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Development of a Novel Real-Time PCR Assay Differentiating Burkholderia mallei From Burkholderia pseudomallei
Ai Koshikawa1, Yoshiki Ichikawa2, Mitsuru Nakase1
1Laboratory of Comparative Pathology, Faculty of Veterinary Medicine, Hokkaido University, Sapporo, Hokkaido, Japan.
Abstract:
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.
Insights
A new real-time PCR method effectively distinguishes glanders (caused by Burkholderia mallei) from melioidosis (caused by Burkholderia pseudomallei). This sensitive assay offers a reliable alternative for diagnosing these related zoonotic diseases.
Area of Science:
- Veterinary Microbiology
- Infectious Diseases
- Molecular Diagnostics
Background:
- Glanders, a zoonotic disease, is caused by Burkholderia mallei (Bm), which is phylogenetically similar to Burkholderia pseudomallei (Bp), the agent of melioidosis.
- Distinguishing between Bm and Bp is challenging, and current diagnostic methods, including the World Organisation for Animal Health (WOAH)-recommended PCR, face limitations due to evolving bacterial strains.
Purpose of the Study:
- To develop and validate a novel real-time PCR assay capable of discriminating between Bm and Bp.
Main Methods:
- Bioinformatic analysis (BLAST) identified a conserved Bm-specific sequence.
- A probe-based real-time PCR assay was optimized to target this sequence.
- The assay's performance was evaluated using bacterial isolates and clinical specimens.
Main Results:
- The novel real-time PCR assay successfully detected Bm DNA in five isolates and clinical samples.
- The assay demonstrated high specificity, not amplifying DNA from closely related Burkholderia species.
- Detection rates in clinical specimens were comparable to the WOAH-recommended method.
Conclusions:
- A novel, highly sensitive real-time PCR method was developed for differentiating glanders from melioidosis.
- This assay shows potential as a reliable alternative diagnostic tool in clinical settings.
- Further validation with diverse and extensive sample sets is recommended.
