Gómez, LiviaBrandão, JoãoNavarro, AnnaMartines, AntoninoSanseverino, IsabellaPorcel-Rodríguez, ElenaMarinov, DimitarAbleitner, OksanaAllerberger, FranzAtsma, AdrieBriancesco, RossellaCenov, ArijanaCíchová, MariannaCoccia, Anna MariaLibera, Simonetta DellaDe Maeyer, KatrienDe Vries, Hendrik JanElsinga, GoffeHeijnen, LeoHokajärvi, Anna-MariaJanská, VeronikaHrabar, JerkoHuse-Kutowska, MonikaJozic, SlavenKlatovsky, StefanieKornschober, ChristianKohls, KatharinaKrom, AdrieLuden, KatrinMikula-Pratschke, ClaudiaPitkänen, TarjaPoje, MatejaProkšová, MiloslavaRetelj, MatjazRozwadowska, BeataRytkönen, AnnastiinaSarekoski, AnniinaSchlager, SabineSpringer, BurkhardStojan, IvaStroobach, ElineŠućić, NevenValério, ElisabeteVan Pelt, GabiVranken, JeromVrdoljak Tomaš, AnaVukić Lušić, DarijaWłos, EwaWubbels, GerhardZiombska, MariaZelenik, KatjaZiuziakowski, MarcinLettieri, Teresa2026-02-032026-02-032025-05-10Environ Res. 2025 Aug 15;279(Pt 2):121786. doi: 10.1016/j.envres.2025.121786. Epub 2025 May 100013-9351http://hdl.handle.net/10400.18/10799The microbiological quality of water intended for human consumption in the EU is regulated by the recast Drinking Water Directive 2020/2184 (DWD), which sets strict parametric values for intestinal enterococci and Escherichia coli (E. coli), allowing no more than zero colony-forming units per 100 mL. Detection and enumeration of E. coli typically rely on culture-based reference methods or the most probable number approach, which require 1–2 days to produce results—potentially delaying timely action during contamination events. In contrast, molecular techniques can deliver results within hours. The DWD permits the use of alternative methods if they are as reliable as the reference method and developing and validating such methods requires multiple laboratory trials in line with ISO standard 16140-2. Following this, we conducted a representative EU study to validate a molecular method based on real-time reverse transcription polymerase chain reaction for rapid E. coli detection in drinking water. In a concerted action, the first of its kind for drinking water, nineteen laboratories across ten Member States participated. To ensure consistency, drinking water was artificially contaminated with E. coli. The alternative method showed slightly lower sensitivity than the reference method (91.1 % vs. 97.2 %) but delivered much faster results, making it a valuable screening tool. It can support rapid decision-making during contamination events, reducing the risk of waterborne outbreaks and protecting public health. For reliable routine performance, appropriate training in this alternative method is strongly recommended.Highlights: -Real-time RT-PCR assay enables E. coli detection in drinking water within 6 h. -Two interlaboratory studies were performed at EU-representative level. -The real-time RT-PCR assay showed a sensitivity higher than 91 %. -RNA extraction is the most critical step of the assay. -Training and practical experience are fundamental to reach the highest sensitivity.engAlternative Method ValidationDetectionDrinking WaterEscherichia ColiFaecal ContaminationReal-Time Reverse Transcription PCRÁgua e SoloAvaliação do Riscojournal article10.1016/j.envres.2025.12178640355062