犬细小病毒实时荧光环介导等温扩增检测方法的建立  

Development of a real-time fluorescent loop-mediated isothermal amplification for the detection of canine parvovirus

作　　者：陈珍容[1] 陈进会[2] 黄杰[1] 龚永平[1] 文继峰[1] 郗立新 颜其贵[1] 

机构地区：[1]四川农业大学动物医学院,四川成都611130 [2]东莞出入境检验检疫局,广东东莞523072

出　　处：《中国兽医科学》2017年第5期577-581,共5页Chinese Veterinary Science

基　　金：成都大熊猫繁育研究基金会项目(CFP研2012-12;CFP研2012-9)

摘　　要：参照GenBank中犬细小病毒(CPV)VP2基因保守区域序列设计合成特殊的内、外引物,在扩增反应体系中加入荧光染料SYBR GreenⅠ,通过恒温荧光检测仪Deaou-308C扩增检测,建立了一种快速检测CPV的实时荧光环介导等温扩增(LAMP)方法。结果显示,这种方法对CPV具有特异性的扩增反应,对照病毒核酸的扩增结果均为阴性。灵敏度试验最低可检出3.72 copies/μL的病毒核酸。重复性试验结果表明,其检测重复性良好。对30份临床宠物犬的粪便样品进行检测,与免疫胶体金检测试纸方法进行比较,符合率为90%。将本方法初步应用于大熊猫粪便中犬细小病毒的检测,结果表明,从大熊猫基地采集的84份粪便样品中有16份为细小病毒阳性,阳性率为19.0%。本方法在63℃下恒温45 min可以完成检测,操作简单、灵敏度高、特异性强,通过恒温实时荧光检测仪可实时检测及自动判读检测结果,对大熊猫的早期诊断非常重要。The special primers were designed based on the published sequence of the VP2 gene of canine parvovirus,and the fluorescent dye SYBR Green I was added into the amplification reaction. By using Deaou-308C constant temperature fluorescence detector,we established a real-time fluorescent loopmediated isothermal amplification （LAMP）method for rapid detection of CPV. The results showed that this method had the specific amplification for CPV,while the results of control of viral nucleic acid amplification were negative,the minimum detectable sensitivity was 3. 72 copies/μL virus nucleic acid. The repeatability test results showed that this method had good reproducibility. Suspected disease materials had been teseted by LAMP,and the LAMP results were compared with immuune colloidal goldtechnique and the coincidence rate was up to 90.0%.This method is further applied for the detection of canine parvovirus using the faeces from the giant panda,and the results displayed that 16 out of 84 fecal samples collected from panda base were parvovirus-positive,and the positive rate was 19%.This method is finished at a constant temperature of 63 ℃ for 45 min,easy to operate and has high sensitivity and specificity. The detecting results can be real-time detection and be automaticlly interpretated from the constant temperature fluorescence detector,which is very important for early diagnosis.

关 键 词：犬细小病毒 实时荧光环介导等温扩增 大熊猫 

分 类 号：S852.659.2[农业科学—基础兽医学]