Development of Fok-I based nested polymerase chain reaction-restriction fragment length polymorphism analysis for detection of hepatitis B virus X region V5M mutation

AIM: To develop a Fok-I nested polymerase chain reaction(PCR)-restriction fragment length polymorphism analysis(PRA) method for the detection of hepatitis B virus X region(HBx) V5 M mutation.METHODS: Nested PCR was applied into DNAs from 198 chronic patients at 2 different stages [121 patients with hepatocellular carcinoma(HCC) and 77 carrier patients]. To identify V5 M mutants, digestion of nested PCR amplicons by the restriction enzyme Fok-I(GGA TGN9↓) was done. For size comparison, the enzymetreated products were analyzed by electrophoresis on 2.5% agarose gels, stained with ethidium bromide, and visualized on a UV transilluminator.RESULTS: The assay enabled the identification of 69 patients(sensitivity of 34.8%; 46 HCC patients and 23 carrier patients). Our data also showed that V5 M prevalence in HCC patients was significantly higher than in carrier patients(47.8%, 22/46 patients vs 0%, 0/23 patients, P < 0.001), suggesting that HBx Ag V5 M mutation may play a pivotal role in HCC generation in chronic patients with genotype C infections.CONCLUSION: The Fok-I nested PRA developed in this study is a reliable and cost-effective method to detect HBx Ag V5 M mutation in chronic patients with genotype C2 infection.

Differentially expressed genes in hepatocellular carcinoma induced by woodchuck hepatitis B virus in mice

INTRODUCTIONHepatocellular carcinoma(HCC)is one of the major causes of death in the word.The mechanism of carcinogenesis is unknown,although it is widely accepted that HBV and HCV are clsely related to liver cancer[1-5[1-5].Previously,a variety of studies have described the differences in gene expression which distinguished tumor from nontumor[6-11].Cloning of the genes,especially the genes associated with HBV and HCV,is still very important to account for the development of liver cancer.

Optimization of competitively differentiated poiymerase chain reaction in detection of HBV basal core promoter mutation

AIM: To improve competitively differentiated polymerase chain reaction (CD-PCR) in detection of HBV basal core promoter mutation.METHODS: Recombinant plasmid of double point mutation A1762T/G1764A in basal core promoter of HBV constructed by site-directed mutagenesis was used as mutant control.To reveal the deficiency mechanism of CD-PCR, relationship between the circle number of PCR and the increased speed of products of each competitive primer was comparatively studied. Diversified amount of dNTPs and mutual primer of the competitive primers were tried to optimize CDPCR. Optimized CD-PCR was evaluated by detecting A1762T/G1764A mutation in recombinant plasmids and clinical sera from patients with HBV infection. RESULTS: The deficiency mechanism of CD-PCR was that the products of mismatched competitive primer grew fast when the amplification of matched primer entered into plateau stage, which led to decrease in or disappearance of the difference in the amount of their products. This phenomenon could be eliminated by reducing dNTPs to10 μmol/L and mutual primer to about 100 nmol/L. Optimized CD-PCR could detect both mutant and wild strain indepe ndent of the amount of templates and the number of PCRcycles. Its detection limit was 103 copies/mL, about 50 copies/reaction. About 10% of mutant DNAs among wild type DNAs could be detected. A1762T/G1764A mutant was detected in 41.8% (51/122) of patients with HBV infection, but not detected in controls with negative HBsAg. CONCLUSION: Optimized CD-PCR can detect mutation independent of the amount of initial templates and the number of PCR cycles.

Establishment of transgenic mouse harboring hepatitis B virus (adr subtype) genomes

INTRODUCTIONHepatitis B virus (HBV) belongs to the group ofhepatovirus, a major pathogen of human acute andchronic hepatitis B[1 4], which has a very closeassociation with human hepatocellular carcinoma(HCC)[5-8], For example, a statistical data from ahospital in Shanghai showed that 80% of HCCpatients were positive for HBsAg ( personalcommunication).

B-cell clonality in the liver of hepatitis C virus-infected patients

AIM:The association of hepatitis C virus(HCV) infection with typeⅡmixed cryoglobulinemia is well established,but the role of HCV in B-cell lymphoma remains controversial.In patients with HCV infection,B-cell clonal expansions have been detected in peripheral blood and bone marrow,and a high prevalence of B-cell non-Hodgkin's lymphomas has been documented.Liver biopsies in chronic HCV infection frequently show portal lymphoid infiltrates with features of B follicles,whose clonality has not yet been investigated.The object of this study was to determine the frequency of liver-infiltrating monoclonal B-cells in 40 patients with HCV infection.METHODS:Eight hundred and forty-eight patients were studied prospectively,including 40 HCV-positive patients and 808 patients with chronic hepatitis B virus(HBV)infection.Immunohistochemical study for B-and T-cell markers was performed on the paraffin-embedded liver tissue sections.The clonality of lymphoid B-cells was tested using a polymerase chain reaction(PCR)approach designed to identify immunoglobulin heavy chain gene(IgH) rearrangements.RESULTS:Liver-infiltrating monoclonal B-cells were detected in the liver for 4(10%)of 40 HCV-positive patients but were present in only 3(0.37%)of 808 liver biopsy specimens with chronic HBV infection.Chi-square testing showed that the monoclonal B-cells infiltration in the liver was more frequent in the HCV-infected patients(P=0.000).A clonal IgH rearrangement was detected in 5(71.4%)of 7 liver biopsy specimens with monoclonal B-cells infiltration.In 2 of 5 patients with both a clonal B-cell expansion and monoclonal B-cells infiltration in the liver,a definite B-cell malignancy was finally diagnosed.CONCLUSION:Liver-infiltrating monoclonal B-cells are detected in the liver of patients with chronic HCV and HBV infection.A high percentage of patients with monoclonal B-cells infiltration and B-cell clonality in the liver were finally diagnosed as having a definite B-cell malignancy.

Detection of pre－C region mutants of hepatitis B virus from HBeAg negative patients in Xi＇an area of China

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Analysis of point mutation in site 1896 of HBV precore and its detection in the tissues and serum of HCC patients

INTRODUCTION Hepatitis B is one of the common infectious diseases,which severely impairs the health of the people in our country and has close relationship

The cloning of 3'-truncated preS/S gene from HBV genomic DNA and its expression in transgenic mice

INTRODUCTIONHepatitis B virus (HBV) is regarded as one of themain etiologic factors involved in the developmentof human hepatocellular carcinoma (HCC).The open reading frame (orf)of X gene of HBVencoded a transactivating factor is the evidence thatstrongly supported the notion that the X gene ofHBV DNA integrated in HCC genomic DNA couldcontribute to the carcinogenesis of liver cells byactivation of some related cellular genes

Mispairing PCR－RFLP：a simple method to detect A_（83） point mutation in HBV preC region

To develop a new method for detecting the A(83) point mutation in HBV preC region, a mispairing primer was designed, which could introduce a Bsu 36I restriction site into the amplified product of A(83) mutant. After polymerase chain reaction(PCR), the amp

ANALYSIS OF POINT MUTATION IN SITE 1896 OF HBV PRECORE AND ITS DETECTION IN THE TISSUES AND SERUM OF HCC PATIENTS

Aim The 3’-base specific polymerase chain reaction (3’- BS- PCR) method was es-tablished to investigate the relationship between the mutation of precore region of Hepatitis B virus(HBV) and the liver damage to the patients caused by HBV and the possibility of HBV precore gene in-tegration in liver cells。 Mdthods According to the DNA sequence of precore region of HBV,themethod of 3’- BS- PCR is applied to analyze the point mutation site 1896 of HBV precore in 126 clini-cal serum specimens and 23 hepatoeellular carcinoma (HCC) patients’ tissues and serum whose trmorshave been surgically excised and pathologically diagnosed.Rdsults The point mutation in site 1896 ofHBV precore has been successfully rates of preore gene of HBV in the 23 patients’ tissues and serum are52.2 % (12/23) and 30.4 % (7/23) respectively.Conclusion The established method for HBV ore-core mutation analysis is simple and results can well repeated.It has provided a new approach to clinicalHBV research and its relationship to liver damage.The results obtained suggested that HBV precoremutation exists in a wide range among serum and tissue of the patients infected by HBV and HCC pa-tients,and the pre-c gene of HBV can not be detected in the serum of 21.8% of the HCC patients(tissue HBV precore gene positive).We may deduce that there may be the integration of HBV precoregenee in the genome of liver cells,which may play an important role in the carcinogenesis of HCC.

慢性HBV感染前C区变异与病毒复制水平关系

探讨HBV前C基因变异与病毒复制水平的关系在慢性HBV感染者中的意义。应用错配聚合酶链反应 (PCR) -限制性片段长度多态性 (RFLP)分析与荧光定量聚合酶链反应检测HBVDNA相结合 ,对 30例HB sAg(+ )、抗 -HBe(+ )及抗 -HBc(+ )慢性HBV感染者 ,其中无症状携带者 (AsC) 9例、慢性乙型肝炎 (CHB) 12例及慢性重症肝炎 (CHF) 9例进行前C区基因变异与HBVDNA水平关系进行分析。AsC组 3例 (33 33 % ) ,CHB组9例 (75 % )及CHF组 8例 (88 89% )有A83(nt 1896 )变异。荧光定量PCR结果表明HBVDNA含量在CHF组中最高。HBV前C变异在HBV不同感染状态中都可见 。

HBeAg阴性慢性乙型肝炎患者乙肝病毒基因变异和分型研究

目的探讨HBeAg阴性的慢性乙肝患者乙肝病毒基因变异情况和分型的关系及其临床意义。方法采用荧光定量聚合酶链反应(PCR)检测乙肝病毒脱氧核糖核酸(HBV-DNA)、实时荧光PCR检测乙肝病毒基因分型、PCR-反向点杂交方法检测乙肝病毒基因变异,并对其关系进行分析。结果在389例HBeAg阴性的慢性乙肝患者血清中HBV-DNA检出阳性214例(55.01%),阴性175例(44.99%),HBV-DNA拷贝数在1×103-水平的例数最高,为102例(26.22%),其次为1×104-水平,41例(10.54%),两者比较有性差异(P<0.001);基因变异方面,HBV-DNA载量在1×105-水平发生前-C区变异比例为71.43%(15例),发生BCP区变异比例为52.38%(11例),两者比较有性差异(P<0.001),并且HBV-DNA载量越高基因变异发生比例越大;214例HBeAg阴性HBV-DNA阳性的慢性乙肝患者基因分型与基因变异结果中,基因分型A型6例(2.80%),B型84例(39.25%),C型106例(49.53),D型7例(3.27),混合型11例(5.14%),两者比较有性差异(P<0.001);而发生前-C区变异结果中A型发生比例为16.67%(1例),B型发生比例为36.90%(31例),C型发生比例为44.34%(47例);在BCP区变异结果中B型发生比例为19.05%(16例),C型发生比例为26.42%(28例),两者比较有性差异(P<0.001),A、D型与混合型均未检出变异。结论 HBeAg阴性HBV-DNA阳性的慢性乙肝患者病毒复制水平处于一个相对较低水平;HBV-DNA载量越高基因变异发生比例越大;本地区乙肝病毒基因分布以B、C型为主,其中B、C基因型HBeAg阴性的慢性乙肝患者基因变异发生率较高。

乙型肝炎病毒C基因启动子和前C基因变异与HBeAg含量和肝炎病情的临床研究

研究乙型肝炎病毒(HBV)C基因启动子(CP)和前C基因变异对HBeAg表达和病情的影响。通过DNA扩增、基因序列分析检测48例慢性乙肝和12例慢性重型乙肝患者血清的HBV CP和前C基因序列,及通过微粒子发光法定量检测血清中HBeAg的含量。(1)前C终止变异(nt1896G→A)在重型乙型肝炎病例中的发生率显著升高(66.7％);CP双变异(nt1762A→T和1764G→A)则在慢性乙型肝炎中度和重度的病例中的发生率显著升高(分别为52.6％和54.5％)。(2)双变异组和终止变异组的HBeAg含量均显著下降,P<0.01。但终止变异组HBeAg含量的下降较双变异组更为明显,P<0.05,且eAb阳性率也显著升高,P<O.05。终止变异联合双变异组的HBeAg含量及eAb阳性率同终止变异组相近。前C终止变异对HBeAg表达的影响较CP双变异更大,对肝炎病情的影响也更明显。

e抗原阴性重症乙型肝炎患者HBV前C区热点变异研究

目的 研究ＨＢＶ信号肽区热点变异与重症乙型肝炎发生及转归的关系。方法 采用错配聚合酶链反应（ＰＣＲ）和限 制性片段长度多态（ＲＦＬＰ）分析方法检测６８例ＨＢｅＡｇ阴性的重症乙型肝炎病人（其中急性重肝６例、亚急性重肝３８ 例、慢性重肝２４例）及４４例慢性乙型肝炎患者的Ｔ１８６２、Ａ１８９６变异情况。结果 急性重肝的Ａ１８９６、Ｔ１８６２变异分别 为６６．７％（４／６）、０（０／６）；亚急性重肝４２．１％（１６／３８）、１５．８％（６／３８）；慢性重肝 ２５．０％（６／２４）、１６．７％（４／２４）；慢性肝炎 ４５．５％（２０／４４）、２．３％（１／４４）。重症乙型肝炎组与慢性肝炎组比较Ｔ１８６２变异率有显著差异（Ｐ＜０．０１），Ａ１８９６变异率无显 著差异（Ｐ>０．０５）。结论 提示Ｔ１８６２变异与乙型肝炎的重症化密切相关。而Ａ１８９６变异与乙型肝炎重症化进程是否有 关还不能确定。

应用错配PCR和限制性片段长度多态性分析技术检测HBV YMDD变异株

目的 建立简便易行的检测乙型肝炎病毒 (HBV)多聚酶基因 (P基因 )上YMDD变异株的方法 ,评价运用拉米夫定治疗慢性乙型肝炎患者对此变异的影响。方法 采用套式 /错配聚合酶链反应 (PCR)结合限制性片段长度多态性 (RFLP)分析技术对 10 8例治疗前HBVDNA(PCR)阳性正在拉米夫定治疗中的慢性乙型肝炎患者的HBVYMDD变异情况进行检测。结果 运用上述技术能有效区分HBVYMDD野生株和变异株 ;上述 10 8例患者经拉米夫定治疗后 ,HBVDNA阴转者 63例 (5 8 3 % ) ,HBVYMDD野生株和变异株分别为 2 3例 (2 1 3 % )和 2 2例 (2 0 4% )。结论 建立的套式 /错配PCR -RFLP分析技术可用于HBVYMDD变异株的临床监测 ;

PCR-ELISA检测血清中HBV聚合酶YMDD基因变异

目的 用 PCR- ELISA检测 HBV聚合酶 YMDD基因的变异 .方法 采用混合引物 ,并通过降低 PCR反应体系中 d NTPs、引物等成分的浓度 ,以及提高退火温度等措施 ,对YMDD基因的各种变异型进行特异扩增 ,再采用 37℃液相杂交和 EL ISA方法对 PCR扩增产物进行检测 ,从而实现 PCR-ELISA方法进行 YMDD基因变异的检测 .结果 该方法可以检测出已知的变异 YMDD基因序列 ,可以检测 2× 1 0个拷贝的模板量 ,同时可以从 1 0 0 0个正常 YMDD基因序列中检测出 1个变异的 YMDD基因序列 ,检测结果不受正常 YMDD基因序列干扰 .对 30例经过 6mo以上拉米夫定治疗的患者血清进行检测 ,检测出 4例 YMDD基因变异 ,并经过测序证实 .结论 该 PCR- ELISA法可以对血清中 HBV

乙型肝炎病毒BCP变异与慢性理症肝炎关系的探讨

目的 :探讨乙型肝炎病毒慢性感染中C基因启动子 (BCP)变异与慢性重症肝炎的关系。方法 :采用PCR微板核酸杂交结合ELISA检测显示技术 ,检测 74例乙型肝炎病毒慢性感染者BCP区核苷酸 (nt) 176 2碱基A→T和 176 4碱基G→A联合突变。结果 :在 74例乙型肝炎病毒慢性感染者中检出BCP区T176 2A突变 2 4例 (32 .4 % ) ,BCP变异在重症肝炎的发生率为 6 3.6 % (7/ 11)显著高于非重症肝炎的发生率 2 7% (17/ 6 3) (P<0 .0 5 )。结论 :BCP变异似可引起病情加重。

抗HBe阳性乙型肝炎和无症状携带者的HBVC基因启动子和前C基因变异

目的 研究抗HBe阳性乙型肝炎病人和抗HBe阳性无症状携带者 (AsC)的乙型肝炎病毒 (HBV)C基因启动子 (CP)和前C基因变异。方法 通过DNA扩增、基因序列分析检测 34例抗HBe阳性和 5 5例抗HBe阴性乙型肝炎病人及 2 8例抗HBe阳性AsC的血清HBVCP和前C基因序列。结果 (1 )抗HBe阳性肝炎组的前C终止变异 (nt1 896G→A)的发生率显著高于抗HBe阴性肝炎组 (6 1 8%和 1 4 5 %) ;而CP双变异 (nt1 76 2A→T和 1 76 4G→A)则在两组中无明显差异 (5 0 0 %和4 5 5 %) ;(2 )同抗HBe阳性AsC组比较 ,抗HBe阳性肝炎组的HBVDNA定量呈高水平 ,前C终止变异的发生率也显著增高 (6 1 8%和 2 8 6 %) ;(3)同抗HBe阳性慢性乙型肝炎 (CHB)组比较 ,抗HBe阳性重型乙型肝炎 (CSH)病人的前C终止变异发生率和CP双变异发生率无明显差异 (70 0 %和5 8 3%,5 0 0 %和 5 0 0 %) ,HBVDNA定量也无明显差异 ,而CPnt 1 75 2A→G变异发生率则显著增高 (80 0 %和 2 9 2 %)。结论 前C终止变异、nt1 84 6突变和病毒复制可能与抗HBe阳性乙型肝炎发病有关 ;而Cpnt1 75 2突变则可能与抗HBe阳性CSH发病有关。

荧光聚合酶链反应检测HBV患者拉米夫定治疗过程中的YMDD变异

目的建立一种在乙型肝炎拉米夫定治疗过程中产生的多聚酶酪氨酸(Y)-蛋氨酸(M)-天门冬氨酸(D)-天门冬氨酸(D)(YMDD主型区)基因变异的检测方法。方法设计包含两个变异位点(I/V)和无变异参照位点的3条下游引物,与同一条上游引物和探针建立3管PCR反应体系,对服用拉米夫定的100位患者的300份血清通过实时荧光PCR反应后的CT值判断是否发生已知(I/V)位点的变异。结果服药6个月检测未发现YMDD变异,服药9个月时检出YIDD变异5例,YVDD变异7例,YIDD/YVDD共同变异3例,变异率分别为5%、7%、3%。服药1年时检出YIDD变异10例,YVDD变异12例,YIDD/YVDD共同变异5例,变异率分别为10%、12%、5%,服药1年总变异率达27%。变异的发生与肝功能异常与HBVDNA病毒的反跳均相关(P<0.05)。结论该法对乙型肝炎拉米夫定治疗引起的基因变异检测方法简便、灵敏、快速、廉价,通过联合HB-VDNA与肝功能的检测对乙型肝炎患者的抗病毒治疗有重要临床意义。

套式PCR检测抗-HBs阳性者血清HBVDNA及序列分析

研究套式PCR在检测抗-HBs阳性不同模式者血清HBVDNA中的意义,从基因角度分析产生的原因。对64例标本采用套式PCR检测HBVDNA并作序列分析,荧光定量法检测HBVDNA含量。抗-HBs阳性不同模式者常规PCR法检测HBVDNA阳性率29.7％,套式PCR检测阳性率84.4％;套式PCR阳性组肝功能损害严重、HBVD-NA与HBsAg值显著高于套式PCR阴性组;套式PCR性阴/常规PCR阴性组48.6％的病例由于S基因变异导致HB-sAg肽氨基酸置换产生的,抗-HBs不能中和病毒而与HBVDNA共存。因此对那些抗-HBs阳性、肝功能反复异常者有必要进行套式PCR检测,以提高HBV感染的诊断率。