Enhanced Detection of Vibrio harveyi Using a Dual-Composite DNAzyme-Based Biosensor

<i>Vibrio harveyi</i> is a serious bacterial pathogen which can infect a wide range of marine organisms, such as marine fish, invertebrates, and shrimp, in aquaculture, causing severe losses. In addition, <i>V. harveyi</i> can be transmitted through food and water, infecting humans and posing a serious threat to public safety. Therefore, rapid and accurate detection of this pathogen is key for the prevention and control of related diseases. In this study, nine rounds of in vitro screening were conducted with Systematic Evolution of Ligands by Exponential Enrichment (SELEX) technology using unmodified DNA libraries, targeting the crude extracellular matrix (CEM) of <i>V. harveyi</i>. Two DNAzymes, named DVh₁ and DVh₃, with high activity and specificity were obtained. Furthermore, a fluorescent biosensor with dual DNAzymes was constructed which exhibited improved detection efficiency. The sensor showed a good fluorescence response to multiple aquatic products (i.e., fish, shrimp, and shellfish) infected with <i>V. harveyi</i>, with a detection limit below 11 CFU/mL. The fluorescence signal was observed within 30 min of reaction after target addition. This simple, inexpensive, highly effective, and easy to operate DNAzymes biosensor can be used for field detection of <i>V. harveyi</i>.