Identification and typing of Francisella tularensis with a highly automated genotyping assay

A PCR assay was developed to genotypically characterize Francisella tularensis and F. novicida. An integrated and partially redundant set of markers was selected to provide positive identification of these species, identify subspecies of F. tularensis and genotype 14 variable number tandem repeat (VNTR) markers. Assay performance was evaluated with 117 Francisella samples. Sample DNA was amplified, and the masses of the PCR products were determined with electrospray ionization/time of flight mass spectrometry (ESI-MS). The base compositions of the PCR amplicons were derived from these high-accuracy mass measurements and contrasted with databased information associated with each of the 25 assay markers. Species and subspecies determinations for all samples were fully concordant with results from established typing methods, and VNTR markers provided additional discrimination among samples. Sequence variants were observed with a number of assay markers, but these did not interfere with sample characterization, and served to increase the genetic diversity detected by the assay. Significance and Impact of the Study: Molecular characterization of pathogenic subspecies of Francisella tularensis can be obscured by their near neighbours, which can be prevalent in environmental samples. This study describes a multilocus PCR assay for characterizing key species and subspecies of Francisella at multiple levels of resolution, using stable markers defining phylogenetic groups, together with more variable strain-defining markers. Target loci were selected to form an integrated and semi-redundant panel enabling robust identification of samples, including unique samples showing novel assay signatures. Analyses of environmental isolates and a diverse panel of curated samples exemplified the applications of this approach in surveillance and outbreak studies. Significance and Impact of the Study: Molecular characterization of pathogenic subspecies of Francisella tularensis can be obscured by their near neighbours, which can be prevalent in environmental samples. This study describes a multilocus PCR assay for characterizing key species and subspecies of Francisella at multiple levels of resolution, using stable markers defining phylogenetic groups, together with more variable strain-defining markers. Target loci were selected to form an integrated and semi-redundant panel enabling robust identification of samples, including unique samples showing novel assay signatures. Analyses of environmental isolates and a diverse panel of curated samples exemplified the applications of this approach in surveillance and outbreak studies.