"Multiplex RT-PCR for SARS-CoV-2 variant surveillance in resource-limited settings: an in-house validation study in Cuba"

Background SARS-CoV-2 genomic surveillance is vital for public health, but whole-genome sequencing (WGS) remains costly and inaccessible in many resource-limited settings. We developed and validated a multiplex real-time RT-PCR assay for rapid, economical detection of key mutations associated with variants of interest (VOI) and concern (VOC). Methodology Two multiplex mixes (M1, M2) targeting eight mutations in the ORF1a and Spike genes were designed. Analytical validation included sensitivity, specificity, reproducibility, and limit of detection (LoD) using WHO international standards and a respiratory pathogen panel. In parallel, an in silico analysis evaluated oligonucleotide efficacy against 10.4 million SARS-CoV-2 genomes from GISAID/NCBI, assessing inclusivity, target-site secondary structure (RNAalifold), and hybridization energy (Primer3Plus). Results The assay demonstrated 100% clinical sensitivity among samples with valid RT-PCR results (41/42 samples yielded interpretable results, with one inhibited sample excluded from sensitivity calculation), a LoD of 5.7 log10 IU/mL, and 100% analytical specificity against 32 non-SARS-CoV-2 respiratory pathogens. Six out of eight oligonucleotide sets showed >96% inclusivity; two sets exhibited reduced inclusivity (94.03%, 90.14%) and structural features potentially affecting binding against emerging variants. The assay enables direct identification of major VOCs (Alpha, Beta, Gamma, Delta, Omicron) and indirect detection of multiple VOIs (P.2, Epsilon, Kappa, Eta, Iota, Lambda). Conclusion This standardized multiplex assay provides a rapid, sensitive, and low-cost alternative for SARS-CoV-2 variant surveillance in Cuba and similar settings. The integration of experimental and in silico validation offers a robust, adaptable framework to sustain diagnostic accuracy amid viral evolution, optimizing the allocation of scarce sequencing resources.