SPADA Draft Documents

a single live organism or inactivated organism strain or extracted/naked DNA/RNA (genomic 635 material) or a surrogate with the assay target or synthetic assay target is used as template at this 636 stage of the assay development process. Minimally, primer concentration optimization, 637 especially if degenerate nucleotides are used, is needed to ensure optimal assay performance. 638 Initial characterization studies could be done in a simple matrix such as water before conducting 639 validation studies in the intended clinical matrix. Analytical sensitivity includes determining the 640 assay limit of detection (LOD), or the lowest concentration of organism that is reproducibly 641 detected (i.e. when 58 of 60 test replicates are positive). 642 Analytical specificity involves both inclusivity and cross-reactivity (exclusivity) testing. For 643 inclusivity testing, all the available strains or variants of the targeted organism are tested at 1.5 644 times the LOD; the strains selected for testing should represent any diversity observed within the 645 assay target region. Synthetic nucleic acid could also be used to fill inclusivity gaps once the 646 assay has been optimized and characterized using whole organism. Exclusivity testing should 647 include other organisms located within the target genus, nucleic acid from the intended matrix 648 (ex. human whole blood), and any organisms that were identified during the initial amplicon 649 BLAST analysis.

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Assay validation

Table 2 presents a summary of best practices for PCR assay validation testing. Assay 653 validation requires a comprehensive analysis of the test system (i.e. the sample to answer process 654 including extraction, testing, and analysis) to define the assay performance characteristics in the 655 intended matrix, and the test system is only validated for that specific use. Both quantitative and 656

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