Research on a Highly Sensitive Aptamer Sensor for Vibrio alginolyticus Based on CRISPR-Cas13a and T7 Transcription Cascade Amplification

Abstract

Vibrio alginolyticus is a prevalent aquatic pathogen that poses significant risks to public health. Thus, the development of rapid and highly sensitive detection methods is imperative. In this study, we developed a novel aptasensor leveraging the CRISPR/Cas13a system. By integrating a tripleamplification strategy comprising "aptamer competition recognition -T7 transcription amplification -Cas13a trans-cleavage", this platform enables efficient and specific detection of V. alginolyticus. The biosensor employs a V. alginolyticus-specific aptamer (Apt) as the recognition element. In the presence of the target bacterium, Apt binds to a surface membrane protein, resulting in the release of a blocking strand (Block). This triggers a conformational change in a hairpin probe (HP), thereby exposing the T7 promoter sequence. Subsequently, T7 RNA polymerase initiates an isothermal transcription reaction, producing abundant RNA products. These RNAs activate the trans-cleavage activity of Cas13a, which cleaves a fluorescent reporter probe to generate a quantifiable signal. This method eliminates the need for nucleic acid extraction and sophisticated instrumentation. It achieves a detection limit as low as 2 CFU/mL, and demonstrates high specificity by effectively distinguishing closely related species (e.g., Vibrio parahaemolyticus). When applied to simulated seawater and seafood samples, the recovery rates ranged from 94.61% to 106.56%, indicating robust anti-interference capacity and reproducibility. This work establishes a highly sensitive and specific biosensing technology for the on-site rapid detection of aquatic pathogens, offering promising applications in environmental monitoring and food safety.