tetano
Editor, Senior Moderator
icrobiol Spectr
. 2023 Mar 21;e0415922.
doi: 10.1128/spectrum.04159-22. Online ahead of print.
Comparison of Reverse Transcription (RT)-Quantitative PCR and RT-Droplet Digital PCR for Detection of Genomic and Subgenomic SARS-CoV-2 RNA
Sara Morón-López[SUP] 1 2 [/SUP], Eva Riveira-Muñoz[SUP] 1 [/SUP], Victor Urrea[SUP] 1 [/SUP], Lucia Gutiérrez-Chamorro[SUP] 1 [/SUP], Carlos Ávila-Nieto[SUP] 1 [/SUP], Marc Noguera-Julian[SUP] 1 2 [/SUP], Jorge Carrillo[SUP] 1 2 3 [/SUP], Oriol Mitjà[SUP] 4 5 6 7 [/SUP], Lourdes Mateu[SUP] 4 5 6 8 [/SUP], Marta Massanella[SUP] 1 2 6 [/SUP], Ester Ballana[SUP] 1 3 [/SUP], Javier Martinez-Picado[SUP] 1 2 3 6 9 [/SUP]
Affiliations
Abstract
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.
Keywords: RT-ddPCR; RT-qPCR; genomic SARS-CoV-2; long COVID; post-COVID-19 condition; subgenomic SARS-CoV-2; viral persistence.
. 2023 Mar 21;e0415922.
doi: 10.1128/spectrum.04159-22. Online ahead of print.
Comparison of Reverse Transcription (RT)-Quantitative PCR and RT-Droplet Digital PCR for Detection of Genomic and Subgenomic SARS-CoV-2 RNA
Sara Morón-López[SUP] 1 2 [/SUP], Eva Riveira-Muñoz[SUP] 1 [/SUP], Victor Urrea[SUP] 1 [/SUP], Lucia Gutiérrez-Chamorro[SUP] 1 [/SUP], Carlos Ávila-Nieto[SUP] 1 [/SUP], Marc Noguera-Julian[SUP] 1 2 [/SUP], Jorge Carrillo[SUP] 1 2 3 [/SUP], Oriol Mitjà[SUP] 4 5 6 7 [/SUP], Lourdes Mateu[SUP] 4 5 6 8 [/SUP], Marta Massanella[SUP] 1 2 6 [/SUP], Ester Ballana[SUP] 1 3 [/SUP], Javier Martinez-Picado[SUP] 1 2 3 6 9 [/SUP]
Affiliations
- PMID: 36943067
- DOI: 10.1128/spectrum.04159-22
Abstract
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.
Keywords: RT-ddPCR; RT-qPCR; genomic SARS-CoV-2; long COVID; post-COVID-19 condition; subgenomic SARS-CoV-2; viral persistence.