TY - JOUR
T1 - Differentiation of francisella tularensis subspecies and subtypes
AU - Larson, Marilynn A.
AU - Sayood, Khalid
AU - Bartling, Amanda M.
AU - Meyer, Jennifer R.
AU - Starr, Clarise
AU - Baldwin, James
AU - Dempsey, Michael P.
N1 - Funding Information:
We thank the Applied Technology and Genomics Division, Wright-Patterson Air Force Base, Ohio, which is the awarding and administering acquisition office that supported this work with a grant (W9113M-07-1-0004) to M.P.D. and that was subcontracted to M.A.L. for assay development. The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication, and the views expressed in this article are those of the authors and do not reflect the official policy or position of the United States Air Force, the Department of Defense, or any agency or institute.
Publisher Copyright:
© 2020 Larson et al.
PY - 2020
Y1 - 2020
N2 - The highly infectious and zoonotic pathogen Francisella tularensis 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 Francisella tularensis subsp. novicida, there are considerable differences in genetic organization. These chromosomal disparities contribute to the substantial differences in virulence observed between the various F. tularensis subspecies and subtypes. The methods currently available to genotype F. tularensis 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 F. tularensis subsp. tularensis subtype A.I, the virulent F. tularensis subsp. tularensis subtype A.II, F. tularensis subsp. holarctica (also referred to as type B), and F. tularensis subsp. mediasiatica, as well as opportunistic F. tularensis subsp. novicida from each other and near neighbors, such as Francisella philomiragia, Francisella persica, and Francisellalike endosymbionts found in ticks. These fluorescence-based singleplex and non-matrix scoring multiplex qPCR assays utilize a hydrolysis probe, providing sensitive and specific F. tularensis subspecies and subtype identification in a rapid manner. Furthermore, sequencing of the amplified F. tularensis 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 F. tularensis subpopulations during epidemiological investigations of tularemia source outbreaks.
AB - The highly infectious and zoonotic pathogen Francisella tularensis 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 Francisella tularensis subsp. novicida, there are considerable differences in genetic organization. These chromosomal disparities contribute to the substantial differences in virulence observed between the various F. tularensis subspecies and subtypes. The methods currently available to genotype F. tularensis 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 F. tularensis subsp. tularensis subtype A.I, the virulent F. tularensis subsp. tularensis subtype A.II, F. tularensis subsp. holarctica (also referred to as type B), and F. tularensis subsp. mediasiatica, as well as opportunistic F. tularensis subsp. novicida from each other and near neighbors, such as Francisella philomiragia, Francisella persica, and Francisellalike endosymbionts found in ticks. These fluorescence-based singleplex and non-matrix scoring multiplex qPCR assays utilize a hydrolysis probe, providing sensitive and specific F. tularensis subspecies and subtype identification in a rapid manner. Furthermore, sequencing of the amplified F. tularensis 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 F. tularensis subpopulations during epidemiological investigations of tularemia source outbreaks.
KW - Diagnostics
KW - Francisella tularensis
KW - Genotyping
KW - Singleplex and multiplex quantitative real-time PCR
KW - Subspeciation
KW - Subspecies and subtype differentiation
KW - Tularemia
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U2 - 10.1128/JCM.01495-19
DO - 10.1128/JCM.01495-19
M3 - Article
C2 - 31941692
AN - SCOPUS:85082542349
SN - 0095-1137
VL - 58
JO - Journal of clinical microbiology
JF - Journal of clinical microbiology
IS - 4
M1 - e01495-19
ER -