兔出血症病毒可视化RT-LAMP检测方法的建立与应用  

Development and application of RT-LAMP for visual detection of rabbit hemorrhagic disease virus

作　　者：刘星星[1] 耿毅[2] 孙娜[1] 陈勤[1] 杨泽晓[2] 李亚军[2] 牟维豪 

机构地区：[1]新疆医科大学厚博学院克拉玛依校区,新疆克拉玛依834000 [2]四川农业大学动物医学院,四川成都611130

出　　处：《中国兽医科学》2017年第6期687-693,共7页Chinese Veterinary Science

基　　金：四川省科技支撑计划项目(No.2016NZ0002)

摘　　要：为建立一种可视化的兔出血症病毒RT-LAMP检测方法,根据兔出血症病毒(rabbit hemorrhagic disease virus,RHDV)的主要结构蛋白VP60基因的序列设计合成4条特异性引物,以重组pMD19-T-VP60质粒为模板,通过反应条件与反应体系优化,建立RHDV的环介导等温扩增(LAMP)检测方法,测定RT-LAMP对RHDV的最低检测限,并与普通RT-PCR进行比较。结果显示,该RT-LAMP的最佳反应温度为62℃,反应时间为40 min,对RHDV的最低检测限为50 copies/L,常规RT-PCR的检测限为5×10~3copies/L,表明建立的RT-LAMP的灵敏度显著高于常规RT-PCR。建立的RT-LAMP的全部反应可在1 h内完成;反应结束后加入荧光染料Et Br可肉眼判定粉色为阳性,而棕色则为阴性,且与兔巴氏杆菌、大肠杆菌、沙门菌、产气荚膜梭菌、无乳链球菌及肺炎克雷伯菌无交叉反应。上述结果表明,建立的RT-LAMP简便、快速、灵敏、特异,且不需昂贵仪器设备,适合在基层推广应用。To establish a visible RT-LAMP for detection of rabbit hemorrhagic disease virus,ac- cording to the sequence of major structural protein VP60 gene of RHDV in GenBank,a set of four specific primers was designed and synthesized. Using recombinant pMDIg-T-VP60 plasmid as a template,the LAMP for detection of RHDV was developed by optimizing its reaction system and conditions, lts detection limit for RHDV was determined by detecting the fold-diluted pMDIg-T-VP60 plasmid. The optimized reac- tion temperature and time of RHDV-LAMP were at 62~Cand 40 min,respectively, rhe detection limit of the LAMP assay was 50 copies/~ L,while the detection limit of the conventional PCR was 5 X 103copies/~ L, suggesting that the RT-LAMP method was more sensitive than the traditional RT-PCR. The LAMP amplifica- tion products was visually inspected after addition of ethidium bromide：positive samples turned pink, while negative samples and no template control reactions remained orange. Moreover,no amplification was found with the samples of Pasteurella,Escherichia coli, Salmonella,Clostridium welchii,Strepto-coccus agalactiae and Klebsiella pneumoniae. The above-mentioned results showed that the developed RT-LAMP in this study was more convenient,rapid,highly sensitive and specific than that of the tradi- tional RT-PCR,and it did not require expensive equipment,which was more suitable for field testing.

关 键 词：兔出血症病毒 RT-LAMP RT-PCR VP60基因 

分 类 号：S852.659.6[农业科学—基础兽医学]