PCR efficiency and the limit of reliable detection (LoRD)

Amplification reactions were carried out on three different PCR platforms; P1, P2, P3. Duplicate PCRs were performed at every dilution of the Candida EDTA-WB reference panel extracts. Quantification cycle (Cq) values were subtracted and melting curves were analyzed to estimate the lowest template DNA concentration by which the appropriate, species descriptive melting peaks were interpretable (LoRD). To study the correlation between the Cq-s and the genomic load standard curves were obtained by plotting Cq values against the log of cell number; 10⁶–10 CFU/1 ml WB which is equivalent to 2 × 10⁵–0.2 GE/PCR. Mean Cq data were calculated and standard curves were built where these plots determined the linear dynamic ranges. Efficiency was calculated per the following formula, E = (10–1/slope) than was converted to percentage efficiency by using the formula, E% = (E-1) × 100 [44]. Amplification efficiency, (correlation coefficient; R²) was also calculated.