Duplex real-time PCR assay for the simultaneous detection of Achromobacter xylosoxidans and Achromobacter spp.

摘要

Several members of the Gram-negative environmental bacterial genus are associated with serious infections, with being the most common. Despite their pathogenic potential, little is understood about these intrinsically drug-resistant bacteria and their role in disease, leading to suboptimal diagnosis and management. Here, we performed comparative genomics for 158 spp. genomes to robustly identify species boundaries, reassign several incorrectly speciated taxa and identify genetic sequences specific for the genus and for . Next, we developed a Black Hole Quencher probe-based duplex real-time PCR assay, Ac-Ax, for the rapid and simultaneous detection of spp. and from both purified colonies and polymicrobial clinical specimens. Ac-Ax was tested on 119 isolates identified as spp. using phenotypic or genotypic methods. In comparison to these routine diagnostic methods, the duplex assay showed superior identification of spp. and , with five isolates failing to amplify with Ac-Ax confirmed to be different genera according to 16S rRNA gene sequencing. Ac-Ax quantified both spp. and down to ~110 genome equivalents and detected down to ~12 and ~1 genome equivalent(s), respectively. Extensive analysis, and laboratory testing of 34 non- isolates and 38 adult cystic fibrosis sputa, confirmed duplex assay specificity and sensitivity. We demonstrate that the Ac-Ax duplex assay provides a robust, sensitive and cost-effective method for the simultaneous detection of all spp. and and will facilitate the rapid and accurate diagnosis of this important group of pathogens.