Identification and evaluation of new specific targets based pan-genome analysis for rapid detection of Burkholderia gladioli pathovar cocovenenans and Burkholderia gladioli in foods

Burkholderia gladioli pathovar cocovenenans (BGC), which is a high morbidity and mortality foodborne pathogens because of produce a lethal toxin (Bongkrekic acid, BA), is one of the best known members of Burkholderia gladioli (BG). Methods for rapid detection and accurate differentiation of BGC and BG are urgently needed because of the difficulty and complexity, especifically no noncultural techniques for discriminating BGC due to lack of specific targets. PCR-based methods with suitable specific targets can achievable rapid detection due to the accuracy, sensitivity, and specificity. In this study, new species and pathovar-specific target genes for rapid detection of BGC and BG in food, was identified using pan-genome analysis. A total of 2098 Burkholderia genome sequences, containing those of 223 Burkholderia gladioli and 31 species of other Burkholderia, were utilized to identify BGC and BG specific targets. One BGC-specific genes (EDD84_14,360) and two BG-specific genes (EDD84_30,365 and EDD84_11,630) were obtained. Based on these specific genes, real-time PCR methods for the rapid detection of BGC and BG were developed respectively. High specificty (100%) were observed when appraised various produce or non-produce bongkrekic acid of BG and 56 non-BG species. Moreover, these real-time PCR methods could accurately detect BGC and BG even when mixed with different concentrations of non-target interfering bacteria (Burkholderia cepacia). The methods could successfully detect 108-103 CFU/g of BGC and BG in artificially contaminated rice noodles without enrichment, and effectively screen of BGC and BG within 24 h from natural food samples with enrichment step in GVC broth which the corresponding sensitivity, specificity and efficiency values were 100%. These results revealed that the real-time PCR methods based on three new pathovar and species-specific molecular targets provide a promising approach with high accuracy and sensitivity to the rapid detection and differentiation of BGC and BG strains in foods.