parapsilosis (marked by arrows) showed doubtful profiles in McRAPD. When their fingerprints were compared to fingerprints of selected C. parapsilosis (CBS 604), orthopsilosis (MCO 456) and metapsilosis (CBS 2916 and MCO 448) strains identified and verified earlier, they clustered unquestionably with
C. metapsilosis. To see whether the strain clustering patterns resulting from McRAPD and conventional RAPD are consistent, McRAPD genotypes were color-coded by ground tint colors in the dendrogram of RAPD fingerprints using different color saturation for different genotypes (additional file 2: Dendrogram of RAPD fingerprints). Whereas McRAPD genotypes correlated very well with RAPD clustering in C. tropicalis, the correlation was limited in C. lusitaniae and no or almost no correlation was observed in C. Torin 2 in vivo albicans, C. krusei, and S. cerevisiae (no McRAPD genotypes were delineated in other species). This is mainly because of different data processing in conventional RAPD versus McRAPD. In RAPD, differences in overall amplification efficiency result in differences in NVP-BSK805 datasheet intensity of banding patterns. Therefore,
it is strongly recommended not to include weak bands into comparison of RAPD fingerprints, because these can appear or disappear in different amplification runs. Also, the relative intensity of strong bands cannot be reliably taken into account for comparison. That is why we used the band-based Jaccard coefficient for processing of RAPD fingerprints, which takes the position of a band into account but neglects its intensity. In contrast, raw fluorescence measured during melting learn more in the McRAPD procedure truly reflects the relative representation of individual RAPD products (bands in electrophoresis) in the sample. Inter-sample and inter-run differences in overall fluorescence of samples are subsequently proportionally equilibrated during numerical normalization of melting data. Then, relative representation of individual RAPD products is reflected in the slope of a normalized curve or in the height of a peak in a derivative curve and this
is taken into account during further evaluation. McRAPD data can be used for automated species identification Since McRAPD data are numerical, the possibility Fenbendazole of automated processing aimed to provide accurate identification is self-intriguing. We considered two approaches to achieve this objective. Firstly, absolute differences between normalized melting curves can be easily calculated as described in Material and Methods; such calculation can be simply automated. This should allow us to compare the McRAPD profile of an unknown isolate to a set of profiles obtained with previously identified yeast strains, revealing the closest match. Performance of such automated identification is summarized in Table 2. Overall accurate identification rate was 80%, varying between 58.5 and 100% in different species.