Comparison between PCR–RFLP and sequencing techniques in the analysis of Paracoccidioides spp. biodiversity: limitations and insights into species and variant differentiation

• Published in: Mycopathologia (1975) Vol. 189; no. 6; p. 97
• Main Authors: Leite, Ingrid Gonçalves Costa, Benard, Gil, Cavalcanti, Sonia Cristina, Bollela, Valdes Roberto, Del Negro, Gilda Maria Barbaro, Martinez, Roberto, Gimenes, Viviane Mazo Fávero, Cocio, Tiago Alexandre
• Format: Journal Article
• Language: English
• Published: Dordrecht Springer Netherlands 01-12-2024; Springer Nature B.V
• Subjects: Biodiversity; Biomedical and Life Sciences; Brazilian culture; Epidemiology; Eukaryotic Microbiology; Evolution; Genetic diversity; Genotypes; Geographical distribution; Glycoprotein gp43; Life Sciences; Medical Microbiology; Microbial Ecology; Microbiology; Molecular biology; New species; Nucleotide sequence; Original Article; Paracoccidioides; Phylogeny; Plant Sciences; Polymerase chain reaction; Strains (organisms); Ecology of; Restriction fragment length polymorphism; DNA sequence analysis; Biodiversity; Molecular biology; species
• ISSN: 0301-486X; 1573-0832; 1573-0832
• DOI: 10.1007/s11046-024-00902-w

Background
The study of
Paracoccidioides
spp. faces significant challenges due to limitations inherent in the molecular biology techniques employed. Recently, new species were described whose geographical and genetic distributions were investigated. The phylogenetic studies have revealed that genotypes originally thought to be exclusive in specific regions from South American countries are now being found in other areas of the continent. This finding indicates a broader geographic distribution of these genotypes than previously recognized.
Objective
To evaluate two molecular biology techniques employed to identify genotypes of
Paracoccidioides
spp. strains from a Brazilian culture collection previously identified only by mycological methods.
Methods
DNA samples from 35
Paracoccidioides
spp. strains maintained in a Brazilian culture collection were subjected to amplification and enzymatic digestion with PCR–RFLP of
tub
1 gene, followed by sequencing of
gp43
Exon 2 loci. Strains with species identification discrepancies had their
tub
1 sequences determined to verify possible nucleotide mutations.
Results
The genotypic characterization of
Paracoccidioides
spp. using PCR–RFLP of the
tub
1 gene identified 22 isolates as
P. brasiliensis
sensu stricto, two as
P. americana
, four as
P. restrepiensis
, and eight as
P. lutzii
. Sequencing of the
gp43
Exon 2 loci revealed discrepancies in the identification of four
P. venezuelensis
isolates, previously characterized as
P. brasiliensis
sensu stricto by PCR–RFLP of
tub
1. The sequencing of
tub
1 from
P. brasiliensis
sensu stricto and
P. venezuelensis
isolates revealed nucleotide differences in the pyrimidine class (C and T) in their sequences, specifically at the position 176 bp.
Conclusion
These molecular tools were able to establish the genetic diversity within the
Paracoccidioides
genus, crucial for taxonomy and epidemiology studies. The finding of presence of
P. venezuelensis
in Brazil, previously thought to be exclusive to Venezuela, highlights genetic connections and evolutionary divergences within the genus. While the PCR–RFLP of
tub
1technique showed limitations in identifying
P. venezuelensis
, sequencing of the
gp43
Exon 2 loci was able to accurately identify this genotype. Thus, our findings contribute to the understanding of the molecular epidemiology of PCM and emphasize the need for precise species characterization in mycological research.