Clinical Validation of a SARS-CoV-2 Real-Time Reverse Transcription PCR Assay Targeting the Nucleocapsid Gene
| Autor: | Jason Y. Park, Jennifer Wagenfuehr, Alejandra Falcon, Laura M. Filkins, Ithiel J. Frame, Jerin Jacob, Midori Mitui, Jeffrey A. SoRelle |
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| Rok vydání: | 2020 |
| Předmět: |
0301 basic medicine
Emergency Medical Services Lysis COVID-19 Vaccines viruses 030106 microbiology Pneumonia Viral Biology medicine.disease_cause Recombinant virus EUA Article Coronavirus Disease 2019 03 medical and health sciences Betacoronavirus COVID-19 Testing Lysis buffer medicine Humans Gene Pandemics Coronavirus Clinical Laboratory Techniques Reverse Transcriptase Polymerase Chain Reaction SARS-CoV-2 RNA COVID-19 Reproducibility of Results General Medicine Nucleocapsid Proteins Virology Reverse transcription polymerase chain reaction 030104 developmental biology Nucleic acid RNA Viral Clinical Validation Coronavirus Infections |
| Zdroj: | The Journal of Applied Laboratory Medicine |
| ISSN: | 2576-9456 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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