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

Persistent Link:
http://hdl.handle.net/10150/610059
Title:
Real-time PCR assays for genotyping of Cryptococcus gattii in North America
Author:
Kelley, Erin; Driebe, Elizabeth; Etienne, Kizee; Brandt, Mary; Schupp, James; Gillece, John; Trujillo, Jesse; Lockhart, Shawn; Deak, Eszter; Keim, Paul; Engelthaler, David
Affiliation:
The Translational Genomics Research Institute, 3051 W. Shamrell Blvd. Ste. 106, Flagstaff, AZ 86001, USA; Centers for Disease Control and Prevention, Atlanta, GA, USA; Department of Pathology and Laboratory Medicine, David Geffen School of Medicine at UCLA, Los Angeles, CA, USA; Center for Microbial Genetics and Genomics, Northern Arizona University, Flagstaff, AZ, USA
Issue Date:
2014
Publisher:
BioMed Central
Citation:
Kelley et al. BMC Microbiology 2014, 14:125 http://www.biomedcentral.com/1471-2180/14/125
Journal:
BMC Microbiology
Rights:
© 2014 Kelley et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0)
Collection Information:
This item is part of the UA Faculty Publications collection. For more information this item or other items in the UA Campus Repository, contact the University of Arizona Libraries at repository@u.library.arizona.edu.
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.
EISSN:
1471-2180
DOI:
10.1186/1471-2180-14-125
Keywords:
Cryptococcus gattii; Genotyping; Real-time PCR; Epidemiology
Version:
Final published version
Additional Links:
http://www.biomedcentral.com/1471-2180/14/125

Full metadata record

DC FieldValue Language
dc.contributor.authorKelley, Erinen
dc.contributor.authorDriebe, Elizabethen
dc.contributor.authorEtienne, Kizeeen
dc.contributor.authorBrandt, Maryen
dc.contributor.authorSchupp, Jamesen
dc.contributor.authorGillece, Johnen
dc.contributor.authorTrujillo, Jesseen
dc.contributor.authorLockhart, Shawnen
dc.contributor.authorDeak, Eszteren
dc.contributor.authorKeim, Paulen
dc.contributor.authorEngelthaler, Daviden
dc.date.accessioned2016-05-20T08:57:38Z-
dc.date.available2016-05-20T08:57:38Z-
dc.date.issued2014en
dc.identifier.citationKelley et al. BMC Microbiology 2014, 14:125 http://www.biomedcentral.com/1471-2180/14/125en
dc.identifier.doi10.1186/1471-2180-14-125en
dc.identifier.urihttp://hdl.handle.net/10150/610059-
dc.description.abstractBACKGROUND: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.en
dc.language.isoenen
dc.publisherBioMed Centralen
dc.relation.urlhttp://www.biomedcentral.com/1471-2180/14/125en
dc.rights© 2014 Kelley et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0)en
dc.subjectCryptococcus gattiien
dc.subjectGenotypingen
dc.subjectReal-time PCRen
dc.subjectEpidemiologyen
dc.titleReal-time PCR assays for genotyping of Cryptococcus gattii in North Americaen
dc.typeArticleen
dc.identifier.eissn1471-2180en
dc.contributor.departmentThe Translational Genomics Research Institute, 3051 W. Shamrell Blvd. Ste. 106, Flagstaff, AZ 86001, USAen
dc.contributor.departmentCenters for Disease Control and Prevention, Atlanta, GA, USAen
dc.contributor.departmentDepartment of Pathology and Laboratory Medicine, David Geffen School of Medicine at UCLA, Los Angeles, CA, USAen
dc.contributor.departmentCenter for Microbial Genetics and Genomics, Northern Arizona University, Flagstaff, AZ, USAen
dc.identifier.journalBMC Microbiologyen
dc.description.collectioninformationThis item is part of the UA Faculty Publications collection. For more information this item or other items in the UA Campus Repository, contact the University of Arizona Libraries at repository@u.library.arizona.edu.en
dc.eprint.versionFinal published versionen
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