副结核分枝杆菌重组酶介导扩增-侧向流试纸条检测方法的建立与初步应用
Establishment and Preliminary Application of Recombinase-mediated Amplification-sidestream Detection for Detection of Mycobacterium paratuberculosis
南岳 1龙美贞 1王元智 1刘一朵 1周向梅1
作者信息
- 1. 中国农业大学动物医学院兽医公共卫生安全全国重点实验室,北京 100193
- 折叠
摘要
旨在建立一种副结核分枝杆菌(Mycobacterium avium subsp.paratuberculosis,MAP)精准快速的检测方法,结合重组酶介导扩增(recombinase-aid amplification,RAA)和侧向流试纸条(lateral flow dipstick,LFD)技术,以MAP的特异性多拷贝插入元件IS900设计引物和探针,通过优化反应时间和温度等条件,建立可用于MAP现场可视化检测的RAA-LFD方法.该检测方法在35 ℃条件下恒温反应30 min即可实现对MAP目的基因的有效扩增.试验结果显示:该方法可特异性地检测出MAP,不与其他常见的病原发生交叉反应;对MAP标准品的最低检测限可达到10 fg·μL-1;使用该方法对149份牛奶样品进行检测,与传统的PCR相比,其敏感性为100％.综上,本研究成功建立了一种针对于MAP的精准快速的试纸条检测方法,并具有广泛的应用前景.
Abstract
To establish a rapid and accurate method for detection of Mycobacterium avium subsp.paratuberculosis(MAP),primers and probes were designed according to the specific multicopy insert element IS900 of MAP.A recombinase-aid amplification combined with lateral flow dipstick(RAA-LFD)method was established for visual detection of MAP in the field within 30 min at 35 ℃ after being optimized.The assay was specific with the target sequence of MAP with a detection limit up to 10 fg·μL-1.One hundred and forty-nine stool samples were tested by this method,showing 100％sensitivity with the PCR.In short,the RAA-LFD assay developed in this study was a rapid,reliable method which can be broadly used.
关键词
副结核分枝杆菌/重组酶介导扩增/侧向流试纸条Key words
Mycobacterium avium subsp.paratuberculosis/recombinase-aid amplification/lateral flow dipstick引用本文复制引用
基金项目
"十四五"重点研发计划(2022YFD1800703)
出版年
2024
国家科技期刊平台
NSTL
万方数据