改良聚合酶链反应检测HBV共价闭合环状DNA  

Detection of hepatitis B virus cccDNA with modif ied polymerase chain reaction

作　　者：汤勃[1] 王宇明[1] 刘俊[1] 张瑞[1] 

机构地区：[1]第三军医大学西南医院全军感染病研究所,重庆市400038

出　　处：《世界华人消化杂志》2005年第18期2188-2192,共5页World Chinese Journal of Digestology

基　　金：国家自然科学基金资助项目;No.30230320~~

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.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 specificity. METHODS： The cccDNA and the relaxed circular DNA （rcDNA） were extracted from HepG2.2.15 cells and supernatant, respectively, and then purified. Two pairs of specific 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 amplified by specific primes and non-specific primes. Whether the digested cccDNA can be amplified by specific primes, without amplifying the digested rcDNA, was observed. The PCR parameters such as substrate amount and circulation times were changed during amplification. 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 were amplified by specific and non-specific primes. More than 104 and 102 rcDNA template molecules were amplified by two pairs of specific and non-specific primes, respectively. The specific primes could not discriminate between rcDNA and cccDNA when the template molecules were overabundant. Before and after the digestion by MBN, different amounts of cccDNA were amplified by specific and non-specific primes; and after the digestion, rcDNA templates were amplified by nonspecific 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 samples from hepatocytes contained mainly cccDNA. CONCLUSION： The combination of MBN selective digestion and specific PCR amplification of the cccDNA is a simple, fast, sensitive and specific method for the detection of HBV cccDNA.

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 

分 类 号：R446.5[医药卫生—诊断学]