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A robust, rapid and flexible real-time PCR assay for hierarchical genetic typing of clinical and environmental isolates of  Francisella  is presented. Typing markers were found by multiple genome and gene comparisons, from which 23 canonical single nucleotide polymorphisms (canSNPs) and 11 canonical insertion-deletion mutations (canINDELs) were selected to provide phylogenetic guidelines for classification from genus to isolate level. The specificity of the developed assay, which uses 68 wells of a 96-well real-time PCR format with a detection limit of 100 pg DNA, was assessed using 62  Francisella  isolates of diverse genetic and geographical origins. It was then successfully used for typing 14  F. tularensis  subsp.  holarctica  isolates obtained from tularemia patients in Sweden in 2008 and five more genetically diverse  Francisella  isolates of global origins. When applied to human ulcer specimens for direct pathogen detection the results were incomplete due to scarcity of DNA, but sufficient markers were identified to detect fine-resolution differences among  F. tularensis  subsp.  holarctica  isolates causing infection in the patients. In contrast to other real-time PCR assays for  Francisella , which are typically designed for specific detection of a species, subspecies, or strain, this type of assay can be easily tailored to provide appropriate phylogenetic and/or geographical resolution to meet the objectives of the analysis.

A Real-Time PCR Array for Hierarchical Identification of Francisella Isolates