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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 .

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