实时荧光定量聚合酶链反应检测鸭乙型肝炎病毒方法的建立及应用  

Establishment and preliminary application of a real-time Fluorescence Quatitative PCR method for detection of DHBV

作　　者：陈宗艳[1] 程安春[1] 汪铭书[1] 徐大为[1] 郭宇飞[1] 陈孝跃[2] 

机构地区：[1]四川农业大学动物科技学院禽病防治研究中心,四川雅安625014 [2]四川省动物疫病与人类健康重点实验室,四川雅安625014

出　　处：《中国兽医学报》2008年第2期140-145,共6页Chinese Journal of Veterinary Science

基　　金：国家科技攻关重大项目(2004BA901A03);四川省生物技术基金资助项目(05NG018-03);四川省重点建设学科基金资助项目(SZD0418)

摘　　要：根据鸭乙型肝炎病毒(duck hepatitis B virus,DHBV)P基因上的保守序列设计并合成引物和荧光标记探针,建立了荧光定量聚合酶链反应检测方法。本试验建立的标准曲线循环阈值(Ct值)与模板浓度具有良好的线性关系,相关系数为0.996;将其检测极限的Ct值通过标准曲线换算成拷贝数约为5 copies/L,相当于5个DHBV颗粒。初步结果表明,FQ-PCR检测DHBV-DNA能直接反映DHBV感染鸭后血清和肝组织中DHBV-DNA水平,在判断体内DHBV是否复制、复制的程度以及是否被清除等方面明显优于常规PCR,为DHBV感染动物模型的建立提供了敏感和可靠的检测手段,有很好的应用前景和研究价值。将所建立的方法运用于检测拉米夫定治疗DHBV感染鸭后外周血和肝组织DHBV-DNA的含量,以评价拉米夫定治疗DHBV的效果。结果表明拉米夫定对DHBV的复制有较好的抑制效果,但停药后出现反跳现象。According to the specific sequence of DHBV P genes,the primers and fluorescent probe were designed and synthesized.The standard curve revealed the linear relationship between CT（cycle threshold） and template concentration with a good correlation（r=0.996）Sensitivity was 5 copies of DHBV genome.The FQ-PCR method for the detection of DHBV was accurate and had a high degree of sensitivity.To investigate the Lamivudine efficacy in treating DHBV infection,the loads of DHBV-DNA from blood and hepatic tissue were measured by FQ-PCR.Lamivudine was proved to be an efficient inhibitor for DHBV replication.Except for the rebound phenomenon after withdrawal.

关 键 词：鸭乙型肝炎病毒 乙型肝炎病毒 FQ-PCR 拉米夫定 

分 类 号：S852.65[农业科学—基础兽医学]