Rapid Detection of Vibrio vulnificus by Loop-mediated Isothermal Amplification Combined with Lateral Flow Dipstick Assay

Abstract

Abstract: A novel and rapid loop-mediated isothermal amplification（LAMP）combined with chromatographic lateral flow dipstick（LFD）assay was developed to detect Vibrio vulnificus. A set of six primers and a fluorescein isothiocyanate（FITC）-labeled probe that recognized V. vulnificus outer membrane protein TolC gene were designed. Biotinylated LAMP amplicons were hybridized exclusively with the FITC-labeled probe and detected by LFD assay. The assay was optimized and could detect V. vulnificus by incubation at 63℃ for only 35 min, and the whole detection procedure from extraction of bacterial genomic DNA to the visualization of the amplicons by LFD last 80 min. V. vulnificus could be accurately detected by LAMP-LFD, and no amplification could be observed when another 9 bacterial genomic DNA were used. The sensitivity for V. vulnificus detection in pure culture was 3.7×10² CFU/mL or equivalent to 7.4 CFU per reaction, which is 100 times higher than that of PCR assay. The results indicate that LAMP-LFD is an accurate, rapid and sensitive tool for V. vulnificus detection and can be used for detection of V. vulnificus in contaminated aquatic foods.

Key words: Vibrio vulnificus, Outer membrane protein, TolC gene, Loop-mediated isothermal amplification, Lateral flow dipstick assay detection

Wang Yaohuan, Wang Ruina, Zhou Qianjin, Chen Jiong. Rapid Detection of Vibrio vulnificus by Loop-mediated Isothermal Amplification Combined with Lateral Flow Dipstick Assay[J]. Biotechnology Bulletin, 2014, 0(6): 81-87.

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