A super convenient and specific CRISPR/Cas12a diagnostic platform for toxigenic Burkholderia gladioli based on virulence genes

In recent years, food poisoning cases caused by toxigenic Burkholderia gladioli pv. cocovenenans (BGC) have been frequently reported in Asian countries like China and Indonesia, which seriously threaten food safety and public health. BGC is the only subspecies within the Burkholderia gladioli species that can cause food poisoning in humans, but there is a high degree of sequence homology between the different subspecies, making detection and differentiation difficult. In this study, a specific DNA target was screened out from the bongkrekic acid biosynthentic gene cluster. A rapid, sensitive, and highly specific CRISPR/Cas12a molecular diagnostic platform was developed for the first time to differentiate between toxigenic and non-toxigenic subspecies. Genomic DNA was extracted from the samples after a simple boiling process. The sensitivity was evaluated from three different levels, the genomic DNA sensitivity was determined to be 10⁻⁴ ng/μL, the target DNA sensitivity was determined to be 14.9 copies/μL, and the bacterial suspension sensitivity was confirmed to be 1.52 CFU/mL. The detection limit of BGC in artificially contaminated coconut water samples was determined to be 1.50 CFU/mL without any microbial enrichment. Including sample pretreatment and DNA extraction process, the whole detection of BGC in samples can be completed within 60 min under the assistance of some small and portable devices. This super convenient diagnostic platform is suitable for food field testing and provides a theoretical basis for the specific identification of BGC.