Analytical sensitivity and clinical performance of a triplex RT-qPCR assay using CDC N1, N2, and RP targets for SARS-CoV-2 diagnosis. (January 2021)

• Record Type: Journal Article
• Authors: Freire-Paspuel, Byron; Garcia-Bereguiain, Miguel Angel
• Is Part Of: International journal of infectious diseases. Volume 102(2021)
• Page Start: 14
• Page End: 16
• Publication Date: 2021-01
• Subjects: Communicable diseases -- Periodicals; Communicable Diseases -- Periodicals; Communicable diseases; Periodicals; Electronic journals; 616.9
• DOI: 10.1016/j.ijid.2020.10.047
• Languages: English
• ISSNs: 1201-9712

Abstract

Highlights: CDC SARS-CoV-2 RT-PCR requires three reactions per sample for N1, N2, and RP targets. Triplex RT-qPCR assay developed for N1, N2, and RP. Assay sensitivity of 97.7% and limit of detection of 1000 copies/mL of sample. 4. Triplex assay saves time and cost effective alternative to standard CDC protocol. Abstract: 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.