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. 2014 May 13:14:125.
doi: 10.1186/1471-2180-14-125.

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

Erin J Kelley  1 , 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
Affiliations

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

Erin J Kelley et al. BMC Microbiol. .

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.

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Figures

Figure 1
Figure 1
VGIIb MAMA plots with VGII DNA show the specificity of VGIIb MAMA for VGIIb DNA. (A) The VGIIb match primers amplify VGIIb DNA efficiently and yield a lower Ct value than the VGIIb mismatch primers, resulting in a VGIIb genotype call. (B) The VGIIb mismatch primers amplify VGIIa DNA more efficiently than the VGIIb match primers, resulting in a non-VGIIb genotype call. (C) VGIIb mismatch primers amplify VGIIc DNA more efficiently than the VGIIb match primers, again resulting in a non-VGIIb genotype call.
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