Specificity of 16S rRNA and IS2404 qPCR assays.

Table 2 shows DNA extracts from closely related mycobacterial species and bacteria potentially contaminating the human skin subjected to the combined 16S rRNA RT/IS2404 qPCR viability assay and the corresponding test results. Mycobacterial species were selected according to their respective genetic contiguousness to M. ulcerans Agy99 (GenBank accession no. CP000325.1) within the 16S rRNA gene sequences as determined by BLASTN analysis (GenBank, NCBI) [13]. M., Mycobacterium; E., Escherichia; P., Propionibacterium; Staph., Staphylococcus; Str., Streptococcus. While in-silico analysis revealed that the combined 16S rRNA RT/IS2404 assay will also amplify mycolactone-producing mycobacteria (MPM) other than M. ulcerans (e.g., M. pseudoshottsii, M. liflandii, and the environmental M. marinum [GenBank accession No. NR_042988.1, AY500838.1, and AF456241.1, respectively]), these MPM species were not included in specificity testing.aDNA extracts that were not available at the DITM were provided by the National Reference Center (NRZ) for Mycobacteria, Borstel, Germany, and the Max von Pettenkofer-Institute (MVP), Ludwig-Maximilians University, Munich, Germany.bThe respective primary patient isolates were considered as ppathogenic bacteria or as ccommensals/contaminants of clinical samples.dResults of the 16S rRNA RT-qPCR of DNA extracts; “+” indicates a positive and “–” a negative test result.eResults of the IS2404 qPCR of DNA extracts; “+” indicates a positive and “–” a negative test result.