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

Iain J MacLeod,Christopher F Rowley,M Essex

doi:10.1038/s42003-021-01751-9

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.

Highlights

Sensitive and reproducible diagnostics are fundamental to containing the spread of existing and emerging pathogens
Sequence divergence within oligonucleotide-binding sites creates primer–template duplex instability and lowers Tm. This can be offset by increasing the primer length or GC content to confer a degree of mismatch tolerance or mitigated using degenerate nucleotides. These approaches must balance the amplification of heterogeneous quasi-species with non-specific amplification by off-target mispriming3. qPCR probe design is more averse to similar modifications because increasing probe Tm through nucleotide degeneracy or sequence lengthening may lead to high Tm variations that reduce specific target discrimination
We challenged the notion that extensive genetic heterogeneity of HIV1 precludes the development of a universal qPCR diagnostic for resistance genotyping[17] by intentionally violating the core tenets of qPCR oligonucleotide design (Table 1)[18]

Summary

Introduction

Sensitive and reproducible diagnostics are fundamental to containing the spread of existing and emerging pathogens. The use of PCR and real-time PCR (qPCR)-based methodologies in clinical diagnostic virology demands high sensitivity and specificity for the selected viral nucleic acid target. Analytical specificity comprises the extent of inclusivity by which all viral phylogenetic variants are captured at the exclusion of its genetic near neighbors This is facilitated by directing primer and probe design to evolutionarily conserved regions identified through multiple sequence alignments that portray geographical and temporal genomic variability. Frequent in silico re-evaluation is necessary to identify escape variants that necessitate assay primer and probe design, assay reoptimization, and clinical validation Attempts to circumvent these labor-intensive re-design processes include performing multiple qPCR assays in parallel (e.g., Lassa fever virus)[4] or sequentially (e.g., Crimean-Congo hemorrhagic fever virus)[5] to capture the majority of phylogenetic lineages and mitigate diagnostic errors on clinical interpretation

Objectives

Methods

Conclusion