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

Abstract

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 betweenlaboratory 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.

Development of a real-time PCR system for the detection of the potential allergen fish in food

“…With the PCR method, a LOD 95 % of 2.5 copies with a 95 % confidence interval of 1.5-4.0 can be reached. According to Poisson statistics, the theoretically lowest achievable LOD 95 % is three copies, which was reached [25]. The difference showing a better LOD 95 % than theoretically expected could be attributed to statistical bias.…”

Screening for six genetically modified soybean lines by an event-specific multiplex PCR method: Collaborative trial validation of a novel approach for GMO detection

“…The mathematical-statistical approach and formulas for calculation of the probability of detection (POD) are described (Uhlig et al 2015).…”

“…Three plasmid combinations (pMON87701 and pMON87708; pMON87769 and CV127; pDP304423 and DAS-68416) had to be serially diluted to obtain solutions with nominal target sequence copy numbers of 4, 2, 1, 0.4 and 0.01 copies/ ll, respectively. Each level had to be analysed in 6 PCR replicates for POD determination (Uhlig et al 2015).…”

“…A statistical analysis based on modelling the POD was performed based on the test result compiled in Table 9 (Uhlig et al 2015). Before calculating the ratios of positive and negative PCR results, the underlying copy numbers were corrected according to the digital PCR estimates (Table 10).…”

“…As a first step, the assay was tested by two laboratories in order to evaluate its inter-laboratory transferability and practicability if used with different equipment and by different operators. Via a collaborative trial with 16 participating laboratories, a further methods performance evaluation of the false positive/negative rate and the inter-laboratory reproducibility of the probability of detection (POD) was conducted (Uhlig et al 2015). We present the results of the validation studies and discuss the potential modularity and applicability of these multiplex assays in other GM crop plants, screening platforms, and applications.…”