SPADA Draft Documents

and what did different models get wrong). To date there has not been an organized effort to 612 provide funding to acquire the needed training and challenge PCR datasets, develop an 613 international committee to referee such contests, or recruit groups of scientists to engage in such 614 contests. Successful completion of such an effort would drive the field forward and have a 615 dramatic effect on the quality of future PCR-based diagnostics.

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Assay development and characterization

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Assay development

Once an optimal assay has been designed in silico , the next step is to assess the performance 620 of the design in wet lab testing using appropriate templates and reagents. Given the increasing 621 genomic diversity resulting from improved sequencing technology, many diagnostic assays 622 require the incorporation of degenerate primers and/or probes for efficacious species-specific 623 detection. Quantitative assays require additional design considerations depending on the intended 624 use of the assay. Specific genomic locations need to be targeted and methodologies employed if 625 the desired readout is genomic copies. The presence of mismatches in either the primer or probe 626 binding locations can affect the PCR kinetics (i.e. the Cq value for a given target concentration) 627 and thus impact the determination of target quantity. Thus, amplification kinetics need to be 628 determined for each new target variant to ensure accurate quantification. Best practices include 629 sequencing the challenge stock to confirm the primers and probe are an exact match to the 630 organism being quantified. 631 When possible, multiple assays are designed and tested, and poorly performing primer 632 candidates are removed from further consideration or submitted for redesign or optimization of 633 salt concentrations of thermocycling conditions before full-scale assay characterization. Usually 634

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