Establishment of a CRISPR-Cas12a based electrochemical detection method for Burkholderia gladioli and its subspecies cocovenenans in fresh noodles and tremella.

Abstract

Burkholderia gladioli pv. cocovenenans is a pathogenic bacterium of concern due to its potential threat to food safety. This study presents a rapid electrochemical detection method based on dual recombinase-aided amplification (RAA) coupled with CRISPR-Cas12a for the sensitive and specific identification of B. gladioli and its toxigenic subspecies B. gladioli pv. cocovenenans in food. The 16 S rDNA and bonA genes were selected as species- and subspecies-specific targets, respectively, and corresponding CRISPR-Cas12a reaction systems were established. An electrochemical biosensor incorporating a gold electrode functionalized with single-stranded DNA probes was constructed, and its specificity and sensitivity were evaluated using artificially contaminated fresh noodles and tremella samples. The method precisely distinguished B. gladioli and its toxigenic cocovenenans subspecies within 1 h, achieving a limit of detection limit of 10² CFU/g in both food matrices. This strategy provides a rapid and field-deployable approach for distinguishing non-toxigenic and toxigenic B. gladioli strains in food, supporting timely screening and food safety monitoring.