Differentiation of Francisella tularensis Subspecies and Subtypes

The highly infectious and zoonotic pathogen is the etiologic agent of tularemia, a potentially fatal disease if untreated. Despite the high average nucleotide identity, which is >99.2% for the virulent subspecies and >98% for all four subspecies, including the opportunistic microbe subsp. , there are considerable differences in genetic organization. These chromosomal disparities contribute to the substantial differences in virulence observed between the various subspecies and subtypes. The methods currently available to genotype cannot conclusively identify the associated subpopulation without using time-consuming testing or complex scoring matrices. To address this need, we developed both single and multiplex quantitative real-time PCR (qPCR) assays that can accurately detect and identify the hypervirulent subsp. subtype A.I, the virulent subsp. subtype A.II, subsp. (also referred to as type B), and subsp. , as well as opportunistic subsp. from each other and near neighbors, such as , , and -like endosymbionts found in ticks. These fluorescence-based singleplex and non-matrix scoring multiplex qPCR assays utilize a hydrolysis probe, providing sensitive and specific subspecies and subtype identification in a rapid manner. Furthermore, sequencing of the amplified targets provides clade confirmation and informative strain-specific details. Application of these qPCR- and sequencing-based detection assays will provide an improved capability for molecular typing and clinical diagnostics, as well as facilitate the accurate identification and differentiation of subpopulations during epidemiological investigations of tularemia source outbreaks.