Assay validation with temperature gradient and standard curve.

Assay development consisted of a temperature gradient approach on standard samples of DNA extracted from <i>Chondria tumulosa</i> tissue and no-template controls (NTC) to monitor for contamination. (a) The ideal annealing temperature (54.7–64°C) was selected among triplicate amplifications that produced a sigmoidal curve in the fewest cycles. (b) Melt curves were verified to only produce a single tall, narrow peak. (c) Threshold cycle (C_q) values are plotted against triplicate DNA serial dilution starting concentrations to generate a best fit standard curve (R² = 0.999, slope = -3.79, intercept = 33.62), using the curve-fitting approach [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0318414#pone.0318414.ref063" target="_blank">63</a>]. Synthetic <i>C</i>. <i>tumulosa</i> DNA was quantified using a fluorometer (Qubit, Invitrogen) and diluted to create 10-fold serial dilutions ranging from 10⁶ to 10⁰ copies per reaction. The limit of detection (LOD, where 95% of technical replicates amplify) is marked with a solid red line and the limit of quantification (LOQ, the lowest initial DNA concentration quantifiable with a coefficient of variation below 35%) is marked with a dashed line. (DOCX)