Field-deployable visual detection of Burkholderia gladioli and its toxigenic pathovar using a one-step RPA-CRISPR/Cas13a assay

ABSTRACT Burkholderia gladioli pathovar cocovenenans produces the potent mitochondrial toxin bongkrekic acid (BA), causing severe foodborne outbreaks. Conventional detection methods are constrained by prolonged turnaround times and the need for sophisticated instruments, limiting their utility for on-site surveillance. In this study, two independent one-step RPA-CRISPR/Cas13a detection systems were established: one targeting a species-level marker for broad screening of B. gladioli and the other targeting the bonA gene cluster for specific identification of the toxigenic pathovar. The complete one-step reaction was performed at 37°C for 30 min, and results were visually interpreted using a portable blue-light transilluminator. The visual detection limit reached 1 × 10⁰ copy/μL for both assays, with no cross-reactivity observed against a panel of six non-target strains. Component validation experiments confirmed that all listed constituents were indispensable for fluorescence signal generation. The practical applicability of the platform was evaluated using 100 naturally contaminated black fungus and mushroom substrate samples. The species-level assay returned positive rates of 78% (39/50) for black fungus and 42% (21/50) for substrate samples, while the bonA -targeted assay yielded positive rates of 38% (19/50) and 8% (4/50), respectively. The visual readout results were in 100% concordance with conventional quantitative real-time PCR. This field-deployable, equipment-free visual detection platform holds considerable promise for on-site surveillance and early warning of BA-producing B. gladioli in food and environmental matrices. IMPORTANCE Bongkrekic acid (BA) poisoning caused by Burkholderia gladioli pv. cocovenenans is a lethal food safety threat, particularly in China and Southeast Asia. Current detection methods depend heavily on laboratory-based instruments, limiting their utility for on-site surveillance. This study developed two independent one-step RPA-CRISPR/Cas13a detection systems: one for broad screening of B. gladioli at the species level and the other for specific identification of the toxigenic pathovar. The one-step reaction was completed at 37°C within 30 min, and results were visually interpreted using only a portable blue-light transilluminator. The visual detection limit reached 1 × 10⁰ copy/μL, and the method exhibited 100% concordance with quantitative real-time PCR when applied to 100 naturally contaminated samples. This field-deployable, equipment-free platform provides a practical tool for on-site surveillance and early warning of BA-producing B. gladioli in food and environmental matrices.