AOAC SPADA Meeting

Traditional Pathway for Development of a Molecular Assay

Reference Materials: live or inactivated organisms or genomic materials 1 prototype strain Multiple strains

Courtesy: Hartman et al 2015. Demonstration of the Pre–Emergency Use Authorization Path Using 3 Minor Groove Binder–Hydrolysis Probe Assays to Detect Escherichia coli O104:H4. Clinical Chemistry 61:11 1391-1398. [Tim Minogue's group]

2018_10_17_SPADA_Brief

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What is the traditional process? Finding a target (user defined- biased- vir factor or agnostic- (just look for unique sequences- K-mer based approach e.g, Uniquemer, BioVelocity) Designing an assay- primer/probe sets (CLC Genomics, Primer BLAST) Checking Specificity; hits target sequences/doesn’t hit anything else (BLAST) Downstream all wet lab (use of BSL2, BSL3 and BSL4 organisms depending on the agent) Assay parameters LoD- one strain used- in buffer Linearity- one strain used – in buffer Inclusivity/Exclusivity- panel of strains – in buffer

Matrix Testing- one strain spiked Interference- one strain spiked

2018_10_17_SPADA_Brief

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