A rapid and sensitive fluorimetric β-galactosidase assay for coliform detection using chlorophenol red-β-D-galactopyranoside.

Clémence Sicard, Norman Shek, Dawn White, Raymond J Bowers, R Stephen Brown, John D Brennan

Analytical and bioanalytical chemistry 2014 Sep

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We report on a new fluorimetric assay for β-galactosidase (β-gal) and faecal coliform bacteria that utilizes a long-wavelength dye, chlorophenol red-β-D-galactopyranoside (CPRG), that has been widely used for colorimetric assays. The novel feature of this new assay is the unexpected development of a large fluorescence response from liberated chorophenol red (CPR) upon complexation with poly-L-arginine (pR) in solution. The binding of CPR to pR occurs through the sulphonate group of CPR, causing formation of a charge-transfer complex and up to a 70-fold increase in emission intensity. A major advantage of the assay is the ability to utilize excitation and emission wavelengths in the red end of the spectrum, which avoids common interferences obtained when using UV-absorbing dyes such as 4-methylumbelliferyl-β-D-galactopyranoside. We provide data on the utility of CPRG as a fluorimetric reporter for both β-gal and Escherichia coli ATCC 25922 and demonstrate optimized reaction conditions for rapid and sensitive detection of E. coli at a level of 1 colony-forming unit (cfu)/10 mL after 12 h of culture followed by a 1-h assay, which is below the regulatory limit for testing of recreational water.

Citation

Clémence Sicard, Norman Shek, Dawn White, Raymond J Bowers, R Stephen Brown, John D Brennan. A rapid and sensitive fluorimetric β-galactosidase assay for coliform detection using chlorophenol red-β-D-galactopyranoside. Analytical and bioanalytical chemistry. 2014 Sep;406(22):5395-403