Nucleic Acid Sequence-Based Amplification (NASBA) serves as a powerful tool for evaluating gene expression and exploring RNA structure and function. This isothermal amplification method employs avian myeloblastosis virus reverse transcriptase (AMV-RT), RNase H, T7 RNA polymerase, transcript-specific primers, and relevant cofactors to efficiently amplify target RNA. In comparison to other RNA analysis techniques such as RT-PCR, RNase protection assays, and Northern blotting, NASBA stands out for its enhanced speed, sensitivity, and versatility. One key advantage of NASBA lies in its ability to facilitate the amplification of large quantities of target RNA through a series of isothermic reactions. Real-time chemistries, including SYBR dyes or molecular beacon probes, enable the detection of target RNA in a closed-tube format, minimizing the risk of contamination. This closed-tube feature is particularly advantageous for applications requiring precise RNA analysis. Recently designed primers and molecular beacon probes for SARS-CoV-2 RNA-dependent RNA polymerase and N genes were employed in a NASBA assay, for testing standard samples with known virus copy numbers, including assessments of clinical sensitivity, precision, and overall clinical performance (Kia V, Tafti A, Paryan M, Mohammadi-Yeganeh S, Ir J Med Sci (1971-) 192:723–729, 2023).