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Multiplexing primer/probe sets for detection of SARS-CoV-2 by qRT-PCR
BACKGROUND: The novel respiratory virus SARS-CoV-2, responsible for over 380,000 COVID-19 related deaths, has caused significant strain on healthcare infrastructure and clinical laboratories globally. The pandemic's initial challenges include broad diagnostic testing, consistent reagent supply...
Autores principales: | , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Elsevier B.V.
2020
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7278635/ https://www.ncbi.nlm.nih.gov/pubmed/32535397 http://dx.doi.org/10.1016/j.jcv.2020.104499 |
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author | Perchetti, Garrett A. Nalla, Arun K. Huang, Meei-Li Jerome, Keith R. Greninger, Alexander L. |
author_facet | Perchetti, Garrett A. Nalla, Arun K. Huang, Meei-Li Jerome, Keith R. Greninger, Alexander L. |
author_sort | Perchetti, Garrett A. |
collection | PubMed |
description | BACKGROUND: The novel respiratory virus SARS-CoV-2, responsible for over 380,000 COVID-19 related deaths, has caused significant strain on healthcare infrastructure and clinical laboratories globally. The pandemic's initial challenges include broad diagnostic testing, consistent reagent supply lines, and access to laboratory instruments and equipment. In early 2020, primer/probe sets distributed by the CDC utilized the same fluorophore for molecular detection - requiring multiple assays to be run in parallel - consuming valuable and limited resources. METHODS: Nasopharyngeal swabs submitted to UW Virology for SARS-CoV-2 clinical testing were extracted, amplified by our laboratory developed test (LDT) - a CDC-based quantitative reverse transcriptase PCR reaction - and analyzed for agreement between the multiplexed assay. Laboratory- confirmed respiratory infection samples were included to evaluate assay cross-reaction specificity. RESULTS: Triplexing correctly identified SARS-CoV-2 in 98.4% of confirmed positive or inconclusive patient samples by single-plex LDT (n = 183/186). All 170 SARS-CoV-2 negative samples tested by single-plex LDT were negative by triplexing. Other laboratory-confirmed respiratory infections did not amplify for SARS-CoV-2 in the triplex reaction. CONCLUSIONS: Multiplexing two virus-specific gene targets and an extraction control was found to be comparable to running parallel assays independently, while significantly improving assay throughput. |
format | Online Article Text |
id | pubmed-7278635 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2020 |
publisher | Elsevier B.V. |
record_format | MEDLINE/PubMed |
spelling | pubmed-72786352020-06-09 Multiplexing primer/probe sets for detection of SARS-CoV-2 by qRT-PCR Perchetti, Garrett A. Nalla, Arun K. Huang, Meei-Li Jerome, Keith R. Greninger, Alexander L. J Clin Virol Article BACKGROUND: The novel respiratory virus SARS-CoV-2, responsible for over 380,000 COVID-19 related deaths, has caused significant strain on healthcare infrastructure and clinical laboratories globally. The pandemic's initial challenges include broad diagnostic testing, consistent reagent supply lines, and access to laboratory instruments and equipment. In early 2020, primer/probe sets distributed by the CDC utilized the same fluorophore for molecular detection - requiring multiple assays to be run in parallel - consuming valuable and limited resources. METHODS: Nasopharyngeal swabs submitted to UW Virology for SARS-CoV-2 clinical testing were extracted, amplified by our laboratory developed test (LDT) - a CDC-based quantitative reverse transcriptase PCR reaction - and analyzed for agreement between the multiplexed assay. Laboratory- confirmed respiratory infection samples were included to evaluate assay cross-reaction specificity. RESULTS: Triplexing correctly identified SARS-CoV-2 in 98.4% of confirmed positive or inconclusive patient samples by single-plex LDT (n = 183/186). All 170 SARS-CoV-2 negative samples tested by single-plex LDT were negative by triplexing. Other laboratory-confirmed respiratory infections did not amplify for SARS-CoV-2 in the triplex reaction. CONCLUSIONS: Multiplexing two virus-specific gene targets and an extraction control was found to be comparable to running parallel assays independently, while significantly improving assay throughput. Elsevier B.V. 2020-08 2020-06-08 /pmc/articles/PMC7278635/ /pubmed/32535397 http://dx.doi.org/10.1016/j.jcv.2020.104499 Text en © 2020 Elsevier B.V. All rights reserved. Since January 2020 Elsevier has created a COVID-19 resource centre with free information in English and Mandarin on the novel coronavirus COVID-19. The COVID-19 resource centre is hosted on Elsevier Connect, the company's public news and information website. Elsevier hereby grants permission to make all its COVID-19-related research that is available on the COVID-19 resource centre - including this research content - immediately available in PubMed Central and other publicly funded repositories, such as the WHO COVID database with rights for unrestricted research re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for free by Elsevier for as long as the COVID-19 resource centre remains active. |
spellingShingle | Article Perchetti, Garrett A. Nalla, Arun K. Huang, Meei-Li Jerome, Keith R. Greninger, Alexander L. Multiplexing primer/probe sets for detection of SARS-CoV-2 by qRT-PCR |
title | Multiplexing primer/probe sets for detection of SARS-CoV-2 by qRT-PCR |
title_full | Multiplexing primer/probe sets for detection of SARS-CoV-2 by qRT-PCR |
title_fullStr | Multiplexing primer/probe sets for detection of SARS-CoV-2 by qRT-PCR |
title_full_unstemmed | Multiplexing primer/probe sets for detection of SARS-CoV-2 by qRT-PCR |
title_short | Multiplexing primer/probe sets for detection of SARS-CoV-2 by qRT-PCR |
title_sort | multiplexing primer/probe sets for detection of sars-cov-2 by qrt-pcr |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7278635/ https://www.ncbi.nlm.nih.gov/pubmed/32535397 http://dx.doi.org/10.1016/j.jcv.2020.104499 |
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