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databases for inclusivity, exclusivity, and background. These are used in the k-mer-based target 481 analysis algorithm as well as the thermodynamics-based scanning of oligonucleotide candidates 482 to determine their coverage and specificity. There is also built-in physical chemistry modeling 483 (e.g., dynamic programming algorithms such as MFOLD or OMP ) to compute thermodynamics 484 for unimolecular folding and bimolecular hybridization, and numerical methods for solving the 485 multi-state coupled equilibrium model to determine the amount bound. There is also the critical 486 component for ranking the results using a weighted scoring equation. Lastly, Table 1 provides a 487 summary of essential design criteria that should be included in a modern primer design pipeline. 488 Improving upon these principles will require implementing the recommendations described in 489 the metrology section below. Combining the designs for different singleplex reactions into a 490 larger multiplexed format is discussed in the next section. In addition, below an iterative process 491 is recommended for performing experimental validation to develop robust assays.

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Figure 7. Modern Design Paradigm for Pathogen Detection by PCR.

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