A 16S rRNA reverse transcriptase-PCR method for rapid detection of intestinal enterococci in distributed drinking water

Abstract Aim To develop and validate a real-time reverse transcription PCR (RT-PCR) method for the rapid and sensitive detection of intestinal enterococci in drinking water. Conventional culture methods require 24–48 h, limiting rapid response to faecal contamination events. Methods An RT-PCR assay targeting 16S rRNA was developed to detect ten Enterococcus species, selected based on their prevalence in human and animal faeces and drinking water samples. Drinking water samples (100 ml) were filtered, treated with lysozyme, followed by RNA extraction and one-step real-time RT-PCR. Validation followed ISO16140-2:2016 with ISO7899-2:2000 as reference method and an interlaboratory study, using artificially contaminated samples and uncontaminated drinking water samples. Results The RT-PCR method demonstrated a sensitivity of 94.6%, slightly higher than the reference culture method (89.2%). The combined RLOD value (0.851) indicated comparable detection capability between methods, with slightly improved sensitivity of the RT-PCR for Enterococcus faecium. Inclusivity testing confirmed detection of all targeted species, while exclusivity testing showed no cross-reactivity with non-target bacteria. Interlaboratory validation confirmed consistent performance across laboratories. Application in drinking water monitoring showed 91.8% agreement between RT-PCR and culture results in samples collected after distribution network repairs, with RT-PCR detecting enterococci more frequently than the reference method. Conclusions The validated RT-PCR method provides reliable detection of intestinal enterococci in drinking water with sensitivity comparable to the reference method, but with a much shorter time-to-result (3–4 h). This rapid detection capability enables faster responses to faecal contamination events and supports improved public health protection and operational efficiency for drinking water utilities.