclonal quasispecies. Conclusion: the results of genotyping showed that the dominant genotype of HBV in Chengdu was B type C, and occasionally D type. This is consistent with the literature reported that the main epidemic genotype in southern China is B type. Further analysis of serovirus sequencing in patients with hepatitis B showed that the amplification product of La-PCR was not a single clone with identical sequence. It is a dominant mutant virus group (quasispecies. S gene direct sequencing method can accurately identify the dominant strain genotype in the related mutant virus group. Compared with the type-specific primer typing method, the false positive interference produced by non-dominant mutant was excluded, and the polyclonal quasispecies characteristics of HBV gene were better reflected. The difference was statistically significant (P < 0.001). The results confirmed the genetic characteristics of high mutation and quasispecies of HBV, and its genetic heterogeneity, especially the evolutionary significance of some important polymorphic loci, and its influence on the diagnosis and treatment of hepatitis B need to be further studied.
【学位授予单位】：四川大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R373

【参考文献】