Figure 2
PCR amplification curves of HMBS standards and canine blood specimens. HMBS standards and DNA of a canine whole blood specimen were logarithmically diluted (103 to 10-1 HMBS template copies or PCR input equivalents of 102 to 10-2 nL canine blood) were amplified in a background of 200 ng pUC19 plasmid DNA. Fluorescence channel F2 normalized by F1 (F2/F1) shows the robust amplification curves of HMBS standards and canine whole blood specimens. The 103 copies or 102 nL blood amplification curves cross the fluorescence threshold at approximately cycle 10, while the 100 copy or 10-1 nL curves (single copy or 0.1 nL blood) cross at approximately cycle 19. In contrast, the 10-1 copy or 10-2 nL curves (0.1 theoretical copy or 0.01 nL blood) do not cross the threshold and are indistinguishable from the negative control. On average for all amplifications, the nL amount of blood was equivalent to 23 × the amount of HMBS copies detected, defining the theoretical detection limit (without consideration of Poisson sampling error) as 0.043 nL blood per 1 HMBS copy.