应用LAMP-LFD技术可视化检测河流弧菌(Vibrio fluvialis)的研究

THE VISUAL DETECTION OF VIBRIO FLUVIALIS BY A NOVEL LAMP-LFD

以河流弧菌(Vibrio fluvialis)为检测对象,将环介导等温扩增技术(loop-mediated isothermal amplification,LAMP)与横向流动试纸条(lateral flow dipstick,LFD)联合应用,建立了快速便捷的LAMP-LFD方法。该方法以河流弧菌的外膜蛋白omp U基因作为检测靶标,在其保守区域设计6条特异性引物(上游内引物由生物素标记),并在两条外引物的有效扩增区段内设计1条特异性探针,由异硫氰酸荧光素(fluorescein isothiocyanate,FITC)标记。使用引物进行有生物素标记的LAMP反应,产物与FITC标记的探针完成杂交,杂交产物在LFD上进行结果展示。结果表明,LAMP-LFD方法可特异性地检出河流弧菌,对近似物种如创伤弧菌等弧菌病原以及迟缓爱德华菌等其他水产养殖病原的检测均呈阴性。经优化,LAMP的反应条件为63°C反应25min,加上5min的探针杂交和3—5min的LFD显色,整个检测时程约35min。利用该方法最低可检测到1.0×10~2 CFU/m L的河流弧菌纯培养物,能够从污染强度为5.0×10~2 CFU/m L的组织样品中检测到该病原。该方法设备依赖性更低,仅需简单的恒温加热设备即可完成。因此,本研究建立的河流弧菌LAMP-LFD方法,具有操作便捷,设备依赖性低、灵敏度高和检测快速等优点,在河流弧菌的基层检测中具有良好的应用前景。

A novel LAMP-LFD method for rapid detection of Vibrio fluvialis was developed based on loop-mediated isothermal amplification （LAMP） integrated with visual detection by a lateral flow dipstick assay （LFD）. Six primers were designed according to the conserved regions of omp U gene （among the primers, the forward inner primer was biotinylated）, and a specific probe was figured out based on the conserved fragment amplified by both outer primers which was labeled by fluorescein isothiocyanate （FITC）. Then a biotinylated LAMP assay was carried out and the amplicon was hybridized exclusively with the FITC-labeled probe, and the hybrid product was visualized via LFD. The results demonstrated that LAMP-LFD could specifically detect E fluvialis, and no characteristic amplification was observed when using genomic DNA of l^brio spp. （like Vibrio vulnificus） and several other aquatic pathogens （like Edwardsiella tarda）. The optimized LAMP assay to detect E fluvialis was performed at 63 ~C for 25rain, and for only 35rain when incorporated 5-min＇ hybridization and 3--5-rain＇ visualization via LFD. The detection limit of LAMP-LFD was 1.0xl02 CFU/mL for V. fluvialis pure cultures and 5x 102 CFU/mL for K fluvialis contaminated tissues of large yellow croaker. The LAMP-LFD was less device-dependent and only simple isothermal equipment was provided. Therefore, the LAMP-LFD method is easy-operation, less device-dependent, sensitive, and rapidly, showing great potential in the routine detection and monitoring of K fluvialis.