Current diagnostic methods for SARS-CoV-2 may detect inactive viral genomic fragments, which can lead to complications. In this study, we aimed to design a Multiplex Real-time RT-PCR panel to detect subgenomic mRNAs (sgRNA) encoding the N and E structural genes of SARS-CoV-2.
 We compared the results of this panel with those of a commercial one-step RT-qPCR kit (Pishtaz Teb Diagnostics, Tehran, Iran) in 147 swab specimens previously classified as positive (Ct ≤ 30) or negative (Ct > 30) by the reference genomic RNA assay. The samples represented different SARS-CoV-2 variants, including Wuhan, Alpha, Delta, and Omicron. We found that samples with a Ct value of 30 or lower in the one-step RT-qPCR commercial kit usually contained active, infectious virus. The agreement between the results of our Multiplex Real-time RT-PCR panel and the one-step RT-qPCR commercial kit was 100% for positive samples, and 40.7% for negative samples. Low agreement for negative samples is due to the detection of non-replicating viral remnants by the genomic RNA assay.
 The developed multiplex real-time RT-PCR assay enables simultaneous detection of SARS‑CoV‑2 sgN, sgE, and RNase‑P targets and showed acceptable analytical and clinical performance across circulating variants. Detection of sgRNA targets may provide complementary information regarding recent or ongoing viral transcription; however, these findings should not be interpreted as definitive evidence of infectivity, and further studies incorporating viral culture are needed to clarify the clinical significance of sgRNA positivity.