兔出血症病毒实时荧光定量RT-PCR检测方法的建立  

Establishment of Real-time RT-PCR for Detecting Rabbit Haemorrhagic Disease Virus

作　　者：肖跃强[1,2] 谢金文[2] 徐二丹[3] 沈志强[2] 丁壮[1] 

机构地区：[1]吉林大学动物医学学院,吉林长春130062 [2]山东省滨州畜牧兽医研究院,山东滨州256600 [3]泰山医学院,山东泰安271016

出　　处：《动物医学进展》2013年第12期69-73,共5页Progress In Veterinary Medicine

基　　金：中国农业科学院兰州兽医研究所家畜疫病病原生物学国家重点实验室开放基金课题

摘　　要：为建立一种敏感、特异、简便的兔出血症病毒(RHDV)检测方法,根据GenBank中公布的RHDV全基因组序列保守区域设计了2对引物,筛选了其中1对建立并优化了PCR条件,扩增产物为331bp,并基于此建立了SYBR GreenⅠ荧光染料real-time RT-PCR检测方法。通过敏感性、特异性、标准曲线建立等表明,该方法可检出101以上拷贝数的模板,只在RHDV有特异性扩增,标准曲线线性相关性好。实际应用结果显示,该方法对疑似RHDV病料检测的阳性率为74.8%,而普通RT-PCR方法检测的阳性率为65.5%。该方法的建立为开展该病的诊断、疫苗制备质控等提供了有效的技术保障。To establish a sensitive,specific and simple diagnosis method for RHDV,2 pairs of primers were designed according to the conservative sequences of rabbit haemorrhagic disease virus（RHDV）published in GenBank,and one pair of them was screened and the reaction system of PCR was optimized,by which a real-time RT-PCR was established based on SYBR Green I fluorescent dye.This method was much more sensitive and specific,a minimum of 101 copies of viral cDNA or more could be detected,only RHDV was detectable among RHDV,rabbit rotavirus and rabbit vesicular stomatitis virus.A standard curve with high linear correlation was also established.The application results showed that the positive detectable ratio of the real-time RT-PCR was 74.8%,but only 65.5% with the RT-PCR.This real-time PCR can be used for diagnosis of RHD and quality control for vaccine production.

关 键 词：兔出血症病毒 实时荧光定量逆转录-聚合酶链反应 建立 

分 类 号：S852.659.6[农业科学—基础兽医学] Q789[农业科学—兽医学]