TaqMan Real-Time PCR Assays for Single-Nucleotide Polymorphisms Which Identify Francisella tularensis and Its Subspecies and Subpopulations

Abstract

Francisella tularensis, the etiologic agent of tularemia and a Class A Select Agent, is divided into three subspecies and multiple subpopulations that differ in virulence and geographic distribution. Given these differences, there is a need to rapidly and accurately determine if a strain is F. tularensis and, if it is, assign it to subspecies and subpopulation. We designed TaqMan real-time PCR genotyping assays using eleven single nucleotide polymorphisms (SNPs) that were potentially specific to closely related groups within the genus Francisella, including numerous subpopulations within F. tularensis species. We performed extensive validation studies to test the specificity of these SNPs to particular populations by screening the assays across a set of 565 genetically and geographically diverse F. tularensis isolates and an additional 21 genetic near-neighbor (outgroup) isolates. All eleven assays correctly determined the genetic groups of all 565 F. tularensis isolates. One assay differentiates F. tularensis, F. novicida, and F. hispaniensis from the more genetically distant F. philomiragia and Francisella-like endosymbionts. Another assay differentiates F. tularensis isolates from near neighbors. The remaining nine assays classify F. tularensis-confirmed isolates into F. tularensis subspecies and subpopulations. The genotyping accuracy of these nine assays diminished when tested on outgroup isolates (i.e. non F. tularensis), therefore a hierarchical approach of assay usage is recommended wherein the F. tularensis-specific assay is used before the nine downstream assays. Among F. tularensis isolates, all eleven assays were highly sensitive, consistently amplifying very low concentrations of DNA. Altogether, these eleven TaqMan real-time PCR assays represent a highly accurate, rapid, and sensitive means of identifying the species, subspecies, and subpopulation of any F. tularensis isolate if used in a step-wise hierarchical scheme. These assays would be very useful in clinical, epidemiological, and/or forensic investigations involving F. tularensis.

Phylogenetic Lineages of Francisella tularensis in Animals

“…Branch length is not representative of evolutionary distance and is not scaled. Data presented in this figure was collected and assembled from the following publications: (Svensson et al, 2009a , b ; Vogler et al, 2009 ; Gyuranecz et al, 2012 ; Birdsell et al, 2014a , b ; Sissonen et al, 2015 ).…”

Optimized MALDI TOF Mass Spectrometry Identification of Francisella tularensis Subsp. holarctica

“…Molecular methods are now the gold standard for identifying Francisella sp. strains at the species, subspecies, and even genotype levels [ 20 , 21 ]. Real-time PCR tests are more adapted for the rapid and accurate detection and identification of F. tularensis .…”

Differences in Blood-Derived Francisella tularensis Type B Strains from Clinical Cases of Tularemia

“…Although the F. tularensis subspecies holarctica (type B) strains have fewer genomic rearrangements than the other clinically relevant subspecies of tularensis [ 18 , 24 ], erythromycin sensitivity versus resistance biovars, canonical SNP (canSNP) analyses, and chromosomal content have been used to identify and classify type B strains into various subgroups [ 18 , 19 , 28 , 29 ]. Karlsson and associates determined that the A2059C SNP in the 23S ribosomal RNA gene was conserved in biovar II strains and conferred the associated resistance to erythromycin [ 28 ].…”