Identification of a highly specific DNA aptamer for Vibrio vulnificus using systematic evolution of ligands by exponential enrichment coupled with asymmetric PCR.

ABSTRACT

Vibrio vulnificus is an important bacterial pathogen that causes serious infections in fish and is also highly pathogenic to humans. Many effective detection methods targeting this pathogen have previously been designed, but many of these methods are time-consuming, complicated and expensive. Thus, these approaches cannot be widely used by small aqacultural concerns. Although DNA aptamers have been used to detect pathogenic bacteria, these have not been applied to marine bacteria, including V. vulnificus. Therefore, we developed a highly specific DNA aptamer for V. vulnificus detection using systematic evolution of ligands by exponential enrichment (SELEX), coupled with asymmetric PCR. After 13 rounds of cross-selection, we identified a novel DNA aptamer (Vapt2). We evaluated the affinity, specificity and limit of detection (LOD) of this aptamer for V. vulnificus. We found that Vapt2 had a high affinity for V. vulnificus (Kd  = 26.8 ± 5.3 nM) and detected this pathogen at a wide range of concentrations (8-2.0 × 108  cfu/ml). Vapt2 bound to V. vulnificus with high selectivity in the presence of other pathogenic bacteria. Our study increases our knowledge of the possible applications of aptamers with respect to marine bacteria. Moreover, our work might provide a framework for the rapid detection of pathogenic bacteria and water pollution.