TaqMan real-time PCR assays for single-nucleotide polymorphisms which identify Francisella tularensis and its subspecies and subpopulations

Dawn N Birdsell; Amy J Vogler; Jordan Buchhagen; Ashley Clare; Emily Kaufman; Amber Naumann; Elizabeth Driebe; David M Wagner; Paul S Keim

doi:10.1371/journal.pone.0107964

TaqMan real-time PCR assays for single-nucleotide polymorphisms which identify Francisella tularensis and its subspecies and subpopulations

PLoS One. 2014 Sep 19;9(9):e107964. doi: 10.1371/journal.pone.0107964. eCollection 2014.

Authors

Dawn N Birdsell¹, Amy J Vogler¹, Jordan Buchhagen², Ashley Clare¹, Emily Kaufman¹, Amber Naumann¹, Elizabeth Driebe², David M Wagner¹, Paul S Keim²

Affiliations

¹ Center for Microbial Genetics and Genomics, Northern Arizona University, Flagstaff, Arizona, United States of America.
² Center for Microbial Genetics and Genomics, Northern Arizona University, Flagstaff, Arizona, United States of America; Translational Genomics Research Institute, Flagstaff, Arizona, United States of America.

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.

Publication types

Research Support, Non-U.S. Gov't
Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

DNA, Bacterial / chemistry
Francisella tularensis / classification
Francisella tularensis / genetics*
Francisella tularensis / isolation & purification
Genotype
Genotyping Techniques
Humans
Polymorphism, Single Nucleotide*
Real-Time Polymerase Chain Reaction / methods
Tularemia / diagnosis

Substances

DNA, Bacterial

Grants and funding

This work was funded by the Department of Homeland Security Science and Technology Directorate via award NBCH2070001 and the Cowden Endowment in Microbiology at Northern Arizona University. Note that the use of products/names does not constitute endorsement by the Department of Homeland Security of the United States. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.