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Clinical evaluation of a multiplex real-time RT-PCR assay for detection of SARS-CoV-2 in individual and pooled upper respiratory tract samples

The aim of this study was to identify and validate a sensitive, high-throughput, and cost-effective SARS-CoV-2 real-time RT-PCR assay to be used as a surveillance and diagnostic tool for SARS-CoV-2 in a university surveillance program. We conducted a side-by-side clinical evaluation of a newly devel...

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Autores principales: Laverack, Melissa, Tallmadge, Rebecca L., Venugopalan, Roopa, Cronk, Brittany, Zhang, XiuLin, Rauh, Rolf, Saunders, Amy, Nelson, William M., Plocharczyk, Elizabeth, Diel, Diego G.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Springer Vienna 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8278812/
https://www.ncbi.nlm.nih.gov/pubmed/34259914
http://dx.doi.org/10.1007/s00705-021-05148-1
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author Laverack, Melissa
Tallmadge, Rebecca L.
Venugopalan, Roopa
Cronk, Brittany
Zhang, XiuLin
Rauh, Rolf
Saunders, Amy
Nelson, William M.
Plocharczyk, Elizabeth
Diel, Diego G.
author_facet Laverack, Melissa
Tallmadge, Rebecca L.
Venugopalan, Roopa
Cronk, Brittany
Zhang, XiuLin
Rauh, Rolf
Saunders, Amy
Nelson, William M.
Plocharczyk, Elizabeth
Diel, Diego G.
author_sort Laverack, Melissa
collection PubMed
description The aim of this study was to identify and validate a sensitive, high-throughput, and cost-effective SARS-CoV-2 real-time RT-PCR assay to be used as a surveillance and diagnostic tool for SARS-CoV-2 in a university surveillance program. We conducted a side-by-side clinical evaluation of a newly developed SARS-CoV-2 multiplex assay (EZ-SARS-CoV-2 Real-Time RT-PCR) with the commercial TaqPath COVID-19 Combo Kit, which has an Emergency Use Authorization from the FDA. The EZ-SARS-CoV-2 RT-PCR incorporates two assays targeting the SARS-CoV-2 N gene, an internal control targeting the human RNase P gene, and a PCR inhibition control in a single reaction. Nasopharyngeal (NP) and anterior nares (AN) swabs were tested as individuals and pools with both assays and in the ABI 7500 Fast and the QuantStudio 5 detection platforms. The analytical sensitivity of the EZ-SARS-CoV-2 RT-PCR assay was 250 copies/ml or approximately 1.75 genome copy equivalents per reaction. The clinical performance of the EZ-SARS-CoV-2 assay was evaluated using NP and AN samples tested in other laboratories. The diagnostic sensitivity of the assay ranged between 94 and 96% across the detection platforms, and the diagnostic specificity was 94.06%. The positive predictive value was 94%, and the negative predictive value ranged from 94 to 96%. Pooling five NP or AN specimens yielded 93% diagnostic sensitivity. The overall agreement between these SARS-CoV-2 RT-PCR assays was high, supported by a Cohen’s kappa value of 0.93. The EZ-SARS-CoV-2 RT-PCR assay performance attributes of high sensitivity and specificity with AN sample matrix and pooled upper respiratory samples support its use in a high-throughput surveillance testing program. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s00705-021-05148-1.
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spelling pubmed-82788122021-07-14 Clinical evaluation of a multiplex real-time RT-PCR assay for detection of SARS-CoV-2 in individual and pooled upper respiratory tract samples Laverack, Melissa Tallmadge, Rebecca L. Venugopalan, Roopa Cronk, Brittany Zhang, XiuLin Rauh, Rolf Saunders, Amy Nelson, William M. Plocharczyk, Elizabeth Diel, Diego G. Arch Virol Original Article The aim of this study was to identify and validate a sensitive, high-throughput, and cost-effective SARS-CoV-2 real-time RT-PCR assay to be used as a surveillance and diagnostic tool for SARS-CoV-2 in a university surveillance program. We conducted a side-by-side clinical evaluation of a newly developed SARS-CoV-2 multiplex assay (EZ-SARS-CoV-2 Real-Time RT-PCR) with the commercial TaqPath COVID-19 Combo Kit, which has an Emergency Use Authorization from the FDA. The EZ-SARS-CoV-2 RT-PCR incorporates two assays targeting the SARS-CoV-2 N gene, an internal control targeting the human RNase P gene, and a PCR inhibition control in a single reaction. Nasopharyngeal (NP) and anterior nares (AN) swabs were tested as individuals and pools with both assays and in the ABI 7500 Fast and the QuantStudio 5 detection platforms. The analytical sensitivity of the EZ-SARS-CoV-2 RT-PCR assay was 250 copies/ml or approximately 1.75 genome copy equivalents per reaction. The clinical performance of the EZ-SARS-CoV-2 assay was evaluated using NP and AN samples tested in other laboratories. The diagnostic sensitivity of the assay ranged between 94 and 96% across the detection platforms, and the diagnostic specificity was 94.06%. The positive predictive value was 94%, and the negative predictive value ranged from 94 to 96%. Pooling five NP or AN specimens yielded 93% diagnostic sensitivity. The overall agreement between these SARS-CoV-2 RT-PCR assays was high, supported by a Cohen’s kappa value of 0.93. The EZ-SARS-CoV-2 RT-PCR assay performance attributes of high sensitivity and specificity with AN sample matrix and pooled upper respiratory samples support its use in a high-throughput surveillance testing program. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s00705-021-05148-1. Springer Vienna 2021-07-14 2021 /pmc/articles/PMC8278812/ /pubmed/34259914 http://dx.doi.org/10.1007/s00705-021-05148-1 Text en © The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature 2021 This article is made available via the PMC Open Access Subset for unrestricted research re-use and secondary analysis in any form or by any means with acknowledgement of the original source. These permissions are granted for the duration of the World Health Organization (WHO) declaration of COVID-19 as a global pandemic.
spellingShingle Original Article
Laverack, Melissa
Tallmadge, Rebecca L.
Venugopalan, Roopa
Cronk, Brittany
Zhang, XiuLin
Rauh, Rolf
Saunders, Amy
Nelson, William M.
Plocharczyk, Elizabeth
Diel, Diego G.
Clinical evaluation of a multiplex real-time RT-PCR assay for detection of SARS-CoV-2 in individual and pooled upper respiratory tract samples
title Clinical evaluation of a multiplex real-time RT-PCR assay for detection of SARS-CoV-2 in individual and pooled upper respiratory tract samples
title_full Clinical evaluation of a multiplex real-time RT-PCR assay for detection of SARS-CoV-2 in individual and pooled upper respiratory tract samples
title_fullStr Clinical evaluation of a multiplex real-time RT-PCR assay for detection of SARS-CoV-2 in individual and pooled upper respiratory tract samples
title_full_unstemmed Clinical evaluation of a multiplex real-time RT-PCR assay for detection of SARS-CoV-2 in individual and pooled upper respiratory tract samples
title_short Clinical evaluation of a multiplex real-time RT-PCR assay for detection of SARS-CoV-2 in individual and pooled upper respiratory tract samples
title_sort clinical evaluation of a multiplex real-time rt-pcr assay for detection of sars-cov-2 in individual and pooled upper respiratory tract samples
topic Original Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8278812/
https://www.ncbi.nlm.nih.gov/pubmed/34259914
http://dx.doi.org/10.1007/s00705-021-05148-1
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