Validation of qualitative PCR methods on the basis of mathematical–statistical modelling of the probability of detection

A new model for the probability of detection (POD curve) for qualitative PCR methods examined in a method validation collaborative study is presented. The model allows the calculation of the POD curve and the limit of detection (LOD 95%), i.e. the number of copies of the target DNA sequence required to ensure 95 % probability of detection. The between-laboratory variability of the limit of detection is used to derive the between-laboratory reproducibility of the PCR method. The model is closely related to the approach for quantitative methods described in ISO 5725.2:2002, and the relative limit of detection approach described in the new standard ISO 16140-2:2014.