环介导等温扩增技术与横向流动试纸条法快速检测鸡贫血病毒的研究  被引量:5

RAPID AND SENSITIVE DETECTION OF CHICKEN ANEMIA VIRUS BY LOOP-MEDIATED ISOTHERMAL AMPLIFICATION COMBINED WITH LATERAL-FLOW DIPSTICK

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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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