AOAC SPADA Meeting

How do we make the call? • Parameters • A positive match between the assay primer/probe set sequences and sequences from the NCBI databases @ > 90% identity over 90% of the primer length is defined as an “assay hit” (2 mismatches allowed for a 20 nt primer) • A positive match between an amplicon sequence from an assay and sequences from the NCBI databases @ > 85% identity over 90% of the amplicon length is defined as an “amplicon hit” (15 mismatches allowed for a 100 bp amplicon) • By comparing the number of assay hits with the number of amplicon hits the final results can be labeled descriptively • Manual validation of negative results: SNPs and indels especially in short primers or probes or at primer ends, will return a ‘no hit’. Validation included looking at the pair- wise alignment for the amplicons and visually inspecting how many SNPs and indels were present; e.g., if a SNP is found on the 2 nd to last bp of a primer then the last 2 bps are typically omitted from the reported BLAST+ hit • Some primers and probes are very short causing no hit call (90% identity over 90% of the length) but still work in the wet lab assay. Based on this assumption these results are manually changed to positive hits.

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How do we make the call?

• Definition of calls • True Positive: assay hit + amplicon hit + correct target organism (Ba assay hit to B. anthracis sequences) • False Positive: assay hit + amplicon hit + hit non-target organism ( B a assay hit to B. cereus sequence) • False Negative: No assay hit + amplicon hit + correct target organism (Ba assay no hit to B. anthracis) • True Negatives: No assay hit + No amplicon hit in all other organisms ( or the amplicon may be there at <85% in a given organism- normally not captured; on occasion we have looked at these)

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