A Novel LAMP-Based Lateral Flow Dipstick Technique for Rapid and Visual Detection of Burkholderia gladioli pv. cocovenenans.

Burkholderia gladioli pv. cocovenenans (B. gladioli pv. cocovenenans), the most representative pathovar of B. gladioli, is capable of producing thermostable and highly virulent bongkrekic acid, which can result in multiple organ failure and an extremely high case fatality rate in humans. This research aims to mine specific targets for B. gladioli pv. cocovenenans and develop a rapid and accurate assay for the detection of B. gladioli pv. cocovenenans in food. Two novel specific target genes (group_0918 and group_0110) were successfully screened through pan-genome analysis, and the excellent specificity and sensitivity of both targets were verified in comparison to PCR assays. Based on these targets, a loop-mediated isothermal amplification (LAMP) method was developed to specifically identify B. gladioli pv. cocovenenans. In pure culture and spiked fresh black fungus samples, the LAMP assay achieved a limit of detection (LOD) of 4.1 × 101 CFU/mL and 4.1 × 102 CFU/g for the detection of B. gladioli pv. cocovenenans, respectively. To enable point of care testing, a LAMP-based lateral flow dipstick(LFD) assay was established with a detection sensitivity of 4.1 × 103 CFU/g in fresh black fungus. Tests on naturally infected food samples demonstrated that the developed LAMP and LAMP-assisted visual detection assays can serve as an effective alternative for the accurate identification of B. gladioli pv. cocovenenans. The novel molecular detection technology established in this study offers an efficient and reliable detection method for the prevention and control of B. gladioli pv. cocovenenans contamination in food, which holds significant application value and promotion prospects.