The following parameters are evaluated during the validation process:
(i) Specificity: Specificity is assessed by spiking tests in environmental matrices.
(ii) Accuracy and Precision: Accuracy of assays is determined using genomic DNA of well-characterized reference strains spiked into complex DNA samples extracted from relevant environmental matrices (drinking water, river water, lake water, and tertiary wastewater effluent, air samples for appropriate agents). Accuracy is assessed at both the probe and the gene level.
(iii) Limit of Detection (LOD) and Limit of Quantification (LOQ): The limit of detection and limit of quantification of our method are established at several levels from cells present in various environmental sample matrices to number of copies added to a known amount of genomic DNA as background.
(iv) Ruggedness and Robustness: The ruggedness and robustness of our assays are evaluated with a standard operating protocol in the hands of more than one technician in order to assess reproducibility of the results. Personnel from laboratories outside the campus may also be involved.
(v) Linearity and Range: This is one of the main parameters that we examine for PCR based diagnostics. We evaluate the range and linearity of the method by spiking varying copies of the target VMGs (e.g., 1 to 10,000 copies) and quantify detection values.
