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

BACKGROUND

Several RT-qPCR kits are available for SARS-CoV-2 diagnosis and some have emergency use authorization from the US Food and Drug Administration. In particular, the nCoV19 CDC kit includes two targets for detecting SARS-CoV-2 (N1 and N2) and an RNaseP (RP) target for RNA extraction quality control, all of which are labeled with FAM, and thus three 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 labeled with FAM, HEX, and Cy5, respectively, so only a single PCR reaction is required for each sample for SARS-CoV-2 diagnosis.

RESULTS

In total, 172 samples were analyzed in both singleplex and triplex assays, where 86 samples tested SARS-CoV-2 negative with both assays, so the triplex assay specificity was 100%. In addition, 86 samples tested SARS-Co-V 2 positive with the singleplex assay and 84 with the triplex assay, so the sensitivity was 97.7%. The limit of detection for the triplex assay was determined as 1000 copies/mL.

CONCLUSIONS

This new triplex RT-qPCR assay based on primers and probes from the CDC protocol is highly reliable for SARS-CoV-2 diagnosis, and it could speed up detection and save reagents during the current SARS-CoV-2 testing supplies shortage.