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The bacterium  Burkholderia pseudomallei  causes melioidosis, a rare but serious illness that can be fatal if untreated or misdiagnosed. Species-specific PCR assays provide a technically simple method for differentiating  B. pseudomallei  from near-neighbor species. However, substantial genetic diversity and high levels of recombination within this species reduce the likelihood that molecular signatures will differentiate all  B. pseudomallei  from other Burkholderiaceae. Currently available molecular assays for  B. pseudomallei  detection lack rigorous validation across large  in silico  datasets and isolate collections to test for specificity, and none have been subjected to stringent quality control criteria (accuracy, precision, selectivity, limit of quantitation (LoQ), limit of detection (LoD), linearity, ruggedness and robustness) to determine their suitability for environmental, clinical or forensic investigations. In this study, we developed two novel  B. pseudomallei  specific assays, 122018 and 266152, using a dual-probe approach to differentiate  B. pseudomallei  from  B. thailandensis ,  B. oklahomensis  and  B. thailandensis -like species; other species failed to amplify. Species specificity was validated across a large DNA panel (>2,300 samples) comprising  Burkholderia  spp. and non -Burkholderia  bacterial and fungal species of clinical and environmental relevance. Comparison of assay specificity to two previously published  B. pseudomallei -specific assays, BurkDiff and TTS1, demonstrated comparable performance of all assays, providing between 99.7 and 100% specificity against our isolate panel. Last, we subjected 122018 and 266152 to rigorous quality control analyses, thus providing quantitative limits of assay performance. Using  B. pseudomallei  as a model, our study provides a framework for comprehensive quantitative validation of molecular assays and provides additional, highly validated  B. pseudomallei  assays for the scientific research community.

Development and Validation of Burkholderia pseudomallei-Specific Real-Time PCR Assays for Clinical, Environmental or Forensic Detection Applications