Clinical Validation of a SARS-CoV-2 Real-Time Reverse Transcription PCR Assay Targeting the Nucleocapsid Gene.
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.
['SoRelle|Jeffrey A|JA|', 'Frame|Ithiel|I|', 'Falcon|Alejandra|A|', 'Jacob|Jerin|J|', 'Wagenfuehr|Jennifer|J|', 'Mitui|Midori|M|', 'Park|Jason Y|JY|', 'Filkins|Laura|L|']
D000073640:Betacoronavirus D000086382:COVID-19 D000086742:COVID-19 Testing D000086663:COVID-19 Vaccines D019411:Clinical Laboratory Techniques D018352:Coronavirus Infections D004632:Emergency Medical Services D006801:Humans D019590:Nucleocapsid Proteins D058873:Pandemics D011024:Pneumonia, Viral D012367:RNA, Viral D015203:Reproducibility of Results D020133:Reverse Transcriptase Polymerase Chain Reaction D000086402:SARS-CoV-2
Clinical Validation SARS-CoV-2 COVID-19 Coronavirus Disease 2019 EUA