Assay verification/validation and optimization are essential, for laboratory developed tests (LDTs) and those that are commercially available (FDA approved). Validation of multiplex PCR assays ensures all targets detect equally. Validation provides the data required to meet regulatory criteria for implementation. The following assay parameters should be evaluated when validating a multiplex PCR assay, in this order:
- Define targets
- Analytical sensitivity
- Analytical specificity
- Accuracy
- Precision
A standard way of validating multiplex PCR assays is to test 10-20 samples using a 3X3X5 method:
- Three technologists/operators
- Three runs: 3 techs run the same set of samples to generate 3 sets of data for each assay parameter
- Five days: Same 3 techs run same sample set over 5 days
Note: The 3X3X5 method can be modified depending on number of samples available for validation and number of technologists participating in the validation.