环介导等温扩增联合横向流动试纸条可视化检测迟缓爱德华菌

Visual detection of Edwardsiella tarda based on loop-mediated isothermal amplification combined with a lateral flow dipstick

将环介导等温扩增(loop-mediated isothermal amplification,LAMP)技术与横向流动试纸条(latera flowdipstick,LFD)联合应用,建立LAMP-LFD方法,用于迟缓爱德华菌(Edwardsiella tarda,E.tarda)的快速检测。根据E.tarda外膜蛋白A(outer membrane protein A,ompA)基因的保守区设计了6套LAMP引物,并筛得1套最适引物用于后续LAMP反应。将该套引物的上游内引物5′端用生物素标记,同时在有效扩增区段内设计1条探针ompAHP,使用异硫氰酸荧光素(fluorescein isothiocyanate,FITC)标记,用于试纸条显色。结果表明,优化后的LAMP反应条件为63℃ 25min,LAMP-LFD对嗜水气单胞菌等致病菌检测结果均呈阴性,对病原菌纯培养物的检测灵敏度为1.0×10~2 CFU/mL,是常规PCR检测灵敏度的100倍。对E.tarda人工污染组织样品的灵敏度为5×10~2 CFU/mL或1.0×10~4 CFU/g。因此,LAMP-LFD可特异地检出E.tarda,且灵敏度高、操作简便、时间短、有潜力成为检测E.tarda的常规方法。

A novel and rapid loop-mediated isothermal amplification （LAMP） method combined with a lateral flow dipstick（LFD） were developed to detect Edwardsiella tarda. Six sets of primers were designed to recognize the highly conserved outer membrane protein A（ompA） gene of E. tar- da . One set of optimal primers was screened out and used to the following（real-time fluorescence） LAMP assay. The picked forward inner primer, ompA-FIP, was biotin-labeled and a DNA probe FITC-ompA-HP was designed and labeled by fluorescein isothiocyanate（FITC）. The biotinylated LAMP amplicons could hybridize with FITC-ompA-HP and visualize on the LFD. The results indi- cated the LAMP LFD assay could robustly and specifically detect E. tarda in 25 min at 63℃, and was negative for other twelve aquatic pathogenic bacteria. The detection limit of LAMP-LFD to pure culture of E. tarda was 1.0×10^2 CFU/mL,which was 100 folds higher than that of the con ventional PCR assay. And the detection limit to artificially contaminated liver tissue of Carassius auratus was 5 ×10^2 CFU/mL（ 1.0 × 10^4 CFU/g）. Therefore, the LAMP-LFD assay was an accu- rate, sensitive, easy-to operate method for the detection of E. tarda ,which has a potential for fu- ture use in detection of E. tarda.