Appendix Q: Recommendations for Developing Molecular Assays for Microbial Pathogen Detection Using Modern In Silico Approaches

Abstract

We describe the use of in silico approaches to improve the process of molecular assay development and reduce time and cost by utilizing available databases of whole genome pathogen sequences combined with modern bioinformatics and physical modeling tools. Well-characterized assays are needed for accurately detecting pathogens in environmental and patient samples and also for evaluation of the efficacy of a medical countermeasure that may be administered to patients. The polymerase chain reaction (PCR) remains the gold standard for pathogen detection due to the simplicity of its instrumentation, low cost of reagents, and outstanding limit of detection (LOD), sensitivity, and specificity. However, creation of such PCR assays often involves iterations of design, preliminary testing, and thorough validation with clinical isolates and testing in relevant matrices, which can be time consuming, costly, and result in suboptimal assays. Since formal validation (e.g., for Emergency Use Authorization [EUA] or Food and Drug Administration [FDA] licensure) of an infectious disease assay can be very expensive and can require extensive time of development, having a well-designed assay up front is a critical first step. Yet, many assays described in the literature utilized limited design capabilities and many initially promising assays fail the validation process, resulting in increased costs and timelines for successful product development. While the computational approaches outlined in this document by no means obviate the need for wet lab testing, they can reduce the amount of effort wasted on empirical optimization and iterative redesigns and also guide validation studies. The proposed computational approaches also result in higher performing assays with better sensitivity, specificity, and lower LOD and reduce the possibility of assay failure due to signature erosion. To provide clarity, an extensive glossary of defined terms is provided.