| 改良聚合酶链反应检测HBV共价闭合环状DNA |
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| 引用本文： | 汤勃,王宇明,刘俊,张瑞. 改良聚合酶链反应检测HBV共价闭合环状DNA[J]. 世界华人消化杂志, 2005, 13(18): 2188-2192 |
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| 作者姓名： | 汤勃 王宇明 刘俊 张瑞 |
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| 作者单位： | 第三军医大学西南医院全军感染病研究所,重庆市,400038 |
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| 基金项目： | 国家自然科学基金资助项目,No.30230320~~ |
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| 摘 要： | 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.
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| 关 键 词： | 肝炎病毒 乙型 共价闭合环状DNA 聚合酶链反应 绿豆芽核酸酶 |
| 修稿时间： | 2005-07-19 |
Detection of hepatitis B virus cccDNA with modified polymerase chain reaction |
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| Bo Tang,Yu-Ming Wang,Jun Liu,Rui Zhang. Detection of hepatitis B virus cccDNA with modified polymerase chain reaction[J]. World Chinese Journal of Digestology, 2005, 13(18): 2188-2192 |
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| Authors: | Bo Tang Yu-Ming Wang Jun Liu Rui Zhang |
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| Affiliation: | Bo Tang,Yu-Ming Wang,Jun Liu,Rui Zhang,Department of Infec-tious Diseases,Southwest Hospital of the Third Military Medical Uni-versity,Chongqing 400038,China |
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| Abstract: | AIM: To establish a simple and fast hepatitis B virus covalently closed circular DNA(cccDNA) detecting method based on polymerase chain reaction(PCR) with satisfactory sensitivity and specifi city.METHODS: The cccDNA and the relaxed circular DNA (rcDNA) were extracted from HepG2.2.15 cells and su-pernatant, respectively, and then purifi ed. Two pairs of specifi c PCR primes were designed to cover the single strand area of rcDNA. And two pairs of non-specific PCR primes were designed to cover the double strand area of rcDNA. Before and after digested by single-strand-specific mung bean nuclease(MBN), cccDNA and rcDNA samples were amplif ied by specif ic primes and non-specif ic primes. Whether the digested cccDNA can be amplifi ed by specif ic primes, without amplifying the digested rcDNA, was observed. The PCR param-eters such as substrate amount and circulation times were changed during amplif ication. The HBV genome plasmid was used as control; and the HBV samples from patient with hepatitis B was used for practical test.RESULTS: Different amounts of rcDNA template wereamplified by specific and non-specific primes. More than 104 and 102 rcDNA template molecules were am-plif ied by two pairs of specif ic and non-specif ic primes, respectively. The specific primes could not discrimi-nate between rcDNA and cccDNA when the template molecules were overabundant. Before and after the digestion by MBN, different amounts of cccDNA were amplifi ed by specif ic and non-specif ic primes; and after the digestion, rcDNA templates were amplif ied by non-specific primes, but not by specific primes. With this strategy, we found the virus samples from the serum of the patient with chronic hepatitis B contained mainly rcDNA and a small quantity of cccDNA, while the sam-ples from hepatocytes contained mainly cccDNA.CONCLUSION: The combination of MBN selective di-gestion and specif ic PCR amplif ication of the cccDNA is a simple, fast, sensitive and specifi c method for the detection of HBV cccDNA. |
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| Keywords: | Hepatitis B virus Covalently closed circular DNA Polymerase chain reaction Mung bean nuclease |
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