ddPCR increases detection of SARS-CoV-2 RNA in patients with low viral loads

Autor: Marchio, Agnès, Batejat, Christophe, Vanhomwegen, Jessica, Feher, Maxence, Grassin, Quentin, Chazal, Maxime, Raulin, Olivia, Farges-Berth, Anne, Reibel, Florence, Estève, Vincent, Dejean, Anne, Jouvenet, Nolwenn, Manuguerra, Jean-Claude, Pineau, Pascal
Přispěvatelé: Organisation Nucléaire et Oncogenèse / Nuclear Organization and Oncogenesis, Institut Pasteur [Paris]-Institut National de la Santé et de la Recherche Médicale (INSERM), Cellule d'Intervention Biologique d'Urgence (Centre National de Référence) - Laboratory for Urgent Response to Biological Threats (National Reference Center) (CIBU), Environnement et Risques infectieux - Environment and Infectious Risks (ERI), Institut Pasteur [Paris]-Institut Pasteur [Paris], Institut Pasteur [Paris], Signalisation antivirale - Virus sensing and signaling, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Centre Hospitalier Compiègne-Noyon, Groupe Hospitalier Nord Essonne [Longjumeau], This work was supported by the « URGENCE COVID-19 » fundraising campaign of Institut Pasteur to PP and NJ., We are grateful to Kenneth Vernick, Karin Eiglmeier, and Corinne Genève from the Unit of Insect Vector Genetics and Genomics at Institut Pasteur for providing access to their ddPCR platform during the lockdown period. We thank Sylvie van der Werf, Olivier Schwartz, and Nicolas Escriou for providing essential reagents, and Jacob Seeler for his critical reading of the manuscript., Institut Pasteur [Paris] (IP)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Paris Cité (UPCité), Université Paris Cité (UPCité)-Environnement et Risques infectieux - Environment and Infectious Risks (ERI), Institut Pasteur [Paris] (IP)-Université Paris Cité (UPCité)-Institut Pasteur [Paris] (IP), Institut Pasteur [Paris] (IP), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité)
Jazyk: angličtina
Rok vydání: 2021
Předmět:
Zdroj: Archives of Virology
Archives of Virology, Springer Verlag, 2021, 166 (9), pp.2529-2540. ⟨10.1007/s00705-021-05149-0⟩
Archives of Virology, 2021, 166 (9), pp.2529-2540. ⟨10.1007/s00705-021-05149-0⟩
ISSN: 1432-8798
0304-8608
DOI: 10.1007/s00705-021-05149-0⟩
Popis: RT-qPCR detection of SARS-CoV-2 RNA still represents the method of reference to diagnose and monitor COVID-19. From the onset of the pandemic, however, doubts have been expressed concerning the sensitivity of this molecular diagnosis method. Droplet digital PCR (ddPCR) is a third-generation PCR technique that is particularly adapted to detecting low-abundance targets. We developed two-color ddPCR assays for the detection of four different regions of SARS-CoV-2 RNA, including non-structural (IP4-RdRP, helicase) and structural (E, N) protein-encoding sequences. We observed that N or E subgenomic RNAs are generally more abundant than IP4 and helicase RNA sequences in cells infected in vitro, suggesting that detection of the N gene, coding for the most abundant subgenomic RNA of SARS-CoV-2, increases the sensitivity of detection during the highly replicative phase of infection. We investigated 208 nasopharyngeal swabs sampled in March-April 2020 in different hospitals of Greater Paris. We found that 8.6% of informative samples (n = 16/185, P < 0.0001) initially scored as “non-positive” (undetermined or negative) by RT-qPCR were positive for SARS-CoV-2 RNA by ddPCR. Our work confirms that the use of ddPCR modestly, but significantly, increases the proportion of upper airway samples testing positive in the framework of first-line diagnosis of a French population. Supplementary Information The online version contains supplementary material available at 10.1007/s00705-021-05149-0.
Databáze: OpenAIRE
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