Comparison of Reverse Transcription (RT)-Quantitative PCR and RT-Droplet Digital PCR for Detection of Genomic and Subgenomic SARS-CoV-2 RNA
Moron-Lopez, Sara (Institut Germans Trias i Pujol. Institut de Recerca de la Sida IrsiCaixa)
Riveira-Muñoz, Eva (Institut Germans Trias i Pujol. Institut de Recerca de la Sida IrsiCaixa)
Urrea, Víctor (Institut Germans Trias i Pujol. Institut de Recerca de la Sida IrsiCaixa)
Gutiérrez-Chamorro, Lucía (Institut Germans Trias i Pujol. Institut de Recerca de la Sida IrsiCaixa)
Avila-Nieto, Carlos (Institut Germans Trias i Pujol. Institut de Recerca de la Sida IrsiCaixa)
Noguera-Julian, Marc (Institut Germans Trias i Pujol. Institut de Recerca de la Sida IrsiCaixa)
Carrillo, Jorge (Institut Germans Trias i Pujol. Institut de Recerca de la Sida IrsiCaixa)
Mitjà, Oriol (Institut Germans Trias i Pujol. Hospital Universitari Germans Trias i Pujol)
Mateu, Lourdes (Institut Germans Trias i Pujol. Hospital Universitari Germans Trias i Pujol)
Massanella, Marta (Institut Germans Trias i Pujol. Institut de Recerca de la Sida IrsiCaixa)
Ballana, Ester (Institut Germans Trias i Pujol. Institut de Recerca de la Sida IrsiCaixa)
Martínez Picado, Francisco Javier (Institut Germans Trias i Pujol. Institut de Recerca de la Sida IrsiCaixa)
Universitat Autònoma de Barcelona

Fecha:	2023
Resumen:	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.
Ayudas:	Agencia Estatal de Investigación PID2019-109870RB-100
Derechos:	Aquest document està subjecte a una llicència d'ús Creative Commons. Es permet la reproducció total o parcial, la distribució, la comunicació pública de l'obra i la creació d'obres derivades, fins i tot amb finalitats comercials, sempre i quan es reconegui l'autoria de l'obra original.
Lengua:	Anglès
Documento:	Article ; recerca ; Versió publicada
Materia:	Genomic SARS-CoV-2 ; Subgenomic SARS-CoV-2 ; RT-qPCR ; RT-ddPCR ; Viral persistence ; Long COVID ; Post-covid-19 condition
Publicado en:	Microbiology Spectrum, Vol. 11 (march 2023) , ISSN 2165-0497

DOI: 10.1128/spectrum.04159-22
PMID: 36943067

8 p, 907.6 KB

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