禽腺病毒4型环介导等温扩增检测方法的建立  

Establishment of LAMP method for detection of fowl adenovirus serotype 4

作　　者：薛晓岩 张振兴 季佳 杨钦鸿 王位 代红炀 李素华 宋建领[2] XUE Xiao-yan;ZHANG Zhen-xing;JI Jia;YANG Qin-hong;WANG Wei;DAI Hong-yang;LISu-hua;ONG Jian-ling(College of Life Sciences,Southwest Forestry University,Kunming 650224,China;Yunnan Academy of Animal Husbandry and Veterinary,Yunnan Tropical and Subtropical Animal Virus Diseases Laboratory,Kunming 650224,China;The Management Bureau of Huize Black Necked Crane National Nature Reserve,Qujing 654200,China)

机构地区：[1]西南林业大学生命科学学院,云南昆明650224 [2]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 [3]会泽黑颈鹤国家级自然保护区管护局,云南曲靖654200

出　　处：《中国兽医科学》2023年第3期285-291,共7页Chinese Veterinary Science

基　　金：云南省应用基础研究计划重点项目(2016FA013);云南国际性高原湿地迁徙型涉禽禽流感等动物疫病监测研究(1963037)。

摘　　要：为了建立禽腺病毒4型(FAdV-4)的环介导等温扩增(LAMP)检测方法,本研究以FAdV-4 hexon基因高度保守的区域设计LAMP引物,并对反应体系和反应条件进行优化,同时评价该方法的特异性、灵敏度及对临床样品的适用性。结果显示,本研究建立的LAMP方法的最佳反应条件为63℃扩增65 min;反应体系中内外引物最佳比例为8∶1,MgSO_(4)和dNTP浓度分别为60 mmol/L和10 mmol/L;与FAdV-10、FAdV-8b、FAdV-11、NDV、IBDV、AIV-H9、IBV、ILTV、ORT、CIAV、ALV等均无交叉反应,具有良好的特异性;本方法可检测的DNA最低浓度为7.5×10^(-8)ng/μL,灵敏度是传统PCR法的100倍。用本研究建立的LAMP法和PCR法对80份临床样品进行检测,结果相符率为90.5%。上述结果表明,本研究建立的FAdV-4 LAMP检测方法具有准确性高、特异性强、灵敏度高和临床应用可行性强的优点,且结果易于判定,便于现场检测,为FAdV-4提供了一种新的现场诊断方法。The purpose of this study is to establish a loop-mediated isothermal amplification(LAMP)detection approach for fowl adenovirus serotype 4(FAdV-4)in grass-roots veterinary stations.Based on the highly conserved region of FAdV-4 hexon gene,the specific primers for LAMP visual detection method were designed.The reaction system and conditions were optimized according to the visualization of the amplification result with the adding of generic intercalating dyes.Furthermore,the specificity,sensitivity,and applicability of primers were evaluated by 80 clinical samples.The optimum reaction conditions of LAMP method established in this study were performed at 63℃ for 65 min,with the optimum ratio of internal and external primers for 8:1,60 mmol/L MgSO_(4) and 10 mmol/L dNTP.No cross-reaction with other avian pathogens was founded by the established LAMP methods here,including FAdV-10、FAdV-8b、FAdV-11、NDV、IBDV、AIV-H9、IBV、ILTV、ORT、CIAV and ALV.It demonstrated that the detecting results of 80 clinical samples were consistent between the established LAMP and conventional PCR assay in a rate of 90.5%.However,the sensitivity of LAMP was 100 times higher than that of PCR,with detection limit of 7.5×10^(-8)ng/μL of DNA template.It could be concluded that that LAMP method established in this study is accurate and reliable for the detection of FAdV-4.A LAMP detection approach for FAdV-4 used in grass-roots veterinary stations was developed in this study,and this FAdV-4 LAMP assay exhibited rapid response,strong specificity and high sensitivity.

关 键 词：禽腺病毒4型 HEXON 环介导等温扩增 可视化 

分 类 号：S852.659.1[农业科学—基础兽医学]