鸭乙型肝炎病毒荧光定量PCR检测方法的建立及应用  

Development and application of real-time PCR assay for detection of duck hepatitis B virus

作　　者：刘扬科[1] 郑立运[2] 常俊标[2] 王川庆[1] 杨霞[1] 王庆端[2] 陈陆[1] 赵军[1] 杨猛[1] 张金来[1] 

机构地区：[1]河南农业大学禽病研究所,河南郑州450002 [2]郑州大学医药科学研究院,河南郑州450052

出　　处：《扬州大学学报（农业与生命科学版）》2010年第3期7-12,共6页Journal of Yangzhou University：Agricultural and Life Science Edition

基　　金：国家新药创制重大专项项目(2009ZX09103-110)

摘　　要：为建立一种快速、特异的鸭乙型肝炎病毒(DHBV)SYBR GreenⅠ荧光定量PCR检测方法,设计一对引物扩增DHBV基因组,将其克隆到pMD-18-T载体上,然后以该重组质粒为模板,选择DHBV S基因保守序列设计一对定量PCR引物,构建标准曲线,进行特异性、敏感性和重复性试验,并用所建立的方法评价拉米夫定和阿德福韦酯在鸭体内对DHBV-DNA复制的抑制效果。结果表明:该方法能特异性检测到DHBV;相关系数为1.00,扩增效率为105%;敏感度高,最低检测限为70个拷贝。拉米夫定和阿德福韦酯对DHBV感染后鸭外周血DHBV-DNA的复制均有很好的抑制效果,但停药后会出现轻度"反跳"现象。建立的DHBV SYBR GreenⅠ荧光定量PCR检测方法具有特异、灵敏、快速等优点,可用于抗HBV药物的评价。A rapid and specific SYBR GreenⅠReal-timePCR assay was established to detect duck hepatitis B virus（DHBV）.A pair of primers was designed for amplifying the genome of DHBV which was cloned into pMD-18-T.Based on the conserved sequences of DHBV S gene,another pair of primers for Real-time PCR was designed and used to amplify the recombinant plasmid for constructing the standard curves.Meanwhile,the specificity,sensitivity and repeatability of the assay were tested.The inhibition rate of DHBV-DNA replication by Lamivudine or adefovir dipivoxil was evaluated with the established method.The results showed that the Real-time PCR assay was highly specific and the regression coefficient was 1.00 with 105% PCR amplification efficiency.It had a detection threshold of 70 copies of plasmid DNA and was highly sensitive.Lamivudine and adefovir dipivoxil were proved to be an efficient inhibitor for the wads of DHBV-DNA from blood and hepatic tissue,except for the rebound phenomenon after withdrawal.The established SYBR GreenⅠReal-time PCR for detecting duck hepatitis B virus is rapid,specific and sensitive,and can be used as a useful tool for evaluating effect of anti-HBV drug.

关 键 词：鸭乙型肝炎病毒 SYBRGreen I 荧光定量PCR 

分 类 号：S852.659.1[农业科学—基础兽医学] R512.62[农业科学—兽医学]