Engineering of an adaptive tandem CRISPR/Cas12a molecular amplifier permits robust analysis of Vibrio parahaemolyticus

Abstract

Seeking new molecular diagnostic method for pathogenic bacteria detection is of utmost importance for ensuring food safety and protecting human health. Herein, we have engineered an adaptive tandem CRISPR/Cas12a molecular amplifier specifically designed for robust analysis of vibrio parahaemolyticus (V. parahaemolyticus), one of the most harmful pathogens. Our strategy involves the integration of three crucial processes: recombinase polymerase amplification (RPA) for copy number amplification, terminal deoxynucleotidyl transferase (TdT) for template-free strand elongation, and CRISPR/Cas12a-mediated trans-cleavage of a reporter molecule. By combining these processes, the target genomic DNA extracted from V. parahaemolyticus is able to activate many CRISPR/Cas12a units (CRISPR/Cas12aⁿ) simultaneously, resulting in a greatly amplified target signal to indicate the presence and concentration of V. parahaemolyticus. This unique model offers more advantages compared to traditional amplification models that use one RPA amplicon to activate one CRISPR/Cas12a unit. Under optimized conditions, our method enables the detection of target V. parahaemolyticus within a linear range of 1 × 10²-1 × 10⁷ CFU/mL, with an impressive limit of detection as low as 12.4 CFU/mL. It is conceivable that the adaptive tandem CRISPR/Cas12a molecular amplifier could be adapted as routine diagnostic kits in future for in-field detection of pathogens.

Keywords: CRISPR/Cas12a system; Molecular diagnosis; Recombinase polymerase amplification (RPA); Terminal deoxynucleotidyl transferase; Vibrio parahaemolyticus.