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

<div><p><i>Francisella tularensis</i>, 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 <i>F. tularensis</i> 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 <i>Francisella</i>, including numerous subpopulations within <i>F. tularensis</i> 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 <i>F. tularensis</i> isolates and an additional 21 genetic near-neighbor (outgroup) isolates. All eleven assays correctly determined the genetic groups of all 565 <i>F. tularensis</i> isolates. One assay differentiates <i>F. tularensis</i>, <i>F. novicida</i>, and <i>F. hispaniensis</i> from the more genetically distant <i>F. philomiragia</i> and <i>Francisella-</i>like endosymbionts. Another assay differentiates <i>F. tularensis</i> isolates from near neighbors. The remaining nine assays classify <i>F. tularensis</i>-confirmed isolates into <i>F. tularensis</i> subspecies and subpopulations. The genotyping accuracy of these nine assays diminished when tested on outgroup isolates (i.e. non <i>F. tularensis</i>), therefore a hierarchical approach of assay usage is recommended wherein the <i>F. tularensis</i>-specific assay is used before the nine downstream assays. Among <i>F. tularensis</i> 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 <i>F. tularensis</i> isolate if used in a step-wise hierarchical scheme. These assays would be very useful in clinical, epidemiological, and/or forensic investigations involving <i>F. tularensis</i>.</p></div

Primers and TaqMan-MGB probes for <i>F. tularensis</i> canSNP assays.

<p>Primers and TaqMan-MGB probes for <i>F. tularensis</i> canSNP assays.</p

Schematic evolutionary tree of <i>Francisella tularensis</i> and <i>Francisella</i> genetic near neighbor species.

<p>Black bars indicate the important canSNP signatures specific to major genetic groups among <i>Francisella</i> species and within <i>F. tularensis</i>. The three recognized subspecies*, as well as divisions within the two major subspecies, <i>tularensis</i> and <i>holarctica</i>, are indicated. The strain representing each genetic group is indicated in blue text.</p