SPADA Meeting Book

performance of different scoring methods (i.e. what did different models get right and wrong). 629 To date there has not been an organized effort to provide funding to acquire the needed training 630 and validation PCR datasets, develop an international committee to referee these contests, or 631 recruit groups of scientists to engage in the contests. Successful completion of this effort would 632 drive the field forward and have a dramatic effect on the quality of future PCR-based 633 diagnostics.

Commented [LN(6]: So the goal isn’t to determine which  models worked best but rather how to put a score on a  model?  That isn’t what it sounded like at the beginning of  this section.  Perhaps the goal should be more clear at the  onset.  Commented [SS7R6]: Gans

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6.0 Assay development and characterization

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6.1 Assay development

Once an optimal assay has been designed in silico , the next step is to assess the performance 638 of the design in wet lab testing using appropriate templates and reagents. Given the increasing 639 genomic diversity represented in genome databases resulting from improved sequencing 640 technology, many diagnostic assays require the incorporation of degenerate primers and/or 641 probes for efficacious species-specific detection. Quantitative assays require additional design 642 considerations depending on the intended use of the assay. Specific genomic locations need to be 643 targeted and methodologies employed if the desired readout is genome copies. The presence of 644 mismatches in either the primer or probe binding locations can affect the PCR kinetics (i.e. the 645 Cq value for a given target concentration) and thus impact the determination of target quantity. 646 Amplification kinetics need to be determined for each new target variant to ensure accurate 647 quantification. Best practices include sequencing the challenge stock to confirm the primers and 648 probe are an exact match to the organism being quantified. 649 When possible, multiple assays are designed and tested, and poorly performing primer 650 candidates are removed from further consideration or submitted for redesign or optimization of 651

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