HIV develops single nucleotide polymorphisms (SNPs), some of which lead to drug resistance mutations (DRMs) that prevent therapeutic viral suppression. Genomic sequencing enables healthcare professionals to select effective combination antiretroviral therapy (ART) to achieve and maintain viral suppression. However, sequencing technologies, which are resource-intensive, are limited in their availability. This report describes the first step toward a highly specific ligation-based SNP discrimination method with endpoint PCR detection, which is more suitable for resource-limited clinics. The approach is based on magnetic bead processing to maximize reaction product transfer and minimize the carryover of incompatible buffer for three consecutive enzymatic reactionsreverse transcription (RT), oligonucleotide ligation assay (OLA), and PCR. The method improved PCR detection following RT → OLA by 8.06 cycles (∼250-fold) compared to direct pipette processing and detected between 103 and 104 RNA copies per reaction. In studies with synthesized nucleic acids based on the well-studied HIV mutation, K103N, the assay successfully differentiated between wild-type and mutant for RNA targets with high specificity. With further development, this design provides a pathway for SNP detection with more accessible PCR instrumentation and is a step toward a self-contained processing approach that incorporates the SNP specificity of the ligation reaction for more effective clinical management of DRMs in resource-constrained settings.

Background COVID‐19 prevalence has remained high throughout the pandemic with intermittent surges, due largely to the emergence of genetic variants, demonstrating the need for more accessible sequencing technologies for strain typing. Methods A ligation‐based typing assay was developed to detect known variants of severe acute respiratory syndrome virus 2 (SARS‐CoV‐2) by identifying the presence of characteristic single‐nucleotide polymorphisms (SNPs). General principles for extending the strategy to new variants and alternate diseases with SNPs of interest are described. Of note, this strategy leverages commercially available reagents for assay preparation, as well as standard real‐time polymerase chain reaction (PCR) instrumentation for assay performance. Results The assay demonstrated a combined sensitivity and specificity of 96.6% and 99.5%, respectively, for the classification of 88 clinical samples of the Alpha, Delta, and Omicron variants relative to the gold standard of viral genome sequencing. It achieved an average limit of detection of 7.4 × 104 genome copies/mL in contrived nasopharyngeal samples. The ligation‐based strategy performed robustly in the presence of additional polymorphisms in the targeted regions of interest as shown by the sequence alignment of clinical samples. Conclusions The assay demonstrates the potential for robust variant typing with performance comparable with next‐generation sequencing without the need for the time delays and resources required for sequencing. The reduced resource dependency and generalizability could expand access to variant classification information for pandemic surveillance.

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