title: Accurate and sensitive real-time PCR assays using intergenic spacer 1 region to differentiate Cryptococcus gattii sensu lato and Cryptococcus neoformans sensu lato.
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.
authors.affiliation: Programa de Pós-graduação em Microbiologia, Londrina, PR, Brazil.
authors.forename: Eliandro Reis
authors.initials: ER
authors.lastname: Tavares
authors.affiliation: Programa de Pós-graduação em Microbiologia, Londrina, PR, Brazil.
authors.forename: Caroline Souza
authors.initials: CS
authors.lastname: Azevedo
authors.affiliation: Departamento de Microbiologia, Ciências Biológicas, Universidade Estadual de Londrina, Londrina, PR, Brazil.
authors.forename: Luciano Aparecido
authors.initials: LA
authors.lastname: Panagio
authors.affiliation: Departamento de Patologia Aplicada, Análise Clínicas e Toxicológicas, Centro de Ciências da Saúde, Universidade Estadual de Londrina, Londrina, PR, Brazil.
authors.forename: Marsileni
authors.initials: M
authors.lastname: Pelisson
authors.affiliation: Departamento de Ciências Patológicas, Centro de Ciências Biológicas, Universidade Estadual de Londrina, Londrina, PR, Brazil.
authors.forename: Phileno
authors.initials: P
authors.lastname: Pinge-Filho
authors.affiliation: Departamento de Ciências Patológicas, Centro de Ciências Biológicas, Universidade Estadual de Londrina, Londrina, PR, Brazil.
authors.forename: Emerson José
authors.initials: EJ
authors.lastname: Venancio
authors.affiliation: Departamento de Análises Clínicas e Toxicológicas, Faculdade de Farmácia, Universidade Federal da Bahia, Salvador, BA, Brazil.
authors.forename: Tânia Fraga
authors.initials: TF
authors.lastname: Barros
authors.affiliation: Departamento de Microbiologia, Ciências Biológicas, Universidade Estadual de Londrina, Londrina, PR, Brazil.
authors.forename: Sueli Fumie
authors.initials: SF
authors.lastname: Yamada-Ogatta
authors.affiliation: Departamento de Microbiologia, Ciências Biológicas, Universidade Estadual de Londrina, Londrina, PR, Brazil lionilmy@uel.br.
authors.forename: Lucy Megumi
authors.initials: LM
authors.lastname: Yamauchi
mesh_terms: D056285:Cryptococcus gattii / Q000145:classification* / Q000235:genetics; D003455:Cryptococcus neoformans / Q000145:classification* / Q000235:genetics; D017931:DNA Primers / Q000235:genetics; D004271:DNA, Fungal / Q000235:genetics; D021903:DNA, Ribosomal Spacer / Q000235:genetics*; D006801:Humans; D008828:Microbiological Techniques / Q000379:methods*; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D044366:Transition Temperature
chemical_list: D017931:DNA Primers; D004271:DNA, Fungal; D021903:DNA, Ribosomal Spacer
keywords: Cryptococcus; IGS1; differentiation; real-time PCR