犬细小病毒环介导等温扩增检测方法的建立  

Development of a loop-mediated isothermal amplification for detection of canine parvovirus

作　　者：关玮琨[1] 何丽丽[1] 冯瑜菲[1] 朱颖[1] 刘文鑫[1] 江馗语[2] 胡文霞[3] 师东方[1] 

机构地区：[1]东北农业大学动物医学学院,黑龙江哈尔滨150030 [2]辽宁省重大动物疫病应急中心,辽宁沈阳110000 [3]哈尔滨维科生物技术开发公司,黑龙江哈尔滨150030

出　　处：《中国兽医科学》2013年第4期407-411,共5页Chinese Veterinary Science

基　　金：国家科技支撑计划项目(2012BAD12B03;2012BAD12B05)

摘　　要：根据GenBank中犬细小病毒(CPV)VP2基因序列,利用Primer Explorer V4软件设计并合成了针对CPV VP2基因序列保守区域的4条寡核苷酸片段(F3/B3,FIP/BIP)作为LAMP引物。以CPV DNA为模板,通过对LAMP反应温度和时间的优化以及特异性试验、敏感性试验和对临床样品的检测,建立了一种针对CPV VP2基因的LAMP快速检测方法。结果显示,该LAMP检测方法只对CPV有梯状扩增条带,对犬瘟热病毒(CDV)、狂犬病病毒(RV)和犬腺病毒Ⅱ型(CAV-Ⅱ)无扩增条带;对CPV的最低检出量为2.1×10-2 TCID50,其敏感性约为常规PCR检测方法(最低检出量为2.1TCID50)的100倍,是犬细小病毒抗原快速检测试纸(最低检出量为101.5 TCID50)的1 500倍。临床样品的LAMP检测结果与PCR检测结果的符合率为100%。The objective of this study was to establish a loop-mediated isothermal amplification （LAMP） method for the detection of VP2 gene in canine parvovirus （CPV）. Primers targeting VP2 gene of CPV were designed by the Primer Explorer version 4 software online. A set of four primers was designed based on the conserved sequence of VP2 gene. Temperature and time condition, specificity test, sensitivity test and clinical samples assay of LAMP were performed. In result, the LAMP method was no cross-reaction with canine distemper virus（CDV）, rabies virus（RV） and canine adenovirus type Ⅱ （CAV-Ⅱ ）. The detection limit equivalent was 2.1 × 10-2TCIDs0 of CPV,which was 10 times more sensitive than the PCR, and 1 500 times more sensitive than rapid CPV Ag test kit. The agreement rate between the LAMP and the PCR was 100% in detection clinical samples. In conclusion, the LAMP maybe became a novel method for the rapid diagnosis of CPV.

关 键 词：犬细小病毒 环介导等温扩增 检测 

分 类 号：S852.659.2[农业科学—基础兽医学]