Table 1 qPCR assay validation parameters
Matrix Calibration curve and regression | Herring sperm DNA, 400 ng per PCR well Pass - Calibration standards ranging from 100 to 1 × 107 pAAV2/2-ND4 copies/well, fitted with a linear regression | |||
|---|---|---|---|---|
Within-run precision and accuracy | Pass - CV% < 15.0, absolute RE% < 8.0 | |||
QC level | Nominal concentration (copies/well) | CV% | RE% | |
QC ULOQ | 1.00E+ 07 | 11.1 | −0.1 | |
QC high | 7.50E+ 06 | 8.0 | −0.8 | |
QC mid | 1.05E+ 04 | 6.2 | −3.5 | |
QC low | 3.00E+ 02 | 6.6 | −7.2 | |
QC LLOQ | 1.00E+ 02 | 14.4 | −2.0 | |
Between run precision and accuracy | Pass - CV% < 9.0, absolute RE% < 6.0 | |||
QC level | Nominal concentration (copies/well) | CV% | RE% | |
QC ULOQ | 1.00E+ 07 | 6.9 | −3.1 | |
QC high | 7.50E+ 06 | 4.4 | 0.1 | |
QC mid | 1.05E+ 04 | 4.9 | 5.4 | |
QC low | 3.00E+ 02 | 8.2 | 4.2 | |
QC LLOQ | 1.00E+ 02 | 4.1 | 2.7 | |
Dynamic range of the assay | 100 to 1 × 107 copies/well corresponding to 250 to 2.5 × 107 copies/μg of DNA | |||
Detection limit of the assay | 6.25 copies/well corresponding to 15.625 copies/μg of DNA | |||
Specificity of the assay | Pass - Tested with DNA extracted from blood samples from six humans | |||
Matrix effect | Pass - Tested with DNA extracted from the six human blood samples spiked with 104 copies of pAAV2/2-ND4 | |||