tetano
Editor, Senior Moderator
J Appl Lab Med
. 2020 Jun 1;jfaa089.
doi: 10.1093/jalm/jfaa089. Online ahead of print.
Clinical Validation of a SARS-CoV-2 Real-Time Reverse Transcription PCR Assay Targeting the Nucleocapsid Gene
Jeffrey A SoRelle[SUP] 1 [/SUP], Ithiel Frame[SUP] 1 [/SUP], Alejandra Falcon[SUP] 2 [/SUP], Jerin Jacob[SUP] 2 [/SUP], Jennifer Wagenfuehr[SUP] 2 [/SUP], Midori Mitui[SUP] 2 [/SUP], Jason Y Park[SUP] 1 2 3 [/SUP], Laura Filkins[SUP] 1 2 [/SUP]
Affiliations
Abstract
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 RT-PCR assay for the SARS-CoV-2 nucleocapsid (N1) gene. Offboard lysis on an automated nucleic acid extraction system (EMAG?) was optimized with endemic Coronaviruses (OC43 and NL63). Genomic RNA and SARS-CoV-2 RNA in a recombinant viral protein coat (Accuplex) 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 to attenuated virus (OC43). SARS-CoV-2 RNA (Accuplex) had more reliable recovery from extraction through amplification compared to genomic RNA. Recovery of genomic RNA was improved by combining lysis buffer with clinical matrix prior to 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 ml (estimated 5 copies per reaction; 35.56 mean Ct 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.
Keywords: COVID-19; Clinical Validation; Coronavirus Disease 2019; EUA; SARS-CoV-2.
. 2020 Jun 1;jfaa089.
doi: 10.1093/jalm/jfaa089. Online ahead of print.
Clinical Validation of a SARS-CoV-2 Real-Time Reverse Transcription PCR Assay Targeting the Nucleocapsid Gene
Jeffrey A SoRelle[SUP] 1 [/SUP], Ithiel Frame[SUP] 1 [/SUP], Alejandra Falcon[SUP] 2 [/SUP], Jerin Jacob[SUP] 2 [/SUP], Jennifer Wagenfuehr[SUP] 2 [/SUP], Midori Mitui[SUP] 2 [/SUP], Jason Y Park[SUP] 1 2 3 [/SUP], Laura Filkins[SUP] 1 2 [/SUP]
Affiliations
- PMID: 32483586
- DOI: 10.1093/jalm/jfaa089
Abstract
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 RT-PCR assay for the SARS-CoV-2 nucleocapsid (N1) gene. Offboard lysis on an automated nucleic acid extraction system (EMAG?) was optimized with endemic Coronaviruses (OC43 and NL63). Genomic RNA and SARS-CoV-2 RNA in a recombinant viral protein coat (Accuplex) 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 to attenuated virus (OC43). SARS-CoV-2 RNA (Accuplex) had more reliable recovery from extraction through amplification compared to genomic RNA. Recovery of genomic RNA was improved by combining lysis buffer with clinical matrix prior to 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 ml (estimated 5 copies per reaction; 35.56 mean Ct 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.
Keywords: COVID-19; Clinical Validation; Coronavirus Disease 2019; EUA; SARS-CoV-2.