Selection of aptamers against inactive Vibrio alginolyticus and application in a qualitative detection assay

Abstract

Aptamers against inactive Vibrio alginolyticus were selected from an 82-nt ssDNA random library by systematic evolution of ligands by exponential enrichment. After 15 rounds of selection, the final pool of aptamers was highly specific for inactivated V. alginolyticus and had a dissociation constant of 27.5 ± 9.2 nM. Using these aptamers and PCR, V. alginolyticus could be detected at 100 cells/ml. Sequencing of the final pool of aptamers revealed that some sequences, termed high-frequency aptamers, appeared more than once; these may be of practical application. All sequences obtained were divided into nine families according to their homology tree, some conserved sequences were also found in each of the six families. One sequence was found in significant proportions of the aptamers, suggesting that this conserved sequence might be important for forming the three-dimensional aptamer structure.

Selection and characterization of ssDNA aptamers specifically recognizing pathogenic Vibrio alginolyticus

“…Data presented as mean and standard deviation and A. veronii) (Figure 3). There are some reports about aptamers against other V. alginolyticus strains (Tang, Zheng, Yan, Li, & Li, 2013;Zheng et al, 2015), but the target V. alginolyticus used in these studies were inactivated by formaldehyde. The target pathogen V. alginolyticus used in this study were live bacteria.…”

Single-Stranded DNA Aptamers against Pathogens and Toxins: Identification and Biosensing Applications

“…Two different species, Vibrio parahaemolyticus and Vibrio alginolyticus were chosen as selection targets. Nine rounds of cell-SELEX using flow cytometry were carried out to identify ssDNA MRE with high affinity and specificity for Vibrio parahaemolyticus ( K d = 16.88 ± 1.92 nM) [ 55 , 56 ]. Tang et al performed 15 rounds of cell-SELEX on inactivated Vibrio alginolyticus .…”

“…Instead, the whole library was characterized to have a K d value of 27.5 ± 9.2 nM and was highly specific toward the target. The enriched library was able to detect 100 CFU/mL of the bacteria based on a PCR amplification assay [ 56 ].…”

Development of novel aptamer‐based enzyme‐linked apta‐sorbent assay (ELASA) for rapid detection of mariculture pathogen Vibrio alginolyticus

“…The aptamers against some severe aquatic viruses, such as Singapore grouper iridovirus (Li et al, ; Li, Wei, et al, ; Li, Zhou, et al, ), red‐spotted grouper nervous necrosis virus (Zhou et al, ), Trachinotus ovatus NNV (Yu, Liu, Wei, et al, ), spring viraemia of carp virus (Yu et al, ), Hirame rhabdovirus virus (Hwang et al, ), viral haemorrhagic septicaemia virus (Punnarak et al, ) and soft‐shelled turtle iridovirus (Li, Wei, et al, ; Li, Zhou, et al, ), have been successfully generated. There are also kinds of aptamers generated against various bacterial targets, such as Lactobacillus acidophilus , V. parahaemolyticus and Salmonella typhimurium (Duan, Wu, Chen, Huang, & Wang, ; Duan et al, ), V. alginolyticus (Yu, Liu, Su, et al, ; Tang, Zheng, Yan, Li, & Li, ; Zheng et al, ), V. harveyi (Zheng, Hao, Song, & Liu, ) and Aeromonas hydrophila (Li, Wang, Chen, & Li, ). Aptamers have been widely applied in different important research areas, including drug‐specific delivery (Loo et al, ; Zhao et al, ), pathogen diagnosis (Li et al, ; Li, Wei, et al, ; Li, Zhou, et al, ; Teng et al, ; Yu, Liu, Wei, et al, ; Zhou et al, ), toxin analysis (Huang et al, ; Yan et al, ) and disease pathogenesis research (Bunka & Stockley, ).…”

“…Data presented as mean ± standard deviation. A pvalue < .05 was considered statistically significant (**p < .01); NS indicates not significant Salmonella typhimurium (Duan, Wu, Chen, Huang, & Wang, 2012;Duan et al, 2013), V. alginolyticus (Yu, Liu, Su, et al, 2019;Tang, Zheng, Yan, Li, & Li, 2013;Zheng et al, 2015), V. harveyi (Zheng, Hao, Song, & Liu, 2014) and Aeromonas hydrophila (Li, Wang, Chen, & Li, 2012). Aptamers have been widely applied in different important research areas, including drug-specific delivery (Loo et al, 2017;Zhao et al, 2015), pathogen diagnosis (Li et al, 2014;Teng et al, 2016;Yu, Liu, Wei, et al, 2019;Zhou et al, 2016), toxin analysis Yan et al, 2018) and disease pathogenesis research (Bunka & Stockley, 2006).…”