Evaluation of rca (rolling circle amplification) method by targeting its rdna gen region for identification of exophiala species

Molecular diagnostics are succesfully applied for the identification of black yeasts, particularly for the genus Exophiala. These techniques are also improve our understanding their epidemiological characteristics from bench to the bedside. We developed two ligase-dependent probe amplification assays based on the rolling circle amplification (RCA) and ligase-dependent reaction (LDR) methods to differentiate the species from the Exophiala genus by targeting the rDNA internal spacer transcribed (ITS) region. We evaluated Exophiala dermatitidis, E. phaeomuriformis, E. heteromorpha, E. xenobiotica, and E. crusticola, as these species had been previously distinguished using ITS sequencing. We investigated 57 reference isolates representing these five species using RCA and LDR methods. Depending on the RCA probes used, the reaction sensitivity was 100% and the specificity ranged from 4.7% to 85.7% (median: 45.2%). In contrast, the sensitivity and specificity of the LDR probes targeting the same isolates were 91.6–100% (median: 95.8%) and 95.4–100% (median 97.7%), respectively. We also assessed 198 additional environmental isolates that represented the same Exophiala species and had been previously identified using ITS sequencing. The overall sensitivity and specificity of the LDR probes that targeted the 255 isolates ranged from 89.9% to 100% (median: 94.1%) and from 93.7% to 100% (median 96.8%), respectively. The assessment of the performance and validation of the LDR probes based on qPCR with SYBR Green revealed high reproducibility and an acceptable range limit in line with the guidelines of the European Network of GMO Laboratories. In conclusion, LDR was found to be more reliable and inexpensive than RCA for species-level identification of Exophiala isolates. To the best our knowledge, this study comprises the first analysis of the efficacy of LDR in the medical mycology field, particularly for the black yeast genus Exophiala identifications.