qPCR efficiency

Plasmid clones, used as standards for efficiency and analytical sensitivity determination, were constructed using LSU qPCR amplicons from B. vogeli, and B. microti-like template DNA with pGEM-T Easy Vector System (Promega, Madison, WI) as recommended by the manufacturer. Plasmids were sequenced and inserts confirmed using M13R primers. Plasmid copy numbers were calculated assuming an average base pair weight of 650 Da and Avogadro’s number (6.022 10²³) using the following equation: copy number = (DNA ng amount × 6.022 10²³ molecules/mol)/(length of DNA in base pairs × 1 × 10⁹ ng/g × 650 g/mol). Duplicate, serial 10-fold dilutions in uninfected canine gDNA (~10–30 ng/μl) resulted in 50–500,000 copies/reaction of plasmid DNA, and standard curves of quantification cycle (C_q) values were plotted against the logarithm of plasmid copy numbers/reaction. PCR efficiency was estimated through linear regression of the dilution curve (10^ (-1/slope)-1) × 100). Coefficients were calculated (R ²) using Bio-Rad CFX Manager™ software. Efficiency reactions were performed using both 2 and 3 primer reactions to establish any potential interference by a third primer.