Identifying Paracoccidioides phylogenetic species by PCR-RFLP of the alpha-tubulin gene

Identifying Paracoccidioides phylogenetic species by PCR-RFLP of the alpha-tubulin gene

Author Roberto, Thiago Nunes Autor UNIFESP Google Scholar
Rodrigues, Anderson Messias Autor UNIFESP Google Scholar
Hahn, Rosane Christine Google Scholar
Camargo, Zoilo Pires de Autor UNIFESP Google Scholar
Abstract Paracoccidioidomycosis is an important systemic fungal infection that occurs throughout Latin America. The etiological agents comprise a species complex that includes two major groups: P. brasiliensis (including subgroups S1, PS2, and PS3) and P. lutzii. A great number of phenotypes may overlap, especially among closely related groups, discouraging the use ofmorphology alone for species recognition. To overcome this problem, here we propose identifying cryptic Paracoccidioides spp. using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) of the alpha-tubulin (TUB1) gene. In silico analysis of 90 TUB1 sequences led to the identification of two restriction enzymes with the potential to identify Paracoccidioides: Bcl I and MspI. A portion of the TUB1 gene was amplified and double digested in vitro with the Bcl I and MspI endonucleases, which generated four different electrophoretic patterns corresponding to the four main genetic groups: S1, PS2, and PS3 of P. brasiliensis and P. lutzii. The major P. brasiliensis group recognized was S1 (n = 17; 42.5%), followed by PS2 (n = 9; 22.5%) and PS3 (n = 6; 15%). A total of eight (20%) P. lutzii isolates were identified, mainly from mid western Brazil. Our data revealed that TUB1-RFLP is an efficient, fast, and inexpensive tool for identifying Paracoccidioides spp., which may be directly applied to the molecular epidemiological studies of paracoccidioidomycosis.
Keywords Paracoccidioides
Paracoccidioidomycosis
PCR-RFLP
Molecular identification
Alpha-tubulin
xmlui.dri2xhtml.METS-1.0.item-coverage Oxford
Language English
Sponsor Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)
Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)
Grant number FAPESP: 2013/05405-9
FAPESP: 2011/07350-1
FAPESP: 2009/54024-2
Date 2016
Published in Medical Mycology. Oxford, v. 54, n. 3, p. 240-247, 2016.
ISSN 1369-3786 (Sherpa/Romeo, impact factor)
Publisher Oxford Univ Press
Extent 240-247
Origin http://dx.doi.org/10.1093/mmy/myv083
Access rights Open access Open Access
Type Article
Web of Science ID WOS:000372105200003
URI https://repositorio.unifesp.br/handle/11600/57888

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