Characterization and validation of a chemiluminescent assay based on a 1,2-dioxetane for rapid detection of enterococci in contaminated water and comparison with standard methods and qPCR

• Published in: Journal of applied microbiology 2011-08, Vol.111 (2), p.407-416
• Main Authors: Bukh, A.S, Roslev, P
• Format: Article
• Language: English
• Subjects: 1,2‐dioxetanes; beta-glucosidase; Biological and medical sciences; chemiluminescence; Colony Count, Microbial; DNA, Bacterial - genetics; early warning; Enterococcus; Enterococcus - genetics; Enterococcus - isolation & purification; enzymatic hydrolysis; faecal indicator bacteria; feces; Feces - microbiology; Fundamental and applied biological sciences. Psychology; Heterocyclic Compounds - analysis; Limit of Detection; Luminescent Measurements - methods; Microbiology; municipal wastewater; Polymerase Chain Reaction; qPCR; quantitative polymerase chain reaction; RNA, Ribosomal, 16S - genetics; Seawater - microbiology; Sensitivity and Specificity; wastewater; water pollution; Water Pollution - analysis; β‐d‐glucosidase
• ISSN: 1364-5072; 1365-2672
• DOI: 10.1111/j.1365-2672.2011.05051.x

Aims: To evaluate the potential for using a novel chemiluminescence-based enzyme assay for rapid detection of enterococci in water contaminated with faecal waste. Methods and Results: The novel assay (EntLight) was based on the enzymatic hydrolysis of the chemiluminescent 1,2-dioxetane [(4-methoxy-4(3-β-d-glucoside-4-chlorophenyl)]spiro[1,2-dioxetane-3-1,3-tricyclo[7·3·1·0²,⁷]tridec-2,7-ene] specific for β-d-glucosidase. The specificity of the proposed EntLight assay was characterized using 26 different Enterococcus strains and 10 bacterial genera other than Enterococcus. With an analysis time of ≤8 h, the assay was found to be sensitive and specific. Validation experiments were carried out using water samples contaminated with raw municipal wastewater in comparison with qPCR and ISO standard methods. EntLight was successfully applied to detect enterococci in contaminated water within ≤8 h, and the proposed assay correlated well with both qPCR and ISO standard methods (R² > 0·776). Conclusions: EntLight can be applied to rapid and simple detection of viable enterococci in water contaminated with faecal matter. Significance and Impact of the Study: The novel EntLight assay and qPCR have the potential to be used as methods for early warning (1-7 h) of faecal pollutions in different water types.