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).