NDLI: Characterization and validation of a chemiluminescent assay based on 1,2-dioxetanes for rapid detection of viable Escherichia coli

Content Provider	Springer Nature Link
Author	Bukh, Annette S. Roslev, Peter
Copyright Year	2010
Abstract	[(4-methoxy-4(3-β-d-galactose-4-chlorophenyl)]spiro[1,2-dioxetane-3-1,3-tricyclo[7.3.1.02,7]tridec-2,7-ene] (“sβ-Gal 102”) and sodium [4-methoxy-4(3-β-d-glucuronic acid-4-chlorophenyl)]spiro[1,2-dioxetane-3-1,3-tricyclo[7.3.1.02,7]tridec-2,7-ene] (“sβ-Glucor 102”) are carbohydrate-containing 1,2-dioxetane compounds that produce chemiluminescence upon enzymatic hydrolysis by β-d-galactosidase, and β-d-glucuronidase, respectively. In this study, we have characterized and validated a sensitive detection principle for viable Escherichia coli based on enzymatic cleavage of sβ-Gal 102 and sβ-Glucor 102 (“ColiLight II”). The proposed chemiluminescent assay was optimized with respect to analytical requirements including incubation time, temperature, pH, enzyme induction, and cell permeabilization. The sensitivity and specificity rates of the assay were tested on ten different bacterial genera. The assay was found to be representative based on low coefficients of variations for both accuracy and precision. The analysis time was less than 1 h and the analytical detection limit was 102 to 103 E. coli cells. In combination with membrane filtration and a brief resuscitation step of 4 h, the proposed assay was capable of detecting low concentrations of stressed E. coli in potable water (<30 CFU 100 ml−1). The proposed chemiluminescent enzyme assay may be used for assessing the metabolic activity of E. coli in oligotrophic environments and for early warning detection of low concentrations of E. coli in water for human consumption.
Starting Page	1947
Ending Page	1957
Page Count	11
File Format	PDF
ISSN	01757598
Journal	Applied Microbiology and Biotechnology
Volume Number	86
Issue Number	6
e-ISSN	14320614
Language	English
Publisher	Springer-Verlag
Publisher Date	2010-03-10
Publisher Place	Berlin, Heidelberg
Access Restriction	Subscribed
Subject Keyword	1,2-dioxetanes Drinking water Early warning β-d-galactosidase β-d-glucuronidase Indicator organisms Microbial Genetics and Genomics Microbiology Biotechnology
Content Type	Text
Resource Type	Article
Subject	Medicine Applied Microbiology and Biotechnology Biotechnology

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