Visual Detection of Vibrio harveyi Based on Loop-mediated Isothermal Amplification Combined with a Lateral Flow Dipstick

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

Abstract

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

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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