title: Real-time PCR assays for genotyping of Cryptococcus gattii in North America.
abstract: BACKGROUND
  Cryptococcus gattii has been the cause of an ongoing outbreak starting in 1999 on Vancouver Island, British Columbia and spreading to mainland Canada and the US Pacific Northwest. In the course of the outbreak, C. gattii has been identified outside of its previously documented climate, habitat, and host disease. Genotyping of C. gattii is essential to understand the ecological and geographical expansion of this emerging pathogen.
  
  
  METHODS
  We developed and validated a mismatch amplification mutation assay (MAMA) real-time PCR panel for genotyping C. gattii molecular types VGI-VGIV and VGII subtypes a,b,c. Subtype assays were designed based on whole-genome sequence of 20 C. gattii strains. Publically available multilocus sequence typing (MLST) data from a study of 202 strains was used for the molecular type (VGI-VGIV) assay design. All assays were validated across DNA from 112 strains of diverse international origin and sample types, including animal, environmental and human.
  
  
  RESULTS
  Validation revealed each assay on the panel is 100% sensitive, specific and concordant with MLST. The assay panel can detect down to 0.5 picograms of template DNA.
  
  
  CONCLUSIONS
  The (MAMA) real-time PCR panel for C. gattii accurately typed a collection of 112 diverse strains and demonstrated high sensitivity. This is a time and cost efficient method of genotyping C. gattii best suited for application in large-scale epidemiological studies.
authors.affiliation: The Translational Genomics Research Institute, 3051 W, Shamrell Blvd, Ste, 106, Flagstaff, AZ 86001, USA. ekelley@tgen.org.
authors.forename: Erin J
authors.initials: EJ
authors.lastname: Kelley
authors.forename: Elizabeth M
authors.initials: EM
authors.lastname: Driebe
authors.forename: Kizee
authors.initials: K
authors.lastname: Etienne
authors.forename: Mary E
authors.initials: ME
authors.lastname: Brandt
authors.forename: James M
authors.initials: JM
authors.lastname: Schupp
authors.forename: John D
authors.initials: JD
authors.lastname: Gillece
authors.forename: Jesse S
authors.initials: JS
authors.lastname: Trujillo
authors.forename: Shawn R
authors.initials: SR
authors.lastname: Lockhart
authors.forename: Eszter
authors.initials: E
authors.lastname: Deak
authors.forename: Paul S
authors.initials: PS
authors.lastname: Keim
authors.forename: David M
authors.initials: DM
authors.lastname: Engelthaler
mesh_terms: D000818:Animals; D003453:Cryptococcosis / Q000382:microbiology / Q000662:veterinary; D056285:Cryptococcus gattii / Q000145:classification* / Q000235:genetics* / Q000302:isolation & purification; D004271:DNA, Fungal / Q000235:genetics; D004783:Environmental Microbiology; D060005:Genotyping Techniques / Q000379:methods*; D006801:Humans; D017720:Molecular Epidemiology / Q000379:methods; D016533:Mycological Typing Techniques / Q000379:methods*; D009656:North America / Q000453:epidemiology; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity
chemical_list: D004271:DNA, Fungal