Analytical sensitivity and clinical performance of a triplex RT-qPCR assay using CDC N1, N2 and RP targets for SARS-CoV-2 diagnosis

Freire-Paspuel, Byron; Garcia-Bereguiain, Miguel Angel

BACKGROUND: Several RT-qPCR kits are available for SARS-CoV-2 diagnosis, some of them with Emergency Use Authorization (EUA) by FDA like de nCoV19 CDC kit that includes 2 targets for detection of SARS-CoV-2 (N1, N2) and one target for RNA extraction quality control (RP), all them labelled with FAM so 3 PCR reactions are required per sample. METHODS: We designed a triplex RT-qPCR assay based on nCoV19 primers and probes where N1, N2 and RP are labelled with FAM, HEX and Cy5, respectively, so a single PCR reaction is required for each sample for SARS-CoV-2 diagnosis. RESULTS: 172 samples where analyzed with both the singleplex and triplex assays; 86 samples tested SARS-CoV-2 negative for both assays, so the triplex assay specificity is 100%; 86 samples tested SARS-Co-V 2 positive for the singleplex assay and 84 tested positive for the triplex assay, so the sensitivity was 97.7%. The limit of detection (LOD) for the triplex assay was found to be 1000 copies/mL. CONCLUSIONS: This new triplex RT-qPCR assay based on primers and probes from the CDC protocol is a highly reliable tool for SARS-CoV-2 diagnosis that could speed up detection and save reagents during current SARS-CoV-2 testing supplies shortage.