环介导等温扩增联合横向流动试纸条可视化检测哈维氏弧菌的研究

doi:10.13560/j.cnki.biotech.bull.1985.2016.06.010

摘要/Abstract

摘要： 以哈维氏弧菌(Vibrio harveyi)为材料，利用环介导等温扩增技术(LAMP)进行核酸扩增，借助横向流动试纸条(LFD)完成产物检测，旨在建立一种可用于哈维氏弧菌快速检测的LAMP-LFD新技术。以哈维氏弧菌的溶血素基因(vhhA)为检测靶标设计了3对特异性引物(其中，上游内引物vhhA-FIP由生物素标记)，进行由生物素标记的LAMP反应；同时设计1条异硫氰酸荧光素(FITC)标记的探针，与获得的LAMP产物进行特异性杂交，杂交产物经LFD完成检测。经优化，LAMP的反应条件为63℃反应40 min，由LFD完成结果判读共需50 min。结果表明，LAMP-LFD方法能特异性地检出哈维氏弧菌，对创伤弧菌等其他9种水产养殖重要病原菌的检测结果呈阴性。利用该方法，针对细菌纯培养物的检测灵敏度为1.0×10² CFU/mL或2 CFU/反应，针对污染有该菌的大黄鱼组织的检测灵敏度为5×10² CFU/mL或20 CFU/反应，均是以LAMP外引物vhhA-F3/vhhA-B3的常规PCR方法的100倍。因此，该方法能够快速、准确地检出哈维氏弧菌，有望在海水养殖过程哈维氏弧菌的监测和即时检测中普及使用。

关键词: 哈维氏弧菌, 溶血素基因, 环介导等温扩增技术, 横向流动试纸条, 检测

Abstract: Based on nucleotide enrichment by a loop-mediated isothermal amplification(LAMP)and chromatographic visualization by a lateral flow dipstick(LFD)assay，this work aims to develop a novel LAMP-LFD method for the rapid detection of Vibrio harveyi. Three pairs of primers were designed using the hemolysin gene(vhhA)of V. harveyi as detection target，and used in LAMP reaction，among which the forward inner primer vhhA-FIP was biotinylated. Similarly，a fluorescein isothiocyanate(FITC)-labeled probe vhhA-HP was designed to specifically hybridize with LAMP products. And then the hybridized LAMP products were visually detected by LFD. The optimized LAMP was performed at 63℃ for 40 min；and visual detection via LFD took 50 min. The results indicated that LAMP-LFD was able to specifically identify V. harveyi from other 9 pathogenic bacteria commonly existing in the aquatic animals，such as V. vulnificus. The detection limit of LAMP-LFD was 1.0×10² CFU/mL for V. harveyi pure cultures(equivalent to 2 CFU per reaction)，and 5×10² CFU/mL for V. harveyi contaminated tissues of large yellow croaker(equivalent to 20 CFU per reaction)，both of which were 100 times lower than that of the conventional PCR method using both outer primers vhhA-F3/vhhA-B3. Therefore，this rapid and accurate LAMP-LFD method is a promising alternative in the surveillance and point-of-care test of V. harveyi in sea farming.

Key words: Vibrio harveyi, hemolysin, loop-mediated isothermal amplification, lateral flow dipstick, assay detection

程蝶, 柴方超, 蔡怡, 周前进, 陈炯. 环介导等温扩增联合横向流动试纸条可视化检测哈维氏弧菌的研究[J]. 生物技术通报, 2016, 32(6): 60-68.

CHENG Die, CHAI Fang-chao, CAI Yi, ZHOU Qian-jin, CHEN Jiong. Visual Detection of Vibrio harveyi Based on Loop-mediated Isothermal Amplification Combined with a Lateral Flow Dipstick[J]. Biotechnology Bulletin, 2016, 32(6): 60-68.

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