Rapid Dual-Gene Detection of Burkholderia gladioli and Subspecies Cocovenenans in Fresh Noodles and Tremella Using CRISPR-Cas12a/Cas13a.

Background: Burkholderia gladioli pv. cocovenenans is a notable foodborne pathogen that poses a significant risk to food safety. Contaminated food requires distinct classification and treatment procedures for non-pathogenic B. gladioli and its pathogenic subspecies cocovenenans. Hence, establishing a rapid and sensitive detection method to distinguish them is necessary.

Objective: In this study, we aimed to establish a method combining the CRISPR-Cas12a/Cas13a (Clustered regularly short palindromic repeats-CRISPR associated proteins 12a and 13a) dual system with recombinase-aided amplification for rapid, specific, and sensitive detection of non-pathogenic B. gladioli and pathogenic subspecies cocovenenans in food.

Methods: First, an RAA (Recombinase-aided amplification)-CRISPR-Cas12a/Cas13a method was developed, and its feasibility was assessed. Next, specificity was analyzed using 23 strains of B. gladioli and 5 non-target strains. Following this, sensitivity was evaluated by preparing gradient dilutions of B. gladioli pv. cocovenenans bacterial solutions. Finally, real food test samples, including fresh noodles and tremella artificially contaminated with B. gladioli pv. cocovenenans, were utilized for method validation and sensitivity comparison.

Results: The established RAA-CRISPR-Cas12a/Cas13a method exhibited high specificity and achieved 100% accuracy in detecting species B. gladioli and its subspecies cocovenenans. This rapid method could be finished within 45 min with a detection limit of 100 CFU/mL (Colony-forming units per millilter) for bacterial concentration. Additionally, it achieved detection limits of 102 CFU/g for fresh noodles and 103 CFU/g for tremella.

Conclusion: The rapid RAA-CRISPR-Cas12a/Cas13a method demonstrated high specificity and sensitivity in detecting and distinguishing species B. gladioli and subspecies cocovenenans in both food test samples and post-cultivation colonies.

Highlights: The RAA-CRISPR-Cas12a/Cas13a method presented in this study offers a novel molecular approach for the rapid, accurate, and sensitive detection of B. gladioli and its subspecies cocovenenans in foods.