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

• Published in: PloS one 2014-09, Vol.9 (9), p.e107964-e107964
• Main Authors: Birdsell, Dawn N, Vogler, Amy J, Buchhagen, Jordan, Clare, Ashley, Kaufman, Emily, Naumann, Amber, Driebe, Elizabeth, Wagner, David M, Keim, Paul S
• Format: Article
• Language: English
• Subjects: Assaying; Bacteria; Biology and life sciences; Chromosomes; Deoxyribonucleic acid; DNA; DNA, Bacterial - chemistry; Endosymbionts; Epidemiology; Etiology; Fish; Forensic engineering; Forensic science; Forensic sciences; Francisella tularensis; Francisella tularensis - classification; Francisella tularensis - genetics; Francisella tularensis - isolation & purification; Genetic aspects; Genetics; Genomics; Genotype; Genotyping; Genotyping Techniques; Geographical distribution; Humans; Laboratories; Low concentrations; Polymerase chain reaction; Polymorphism, Single Nucleotide; Real time; Real-Time Polymerase Chain Reaction - methods; Research and Analysis Methods; Single nucleotide polymorphisms; Single-nucleotide polymorphism; Subpopulations; Tularemia; Tularemia - diagnosis; Virulence
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0107964

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.