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

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