Development of a Novel Real‐Time PCR Assay Differentiating Burkholderia mallei From Burkholderia pseudomallei

Glanders is a zoonotic disease caused by Burkholderia mallei (Bm). Bm is phylogenetically similar to B. pseudomallei (Bp), the causative agent of melioidosis, making it difficult to differentiate between them. Recently, the PCR method recommended by the World Organisation for Animal Health (WOAH) failed to detect a Kuwait strain of Bm, and the WOAH has cautioned that ongoing Bm evolution may lead to the emergence of variants that escape standard assays. Therefore, this study aimed to develop a novel real-time PCR method to discriminate between Bm and Bp. Local and web-based BLAST searches identified a 494-bp sequence conserved across Bm but absent from other bacterial genomes, including Bp, except for one B. thailandensis strain. The optimized probe-based real-time PCR targeting this sequence detected the genomes of five Bm isolates but did not amplify DNA from closely related Burkholderia species. In clinical specimens from Mongolia, this assay amplified Bm DNA at an equivalent detection rate to the WOAH-recommended real-time PCR method. These findings provide a novel, highly sensitive diagnostic method for differentiating glanders from melioidosis and suggest its potential use as an alternative method in clinical applications. Further verification using a large number of samples collected from various regions will be necessary in the future.