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Med Mycol. 2016 Jan;54(1):89-96. doi: 10.1093/mmy/myv078. Epub 2015 Sep 21.

Accurate and sensitive real-time PCR assays using intergenic spacer 1 region to differentiate Cryptococcus gattii sensu lato and Cryptococcus neoformans sensu lato.

Author information

1
Programa de Pós-graduação em Microbiologia, Londrina, PR, Brazil.
2
Departamento de Microbiologia, Ciências Biológicas, Universidade Estadual de Londrina, Londrina, PR, Brazil.
3
Departamento de Patologia Aplicada, Análise Clínicas e Toxicológicas, Centro de Ciências da Saúde, Universidade Estadual de Londrina, Londrina, PR, Brazil.
4
Departamento de Ciências Patológicas, Centro de Ciências Biológicas, Universidade Estadual de Londrina, Londrina, PR, Brazil.
5
Departamento de Análises Clínicas e Toxicológicas, Faculdade de Farmácia, Universidade Federal da Bahia, Salvador, BA, Brazil.
6
Departamento de Microbiologia, Ciências Biológicas, Universidade Estadual de Londrina, Londrina, PR, Brazil lionilmy@uel.br.

Abstract

In this work, two accurate and sensitive real-time polymerase chain reaction (PCR) assays to differentiate pathogenic Cryptococcus gattii sensu lato (s.l.) and C. neoformans sensu lato (s.l.) targeting the intergenic spacer 1 (IGS1) region from rDNA locus were developed. Specific primers were designed based on their IGS1 sequence analyses and the optimal real-time PCR assays showed that the dissociation curves generated two different melting peaks, at 82.8 and 84.2ºC for C. gattii s.l. and C. neoformans s.l., respectively. No amplifications were observed in the negative template control. The minimum limit of detection of both primers was 100 plasmid copies per reaction, and they were highly specific when tested with a range of fungal DNAs. Overall, the results showed that the designed primers completely differentiated C. gattii s.l. and C. neoformans s.l. from clinical and environmental sources with great accuracy when compared to phenotypic identification, with no cross-reactivity to other fungal DNA.

KEYWORDS:

Cryptococcus; IGS1; differentiation; real-time PCR

PMID:
26392390
DOI:
10.1093/mmy/myv078
[Indexed for MEDLINE]

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