Fig. 2.

Graphical representation of the principle of Pairwise Efficiency method and its application to six dilution curves. (a) A graphical illustration of the Pairwise Efficiency method. Small portions of three amplification curves, with three fluorescence data points on each, are shown. Dashed line connects point A to point F on separate curves, and represents a single, unique pairwise E determination (pair AF). All possible pairs, each one representing a unique pairwise E value, are shown on the right. Since some of the values occur on the same cycle (for example, AE, BF), such values are excluded from the determinations, and are denoted in gray. (b) The amplification curves from the wells C1 through C6 are shown (RFU data taken from Additional file 2: Dataset 1). Different shapes (circles, squares, triangles etc.) represent fluorescence readings taken by the machine after each PCR cycle. Horizontal lines denote the region of amplification curves from which the fluorescence data points were taken for analysis. Upper cutoff was set at 180 RFU, and lower cutoff was set at 20 RFU. In this experiment, the total of 24 fluorescence data points fall inside the denoted region, and unique pairs formed by these 24 points, excluding repetitive values occurring on the same cycle, are taken for analysis