Supplemental Figure S2.

Validation of 25-nt target PCR. A representative calibration curve is given for amplification of the 25-nt target, but individual calibration curves were run for each experiment to account for any minor variances in template dilution. Amplification of the 25-nt target has an efficiency of 95.8% (R² = 0.999) and a linear dynamic range of 10² to 10⁶ copies. Amplification of 5 to 10 copies was possible, but with reduced efficiency. The quantification cycle (C_q) of the no template control (NTC) for the 25-nt assay is >40 cycles (not including the 10 first-stage preamplification cycles). In the representative calibration curve, four of six NTCs did not amplify above the 100 relative fluorescence unit (RFU) threshold. The other two NTCs had C_qs of 42.5 and 44.8 (effectively, 52.5 and 54.8 cycles, respectively). Specificity of the 25-nt target amplification is ensured by using a sequence-specific probe. n = 6 per standard concentration. y-int, y-intercept.