摘要
为了建立一种新型、稳定、可普遍应用的诊断犬瘟热病毒(Canine distemper virus,CDV)的方法,试验采用环介导等温扩增技术(loop-mediated isothermal amplification,LAMP)-横向流动试纸条(lateral flow dipstick,LFD)相结合的方法,根据CDV N基因序列中同源性相对较高的保守区设计一套标有生物素(biotin)的特异性引物,结合异硫氰酸(FITC)荧光素标记的扩增产物,建立RT-LAMPLFD检测方法,对该方法的反应条件和反应体系进行优化,同时分析敏感性及特异性,最后检测临床应用情况。结果表明:试验成功构建CDV RT-LAMP-LFD方法,此方法的最佳反应条件为60℃反应60 min;最佳的反应体系为镁离子浓度12 mmol/L,甜菜碱浓度0. 2 mmol/L,Bst DNA聚合酶活性8 U,AMV酶活性5 U,外引物与环引物比例1∶2;最低检测下限为1×10~2个拷贝/m L,能够特异地检测出CDV的存在,犬细小病毒(CPV)等其他病毒均为阴性,可以检测出病犬口鼻腔黏液中的CDV。说明试验建立的CDV RT-LAMP-LFD检测方法特异性强,敏感性高,操作简单,可广泛应用。
In order to establish a new,stable and widely applicable method for the diagnosis of Canine distemper virus(CDV),a combination of loop-mediated isothermal amplification(LAMP)and lateral flow dipstick(LFD)was used in this experiment.According to CDV N gene sequence homology in the relatively conservedregion,a biotin-marked specific primers were designed,combined with the isothiocyanate(FITC)of the labeled amplification products,for the establishment of a RT-LAMP-LFD detection method.The reaction conditions of the method and the reaction system were optimized,the sensitivity and specificity were analyzed,and it was used finally for clinical application.The results showed that CDV RT-LAMP-LFD method was successfully constructed in this experiment.The optimum reaction conditions were 60 min at 60℃.The optimum reaction system was 12 mmol/L of magnesium ion,0.2 mmol/L of betaine,8 U of Bst DNA polymerase,5 U of AMV enzyme,and 1∶2 ratio of external primers to ring primers.The lowest detection limit was 1×102 copies/m L,which could specifically detect the existence of CDV.Other virusessuch as Canine parvovirus(CPV)were negative,and CDV could be detected in the mouth and nasal mucus of diseased dogs.The results suggested that the CDV RT-LAMP-LFD method established in this experiment had strong specificity,high sensitivity,simple operation,and could be widely used.
作者
郝镯
李楠
张珊珊
李吉平
何洪彬
孟轲音
李筱鑫
万忠海
穆玉堂
李忠义
田多
李晓娜
王承宇
HAO Zhuo;LI Nan;ZHANG Shanshan;LI Jiping;HE Hongbin;MENG Keyin;LI Xiaoxin;WAN Zhonghai;MU Yutang;LI Zhongyi;TIAN Duo;LI Xiaona;WANG Chengyu(Jilin Province Key Laboratory of Zoonosis Prevention and Control,Institute of Military Medical Sciences,Academy of Military Sciences,Changchun 130122,China;Shandong Normal University,Jin’an 250014,China)
出处
《黑龙江畜牧兽医》
CAS
北大核心
2019年第17期1-6,180,共7页
Heilongjiang Animal Science And veterinary Medicine
基金
国家重点研发计划项目(2016YFD0501001)
