A real-time PCR array for hierarchical identification of Francisella isolates

Abstract

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.

Figure 1. Phylogenies of Francisella based on 16S rDNA and MLVA, respectively.

A) Phylogeny of Francisella and representative relatives based on alignment of 1,070 bp of the 16S rDNA gene. Bootstrap values are indicated at the branching points. The scale bar indicates 0.02 nucleotide changes per site. Modified from . B) Phylogeny of Francisella based on MLVA. Subspecies and major genetic branches (A1-A2, B1-B5) are indicated. Currently available genome sequences are in black boxes. Multiple strains are indicated by triangles at the branch edges. Modified from Johansson et al 2004 .

Figure 2. Example of plate design and interpretation of results for the genetic classification of F. tularensis strain LVS.

A) The allelic state of each marker in the LVS strain is indicated in boldface. A colored well corresponds to a phylogenetically determining (canonical) marker for a specific genetic subclade. B) A phylogenetic tree is generated from hierarchical analysis of the typing results. Thick lines indicate the inferred evolutionary history of strain LVS. D = derived state, A = ancestral state.

Figure 3. Schematic SNP and INDEL phylogeny, indicating genetic markers and Francisella subclades.

Markers presented in this study are indicated in black and, for comparison, SNP markers developed in a recent study by Vogler et al 2009 are indicated in gray. The branch names of Vogler et al. have been abbreviated to simplify the nomenclature. Stars indicate terminal subclades defined by Francisella genomes and circles represent collapsed branch points along the genetic lineages that contain isolates of a particular genotype (a subclade). The subclades are named for the flanking SNPs and INDELs. The branch lengths do not represent true phylogenetic distances. The position of B.15/Ftind47 (marked by the asterisk in the figure) could not be definitively determined; it could be either where shown, or be descendant from B.1/2.

Figure 4. Example of use.

The subclade names for 14 isolates and six ulcer specimens from tularemia patients in Sweden 2008 (Table 4) genotyped by the developed hierarchical real-time PCR array, and the location of the receiving hospitals.