12.2.2. PCR detection limit

The detection limit of a qPCR assay is the lowest number of nucleic acid targets in a given template volume that can be detected in at least 95% of replicate assays. The detection limit is established by performing at least three independent trials, each with trial consisting of three independent two-fold serial dilutions of a template of known concentration. At each dilution level in each serial dilution series, eight replicate qPCR assays are run, i.e. a total of 24 replicate assays at each dilution level. The detection limit is the highest dilution level (i.e. lowest amount of target nucleic acid template) giving at least 23 positive results from the 24 assays (95% of the replicates).