Real-time PCR assays for genotyping of Cryptococcus gattii in North America

Erin J. Kelley, Elizabeth M. Driebe, Kizee Etienne, Mary E. Brandt, James M. Schupp, John D. Gillece, Jesse S. Trujillo, Shawn R. Lockhart, Eszter Deak, Paul S Keim, David M. Engelthaler

Research output: Contribution to journalArticle

2 Citations (Scopus)

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.

Original languageEnglish (US)
Article number125
JournalBMC Microbiology
Volume14
Issue number1
DOIs
StatePublished - May 13 2014

Fingerprint

Cryptococcus gattii
North America
Real-Time Polymerase Chain Reaction
Multilocus Sequence Typing
Disease Outbreaks
Northwestern United States
Mutation
British Columbia
DNA
Climate
Islands
Canada
Ecosystem
Epidemiologic Studies
Genome
Costs and Cost Analysis

Keywords

  • Cryptococcus gattii
  • Epidemiology
  • Genotyping
  • Real-time PCR

ASJC Scopus subject areas

  • Microbiology (medical)
  • Microbiology

Cite this

Kelley, E. J., Driebe, E. M., Etienne, K., Brandt, M. E., Schupp, J. M., Gillece, J. D., ... Engelthaler, D. M. (2014). Real-time PCR assays for genotyping of Cryptococcus gattii in North America. BMC Microbiology, 14(1), [125]. https://doi.org/10.1186/1471-2180-14-125

Real-time PCR assays for genotyping of Cryptococcus gattii in North America. / Kelley, Erin J.; Driebe, Elizabeth M.; Etienne, Kizee; Brandt, Mary E.; Schupp, James M.; Gillece, John D.; Trujillo, Jesse S.; Lockhart, Shawn R.; Deak, Eszter; Keim, Paul S; Engelthaler, David M.

In: BMC Microbiology, Vol. 14, No. 1, 125, 13.05.2014.

Research output: Contribution to journalArticle

Kelley, EJ, Driebe, EM, Etienne, K, Brandt, ME, Schupp, JM, Gillece, JD, Trujillo, JS, Lockhart, SR, Deak, E, Keim, PS & Engelthaler, DM 2014, 'Real-time PCR assays for genotyping of Cryptococcus gattii in North America', BMC Microbiology, vol. 14, no. 1, 125. https://doi.org/10.1186/1471-2180-14-125
Kelley EJ, Driebe EM, Etienne K, Brandt ME, Schupp JM, Gillece JD et al. Real-time PCR assays for genotyping of Cryptococcus gattii in North America. BMC Microbiology. 2014 May 13;14(1). 125. https://doi.org/10.1186/1471-2180-14-125
Kelley, Erin J. ; Driebe, Elizabeth M. ; Etienne, Kizee ; Brandt, Mary E. ; Schupp, James M. ; Gillece, John D. ; Trujillo, Jesse S. ; Lockhart, Shawn R. ; Deak, Eszter ; Keim, Paul S ; Engelthaler, David M. / Real-time PCR assays for genotyping of Cryptococcus gattii in North America. In: BMC Microbiology. 2014 ; Vol. 14, No. 1.
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