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...
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| Format: | Article |
| Language: | English |
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Elsevier
2021-01-01
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| Series: | International Journal of Infectious Diseases |
| Online Access: | http://www.sciencedirect.com/science/article/pii/S1201971220322505 |
| _version_ | 1818921262081114112 |
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| author | Byron Freire-Paspuel Miguel Angel Garcia-Bereguiain |
| author_facet | Byron Freire-Paspuel Miguel Angel Garcia-Bereguiain |
| author_sort | Byron Freire-Paspuel |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-12-20T01:34:51Z |
| format | Article |
| id | doaj.art-337c61392df2458494aa5c7edf2997f4 |
| institution | Directory Open Access Journal |
| issn | 1201-9712 |
| language | English |
| last_indexed | 2024-12-20T01:34:51Z |
| publishDate | 2021-01-01 |
| publisher | Elsevier |
| record_format | Article |
| series | International Journal of Infectious Diseases |
| spelling | doaj.art-337c61392df2458494aa5c7edf2997f42022-12-21T19:58:01ZengElsevierInternational Journal of Infectious Diseases1201-97122021-01-011021416Analytical sensitivity and clinical performance of a triplex RT-qPCR assay using CDC N1, N2, and RP targets for SARS-CoV-2 diagnosisByron Freire-Paspuel0Miguel Angel Garcia-Bereguiain1One Health Research Group, Universidad de Las Americas, Quito, EcuadorCorresponding author.; One Health Research Group, Universidad de Las Americas, Quito, EcuadorBackground: 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.http://www.sciencedirect.com/science/article/pii/S1201971220322505 |
| spellingShingle | Byron Freire-Paspuel Miguel Angel Garcia-Bereguiain Analytical sensitivity and clinical performance of a triplex RT-qPCR assay using CDC N1, N2, and RP targets for SARS-CoV-2 diagnosis International Journal of Infectious Diseases |
| title | Analytical sensitivity and clinical performance of a triplex RT-qPCR assay using CDC N1, N2, and RP targets for SARS-CoV-2 diagnosis |
| title_full | Analytical sensitivity and clinical performance of a triplex RT-qPCR assay using CDC N1, N2, and RP targets for SARS-CoV-2 diagnosis |
| title_fullStr | Analytical sensitivity and clinical performance of a triplex RT-qPCR assay using CDC N1, N2, and RP targets for SARS-CoV-2 diagnosis |
| title_full_unstemmed | Analytical sensitivity and clinical performance of a triplex RT-qPCR assay using CDC N1, N2, and RP targets for SARS-CoV-2 diagnosis |
| title_short | Analytical sensitivity and clinical performance of a triplex RT-qPCR assay using CDC N1, N2, and RP targets for SARS-CoV-2 diagnosis |
| title_sort | analytical sensitivity and clinical performance of a triplex rt qpcr assay using cdc n1 n2 and rp targets for sars cov 2 diagnosis |
| url | http://www.sciencedirect.com/science/article/pii/S1201971220322505 |
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