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

Scientists developed and validated a multiplex RT-PCR test for rapid and economical detection of key mutations associated with SARS-CoV-2 variants. The test consists of two multiplex mixes targeting eight mutations in the ORF1a and Spike genes. Validation demonstrated 100% clinical sensitivity among samples with valid results (41 of 42 samples), a limit of detection of 5.7 log10 IU/mL, and 100% analytical specificity against 32 non-COVID respiratory pathogens. Six of eight oligonucleotide sets showed greater than 96% inclusivity, while two sets exhibited reduced inclusivity (94.03% and 90.14%). The test enables direct identification of major variants (Alpha, Beta, Gamma, Delta, Omicron) and indirect detection of multiple other variants. This method represents a rapid, sensitive, and low-cost alternative for SARS-CoV-2 variant surveillance in Cuba and similar resource-limited settings.