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. 2020 Dec 16;14(12):e0008897.
doi: 10.1371/journal.pntd.0008897. eCollection 2020 Dec.

The development of a novel diagnostic PCR for Madurella mycetomatis using a comparative genome approach

Affiliations

The development of a novel diagnostic PCR for Madurella mycetomatis using a comparative genome approach

Wilson Lim et al. PLoS Negl Trop Dis. .

Abstract

Background: Eumycetoma is a neglected tropical disease most commonly caused by the fungus Madurella mycetomatis. Identification of eumycetoma causative agents can only be reliably performed by molecular identification, most commonly by species-specific PCR. The current M. mycetomatis specific PCR primers were recently discovered to cross-react with Madurella pseudomycetomatis. Here, we used a comparative genome approach to develop a new M. mycetomatis specific PCR for species identification.

Methodology: Predicted-protein coding sequences unique to M. mycetomatis were first identified in BLASTCLUST based on E-value, size and presence of orthologues. Primers were then developed for 16 unique sequences and evaluated against 60 M. mycetomatis isolates and other eumycetoma causing agents including the Madurella sibling species. Out of the 16, only one was found to be specific to M. mycetomatis.

Conclusion: We have discovered a predicted-protein coding sequence unique to M. mycetomatis and have developed a new species-specific PCR to be used as a novel diagnostic marker for M. mycetomatis.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Presence of the 16 PCR amplicons in 60 M.
mycetomatis isolates tested. Most PCR reactions resulted in amplification in all isolates tested except PCR 4, 5 and 12.
Fig 2
Fig 2. The specificity of Mmy-Fw and Mmy-Rv on DNA isolated from eumycetoma grains.
Lane 1, 100 bp DNA ladder; Lane 2, negative control; Lane 3, 4, 6, 8, 11 and 12, Madurella mycetomatis DNA extracted from grains; Lane 5 and 7, Falciformispora senegalensis DNA extracted from grains; Lane 9 and 10, Falciformispora tompkinsii DNA extracted from grains; Lane 13, Madurella mycetomatis DNA from isolate as a positive control. Presence of amplicons on lane 3, 4, 5, 6, 8, 11 and 12 and none on the other lanes confirms the specificity of Mmy-Fw and Mmy-Rv towards M. mycetomatis.

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