Optimized protocol for direct extraction of SARS-CoV-2 RNA from raw wastewater samples (ANRS 0160)
This study aimed to evaluate whether a nucleic acid extraction protocol specifically designed for raw wastewater (WW) provides a measurable advantage over protocols not originally intended for WW matrices. Three laboratories have independently compared two RNA extraction protocols, using paired WW samples. A common WW-designed protocol (Z), based on silica columns was tested across all labs. As comparators, three non-WW designed protocols were used: NS1 and NS2 both silica beads-based, with NS2 including an additional phenol-chloroform step; and SB a homemade protocol using also silica beads but differing in its formulation. As different samples were used across labs, direct statistical comparison was difficult; instead, paired comparisons within each lab were used to rank SARS-CoV-2 RNA detection and RT-qPCR inhibitor removal. NS1 yielded significantly higher SARS-CoV-2 RNA concentrations than Z (Logrank test, <i>p</i> ≤ 0.001), though with RT-qPCR inhibition in one sample. NS2 also showed higher SARS-CoV-2 RNA detection than Z (Wilcoxon test, <i>p</i> < 0.0001), with both protocols showing complete inhibitor removal. SB performed worse than Z for SARS-CoV-2 RNA detection (Logrank test, <i>p</i> ≤ 0.05), and showed inhibition in one sample. NS2 was the most effective option for both RNA detection and inhibitor removal.
Authors
- Julie Loutreul
- Florian Sandron (ORCID: https://orcid.org/0000-0001-7491-0283)
- Isabelle Bertrand (ORCID: https://orcid.org/0000-0003-0743-4402)
- Zuzana Gerber (ORCID: https://orcid.org/0000-0003-0846-3619)
- Robert Olaso (ORCID: https://orcid.org/0000-0001-7631-9657)
- Jean‐François Deleuze (ORCID: https://orcid.org/0000-0002-5358-4463)
- Christophe Gantzer (ORCID: https://orcid.org/0000-0003-2287-4852)
- Ahlam Chaqroun
- Nicolas Boudaud (ORCID: https://orcid.org/0000-0003-4031-4381)
- Nicolas Cluzel (ORCID: https://orcid.org/0000-0001-7880-8913)
- Olivier Rohr (ORCID: https://orcid.org/0000-0001-8992-2025)
- Damien Delafoy (ORCID: https://orcid.org/0000-0002-0586-4655)
- Julie Challant (ORCID: https://orcid.org/0009-0004-5397-0788)
- Clémentine Wallet (ORCID: https://orcid.org/0000-0002-2388-4510)
- Ghina El Soufi (ORCID: https://orcid.org/0009-0005-2945-7914)
- Léo Di Jorio (ORCID: https://orcid.org/0009-0005-9198-4942)
- Stéphanie Raffestin
- Vincent Maréchal
Institutions
- Centre National de la Recherche Scientifique (FR)
- Inserm (FR)
- Commissariat à l'Énergie Atomique et aux Énergies Alternatives (FR)
- Université Paris-Saclay (FR)
- Sorbonne Université (FR)
- Centre National de Recherche en Génomique Humaine (FR)
- Institut Pasteur de la Guyane (GF)
- CEA Paris-Saclay (FR)
- Centre de Recherche Saint-Antoine (FR)
- Laboratoire de Chimie Physique et Microbiologie pour les Matériaux et l'Environnement (FR)
- Actalia (France) (FR)
- Unité de Modélisation Mathématique et Informatique des Systèmes Complexes (FR)
- SUMMIT
- Université de Strasbourg (FR)
- Université de Lorraine (FR)
Publication Details
- Journal
- MethodsX
- Published
- 2025-04-18
- DOI
- https://doi.org/10.1016/j.mex.2025.103323
- Citations
- 2
- Primary Topic
- SARS-CoV-2 detection and testing
- Type
- article
- Field-Weighted Citation Impact
- 2.79
Funders
- Agence Nationale de la Recherche