非洲马瘟病毒可视化RT-LAMP现场检测方法的建立  

Development of visual reverse transcription loop-mediated isothermal amplification assay for field detection of the African horse sickness virus

作　　者：李富祥[1] 赵文华[1] 杨仕标[1] LI Fu-xiang;ZHAO Wen-hua;YANG Shi-biao(Yunnan Tropical and Subtropical Animal Virus Diseases Laboratory/Yunnan Animal Science and Veterinary Institute,Kunming 650224,China)

机构地区：[1]云南省畜牧兽医科学院云南省热带亚热带动物病毒病重点实验室,云南昆明650224

出　　处：《中国预防兽医学报》2020年第6期579-583,共5页Chinese Journal of Preventive Veterinary Medicine

基　　金：国家重点研发计划项目(2017YFD0501805、2017YFD0501801);云南省创新人才计划项目(2017HB090)。

摘　　要：为建立非洲马瘟病毒(AHSV)可视化RT-LAMP检测方法,本研究根据AHSV外衣壳蛋白(VP7)基因保守序列设计2对特异性引物,通过反应条件优化建立了AHSV可视化RT-LAMP检测方法。结果显示,本实验所建立RT-LAMP方法的最佳反应条件为62℃1 h;利用该方法检测进口灭活冻干疫苗中的9个血清型AHSV以及马流感病毒、马传贫病毒、马动脉炎病毒和蓝舌病病毒的RNA,结果显示所有血清型AHSV RNA检测均为阳性,其它病毒RNA检测为阴性,该方法特异性较强;该方法最低可检测到3.2×10-9 ng/μL的RNA,敏感性是普通RT-PCR方法的1000倍。利用建立的RT-LAMP方法和普通RT-PCR方法对35份马血和5份驴血样品同时进行检测,结果显示二者阳性和阴性符合率均为100%。本研究在国内外首次建立了AHSV 9种血清型的可视化RT-LAMP检测方法,其具有特异性强、敏感性高、快速和高通量检测的优点,为非洲马瘟(AHS)快速的可视化现场诊断提供可行方法。To establish a visual detection method for African horse sickness virus(AHSV),two pairs of specific primers were designed according to the conserved region of the outer capsid proteins VP7 gene of AHSV,and the RT-LAMP assay was developed by optimization of reaction conditions.The established RT-LAMP reaction was carried out at 62℃for 1 h.The method was used to detect RNA samples of all 9 serotypes of AHSV,which were from imported,inactivated and freeze-dried AHSV vaccines,as well as equine influenza virus(EIV),equine infectious anemia virus(EIAV),equine arteritis virus(EAV)and bluetongue virus(BTV),respectively,and the results showed that the method had high specificity which could detected RNA samples of all 9 serotypes AHSV and had no cross-amplifications with RNA samples of the other viruses.The RT-LAMP assay has high sensitivity with the detection limit of 3.2×10-9 ng/μL of the RNA,1000-fold more sensitive than conventional RT-PCR method.The RT-LAMP and conventional RT-PCR were used simultaneously to detect 35 horse blood and 5 donkey blood samples,and the results showed that the positive and negative coincidence rates were 100%.In this study,visualization RT-LAMP that detected nine serotypes of AHSV were established for the first time at home and abroad,which has the advantages of strong specificity,high sensitivity,fast and high throughput detection,it provided a feasible method for the rapid visual field diagnosis of AHS.

关 键 词：非洲马瘟病毒 反转录环介等温扩增 可视化现场检测 

分 类 号：S855.3[农业科学—临床兽医学]