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 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.
| Medienart: |
E-Artikel |
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| Erscheinungsjahr: |
2023 |
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| Erschienen: |
2023 |
| Enthalten in: |
Zur Gesamtaufnahme - year:2023 |
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| Enthalten in: |
Microbiology spectrum - (2023) vom: 21. März, Seite e0415922 |
| Sprache: |
Englisch |
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| Beteiligte Personen: |
Morón-López, Sara [VerfasserIn] |
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| Links: |
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| Themen: |
Genomic SARS-CoV-2 |
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| Anmerkungen: |
Date Revised 20.10.2023 published: Print-Electronic Citation Status Publisher |
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| doi: |
10.1128/spectrum.04159-22 |
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| funding: |
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| Förderinstitution / Projekttitel: |
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NLM354484192 |
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| 245 | 1 | 0 | |a Comparison of Reverse Transcription (RT)-Quantitative PCR and RT-Droplet Digital PCR for Detection of Genomic and Subgenomic SARS-CoV-2 RNA |
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| 520 | |a 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 | ||
| 650 | 4 | |a Journal Article | |
| 650 | 4 | |a RT-ddPCR | |
| 650 | 4 | |a RT-qPCR | |
| 650 | 4 | |a genomic SARS-CoV-2 | |
| 650 | 4 | |a long COVID | |
| 650 | 4 | |a post-COVID-19 condition | |
| 650 | 4 | |a subgenomic SARS-CoV-2 | |
| 650 | 4 | |a viral persistence | |
| 700 | 1 | |a Riveira-Muñoz, Eva |e verfasserin |4 aut | |
| 700 | 1 | |a Urrea, Victor |e verfasserin |4 aut | |
| 700 | 1 | |a Gutiérrez-Chamorro, Lucia |e verfasserin |4 aut | |
| 700 | 1 | |a Ávila-Nieto, Carlos |e verfasserin |4 aut | |
| 700 | 1 | |a Noguera-Julian, Marc |e verfasserin |4 aut | |
| 700 | 1 | |a Carrillo, Jorge |e verfasserin |4 aut | |
| 700 | 1 | |a Mitjà, Oriol |e verfasserin |4 aut | |
| 700 | 1 | |a Mateu, Lourdes |e verfasserin |4 aut | |
| 700 | 1 | |a Massanella, Marta |e verfasserin |4 aut | |
| 700 | 1 | |a Ballana, Ester |e verfasserin |4 aut | |
| 700 | 1 | |a Martinez-Picado, Javier |e verfasserin |4 aut | |
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