| Summary: | Standardized, reproducible and validated Mucorales quantitative PCR (qPCR) assays are needed in the context of routine testing in diagnostic labs. We, therefore, compared the commercial MucorGenius<sup>®</sup> assay (PathoNostics, Maastricht) targeting five genera of Mucorales to our in-house qPCR targeting <i>Rhizomucor</i> spp., <i>Lichtheimia</i> spp. and <i>Mucor/Rhizopus</i> spp. To assess their analytical sensitivity, 25 frozen leftover serum specimens, which had already tested positive based on our in-house assay, were selected. These sera were from 15 patients with probable or proven mucormycosis. For analytical specificity, 0.5 pg from 15 purified fungal DNAs from nine different Mucorales genera were spiked into pooled qPCR-negative leftover serum specimens. All samples were tested in parallel with both assays and the quantitative cycles (Cq) were compared. A total of 13/25 (52%) serum samples were amplified by one of the two assays with only four of them detected with the MucorGenius<sup>®</sup> assay. In spiked specimens, all targeted strains were successfully amplified by our in-house qPCR. The MucorGenius<sup>®</sup> assay was not able to detect <i>Lichtheimia</i> <i>corymbifera</i> but successfully amplified all other species targeted by the kit and two additional non-targeted species (<i>Syncephalastrum</i> <i>monosporum</i> and <i>Saksenaea</i> <i>vasiformis</i>). The MucorGenius<sup>®</sup> assay showed lower analytical sensitivity compared to our in-house assay. Indeed, the MucorGenius<sup>®</sup> assay amplified more species, as expected, but showed a decreased detection of the frequent species <i>Lichtheimia</i> <i>corymbifera</i>.
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