Open Access Methodology article

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

Erin J Kelley1*, Elizabeth M Driebe1, Kizee Etienne2, Mary E Brandt2, James M Schupp1, John D Gillece1, Jesse S Trujillo1, Shawn R Lockhart2, Eszter Deak23, Paul S Keim14 and David M Engelthaler1

Author Affiliations

1 The Translational Genomics Research Institute, 3051 W. Shamrell Blvd. Ste. 106, Flagstaff, AZ 86001, USA

2 Centers for Disease Control and Prevention, Atlanta, GA, USA

3 Department of Pathology and Laboratory Medicine, David Geffen School of Medicine at UCLA, Los Angeles, CA, USA

4 Center for Microbial Genetics and Genomics, Northern Arizona University, Flagstaff, AZ, USA

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BMC Microbiology 2014, 14:125  doi:10.1186/1471-2180-14-125

Published: 13 May 2014

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.

Keywords:
Cryptococcus gattii; Genotyping; Real-time PCR; Epidemiology