环介导等温扩增联合横向流动试纸条快速检测单核细胞增生李斯特菌的研究

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

将环介导等温扩增技术(loop-mediated isothermal amplification,LAMP)与横向流动试纸条检测技术(lateral flow dipstick,LFD)联合,建立了1种可应用于单核细胞增生李斯特菌快速检测的新方法。针对单核细胞增生李斯特菌的actA基因设计3对引物(其中,上游内引物由生物素标记)和1条异硫氰酸荧光素(fluorescein isothiocyanate,FITC)标记的探针,进行由生物素标记的LAMP扩增反应,并利用LFD对经FITC标记探针杂交的扩增产物完成检测。优化后的LAMP反应条件为65℃反应40min,从细菌基因组DNA提取到LFD结果判断只需90min左右,比常规PCR技术缩短近2h。LAMP-LFD可特异性地检出单核细胞增生李斯特菌,对哈维弧菌等常见弧菌及嗜水气单胞菌等的检测结果为阴性。灵敏性试验表明,LAMP-LFD针对病原纯培养物的检测灵敏度为3.2×101 CFU/mL或0.64CFU/反应,是LAMP检测的10倍,常规PCR检测的100倍;针对人工污染单核细胞增生李斯特菌的原料奶样品的检测灵敏度为1.6×102 CFU/mL或3.2CFU/反应。利用本方法可从采集样品中检测到单核细胞增生李斯特菌,检测结果与传统的细菌分离培养方法结果一致。试验表明,本研究建立的LAMP-LFD技术可特异、准确地应用于单核细胞增生李斯特菌检测,而且灵敏度高、操作简单、检测成本低,有望发展成为单核细胞增生李斯特菌快速检测的有效手段。

A new rapid loop-mediated isothermal amplification(LAMP)combined with a lateral flow dipstick(LFD)method was developed to detect Listeria monocytogenes.Three pairs of primers(the forward inner primer was biotinylated)and a fluorescein isothiocyanate(FITC)labeled DNA probe were designed specifically to recognize the actA gene of L.monocytogenes.The biotinylated LAMP was performed and then the LAMP products were hybridized with FITC-labeled probe and detected by LFD.The assay was optimized and could detect L.monocytogenes by incubation at 65℃for 40 min,and the whole detection procedure from extraction of bacterial genomic DNA to the visualization of the amplicons by LFD lasts only 90 min,which save by almost 2hours when compared to conventional PCR detection.The LAMP-LFD could specifically detect 3 L.monocytogenes isolates and did not detect other non-monocytogenes.The detection limit of LAMPLFD in pure culture of L.monocytogenes was 3.2×101 CFU/mL or 0.64CFU/reaction,which was 10 times than LAMP and 100 times higher than the conventional PCR detection.In the case of contaminated raw milk without enrichment,the detection limit was 1.6 × 102 CFU/mL or 3.2CFU/reaction.The LAMP-LFD assays could positively detect L.monocytogenes from gathered field samples,which was quite coincident with what was observed by conventional culture method.The results indicated that this method was a distinctive,accurate and easy-to-operate tool for the identification of L.monocytogenes,and could be used efficiently for rapid detection of L.monocytogenes in contaminated foods.