HBV基因分型的研究现状

乙型肝炎病毒（hepatitisBvirus,HBV）是重要的肝脏疾病致病因子,HBV感染能够引起急性、慢性乙型肝炎、肝硬化、爆发性肝炎、重型肝炎、原发性肝癌等。目前,全世界慢性乙型肝炎感染患者有3．8亿左右,而我国就占了1．5亿,其中有2900万人为慢性乙型肝炎患者,每年因此病死亡的人将近50万。我国是乙型肝炎多发的大国,研究乙型肝炎病毒对我国及人类都有重要的意义。近年研究结果发现,     

载脂蛋白E基因的PCR快速分型法的建立

目的建立载脂蛋白E基因的PCR快速分型法.方法采用以PCR扩增为核心的载脂蛋白E(apoE)基因型分析方法,扩增其中含有112位和158位两个氨基酸位点之间的编码序列,产物为267bp apoE基因经扩增后,扩增产物经HhaI 内切酶消化后,然后进行5%琼脂糖凝胶电泳检测,快速鉴定apoE的基因型.结果本法检测了85名健康人apoE 基因型,求出三个等位基因E2、E3、E4的频率分别为0.0824、0.8412、0.0764.结论该方法简单、准确,适合于一般实验室开展.     

乙型肝炎病毒基因分型及其应用的研究进展

乙型肝炎病毒(HBV)基因分型作为HBV感染的分子流行病学调查,鉴定传染源,确定传播关系及发病机制和临床流行病学研究的一种有力工具,日益受到广泛重视。随着HBV基因分型方法的日趋完善及新基因型的发现,人们对HBV基因型有了进一步认识。本文就HBV基因型的分型和地理分布,HBV基因型与血清亚型的关系,HBV 基因型和变异的关系及与临床治疗和转归的关系等相关研究进展作一综述。     

载脂蛋白E基因的PCR快速分型法的建立

目的　建立载脂蛋白E基因的PCR快速分型法 .方法　采用以PCR扩增为核心的载脂蛋白E(apoE)基因型分析方法 ,扩增其中含有 112位和 15 8位两个氨基酸位点之间的编码序列 ,产物为 2 6 7bpapoE基因经扩增后 ,扩增产物经HhaI内切酶消化后 ,然后进行 5 %琼脂糖凝胶电泳检测 ,快速鉴定apoE的基因型 .结果　本法检测了 85名健康人apoE基因型 ,求出三个等位基因E2、E3、E4的频率分别为 0 0 82 4、0 8412、0 0 76 4 结论　该方法简单、准确 ,适合于一般实验室开展 .     

登革热病毒多重荧光PCR检测及基因分型方法的研究

目的建立一种登革热病毒双靶基因多重荧光PCR检测方法,用于登革热病毒的实验室诊断和基因分型。方法选取登革热病毒Ⅰ-Ⅳ型病毒保守区设计型特异性引物探针和通用型引物探针。评估多重荧光PCR检测方法的特异性、重复性和检测限;并对20份阳性样本进行检测。结果20个登革热阳性核酸标本在通用型检测全部为阳性,特异性型别检测发现登革热病毒Ⅰ型10例、登革热病毒Ⅱ型3例、登革热病毒Ⅲ型3例、登革热病毒Ⅳ型4例;20名正常无症状人群标本提取的核酸和HIV、HCV和HEV通用型和特异性型别检测全部为阴性。梯度检测的变异系数均小于5％。对登革热Ⅰ-Ⅳ型病毒检测最低检测限达10^3 eopies／ml。结论本研究建立的登革热病毒双靶基因多重荧光PCR检测及分型方法具有特异性好、重复性好、快速易操作等优点,可用于登革热病毒的快速检测和基因分型鉴定。     

HBV基因型与拉米夫定疗效相关性的研究

根据聚合酶链反应邛艮制性片断长度多态性(PCR-RFLP)基因型分型法，目前已将前S基因分为A、B、C、D、E和F六种基因型。本文就HBV基因型分型方法，基因型与疾病的关系以及与拉米夫定抗病毒疗效相关性的研究作一综述。     

高通量apoE基因分型技术的建立及应用

目的建立一种准确、快速、高通量的apoE基因分型技术。方法从外周血样品提取基因组DNA，PCR扩增覆盖第112和158密码子的apoE基因片段；构建apoE基因片段重组质粒，并进行定点诱变，以得到3种等位基因型的对照样品；PCR产物消化处理，以除去残余的引物和dNTPs；进行模板指导的荧光染料标记终止碱基的掺入反应，应用荧光偏振检测仪分析荧光偏振值的变化；检测79例阿尔茨海默病(Alzheimer’s disease，AD)患者和63名健康老年人的apoE基因型，分析基因型与AD易感性之间的关系。结果对分析结果进行测序验证，表明模板指导的荧光染料标记终止碱基掺入-荧光偏振检测技术分析结果与测序结果完全相符。AD组和健康对照组样品的基因分型结果提示apoEε4等位基因是迟发型AD的危险因素。结论应用此技术进行apoE基因多态性的基因分型分析，具有准确、简易和高通量等优势，可以作为AD风险分析的一种新技术．也适于apoE基因与其他疾病相关性研究时的大规模基因筛查分析。     

一种新的土拨鼠α干扰素基因分型方法的建立及其意义

目的：建立新的土拨鼠α干扰素（IFN-α）基因分型方法，分析急性和慢性土拨鼠肝炎病毒（Woodchuck hepati-tis virus，WHV）感染的土拨鼠肝细胞中不同型别的IFN-α基因表达谱及其意义。方法：利用已知序列和基因型的土拨鼠IFN-α基因克隆质粒，建立基于土拨鼠IFN-α基因序列的限制性片段长度多态性的土拨鼠IFN-α基因分型方法。PolyIC体外刺激正常土拨鼠外周血单个核细胞（PBMC），调查正常土拨鼠PBMC受PolyIC刺激后IFN-α基因的表达谱。提取急性和慢性土拨鼠肝炎病毒感染的土拨鼠肝组织mRNA，RT-PCR扩增土拨鼠IFN-α基因，分子克隆后调查急性和慢性感染的土拨鼠肝细胞中IFN-α基因表达谱。结果：建立了土拨鼠IFN-α基因限制性片段长度多态性分型方法。急性和慢性感染的土拨鼠肝细胞中IFN-α基因的表达谱明显不同于正常动物的表达谱，假基因表达所占比例大。结论：新建立的土拨鼠IFN-α基因分型方法可用于分析土拨鼠不同组织中IFN-α的基因表达谱。     

海府地区慢性HBV感染不同免疫状态患者基因分型、病毒变异研究

目的 了解海府地区慢性HBV感染不同免疫状态患者基因分型、病毒变异及其相互之间的关联性.方法 入选对象为海府地区慢性HBV感染患者,依照HBV感染自然史分为4组:慢性无症状HBV携带组、HBeAg(+)CHB组、HBsAg-IaC组、HBeAg(-)CHB组,各50例,PCR-RELP、PCR-反向点杂交法检测所有样本基因型、病毒变异.结果 ①4组患者B基因型感染分布比率分别为:60％、56％、62％、60％;C基因为:38％、38％、34％、32％;D基因为:2％、6％、0、6％;B+C基因为:0、0、4％、2％.各型基因在4组患者中感染同比分布差异无统计学意义(P＞0.05).4组之间优势基因均为B、C基因,尤以B基因感染为主.②4组患者均存在PC、BCP区变异,其中HBeAg(-)CHB组患者变异比例最高,其次为HBeAg(+)CHB组,与HBsAg-IaC组相较,差异有统计学意义(x2=24.73、18.32、6.78、3.84;P=6.59E-07、1.87E-05、0.009、0.049).③B、C型基因感染患者均存在PC、BCP区变异.PC、BCP区变异结果中C基因型发生比例较B基因型高,分别为:43.66％比15.97％、33.80％比10.92％,两者相较差异有统计学意义(x2=17.59、14.84,P=2.74E-05、0.000).结论 海府地区慢性HBV感染优势基因为B、C基因,尤以B基因为主,4种免疫状态均存在PC、BCP区变异.C基因型感染患者及HBeAg(-)CHB患者存在高PC、BCP区变异,可能更易引起严重的肝细胞炎症、坏死和纤维化修复、病毒的高复制及流行,因此,对患者基因分型及病毒变异进行有效监控,将为海府地区慢性HBV感染者的管理开辟新的途径,具有很高的临床实用价值.     

HBV基因型芯片检测方法的研究

HBV属于嗜肝DNA病毒科，基因组经反转录过程进行复制。由于反转录酶缺乏自我校读功能，且感染过程中，在宿主免疫反应的抗病毒压力或特异性治疗的作用下，病毒基因组发生变化，造成HBV核苷酸序列的显著差别。HBV基因分为A～H 8个基因型，能更好的刻画病毒致病性的不同。基因芯片技术的出现，对于像HBV这样高变异性病原体的基因分型检测无疑是一大改进。     

HBV subtype as a marker of the clinical course of chronic HBV infection in Japanese patients

Hepatitis B virus (HBV) genotype C is predominant in Japan. However, many HBV subtypes are involved in each genotype, and the clinical manifestations in the patients associated with each subtype remain unknown. Therefore, we investigated the relationship between HBV subtype and clinical aspects of chronic HBV infection. The subtype of 237 patients with chronic HBV infection, including 74 asymptomatic carriers, was determined. The subtypes of 110 HBV carriers undergoing long-term follow-up management were determined twice to detect subtypic changes. The clinical features of the patients were also studied with regard to presence or absence of subtypic change. The subtypic distribution in the 237 HBV carriers was as follows: subtype adr, 161 (68%); subtype adw, 25 (11%); subtype adwr, 12 (5%); subtype ar, 24 (10%); subtype adyr, 4 (2%); and unclassified, 8 (3%). The proportion of asymptomatic carriers in patients with subtype adw was significantly higher than those in patients with subtype adr (56% vs. 28%, P < 0.05). In addition, the proportion of HCC in patients with subtype adwr was significantly higher than those in patients with subtype adr (25% vs. 6%, P < 0.05). The prevalence of subtype adr in 74 asymptomatic carriers tended to decrease with age (82% in carriers aged < or =35 years vs 43% in those aged > or =61 years, P < 0.05). The subtypic change and the course of chronic HBV infection had no significant correlation. These results suggest that HBV subtypes are associated with the clinical course of chronic HBV infection.     

The influence of HLA alleles and HBV subgenotyes on the outcomes of HBV infections in Northeast China

Hepatitis B virus (HBV) infection has a wide variety of clinical outcomes, it could be spontaneouly recovered and also could develop fulminant liver failure or cirrhosis with hepatocellular carcinoma. Human leukocyte antigen (HLA) polymorphism and HBV (sub)genotypes have been speculated to associate with the outcome of HBV infection because the data obtained from various populations who bear different HLA alleles have shown a HLA polymorphism associated outcome of HBV infection. However, as the most important viral and host genetic factors, the impact of HBV (sub)genotypes in combination with HLA polymorphism on the clinical outcomes of HBV infections remains unclear. To demonstrate the association of HLA allele polymorphism in combination with HBV subgenotypes with the outcome of HBV infection in Northeastern Han Chinese population, a total of 230 HBV-infected individuals (Infection group) were compared to 210 random selected controls (Control group) who are negative for HBV infection for their HLA alleles frequency as well as the associations with the virus infection, clearance and persistence in combination with HBV subgenotypes. Of the 230 HBV-infected subjects, 54 were acute self-limited hepatitis (ASH) with HBV subgenotype C2 (ASH-C2), 144 were chronic hepatitis (CH) with HBV subgenotype C2 and B2 (CH-C2 and CH-B2), and 32 were spontaneously recovered (SR) without subgenotype results. When two groups are compared, the results suggest that B*48, B*51 and DRB1*12 carrier may have a high risk for HBV infection, but B*51 is likely association with spontaneous recovery and DRB1*07, 12 may be implied in viral persistence. HLA-B*15, DRB1*11 and 14 associated with viral clearance in the cases of HBV-C2 infection; HLA-B*54 carriers in chronic group are more sensitive to with the infection of HBV subgenotype B2; HLA-B*07 and DRB1*13 may protect subjects from HBV infection. The data presented a link between HLA polymorphism and HBV pathogenesis and suggested potential therapeutic targets for hepatitis B.     

TR-FIA与RIA法检测HBV标志物的比较分析

铕(Eu)标记时间分辨荧光免疫技术(TR-FIA)是新近推出的一种非放射性标记微量检测技术, 将其与RIA比较分析, 结果表明: TR-FIA法较RIA法灵敏度高, 测量范围宽, 准确度相近; 在乙型肝炎病毒标志物(HBV M)常见的8种组合模式中, 有7种完全符合(符合率为100%),只有HBSAg 抗-Hbe 抗-HBc组合模式符合率为73.3%,而HBeAg值差异较大.     

Perturbation Analysis: A Simple Method for Filtering SNPs with Erroneous Genotyping in Genome-Wide Association Studies

We introduce a simple and yet scientifically objective criterion for identifying SNPs with genotyping errors due to poor clustering. This yields a metric for assessing the stability of the assigned genotypes by evaluating the extent of discordance between the calls made with the unperturbed and perturbed intensities. The efficacy of the metric is evaluated by: (1) estimating the extent of over‐dispersion of the Hardy‐Weinberg equilibrium chi‐square test statistics; (2) an interim case‐control study, where we investigated the efficacy of the introduced metric and standard quality control filters in reducing the number of SNPs with evidence of phenotypic association which are attributed to genotyping errors; (3) investigating the call and concordance rates of SNPs identified by perturbation analysis which have been genotyped on both Affymetrix and Illumina platforms. Removing SNPs identified by the extent of discordance can reduce the degree of over‐dispersion of the HWE test statistic. Sensible use of perturbation analysis in an association study can correctly identify SNPs with problematic genotyping, reducing the number required for visual inspection. SNPs identified by perturbation analysis had lower call and concordance rates, and removal of these SNPs significantly improved the performance for the remaining SNPs.     

乙肝病毒基因组中U5序列在乙肝患者垂直传递中的分析研究

目的研究乙肝病毒在乙肝患者垂直传递中的情况，方法运用分子遗传学的技术方法：单核苷酸多态性（SNP）及多聚酶链反应-单链构相多态性（PCR-SSCP），对30个家庭（68个病例）的乙肝患者及其子女进行了研究。结果在乙肝患者及其受感染后出生子女中游离型及整合型乙肝病毒持续增高，与感染前所生子女间比较差异显著（P〈0．05），SNP分析结果显示在乙肝病毒基因组中，U5序列和非U5序列中许多基因位点发生了碱基替换，插入或缺失，男性乙肝患者及其受感染子女的SNP分析结果被确定。结论乙肝病毒可通过男性患者传递至子女；至此，又一个分子遗传学证据证实了了乙肝病毒的遗传性传递。     

High prevalence of HBV and HCV infection among intravenous drug users in Korea

Although intravenous drug users are a well-known route of hepatitis C virus (HCV) and hepatitis B virus (HBV) transmission, there is no data on the prevalence of HBV and HCV infection among intravenous drug users in Korea. In order to describe the prevalence of HBV and HCV infection, and to determine HCV genotypes in the population, serum samples were collected from 107 intravenous drug users during 2005-2006. Fifty-seven percent (n = 61) were HCV RNA positive and 51% (n = 55) were HBV DNA positive. Co-infection of HBV and HCV were found in 23% (n = 25). HCV genotypes 1b, 2a/2c, 2, 2b, and 3a were found in 38% (n = 23), 44% (n = 27), 8% (n = 5), 2% (n = 1), and 3% (n = 2), respectively. Moreover, mixed infections of genotypes 1b and 2a/2c were found in 5% (n = 3). When the number of patients with HCV genotype 1b compared with that of patients with genotype 2a/2c, HBV DNA titer was not significantly different by independent t-test (t = -0.881, P = 0.392 > 0.05) between the two patient groups. These results suggest that the prevalence of HBV and HCV infection among intravenous drug users is high showing over 50% in Korea and a strategic prevention program should be performed in this group to prevent further infection into local community.     

奉贤地区HBsAg阴性的HBV自然感染母亲对新生儿影响的研究

目的　为揭示HBsAg阴性的乙型肝炎病毒 (HBV)自然感染孕妇的宫内感染及其危险因素。方法　采用多聚酶链反应 (PCR)技术结合酶联免疫吸附法 (ELISA) ,对奉贤地区 131例HBsAg阴性的HBV自然感染孕妇外周血 ,及其分娩后的脐带血进行HBV血清学标志物 (HBVM)和HBVDNA检测。结果　HBsAg阴性的HBV自然感染孕妇宫内的感染率 (除外单一抗 -HBs阳性 )为 5 2 6 7% ;脐血中不同HBVM组合的HBVDNA检出率依次为 :抗 -HBe( )、抗 -HBc( ) >抗 -HBs( )、抗 -HBe( )、抗 -HBc( ) >抗 -HBs( )、抗 -HBe( ) >抗 -HBs( )、抗 -HBc( ) >抗 -HBs( ) ;脐血HBVDNA总检出率为 16 79%。结论　HBsAg阴性的HBV自然感染孕妇也可能发生宫内感染。提议HBsAg阴性的HBV自然感染孕妇和新生儿有进行自动和被动免疫接种的必要性     

HBV pgRNA在HBV相关疾病中的表达及其临床意义

目的 探讨血清HBV 前基因组RNA(pgRNA)在HBV相关疾病中的表达及其临床意义。方法 收集90例慢性乙型肝炎、54例乙肝肝硬化和44例HBsAg阳性原发性肝癌患者血清样本,进行HBsAg、HBeAg、HBV DNA和HBV pgRNA检测。结果 血清HBV pgRNA水平在慢性乙型肝炎组、肝硬化组、肝癌组患者中的中位数分别为7.08、5.16 和4.08 log copies/mL ,差异有统计学意义(χ^2=24.743, P <0.001)。在HBeAg阳性患者中,HBV pgRNA水平在慢性乙型肝炎组、肝硬化组、肝癌组患者中逐渐降低,差异有统计学意义(χ^2=14.864, P <0.001);且血清HBV pgRNA水平与血清HBV DNA、HBsAg水平及HBeAg滴度呈正相关。而在HBeAg阴性患者中,血清HBV pgRNA水平与血清HBV DNA、HBsAg水平及HBeAg滴度无相关性。结论 血清HBV pgRNA水平在慢性乙型肝炎、肝硬化和肝癌患者中逐渐降低,且与HBV DNA、HBsAg水平及HBeAg滴度呈正相关。