Amplification profiles and determination of the number molecules with perfect match and mismatch primers. (A) Data generated from amplification profiles presented in (B). The number of molecules calculated with different methods is presented. The respective standard curve (SC) data is directly linked to the amplification profiles shown in (B) and input number of molecules. The LRE method is dependent only on fluorescence data (B) and instrument calibration. The Avg SC data is derived from an average standard curves obtained with perfect match primer pairs. Accuracy is calculated with the respective standard curves and represents the average ratio of observed to input molecules. Specificity is calculated with LRE or Avg SC data and represents the average ratio of observed to input molecules. Sensitivity is the limit of detection of an assay and is defined as the lowest input number of molecules generating a complete amplification profile . The input number of molecules was determined optically using a spectrophotometer. (C) Melting profiles associated to the amplification profiles presented in (B).