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HBV cccDNA检测方法及其研究意义 总被引:4,自引:0,他引:4
HBV cccDNA是HBV mRNA和前基因组RNA的合成模板,是HBV持续感染的关键因素,检测HBV cccDNA对进一步认识HBV生活周期及指导抗病毒治疗等有重要意义.最近,利用PCR方法检测HBV cccDNA受到越来越多的重视.现就检测HBV cccDNA的各种PCR方法及其研究意义进行综述. 相似文献
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HBV感染者血清中HBVcccDNA、HBeAg及HBV DNA的关系 总被引:6,自引:0,他引:6
探讨HBV感染者血清HBVcccDNA与血清HBV DNA及HBeAg的关系。分别以PER分子信标技术和ELISA方法对非HBV相关肝炎、HBV健康携带者、急性乙型肝炎(AHB)、慢性乙型肝炎(CHB)、乙肝肝硬化、乙肝患者血清中HBVcccDNA HBV DNA含量及HBeAg进行了检测。HBVcccDNA仅见于HBV DNA阳性血清中;HBeAg阳性组的HBVcccDNA阳性率显著高于HBeAg阴性组(P〈0.05);145例HBV DNA阳性患者中,HBVcccDNA阳性组HBVD—NA水平显著高于HBVcccDNA阴性组(P〈0.01)。血清HBVcccDNA可能是乙肝病毒在患者体内大量复制的血清标志。 相似文献
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现有的抗病毒治疗方案很难清除HBV感染而达到"完全"治愈,导致许多患者需要长期、甚至终身接受核苷(酸)类似物抗病毒治疗,其根本的原因是肝细胞核内存在具有转录活性的共价闭合环状DNA(cccDNA)。为了让读者了解HBV cccDNA方面的研究进展,本期邀请了国内权威专家,就抗HBV治疗与慢性乙型肝炎的功能性治愈、靶向HBV cccDNA的药物及生物技术、HBV cccDNA转录调控机制与抗HBV治疗前景、HBV cccDNA的体外细胞模型和实验小鼠模型、原位杂交技术在检测HBV核酸和cccDNA中的应用、HBV cccDNA定量检测方法等6个方面的研究进展分别加以介绍。 相似文献
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目的探讨肝移植术后患者肝组织内乙型肝炎病毒共价闭合环状DNA(HBV cccDNA)的表达及其临床意义。方法应用原位聚合酶链反应及免疫组织化学法检测2004年4月至2007年4月在北京地坛医院肝移植术后出现HBV再感染的25例患者及10例肝移植术后未出现HBV再感染者肝组织中HBV cccDNA及乙型肝炎表面抗原(HBsAg)、乙型肝炎核心抗原(HBcAg)、前-S1的表达。结果在35例标本中检测到cccDNA阳性的有22例(62.9%),阳性信号为紫蓝色呈块状或颗粒状,定位于细胞核。25例术后出现HBV再感染的患者其HBV cccDNA阳性率显著高于未出现HBV再感染者(84%对10%,P<0.01)。HBV cccDNA和HBcAg均为阳性的有20例,有2例患者肝组织中HBcAg阴性,但HBV cccDNA检测为阳性。肝组织中HBV cccDNA的表达阳性率与HBcAg具有高度一致性(Kappa=0.755,P<0.01)。结论肝移植患者肝组织中HBV cccDNA阳性表达可能与乙型肝炎再感染具有密切关系。 相似文献
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目的探讨慢性乙型肝炎患者肝组织HBV共价闭合环状DNA(cccDNA)、肝组织总HBV DNA(HBV tDNA)与血清HBV DNA之间的相关性及其与临床的关系。方法 78例慢性乙型肝炎患者入选本研究。肝组织β- globinDNA、HBV cccDNA和HBV tDNA采用实时荧光定量PCR方法检测,平均每个肝细胞HBV cccDNA和HBV tDNA含量(拷贝/cell)=HBV cccDNA(实测值)/β-globin DNA(实测值)和HBV tDNA(实测值)/β3-globin DNA(实测值),肝组织HBV cccDNA和HBV tDNA含量的计算单位定义为log10拷贝/106cell;采用实时荧光定量PCR、ELISA法检测血清HBVDNA和HBV标志物;采用免疫组织化学方法检测肝细胞中HBsAg和HBcAg的表达。统计分析采用pearson相关分析及t检验。结果 (1)肝组织HBV cccDNA与HBV tDNA定量呈正相关(r=0.696,P<0.001);肝组织HBV cccDNA与血清HBV DNA定量呈正相关(r=0.304,P<0.01);肝组织HBV tDNA与血清HBV DNA定量呈正相关(r=0.341,P<0.01);(2)肝细胞内HBcAg定性检测阳性患者的血清HBV DNA定量明显高于阴性患者,且差异有统计学意义(P<0.05);肝细胞内HBcAg定性检测阳性患者与阴性患者的肝组织HBV cccDNA和HBV tDNA定量差异均无统计学意义和(P均>0.05);(3)肝细胞内HBsAg定性检测阳性患者的血清HBV DNA定量明显高于阴性患者,且差异有统计学意义(P<0.05);肝细胞内HBsAg定性检测阳性患者与阴性患者的肝组织HBV cccDNA和HBV tDNA定量差异均无统计学意义(P>0.05);(4)HBeAg(+)/抗-HBe(-)患者血清HBV DNA定量明显高于HBeAg(-)/抗-HBe(+)患者,且差异有统计学意义(P<0.05);HBeAg(+)/抗-HBe(-)患者肝组织HBV cccDNA和HBV tDNA定量与HBeAg(-)/抗-HBe(+)患者比较差异均无统计学意义(均P>0.05);(5)肝组织HBV cccDNA、HBV tDNA以及血清HBV DNA三者与肝脏炎症活动度及纤维化程度均无显著相关性(P>0.05)。结论 (1)血清HBV DNA定量结果并不一定能完全反映患者肝组织中HBV cccDNA和HBV tDNA含量,尤其在血清HBV DNA<500拷贝/mL时,肝组织中仍存在HBV cccDNA和HBV tDNA,且含量大小不等。(2)肝细胞内HBcAg定性检测阳性或者HBsAg定性检测阳性患者的血清HBV DNA定量均明显高于阴性患者;而两者的肝组织HBV cccDNA和HBV tDNA定量均没有显著差异;(3)HBeAg(+)/抗-HBe(-)患者血清HBV DNA定量明显高于HBeAg(-)/抗-HBe(+)患者,而两者的肝组织HBV cccDNA和HBV tDNA均没有显著差异;(4)肝组织HBV cccDNA、HBV tDNA及血清HBV DNA与肝脏炎症活动度和纤维化程度均无显著相关性。 相似文献
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乙型肝炎病毒cccDNA定量与乙型肝炎临床及病理关系 总被引:4,自引:1,他引:4
目的探讨慢性乙型肝炎(CHB)肝组织HBVcccDNA定量与乙型肝炎的关系。方法分别采用荧光定量PCR、酶联免疫吸附分析法(ELISA)检测48例CHB肝组织HBVcccDNA定量、肝组织和血清HB VDNA定量、乙型肝炎病毒标志物。同时用链霉菌抗生素蛋白-过氧化物酶连接法(SP)检测肝细胞中HBcAg表达。分析肝组织HBVcccDNA与组织和血清HBV DNA、HBeAg、肝细胞内HBcAg水平及肝脏炎症活动度的关系.结果1.肝组织HBVcccDNA定量与组织和血清HBV DNA定量呈正相关(r=0.837,P〈0.001;r=0.627,P〈0.005);2.肝组织HBV cccDNA定量与肝细胞内HBcAg半定量呈正相关(r=0.618,P〈0.005);3.肝组织HBV cccDNA定量与肝脏炎症活动度尤明显相关(P〉0.05):4.HBeAg阳性较抗-HBe阳性患者肝组织HBV cccDNA定量、肝组织和血清HBV DNA定量高(P〈0.05)。结论荧光定量PCR法检测肝组织HBV cccDNA定量是评价HBV复制最直接可靠的指标,在CHB的诊断和抗病毒治疗中有重要意义。但与肝组织炎症无明显相关。 相似文献
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从慢性HBV感染的肝细胞中清除HBV cccDNA被认为是根除HBV的关键。在抗病毒治疗之前、期间和之后监测HBV cccDNA对于慢性乙型肝炎患者的疗效评估极为重要;随着靶向HBV cccDNA的抗HBV新药研发的不断推进,也迫切需要准确而灵敏的HBV cccDNA检测方法,以评价其疗效。近年来,为了提高HBV cccDNA定量检测的特异度,在传统的PCR定量检测方法的基础上进行了改进。同时,为了提高敏感度,还推出了HBV cccDNA的数字PCR定量检测方法等。本文综述了引入酶切的qPCR法、磁珠捕获杂交法、滚环扩增结合原位PCR法、数字PCR法和单细胞内数字PCR法等方法在HBV cccDNA定量检测方面的应用进展。 相似文献
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肝病患者血清中HBVcccDNA检测的临床意义 总被引:6,自引:0,他引:6
目的 了解cccDNA与病毒复制及拉米夫定耐药突变(YMDD)及肝脏病变的关系.方法 采用分子信标PCR技术检测HBV携带者、慢性乙型肝炎、乙型肝炎肝硬化、肝癌患者血清中cccDNA与HBVDNA及YMDD突变.结果 对283例不同HBV感染者血清进行了cccDNA检测,阳性123例(43.46%),全部为HBVDNA阳性标本;cccDNA与血清HBV-DNA相关(x2=28.27,P<0.01)及ALT相关(x2=48.46,P<0.01).68例接受拉米夫定治疗半年以上患者复查血清ALT、HBVDNA、YMDD及ccDNA,显示ALT异常32例(与cccDNA相关x2=48.46,P<0.01),HBVDNA阳性24例(与cccDNA相关x2=28.27,P<0.01),其中包括YMDD阳性18例与cccDNA阳性16例(P=0.046).结论 血清cccDNA,是反映HBV复制及肝脏细胞损伤的血清标志.监测YMDD与血清cccDNA可以提示抗病毒治疗中HBV复制状态及病变进展情况. 相似文献
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Liu MC Yu M Zhang NL Gong WB Wang Y Piao WH Wang QH Wang GQ 《Journal of viral hepatitis》2004,11(2):124-129
The 2.2.15 cells-derived from HepG2 cells transfected with a plasmid containing hepatitis B virus (HBV) DNA secrete surface antigen (HBsAg) particles, nucleocapsids and virions (Proc Natl Acad Sci U S A 1987; 84: 1005-1009). The latter elicit acute hepatitis in chimpanzees (Proc Natl Acad Sci U S A 1987; 84: 4641-4644). We studied the presence of intracellular and extracellular HBV covalently closed circular (ccc) DNA in this culture system by polymerase chain reaction (PCR), kinetically analysed HBsAg and hepatitis B e antigen (HBeAg) released in the culture media by quantitative enzyme-linked immunosorbent assay and quantitated by real-time PCR but HBV DNA from intracellular and extracellular HBV-DNA. HBV cccDNA was found both intracellularly and extracellularly. A significant correlation was seen between the extracellular HBV DNA levels and virus antigens (r = 0.833; P = 0.01 and r = 0.939; P < 0.01 for HBsAg and HBeAg, respectively), whereas there was no statistical correlation between intracellular HBV DNA levels and virus antigen levels (r = 0.024; P = 0.955 and r = 0.177; P = 0.625 for HBsAg and HBeAg, respectively). These data would be valuable in studies of the HBV life cycle and of potential anti-viral agents. 相似文献
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改良聚合酶链反应检测HBV共价闭合环状DNA 总被引:7,自引:0,他引:7
目的:建立一种基于聚合酶链反应(PCR)的简便快速、具有较高敏感性和特异性的检测乙型肝炎病毒(HBV)共价闭合环状DNA(cccDNA)的方法.方法:分别提取HepG2.2.15细胞内的cccDNA及培养上清中的松驰环DNA(rcDNA)样品,试剂盒纯化;设计2对特异性引物,其扩增区域跨越rcDNA单链区;设计2对非特异性引物,扩增区域位于rcDNA双链区.经单链特异性绿豆芽核酸酶(MBN)分别消化cccDNA及rcDNA样品;以特异性引物和非特异性引物对消化前后的两种样品分别进行PCR扩增,并改变PCR扩增参数如底物数量、循环次数等,观察特异性引物能否顺利扩增消化后的cccDNA,同时又不扩增消化后的rcDNA.HBV基因组质粒样品作为对照.此外还采用实际乙型肝炎患者体内病毒样本检验此策略的实用性.结果:分别以非特异性引物和特异性引物扩增不同模板数的HBVrcDNA样品,2对非特异性引物可扩增出模板数在102以上的HBVrcDNA样品,2对cccDNA特异性引物也可以扩增出模板数在104以上的样品.特异性引物在PCR反应模板数较多时将不能区分消化前的rcDNA和cccDNA.不同数量HBVcccDNA和rcDNA模板在MBN消化前后,分别应用非特异性引物和特异性引物进行PCR扩增,发现不同数量的cccDNA模板分子经过MBN消化后,仍可用特异性引物和非特异性引物扩增出相应条带;rcDNA样品经过MBN消化后,非特异性引物可扩增出产物条带,而特异性引物无法扩增出条带.采用此种策略,我们发现慢性乙肝患者血清HBV核酸样品主要成份为rcDNA,并带有少量cccDNA,而肝细胞内HBV核酸样品富含cccDNA,与实际情况一致.结论:联合应用MBN选择性消化和cccDNA特异性引物的PCR检测法简便快速,敏感性和特异性均较满意. 相似文献
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Liaw YF 《Journal of gastroenterology and hepatology》2002,17(Z3):S333-S337
Lamivudine is a nuleoside analog with potent inhibitory effects on hepatitis B virus (HBV) replication. Prolonged therapy is required for sustained suppression. However, HBV species with mutations in the tyrosine-methionine-aspartate-aspartate (YMDD) locus of the HBV-RNA-dependent DNA polymerase confering resistance to lamivudine may emerge after 6-9 months therapy with an incidence of 38% and 67% after 2 and 4 years of lamivudine therapy, respectively. During continued lamivudine therapy, patients with YMDD mutant HBV usually show serum alanine aminotransferase (ALT) and HBV-DNA elevations at lower median levels than their baseline. Marked flare of serum ALT or acute exacerbation may occurred as the results of cytotoxic T lymphocyte mediated immune response directed against YMDD mutant. Although viral clearance with or without emergence of distinct lamivudine resistant mutants may occur after such exacerbations, 20% of the exacerbations are complicated with decompensation or even fatality. The exacerbations appear to be more severe than those occur during the natural course of wild type HBV chronic infection. The current practice of continuing lamivudine therapy, therefore, requires careful evaluation. Alternatives include interferon therapy but this seems ineffective. Adefovir dipivoxil and entecavir may effectively suppress the YMDD mutant but these treatments have not yet been available for use. Recent studies have shown no benefit to continuing lamivudine therapy in patients with YMDD mutantions. Before a rescue drug becomes available, the most cost-effective strategy is to select patients with stronger endogenous anti-HBV immunity to increase efficacy and to shorten the duration of lamivudine therapy to avoid the emergence of YMDD mutations. 相似文献
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Pisit Tangkijvanich Piyawat Komolmit Varocha Mahachai Pattaratida Sa‐nguanmoo Apiradee Theamboonlers Yong Poovorawan 《Hepatology research》2010,40(3):269-277
Aim: The aim of this study was to compare the clinical applicability of quantitative serum hepatitis B surface antigen (HBsAg), hepatitis B e‐antigen (HBeAg) and hepatitis B virus (HBV) DNA for predicting virological response (VR) to pegylated interferon (PEG‐IFN) therapy. Methods: Thirty HBeAg‐positive chronic hepatitis B patients who received PEG‐IFN‐α‐2b for 48 weeks were enrolled. Quantitative HBsAg, HBeAg and HBV DNA were measured before, during and after the therapy. Paired liver biopsies were performed before and after treatment for covalently closed circular (ccc)DNA and intrahepatic HBV DNA analysis. Results: VR at 48 weeks post‐treatment, defined as HBeAg seroconversion and HBV DNA less than 10 000 copies/mL was achieved in 10 (33.3%) patients. Responders had significantly lower baseline HBsAg, HBeAg, cccDNA and intrahepatic HBV DNA levels than non‐responders. Baseline and reduced levels of log10 HBsAg and log10 HBeAg correlated well with those of log10 cccDNA and log10 total intrahepatic HBV DNA. Responders showed consistent decrease in serum HBsAg, HBeAg and HBV DNA levels during therapy. HBeAg level of 2.0 log10 sample to cut‐off ratio at week 24 on therapy provided the best prediction of sustained virological response, with sensitivity and negative predictive values of 85% and 92%, respectively. One patient (3.3%) who cleared HBsAg at follow up exhibited a more rapid decline in serum HBsAg during therapy than those who developed VR without HBsAg clearance. Conclusion: Quantitative measurement of serum HBeAg during therapy may be superior to serum HBsAg and HBV DNA as a prediction of HBeAg seroconversion. Kinetics of HBsAg levels on therapy may help predict HBsAg clearance after treatment. 相似文献
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目的 检测慢性乙型肝炎患者肝组织HBV共价闭合环状DNA (cccDNA)和血清HBsAg,分析两种定量指标之间及其与血清HBV DNA载量的相关性.方法 应用PSAD消化+滚环扩增+跨缺口实时荧光PCR方法,定量检测54例慢性乙型肝炎患者甲醛固定石蜡包埋肝组织HBV cccDNA水平;用化学发光试剂定量检测患者血清HBsAg.用Pearson检验及直线回归分析方法 对数据进行分析.结果 患者肝组织HBV cccDNA与血清HBsAg定量水平之间呈正相关(r=0.459,P<0.01),但与血清HBV DNA载量相关性无统计学意义;血清HBsAg定量水平与血清HBV DNA载量呈正相关(r=0.328,P< 0.05),与病毒复制效率呈负相关(r=-0.373,P<0.05).结论 慢性乙型肝炎患者肝组织HBV cccDNA载量与血清HBsAg定量水平相关,结合血清HBVDNA定量检测,可以更全面的反映HBV的复制水平,评价抗病毒疗效. 相似文献
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SUSUMU NAKAHORI OSAMU YOKOSUKA TOSHIKI EHATA WANG-LONG CHUANG FUMIO IMAZEKI YOSHIMI ITO MASAO OHTO 《Journal of gastroenterology and hepatology》1995,10(4):419-425
The precore region of hepatitis B virus (HBV) is indispensable for secretion of e antigen protein. Therefore, the precore stop codon mutants may play an important role in the process of e antigen seroconversion. However, the presence of the mutants in hepatitis B e antigen positive carriers has not been fully studied because of difficulties in detecting the mutants in the presence of large amounts of wild-type viruses. To overcome this, a sensitive method has been developed to detect the presence of G to A stop codon mutants at codon 28 of precore region. Primers for polymerase chain reaction (PCR) were devised to introduce restriction enzyme site Sty I for wild-type viruses and Dde I for the mutants. The amplification products with these primers were digested with Sty I to exclude the products from wild-type viruses. The remaining amplicon from precore mutants were re-amplified, and the presence of precore mutant was confirmed with Dde I digestion. The presence of precore mutants was examined in 61 HBV carriers by the method combining PCR and restriction enzyme digestion. Approximately 0.1% of precore mutant DNA among 106 copies of wild-type virus DNA was detectable by this method. The presence of the precore mutants was detected in seven of 10 (70%) e antigen positive asymptomatic carriers, and in 29 of 36 (81%) e antigen positive patients with chronic liver diseases, and in all 15 (100%) anti-e antibody positive patients with chronic liver diseases. This study revealed that a small amount of the precore mutants was present in the majority of HBV carriers. 相似文献