SPADA Docs

Positive control

Add to your analyte target

Verify that PCR is working, so that a negative for the target analyte is meaningful Verify that primers are compatible with each other

Multiplex testing

Combine validated singleplexes into larger multiplexes Use synthetic gBlocks for initial testing Use actual patient samples, known positives and negatives

Synthetic target

Cheap, non-pathogenic

Testing with patient samples

Determine sensitivity, specificity, and LOD

Gather assay information

Use MIQE standards

Ensure assay is reproducible and documented

721 722 6.3 Assay Stewardship – when new viral or bacterial strains are discovered, will the existing 723 assay work? 724 After an assay is designed and validated, it is relatively common for the scientific community 725 to discover a new strain of a pathogen that has a mutation at a site that causes the assay to fail. 726 This process is termed “signature erosion” (9). To date, there have not been any good methods 727 for anticipating new mutations. One general recommendation is to design primers to be a little 728 longer than needed for a perfect match. In the event that a single or double mutation occurs at a 729 later time, the primers will still work as long as the new mutation does not occur at the 3’-end of 730 either primer. In addition, there is a need to have software that can computationally test any new 731 sequenced isolate against existing validated assays to predict if the new isolate is likely to be 732 “covered” by the existing assay (i.e. if the existing assay is predicted to give an amplicon for the 733 new isolate with high efficiency), or if the existing assay is likely to fail for the new isolate (i.e. 734 the existing assay is predicted to give an amplicon for the new isolate with low efficiency). Even 735 better would be for software to constantly monitor existing databases for new entries and to 736 automatically alert a user or agency if a new isolate is “high risk” for assay failure. This 737 capability would allow for real-time monitoring of assays to alert agencies in charge of 738 protecting the public about potential assay failures. The agencies could then focus their ongoing 739 validation efforts on those assays that have a high likelihood of failure rather than using their 740

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