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quantification. Best practices include sequencing the challenge stock to confirm the primers and 661 probe are an exact match to the organism being quantified. 662 When possible, multiple assays are designed and tested, and poorly performing primer 663 candidates are removed from further consideration or submitted for redesign or optimization of 664 salt concentrations or thermocycling conditions before full-scale assay characterization. Usually 665 a single live organism or inactivated organism strain or extracted/naked DNA/RNA (genomic 666 material) or a surrogate with the assay target or synthetic assay target is used as template at this 667 stage of the assay development process. Minimally, primer concentration optimization, 668 especially if degenerate nucleotides are used, is needed to ensure optimal assay performance. 669 Initial characterization studies can be done in a simple matrix such as water before conducting 670 validation studies in the intended sample matrix. Analytical sensitivity includes determining the 671 assay limit of detection (LOD), or the lowest concentration of organism that is reproducibly 672 detected (e.g., when 58 of 60 test replicates are positive (27, 28). 673 Analytical specificity involves both inclusivity and cross-reactivity (exclusivity) testing. For 674 inclusivity testing, all the available strains or variants of the targeted organism are tested at levels 675 above LOD. The strains selected for testing should represent any diversity observed within the 676 assay target region. Synthetic nucleic acid could also be used to fill inclusivity gaps once the 677 assay has been optimized and characterized using whole organism. Exclusivity testing should 678 include other organisms located within the target genus, nucleic acid from the intended matrix 679 (e.g., human whole blood), and any organisms that were identified during the initial amplicon 680 BLAST analysis that have significant sequence identity. Such BLAST -identified targets that 681 warrant further testing include organisms that have 90-95% sequence identity.

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