Qualitative detection of E. coli in distributed drinking water using real-time reverse transcription PCR targeting 16S rRNA: Validation and practical experiences

• Published in: Water research (Oxford) 2024-08, Vol.259, p.121843, Article 121843
• Main Authors: Heijnen, Leo, de Vries, Hendrik Jan, van Pelt, Gabi, Stroobach, Eline, Atsma, Adrie, Vranken, Jerom, De Maeyer, Katrien, Vissers, Liesbeth, Medema, Gertjan
• Format: Article
• Language: English
• Subjects: Belgium; chlorine; detection limit; disinfection; Drinking water; E. coli; Escherichia coli; Escherichia fergusonii; Fecal contamination; hygiene; microbial contamination; Netherlands; ozone; Rapid detection; rapid methods; reverse transcriptase polymerase chain reaction; ribosomal RNA; RT-PCR; selective media; serotypes; Shigella; species; water; Water quality
• ISSN: 0043-1354; 1879-2448; 1879-2448
• DOI: 10.1016/j.watres.2024.121843

•A validated RT-PCR method targeting 16S ribosomal RNA to qualitatively E. coli in drinking water at a sensitivity of 1 CFU/100 ml.•The method is as sensitive as the reference culture method.•The E. coli RT-PCR showed high interlaboratory reproducibility.•An extensive comparison on practical samples showed good agreement between RT-PCR and the culture method.•Application of the RT-PCR will improve the safety of drinking water consumers. Escherichia coli (E. coli) plays a central role as an indicator for fecal contamination to predict the possible presence of microbial pathogens in drinking water. Current detection methods for E. coli are based on time-consuming culture-based techniques. There is a strong need for methods to detect fecal contamination rapidly in distributed drinking water to prevent outbreaks of waterborne disease and support water utilities to efficiently manage their operations like actions to repair or maintain distribution pipes, to minimize impact on consumers. This study describes the validation and application of a qualitative real time reverse transcription PCR (RT-PCR) method targeting 16S ribosomal RNA (rRNA) for rapid detection of E. coli in distributed drinking water. The RT-PCR assay targets 16S rRNA, a highly abundant RNA in viable cells, enabling robust detection at the required sensitivity of 1 CFU/100 ml. The validation was performed by comparing the RT-PCR method with the culture-based chromogenic reference method (CCA) using the protocol and criteria described in ISO 16,140–2:2016. The validation demonstrated that this RT-PCR method can be used to specifically detect E. coli in a broad range of drinking water samples with at least the same limit of detection as the culture method (Relative Limit Of Detection = 0.75, range 0.43–1.43). The inclusivity study showed that the RT-PCR method was able to detect a broad range of E. coli strains derived from different sources and geographic areas, including pathogenic serotype O157 strains that are not detected with the culture method. The exclusivity study determined that other bacterial genera are not detected with this RT-PCR. However, Escherichia fergusonii was detected and, based on “in silico” analysis, it is expected that also E. albertii and E. marmotae and Shigella species will be detectable using this RT-PCR. An interlaboratory study confirmed that the RT-PCR and culture method have comparable sensitivities when tested by different participants at different laboratories.