SPADA Meeting Book

potential false positive organisms, but this exhaustive approach is not optimal because it detects 203 many hits that are not relevant to sequences that might be found in relevant sample matrices 204 (e.g., body fluids or soil). In other words, screening primers against all known sequences is 205 overly restrictive to proper design. In contrast, a modern approach uses an unbiased search of all 206 available sequence data for the organism of interest to identify potential targets and then 207 validates those targets/genes against well-defined inclusivity, exclusivity, and environmental 208 background sequence panels (e.g., SPADA environmental panel list of organisms). 212 relatively neglected compared to other parts of the process such as instrumentation, enzymes, 213 buffer additives, and data analysis. However, high quality primer design offers a tremendous 214 opportunity to improve diagnostic performance (i.e. sensitivity, specificity, and limit of 215 detection), as well as reduce the development time and cost. Figure 5 shows a traditional primer 216 design approach. Such a design pipeline brings together numerous tools that work well for their 217 intended uses, but were not specifically optimized to be used together for primer design. As a 218 result of the deficiencies of such traditional approaches, developing a high-performing assay 219 requires extensive experimentation with numerous cycles of re-design and testing and even after 220 significant financial investment, the resulting assays are often fragile and prone to failure (8). 221 Below, modern methods are recommended for each step in the assay development pipeline. 222 These methods are database-driven, apply physical chemistry modeling, and utilize modern 223 design algorithms and computational resources to overcome some of the weaknesses associated 224 with the traditional approach. 225 209 210 4.2 Traditional primer design paradigm 211 Primer design is a critical aspect in the development of diagnostic assays that has been

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