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, p ≤ 0.001), though with RT-qPCR inhibition in one sample. NS2 also showed higher SARS-CoV-2 RNA detection than Z (Wilcoxon test, p < 0.0001), with both protocols showing complete inhibitor removal. SB performed worse than Z for SARS-CoV-2 RNA detection (Logrank test, p ≤ 0.05), and showed inhibition in one sample. NS2 was the most effective option for both RNA detection and inhibitor removal.