Identification and characteristics of aptamers against inactivated Vibrio alginolyticus

Abstract

Vibrio alginolyticus is an opportunistic pathogen that can infect many aquatic animals and cause economical losses to aquaculture and the food industry. Therefore, sensitive and rapid detection of V. alginolyticus is urgently needed. In the present paper, three aptamers were proved to have good specificity towards inactivated V. alginolyticus, and they could distinguish inactivated V. alginolyticus from inactivated Vibrio harveyi, Vibrio parahaemolyticus, Vibrio anguillarum, Edwardsiella tarda, Aeromonas hydrophila and Escherichia coli. Incubating heat- and formaldehyde-treated cells with aptamers #7, #8, and #23, washes with binding buffer, heat-induced release of bound aptamers, and application of the polymerase chain reaction to amplify the aptamers allowed the detection of 10, 10 and 103 cells/ml, respectively. Aptamer #8 had good affinity with both live and inactivated V. alginolyticus. The combination of aptamers #8 and #7, or aptamers #8 and #23, could distinguish live V. alginolyticus from inactivated V. alginolyticus based on their different affinities with the microorganism. The putative secondary structures were determined for these aptamers using software RNAstructure 5.3 and the affinity constants (Kds) 28.39 ± 11.46, 12.82 ± 6.00 and 46.89 ± 15.87 nM, were calculated respectively by employing their saturation curves. The results demonstrated that an aptamer-based method can be used for the detection of V. alginolyticus.