Clinical Validation of a SARS-CoV-2 Real-Time Reverse Transcription PCR Assay Targeting the Nucleocapsid Gene
© American Association for Clinical Chemistry 2020. All rights reserved. For permissions, please email: journals.permissionsoup.com..
BACKGROUND: Detection of SARS-CoV-2 viral RNA is important for the diagnosis and management of COVID-19.
METHODS: We present a clinical validation of a reverse transcription PCR (RT-PCR) assay for the SARS-CoV-2 nucleocapsid (N1) gene. Off-board lysis on an automated nucleic acid extraction system was optimized with endemic coronaviruses (OC43 and NL63). Genomic RNA and SARS-CoV-2 RNA in a recombinant viral protein coat were used as control materials and compared for recovery from nucleic acid extraction.
RESULTS: Nucleic acid extraction showed decreased recovery of endemic Coronavirus in vitro transcribed RNA (NL63) compared with attenuated virus (OC43). SARS-CoV-2 RNA had more reliable recovery from extraction through amplification than genomic RNA. Recovery of genomic RNA was improved by combining lysis buffer with clinical matrix before adding RNA. The RT-PCR assay demonstrated 100% in silico sensitivity and specificity. The accuracy across samples was 100% (75 of 75). Precision studies showed 100% intra-run, inter-run, and inter-technologist concordance. The limit of detection was 264 copies per milliliter (estimated 5 copies per reaction; 35.56 mean threshold cycle value).
CONCLUSIONS: This SARS-CoV-2 assay demonstrates appropriate characteristics for use under an Emergency Use Authorization. Endemic coronavirus controls were useful in optimizing the extraction procedure. In the absence of live or attenuated virus, recombinant virus in a protein coat is an appropriate control specimen type for assay validation during a pandemic.
| Medienart: |
E-Artikel |
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| Erscheinungsjahr: |
2020 |
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| Erschienen: |
2020 |
| Enthalten in: |
Zur Gesamtaufnahme - volume:5 |
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| Enthalten in: |
The journal of applied laboratory medicine - 5(2020), 5 vom: 01. Sept., Seite 889-896 |
| Sprache: |
Englisch |
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| Beteiligte Personen: |
SoRelle, Jeffrey A [VerfasserIn] |
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| Links: |
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| Themen: |
COVID-19 |
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| Anmerkungen: |
Date Completed 23.09.2020 Date Revised 18.12.2020 published: Print Citation Status MEDLINE |
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| doi: |
10.1093/jalm/jfaa089 |
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| funding: |
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| Förderinstitution / Projekttitel: |
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| PPN (Katalog-ID): |
NLM310639271 |
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| 100 | 1 | |a SoRelle, Jeffrey A |e verfasserin |4 aut | |
| 245 | 1 | 0 | |a Clinical Validation of a SARS-CoV-2 Real-Time Reverse Transcription PCR Assay Targeting the Nucleocapsid Gene |
| 264 | 1 | |c 2020 | |
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| 500 | |a Date Completed 23.09.2020 | ||
| 500 | |a Date Revised 18.12.2020 | ||
| 500 | |a published: Print | ||
| 500 | |a Citation Status MEDLINE | ||
| 520 | |a © American Association for Clinical Chemistry 2020. All rights reserved. For permissions, please email: journals.permissionsoup.com. | ||
| 520 | |a BACKGROUND: Detection of SARS-CoV-2 viral RNA is important for the diagnosis and management of COVID-19 | ||
| 520 | |a METHODS: We present a clinical validation of a reverse transcription PCR (RT-PCR) assay for the SARS-CoV-2 nucleocapsid (N1) gene. Off-board lysis on an automated nucleic acid extraction system was optimized with endemic coronaviruses (OC43 and NL63). Genomic RNA and SARS-CoV-2 RNA in a recombinant viral protein coat were used as control materials and compared for recovery from nucleic acid extraction | ||
| 520 | |a RESULTS: Nucleic acid extraction showed decreased recovery of endemic Coronavirus in vitro transcribed RNA (NL63) compared with attenuated virus (OC43). SARS-CoV-2 RNA had more reliable recovery from extraction through amplification than genomic RNA. Recovery of genomic RNA was improved by combining lysis buffer with clinical matrix before adding RNA. The RT-PCR assay demonstrated 100% in silico sensitivity and specificity. The accuracy across samples was 100% (75 of 75). Precision studies showed 100% intra-run, inter-run, and inter-technologist concordance. The limit of detection was 264 copies per milliliter (estimated 5 copies per reaction; 35.56 mean threshold cycle value) | ||
| 520 | |a CONCLUSIONS: This SARS-CoV-2 assay demonstrates appropriate characteristics for use under an Emergency Use Authorization. Endemic coronavirus controls were useful in optimizing the extraction procedure. In the absence of live or attenuated virus, recombinant virus in a protein coat is an appropriate control specimen type for assay validation during a pandemic | ||
| 650 | 4 | |a Journal Article | |
| 650 | 4 | |a Validation Study | |
| 650 | 4 | |a COVID-19 | |
| 650 | 4 | |a Clinical Validation | |
| 650 | 4 | |a Coronavirus Disease 2019 | |
| 650 | 4 | |a EUA | |
| 650 | 4 | |a SARS-CoV-2 | |
| 650 | 7 | |a COVID-19 Vaccines |2 NLM | |
| 650 | 7 | |a Covid-19 aAPC vaccine |2 NLM | |
| 650 | 7 | |a Nucleocapsid Proteins |2 NLM | |
| 650 | 7 | |a RNA, Viral |2 NLM | |
| 700 | 1 | |a Frame, Ithiel |e verfasserin |4 aut | |
| 700 | 1 | |a Falcon, Alejandra |e verfasserin |4 aut | |
| 700 | 1 | |a Jacob, Jerin |e verfasserin |4 aut | |
| 700 | 1 | |a Wagenfuehr, Jennifer |e verfasserin |4 aut | |
| 700 | 1 | |a Mitui, Midori |e verfasserin |4 aut | |
| 700 | 1 | |a Park, Jason Y |e verfasserin |4 aut | |
| 700 | 1 | |a Filkins, Laura |e verfasserin |4 aut | |
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