Species-specific qPCR performance
Using our qPCR assay, N. barbatae DNA from two different isolates was successfully detected in the absence of all otherNannizziopsis species in this study, including the closely related N. crocodili . These results suggest that mtDNA targets can be used to distinguish between species of Nannizziopsisfungi. A small degree of non-specific fluorescence was observed fromN. dermatitidis DNA at the optimal probe annealing Tm of 60oC owing to a lower amount of variation in this species at the probe target location (FIGURE 1). This was resolved without sacrificing reaction efficiency by raising the annealing temperature to 63oC. The probe sequence confers the specificity in the assay as the PCR primer sequences were conserved across Nannizziopsis species and could produce an amplicon from all but N. vriesii (Supplementary Figure 1).
Standard curve DNA was diluted across genome equivalent (GE) values 400,000 GE, 40,000 GE, 4,000 GE, 400 GE, 40 GE, 4 GE and 0.4 GE with each standard prepared in triplicate reactions in two separate qPCR runs to evaluate for reproducibility (FIGURE 4). The reaction efficiency ranged from 0.89 to 1.01 and the R2 values were greater than 0.99 for both runs (ranging from 0.996 – 0.999). TheC t values of the quantitation standards were also consistent between runs with each dilution crossing the cycle threshold at approximately the same cycle number.
To test the performance of this assay to detect the presence of N. barbatae from field collected samples, a total of 96 skin swab samples were obtained from 85 individual animals that were either healthy or displaying various levels of disease severity (see example in Supplementary Figure 2) over the course of 2021 and tested using this protocol. A total of 67 swab samples returned a positive result for infection with N. barbatae (TABLE 2).