环介导等温扩增技术与横向流动试纸条法快速检测单增李斯特菌的研究

Rapid and sensitive detection of Listeria monocytogenes by loop-mediated isothermal amplification combined with a lateral-flow dipstick

为建立一种快速、便捷的单增李斯特菌检测方法,本研究针对单增李斯特菌hlyA基因设计了4条特异性引物和1条异硫氰酸荧光素标记的探针,采用环介导等温扩增(LAMP)与横向流动试纸条检测技术(LFD)联合应用,建立了一种快速、便捷的单增李斯特菌LAMP-LFD检测方法。通过对其反应条件进行优化,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。该方法仅可以特异性地检出单增李斯特菌,而对其它6种常见食源性致病菌的检测均呈阴性。对纯细菌培养物的检测灵敏度为3.6 cfu/m L,是利用外引物建立的PCR方法的100倍。该方法重复性好。对15份牛奶和15份猪肉的细菌分离鉴定和LAMP-LFD检测表明,二者符合率为100%。研究结果表明,本研究建立的LAMP-LFD方法能够快速、特异地检测单增李斯特菌,适合基层实验室、应急检测或现场监测等使用,具有较高的推广价值。

In this study, we established a rapid Listeria monocytogenes detection method by combining loop-mediated isothermal amplification （LAMP） and lateral flow dipstick （LFD） examination. The method used four specific primers targeting to hlyA gene of L.monocytogenes for LAMP and one fluorescein isothiocyanate-labeled probe to specifically hybrid to the biotin-labeled LAMP product for LFD examination. The optimized amplification temperature and time were 63 ℃ and 50 min, respectively, and the total procedure time was only 80 min by taking sample process into account. The established LAMP-LFD assay was able to specifically detect L.monocytogenes and was negative to other 6 most common foodborne pathogens. In addition, detection sensitivity for pure bacterial culture reached 3.6 cfu/mL, 100-fold higher than that of conventional PCR. The method was repeatability and stability. Tests of pathogen isolation and identification and the LAMP-LFD in 15 milk and 15 porky samples showed the coincidence rate of both was 100%. Overall, the established LAMP-LFD method, characterized for easy to perform and visualize the results judgment, was rapid and specific for L.monocytogenes detection Thus it is suitable for basic laboratory assay, emergency use and in on-site monitoring the L.monocytogenes infection.