SPADA Draft Documents

from lab-specific research interests on specific genes of given pathogens or from known 186 (literature-based), or suspected, virulence factor genes. The resulting assays are then screened 187 (either computationally or “by eye”) for inclusivity and exclusivity using hand-selected 188 sequences. The resulting assays are also often broadly screened computationally against all 189 known sequences (e.g., by using BLAST ) to identify potential false positive organisms, but this 190 exhaustive approach is not optimal because it detects many hits that are not relevant to actual 191 sequences that might be found in actual sample matrices (e.g., body fluids or soil). In other 192 words, screening primers against all known sequences is overly restrictive to proper design. In 193 contrast, a modern approach uses an unbiased search of all available sequence data to identify 194 potential targets and then validate those targets/genes against well-defined inclusivity, 195 exclusivity, and environmental background sequence panels. 199 relatively neglected compared to other parts of the process such as instrumentation, enzymes, 200 buffer additives, and data analysis. However, using high quality primer design is a tremendous 201 opportunity to improve diagnostic performance (i.e. sensitivity, specificity, and limit of 202 detection), as well as reduce the development time and cost. Figure 5 shows a traditional primer 203 design approach. Such a design pipeline brings together numerous tools that work well for their 204 intended uses, but were not specifically optimized for primer design. As a result of the 205 deficiencies of such traditional approaches, developing a high-performing assay requires 206 extensive experimentation with numerous cycles of re-design and testing and even after 207 significant financial investment, the resulting assays are often fragile and prone to failure (8). 208 196 197 Traditional primer design paradigm 198 Primer design is a critical aspect in the development of diagnostic assays that has been

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