Sensitivity, specificity and reproducibility

The specificity of amplification was performed using agarose gel electrophoresis to verify the size amplicon. The limit of detection was estimated by testing a sextuplicate of the diluted plasmids at 50, 25, 12.5 and 5 copies per reaction. The analytical specificity of real-time PCR was estimated by screening DNA extracts from the bacterial and amoebal strains mentioned in Table ​Table1.1. To measure inter-assay variability, six different concentrations of plasmid DNA ranging from 5 to 5 × 10⁵ copies per reaction were assayed in duplicate in five separate runs that were carried out on different days.

Intra-assay variability was assessed by performing a single q-PCR assay using five replicates of each of the following plasmid copy numbers per reaction: 5 × 10⁵, 5 × 10³, 5 × 10², 5 × 10 and 5. Analysis of the results was carried out based on the mean of Ct values, coefficient of variation (CV) and the standard deviation. Coefficient of variation is equal to the standard deviation divided by the mean of Ct. Each conducted run contained negative controls. A standard curve was developed through tenfold serial dilutions of plasmid DNA (5 × 10⁵–5 copies/reaction).