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Comparison of Reverse Transcription (RT)-Quantitative PCR and RT-Droplet Digital PCR for Detection of Genomic and Subgenomic SARS-CoV-2 RNA
Most individuals acutely infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) exhibit mild symptoms. However, 10 to 20% of those infected develop long-term symptoms, referred to as post-coronavirus disease 2019 (COVID-19) condition (PCC). One hypothesis is that PCC might be exa...
Autores principales: | , , , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
American Society for Microbiology
2023
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10100669/ https://www.ncbi.nlm.nih.gov/pubmed/36943067 http://dx.doi.org/10.1128/spectrum.04159-22 |
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author | Morón-López, Sara Riveira-Muñoz, Eva Urrea, Victor Gutiérrez-Chamorro, Lucia Ávila-Nieto, Carlos Noguera-Julian, Marc Carrillo, Jorge Mitjà, Oriol Mateu, Lourdes Massanella, Marta Ballana, Ester Martinez-Picado, Javier |
author_facet | Morón-López, Sara Riveira-Muñoz, Eva Urrea, Victor Gutiérrez-Chamorro, Lucia Ávila-Nieto, Carlos Noguera-Julian, Marc Carrillo, Jorge Mitjà, Oriol Mateu, Lourdes Massanella, Marta Ballana, Ester Martinez-Picado, Javier |
author_sort | Morón-López, Sara |
collection | PubMed |
description | Most individuals acutely infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) exhibit mild symptoms. However, 10 to 20% of those infected develop long-term symptoms, referred to as post-coronavirus disease 2019 (COVID-19) condition (PCC). One hypothesis is that PCC might be exacerbated by viral persistence in tissue sanctuaries. Therefore, the accurate detection and quantification of SARS-CoV-2 are not only necessary for viral load monitoring but also crucial for detecting long-term viral persistence and determining whether viral replication is occurring in tissue reservoirs. In this study, the sensitivity and robustness of reverse transcription (RT)-droplet digital PCR (ddPCR) and RT-quantitative PCR (qPCR) techniques have been compared for the detection and quantification of SARS-CoV-2 genomic and subgenomic RNAs from oropharyngeal swabs taken from confirmed SARS-CoV-2-positive, SARS-CoV-2-exposed, and nonexposed individuals as well as from samples from mice infected with SARS-CoV-2. Our data demonstrated that both techniques presented equivalent results in the mid- and high-viral-load ranges. Additionally, RT-ddPCR was more sensitive than RT-qPCR in the low-viral-load range, allowing the accurate detection of positive results in individuals exposed to the virus. Overall, these data suggest that RT-ddPCR might be an alternative to RT-qPCR for detecting low viral loads in samples and for assessing viral persistence in samples from individuals with PCC. IMPORTANCE We developed one-step reverse transcription (RT)-droplet digital PCR (ddPCR) protocols to detect SARS-CoV-2 RNA and compared them to the gold-standard RT-quantitative PCR (RT-qPCR) method. RT-ddPCR was more sensitive than RT-qPCR in the low-viral-load range, while both techniques were equivalent in the mid- and high-viral-load ranges. Overall, these results suggest that RT-ddPCR might be a viable alternative to RT-qPCR when it comes to detecting low viral loads in samples, which is a highly relevant issue for determining viral persistence in as-yet-unknown tissue reservoirs in individuals suffering from post-COVID conditions or long COVID. |
format | Online Article Text |
id | pubmed-10100669 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2023 |
publisher | American Society for Microbiology |
record_format | MEDLINE/PubMed |
spelling | pubmed-101006692023-04-14 Comparison of Reverse Transcription (RT)-Quantitative PCR and RT-Droplet Digital PCR for Detection of Genomic and Subgenomic SARS-CoV-2 RNA Morón-López, Sara Riveira-Muñoz, Eva Urrea, Victor Gutiérrez-Chamorro, Lucia Ávila-Nieto, Carlos Noguera-Julian, Marc Carrillo, Jorge Mitjà, Oriol Mateu, Lourdes Massanella, Marta Ballana, Ester Martinez-Picado, Javier Microbiol Spectr Research Article Most individuals acutely infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) exhibit mild symptoms. However, 10 to 20% of those infected develop long-term symptoms, referred to as post-coronavirus disease 2019 (COVID-19) condition (PCC). One hypothesis is that PCC might be exacerbated by viral persistence in tissue sanctuaries. Therefore, the accurate detection and quantification of SARS-CoV-2 are not only necessary for viral load monitoring but also crucial for detecting long-term viral persistence and determining whether viral replication is occurring in tissue reservoirs. In this study, the sensitivity and robustness of reverse transcription (RT)-droplet digital PCR (ddPCR) and RT-quantitative PCR (qPCR) techniques have been compared for the detection and quantification of SARS-CoV-2 genomic and subgenomic RNAs from oropharyngeal swabs taken from confirmed SARS-CoV-2-positive, SARS-CoV-2-exposed, and nonexposed individuals as well as from samples from mice infected with SARS-CoV-2. Our data demonstrated that both techniques presented equivalent results in the mid- and high-viral-load ranges. Additionally, RT-ddPCR was more sensitive than RT-qPCR in the low-viral-load range, allowing the accurate detection of positive results in individuals exposed to the virus. Overall, these data suggest that RT-ddPCR might be an alternative to RT-qPCR for detecting low viral loads in samples and for assessing viral persistence in samples from individuals with PCC. IMPORTANCE We developed one-step reverse transcription (RT)-droplet digital PCR (ddPCR) protocols to detect SARS-CoV-2 RNA and compared them to the gold-standard RT-quantitative PCR (RT-qPCR) method. RT-ddPCR was more sensitive than RT-qPCR in the low-viral-load range, while both techniques were equivalent in the mid- and high-viral-load ranges. Overall, these results suggest that RT-ddPCR might be a viable alternative to RT-qPCR when it comes to detecting low viral loads in samples, which is a highly relevant issue for determining viral persistence in as-yet-unknown tissue reservoirs in individuals suffering from post-COVID conditions or long COVID. American Society for Microbiology 2023-03-21 /pmc/articles/PMC10100669/ /pubmed/36943067 http://dx.doi.org/10.1128/spectrum.04159-22 Text en Copyright © 2023 Morón-López et al. https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license (https://creativecommons.org/licenses/by/4.0/) . |
spellingShingle | Research Article Morón-López, Sara Riveira-Muñoz, Eva Urrea, Victor Gutiérrez-Chamorro, Lucia Ávila-Nieto, Carlos Noguera-Julian, Marc Carrillo, Jorge Mitjà, Oriol Mateu, Lourdes Massanella, Marta Ballana, Ester Martinez-Picado, Javier Comparison of Reverse Transcription (RT)-Quantitative PCR and RT-Droplet Digital PCR for Detection of Genomic and Subgenomic SARS-CoV-2 RNA |
title | Comparison of Reverse Transcription (RT)-Quantitative PCR and RT-Droplet Digital PCR for Detection of Genomic and Subgenomic SARS-CoV-2 RNA |
title_full | Comparison of Reverse Transcription (RT)-Quantitative PCR and RT-Droplet Digital PCR for Detection of Genomic and Subgenomic SARS-CoV-2 RNA |
title_fullStr | Comparison of Reverse Transcription (RT)-Quantitative PCR and RT-Droplet Digital PCR for Detection of Genomic and Subgenomic SARS-CoV-2 RNA |
title_full_unstemmed | Comparison of Reverse Transcription (RT)-Quantitative PCR and RT-Droplet Digital PCR for Detection of Genomic and Subgenomic SARS-CoV-2 RNA |
title_short | Comparison of Reverse Transcription (RT)-Quantitative PCR and RT-Droplet Digital PCR for Detection of Genomic and Subgenomic SARS-CoV-2 RNA |
title_sort | comparison of reverse transcription (rt)-quantitative pcr and rt-droplet digital pcr for detection of genomic and subgenomic sars-cov-2 rna |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10100669/ https://www.ncbi.nlm.nih.gov/pubmed/36943067 http://dx.doi.org/10.1128/spectrum.04159-22 |
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