“Multiplex RT-PCR for SARS-CoV-2 variant surveillance in resource-limited settings: an in-house validation study in Cuba”
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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.
Author Summary
Genomic surveillance is a cornerstone of the public health response to SARS-CoV-2, yet whole-genome sequencing capacity remains out of reach for many laboratories in low- and middle-income countries. To bridge this gap, we developed and validated a multiplex real-time RT-PCR assay that detects eight key mutations in the viral genome, enabling the identification of 11 priority lineages. The test costs approximately 5 USD per sample, produces results in a few hours, and uses standard thermocyclers — making it suitable for decentralized deployment. Laboratory validation confirmed high diagnostic performance with no cross-reactivity against 32 other respiratory pathogens. Crucially, we combined this experimental work with a large-scale computational analysis of over 10 million publicly available viral genomes. This in silico step allowed us to verify that most of our primer and probe sets remain effective against global viral diversity, and to identify two sets that may require future optimization. Our work provides a practical, sustainable model for variant monitoring in settings with constrained sequencing capacity, as exemplified by Cuba. By integrating robust laboratory validation with ongoing bioinformatic surveillance, this framework optimizes the use of scarce genomic resources and strengthens pandemic preparedness in similar regions worldwide.
