犬瘟热病毒反转录环介导等温扩增诊断方法的建立  

Development of a reverse transcriptase loop-mediated isothermal amplification(RT-LAMP) system for diagnosis of canine distemper virus

作　　者：刘彩玉[1,2] 王吉贵[2] 袁道莉[2] 王爽[2] 朱新产[1] 刘维全[2] 

机构地区：[1]青岛农业大学生命科学学院,山东青岛266109 [2]中国农业大学生物学院,北京100193

出　　处：《中国兽医科学》2010年第4期368-372,共5页Chinese Veterinary Science

摘　　要：根据GenBank中犬瘟热病毒H基因保守序列设计合成了内(FIP,BIP)、外(F3,B3)2对引物,其中外引物扩增片段的大小为247 bp。通过对反应条件中内、外引物的比例、dNTP浓度、Mg2+浓度、反应温度和时间等条件逐一优化,成功建立了犬瘟热病毒的RT-LAMP检测方法。该方法在60℃下保温50 min即可完成,可检测至10-1TCID50的病毒,与常规RT-PCR检测方法相比灵敏度高100倍。对犬腺病毒、犬细小病毒、狂犬病病毒、犬冠状病毒、犬瘟热病毒同时检测,结果显示,本方法具有高度的特异性。Based on conserved sequence of canine distemper virus（CDV） H gene in GenBank,two pairs of primers（external primers F3 and B3,and internal primers FIP and BIP）were designed and synthesized.The fragment of amplification was 247bp in size using the external primers.After optimization of the ratio of the internal to the external primers,concentration of dNTP and Mg^2 ＋,temperature and time of reaction,a RT-LAMP method for specific detection of CDV was established successfully.It was carried out at 60℃ for 50min,and its sensitivity was 10^-1 TCID50,which was 100 times more than the traditional RT-PCR method.The detection of canine adenovirus,canine parvovirus,rabies virus and canine coronavirus by the developed RT-LAMP was negative,indicating that the RT-LAMP possessed good specificity.

关 键 词：犬瘟热病毒 反转录环介导等温扩增反应 诊断 

分 类 号：S852.659.5[农业科学—基础兽医学]