应用竞争性荧光定量聚合酶链反应检测乙型肝炎病毒DNA  

Detection of hepatitis B virus DNA by competitive fluorescence quantitative polymerase chain reaction

作　　者：刘蓉[1] 李明远[1] 张发强[2] 夏增亮[2] 闫乃红[2] 王玲[2] 卢亦路[2] 夏庆杰[2] 陈清英 卢军 夏军 

机构地区：[1]四川大学华西基础医学与法医学院微生物学教研室,四川省成都市610041 [2]四川大学华西分子遗传实验室,四川省成都市610041 [3]重庆市涪陵区生殖健康保健中心,重庆市40800

出　　处：《世界华人消化杂志》2007年第12期1421-1424,共4页World Chinese Journal of Digestology

基　　金：国家自然科学基金资助课题;No.39880025~~

摘　　要：目的:建立竞争性荧光定量聚合酶链反应(CFQ-PCR),并探讨CFQ-PCR在乙型肝炎病毒(HBV)临床检测中的意义.方法:根据HBV病毒adr亚型基因组序列合成一对HBV特异的引物,和一条特异的TaqMan探针;根据上述引物序列,采用分子克隆技术制备内对照DNA;再根据内对照序列合成一条内对照DNA特异的与上述TaqMan探针不同标记的TaqMan探针;将适量的内对照DNA加入到PCR反应体系中,使其与HBV靶序列共扩增.结果:在30μL CFQ-PCR反应体系中,加入约20拷贝内对照DNA能够稳定地获得共扩增曲线;经琼脂糖凝胶电泳分析,加入约100-500拷贝内对照DNA能够有效地获得共扩增产物条带信号;在210个临床HBsAg阳性血清标本的CFQ-PCR扩增中识别出8个未能有效扩增的标本,60份HBsAg阴性血清标本中识别出2个内对照未能有效扩增的标本,后经DNA纯化处理,上述全部标本的内对照均获得阳性扩增结果,其中有7个HBsAg阳性血清标本获得HBV DNA扩增阳性结果.结论:CFQ-PCR能够有效地提示临床标本HBV DNA体外扩增时由于扩增失败导致的假阴性,适合临床推广应用.AIM： To set up a competitive fluorescence quantitative polymerase chain reaction （CFQ- PCR） for decreasing the false negative ratio in HBV DNA detection. METHODS： According to HBV adr subtype gene, a pair of HBV-specific primer as well as a specific TaqMan probe was synthesized. Based on the above primer sequences, a constructed inner control DNA and an inner TaqMan probe was constructed. Right amount of inner control DNA was added into the HBV FQ-PCR system for co-amplification with the target HBV DNAs. RESULTS： In a 30-1μL CFQ-PCR system, about 20 copies of inner control DNA produced a stable amplification curve. Electrophoresis showed co-amplification products bands as about 100 to 500 copies of inner control DNA were used. In 8 of 210 （4.3%） cases with HBsAg positive in serum, both the inner control DNA and HBV DNA were not amplified, while in 2 of 60 （3.3%） samples with HBsAg negative in serum, the inner control DNA was not amplified. But after purification, all the above cases that failed in amplification succeeded in the inner DNA amplification, of which 7 HBsAg-positive cases succeeded in HBV DNA amplification. CONCLUSION： CFQ-PCR has a big advantage in telling out the false negative result in the HBV DNA PCR assay.

关 键 词：乙型肝炎病毒 竞争性荧光定量聚合酶链反应 假阴性 

分 类 号：R450[医药卫生—治疗学]