2.5. In Silico ITS-RFLP

In order to identify enzymes with the potential for species differentiation among the etiological agents of chromoblastomycosis and phaeohyphomycosis, in silico screening was conducted using sequences from the ITS region of the study’s target species (N = 60) available in the GenBank database (Table S1). These sequences were downloaded and compiled into a MultiFasta file and aligned with the online software Mafft 7 “https://mafft.cbrc.jp/alignment/server/index.html (accessed on 20 August 2023)” [19].

Subsequently, the presence of restriction sites for commercially available enzymes was analyzed using the CLC Sequence Viewer software 8.0^® [20], with the goal of generating fragments of distinct sizes suitable for species differentiation while ensuring the consistency of restriction sites within the same species. Following the screening and identification of potential enzymes, the online tool NEBcutter v.3^® from New England Biolabs “https://nc3.neb.com/NEBcutter/ (accessed on 25 August 2023)” was employed for visualizing the fragmentation pattern in a virtual agarose gel [21].