A novel method for detecting Burkholderia gladioli pv. cocovenenans by fluorescent probe-based loop-mediated isothermal amplification

Abstract:

[Objective] To establish a fluorescent probe-based loop-mediated isothermal amplification method for the specific detection of Burkholderia gladioli pv. cocovenenans. [Methods] According to the results of the multiple sequence alignment analysis, a conserved sequence, bonA, was selected for the specific detection of B. gladioli pv. cocovenenans. The primers and fluorescent probes were designed according to the conserved sequence for the establishment of a fluorescent probe-based loop-mediated isothermal amplification method. The established method was employed to detect 19 strains of B. gladioli and 3 non-target strains, and thus the specificity of this method was examined. Furthermore, the specificity of this method was compared with that of the real-time fluorescent PCR. Additionally, we investigated the limit of detection of this method for genomic DNA. [Results] The established method exhibited specificity towards B. gladioli pv. cocovenenans and could obtain the result within 30 min. Moreover, this method could visualize the detection results. The limits of detection for the genomic DNA and bacterial suspension were 1.98 pg/μL and 2.7×10² CFU/mL, respectively. In addition, this method can be applied to the detection of B. gladioli pv. cocovenenans in food samples. [Conclusion] A rapid, visualizable, and specific method was established for detecting B. gladioli pv. cocovenenans and can serve as a rapid and accurate molecular detection method for ensuring food safety.