Author(s): Coburn JT, Lytle FE, Huber DM
A rapid, laser-based method of pathogen identification has been developed. The method relies on the extent of amino-peptidase hydrolysis of a series of nonfluorescent L-amino acid β-naphthylamides to produce the highly fluorescent β-naphthylamlne by the pathogen of interest. The luminescence background was composed of Raman and Rayleigh scatter, fluorescent impurities in the buffer, β-naphthylamine fluorescence due to substrate decomposition, and emission of the biological matrix. The blank levels have been systematically examined and reduced to levels which allow the measurement of the fluorophore in the 0.1 nM range. Thus, unambiguous identification of pathogens at the 50 000 cell/mL level has been achieved. This corresponds to 2 to 3 orders of magnitude fewer cells than has been achieved by other techniques. Identification of pathogens at this level will drastically reduce the cell growth period from 48 h to 6 h and afford a more rapid turnaround time for bacterial identification.