TAPPR PCR Assay Design – Targeted, Automated, Primer and Probe Retrieval for Scalable Molecular Assay Design

Abstract Motivation The rapid and reliable detection of infectious disease agents is critical for effective biosurveillance, diagnostics, and outbreak response. However, existing molecular assay design methods face significant limitations in scalability, speed, and adaptability to rapidly evolving pathogens, often leading to outdated or suboptimal assays. Here, we present Targeted, Automated Primer and Probe Retrieval (TAPPR), a novel, automated pipeline for scalable molecular assay design. TAPPR employs alignment-free methods to identify conserved regions and marker sequences across large-scale genomic datasets, supporting customizable design parameters for inclusivity, exclusivity, and assay specificity. To evaluate TAPPR's performance, assays were designed for diverse microbial targets, including Mpox, Mycobacterium tuberculosis, SARS-CoV-2, and Candida albicans, representing viral, bacterial, and fungal pathogens. The pipeline incorporates k-mer set operations and clustering strategies to address sequence diversity and streamline conserved region identification. Designed assays underwent in silico PCR simulations and laboratory testing to assess specificity, sensitivity, and exclusivity. Results demonstrated high accuracy across targets, with superior sensitivity to existing fielded assays where available. Additionally, we compare TAPPR to alternative available molecular assay design tools to demonstrate its advantages. This work highlights TAPPR’s capability to accelerate the development of molecular diagnostics by efficiently leveraging vast genomic datasets and addressing computational bottlenecks. TAPPR represents a scalable, adaptable tool for rapidly designing high-quality molecular assays, positioning itself as a critical asset for biosurveillance and public health response to emerging and re-emerging infectious disease threats. Results TAPPR demonstrates rapid and scalable data-driven molecular assay design through alignment-free estimations of conserved regions and marker regions. Through both in silico and lab bench evaluation, TAPPR assays are demonstrated to perform equivalently or better than previously utilized publicly available qPCR assays for emergent disease diagnostics. TAPPR is also shown to produce results where other available automated molecular assays design solutions fail to do so on the order of hours. Availability and implementation TAPPR is available at https://github.com/mriglobal/tappr under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International Public License.