PANDAA intentionally violates conventional qPCR design to enable durable, mismatch-agnostic detection of highly polymorphic pathogens

Abstract

Sensitive and reproducible diagnostics are fundamental to containing the spread of existing and emerging pathogens. Despite the reliance of clinical virology on qPCR, technical challenges persist that compromise their reliability for sustainable epidemic containment as sequence instability in probe-binding regions produces false-negative results. We systematically violated canonical qPCR design principles to develop a Pan-Degenerate Amplification and Adaptation (PANDAA), a point mutation assay that mitigates the impact of sequence variation on probe-based qPCR performance. Using HIV-1 as a model system, we optimized and validated PANDAA to detect HIV drug resistance mutations (DRMs). Ultra-degenerate primers with 3’ termini overlapping the probe-binding site adapt the target through site-directed mutagenesis during qPCR to replace DRM-proximal sequence variation. PANDAA-quantified DRMs present at frequency ≥5% (2 h from nucleic acid to result) with a sensitivity and specificity of 96.9% and 97.5%, respectively. PANDAA is an innovative advancement with applicability to any pathogen where target-proximal genetic variability hinders diagnostic development.