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作 者:李秀梅[1] 石瑜[1] 朱雅宁[1] 袁雪涛[1] 杨爱华[1] 杨颖[1]
机构地区:[1]天津市动物疫病预防控制中心,天津300402
出 处:《中国动物传染病学报》2018年第1期25-31,共7页Chinese Journal of Animal Infectious Diseases
摘 要:本研究将环介导等温扩增技术(loop-mediated isothermal amplification,LAMP)与横向流动试纸条技术(lateral flow dipstick,LFD)联合应用,针对鸡贫血病毒Cux-1 VP2基因设计了4条特异性引物和1条异硫氰酸荧光素(fluorescein isothiocyanate,FITC)标记的探针,并对其反应条件进行优化,建立了一种快速、便捷的鸡贫血病毒LAMP-LFD检测方法。结果显示,优化后的扩增温度和时间为63℃反应50 min,完成检测全过程仅需80 min。该方法检测灵敏度为10 fg,是利用外引物建立的PCR方法的100倍;可特异性地检出鸡贫血病毒;重复性、稳定性好。对30份样品的病毒分离鉴定和LAMP-LFD检测表明,二者符合率为100%。本研究建立的LAMP-LFD方法能够快速、特异地检测鸡贫血病毒,并且不需要其他辅助仪器,结果可直观判断,适合基层实验室、应急检测或现场监测等使用。In the present study, we established a rapid Chicken anemia virus (CAV) detection method by combining loop-mediated isothermal ampliflcation (LAMP) with lateral flow dipstick (LFD) examination. The method utilized four speciflc primers targeting CAV Cux-1 VP2 gene for LAMP and fluorescein isothiocyanate (FITC)-labeled probe to speciflcally hybrid to the biotin-labeled LAMP product for LFD examination. The optimized ampliflcation temperature and time were 63℃ and 50 min, respectively and whole procedure time was only 80 min including sample process. The LAMP-LFD method developed in this study speciflcally detected CAV and showed no reaction with other 6 immunosuppressive viruses. The detection sensitivity was 10 fg, i.e. 100-fold lower than that of conventional PCR using the outer primer set. This method had good repeatability and stability. The LAMP-LFD method was used to test 30 specimens and compared with virus isolation and identiflcation method. As a result, both methods had 100% agreement. Overall, the LAMP-LFD method rapidly and speciflcally detected CAV and did not required other auxiliary equipment, thus suitable for basic laboratory testing, emergency use and on-site monitoring.
关 键 词:鸡贫血病毒 VP2基因 环介导等温扩增 横向流动试纸条 检测
分 类 号:S852.659.2[农业科学—基础兽医学]
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