犬瘟热病毒N基因环介导等温扩增检测方法的建立  

Establishment of method for detecting the N gene of canine distemper virus by loop-mediated isothermal amplification

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

出　　处：《中国预防兽医学报》2013年第2期138-141,共4页Chinese Journal of Preventive Veterinary Medicine

基　　金：国家科技支撑计划(2012BAD12B03)

摘　　要：为建立犬瘟热病毒(CDV)环介导等温扩增(LAMP)检测方法,本研究根据GenBank中CDVN基因保守序列设计合成了2对LAMP引物,内引物FIP、BIP和外引物F3、B3。以CDVN基因重组质粒(pMD-CDV-N)为模板,经反应条件优化、特异性试验、敏感性试验及临床样品检测。检测结果显示,该方法于61℃水浴60min即可完成反应,最低检出量为70个拷贝,比RT-PCR灵敏10倍。该检测方法仅对CDV产生阳性扩增,而对狂犬病毒,犬细小病毒,犬腺病毒Ⅱ型扩增结果均为阴性。用建立的LAMP方法检测临床样品,检测结果与RT-PCR检测结果一致。本方法简便,特异,有利于CDV的临床检测。In this study, a loop-mediated isothermal amplification （LAMP） method for the rapid detection of canine distemper virus （CDV） was developed with a set of primers to amplify nucleocapsid （N） gene of CDV. Under the optimized conditions of LAMP, the results showed that CDV N gene was spciefically amplified at 61 ~C for 60 min in water bath with a detection limit of approximately 70 copies of CDV, which was ten times more sensitive than RT-PCR, and no cross-reaction with rabies virus, canine parvovirus and canine adenovirus II （CAV II）. In addition, the results gathered by detecting the clinical samples using the LAMP were totally in agreement with the RT-PCR assay. As it is an easy and simple method, the LAMP is potentially applicable for canine distemper diagnosis.

关 键 词：犬瘟热病毒 N基因 环介导等温扩增 

分 类 号：S852.65[农业科学—基础兽医学]