Serial dilutions of the target sequence DNA sequence, unique to emydid mycoplasma, versus corresponding cycle threshold (Ct) values in the quantitative PCR assay used to test nasal lavage samples obtained from three-toed box turtles (Terrapene carolina triunguis) for emydid mycoplasma. These dilutions were run as calibration curves on each plate to convert Ct values into copy numbers, and to generate a mean calibration curve of y=41.4–3.386x (see Table 1 for additional specifications). Each point is the mean of three replicates, and each dilution curve was run on a separate plate. Although the detection limit was roughly 50 copies, the assay was optimized for DNA concentrations ranging from 5×108 to 5×102 copies. Both intra- and interassay variability increased the most below 5×102 copies, but also above 5×106 copies.
