Fluorescence assay for microbial beta-lactamase
AssigneeWashington Research Foundation
InventorsKirk C. S. Chen, Florence J. Knapp, King K. Holmes
Life of this patent
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Abstract
A rapid method for the specific detection of the presence of Beta-lactamase from microbial sources is disclosed. The method utilizes a Beta-lactam ring containing substrate whose amide bond is hydrolyzed in the presence of Beta-Lactamase. A substrate, which includes a Beta-Lactam antibiotic with an acyl side chain containing an .alpha.-amino group and a .alpha.-phenyl group and/or its derivatives is first contacted with an organism thought to produce Beta-lactamase or a cell-free Beta-lactamase preparation, and, subsequently, it is determined whether the reaction product between the substrate and the organism or the preparation fluoresces.
Claims27
A rapid method of detecting the presence of Beta-lactamase from microbial sources using a Beta-lactam ring containing substrate whose amide bond is hydrolyzed in the presence of Beta-lactamase, comprising:
- (1) contacting a non-fluorescing substrate which comprising a Beta-lactam antibiotic with an acyl side chain containing an .alpha.-amino group and .alpha.-phenyl group or a derivative of an .alpha.-phenyl group, with either an organism thought to produce Beta-lactamase or a cell-free Beta-lactamase preparation;
- (2) incubating the substrate and the organism or preparation under conditions and for a period of time sufficient to effect hydrolysis of the substrate;
- and
- (3) determining whether the reaction product between the substrate and either the organism or the preparation fluoresces as a result of Beta-lactamase hydrolysis of said substrate.
A method of differentiating Beta-lactamase activity from acylase activity, comprising:
- (1) contacting a non-fluorescing Beta-lactam ring containing substrate whose amide bond is hydrolyzed in the presence of Beta-lactamase, the substrate comprising a Beta-lactam antibiotic with an acyl side chain containing an .alpha.-amino group and an .alpha.-phenyl group or a derivative of an .alpha.-phenyl group, with either an organism thought to produce Beta-lactamase or a cell-free Beta-lactamase preparation;
- (2) incubating the substrate and the organism or preparation under conditions and for a period of time sufficient to effect hydrolysis of the substrate;
- and
- (3) determining whether the reaction product between the substrate and either the organism or the preparation fluoresces, as a result of Beta-lactamase hydrolysis of said substrate, said fluorescence acting as an indicator of the presence of Beta-lactamase activity.
A method of semi-quantitatively determining the amount of Beta-lactamase activity from a microbial source, comprising:
- (1) contacting a non-fluorescing Beta-lactam ring containing substrate whose amide bond is hydrolyzed in the presence of Beta-lactamase, the substrate comprising a Beta-lactam antibiotic with an acyl side chain containing an .alpha.-amino group and an .alpha.-phenyl group or a derivative of an .alpha.-phenyl group, with either an organism thought to produce Beta-lactamase or a cell-free Beta-lactamase preparation;
- (2) incubating the substrate and the organism or preparation under conditions and for a period of time sufficient to effect hydrolysis of the substrate;
- and
- (3) determining the amount of Beta-lactamase activity as a measure of fluorescence intensity generated as a result of Beta-lactamase hydrolysis of said substrate.
A method of differentiating between the presence of penicillinase and cephalosporinase from microbial sources, comprising:
- (1) contacting a non-fluorescing penicillinase substrate with an acyl side chain containing an .alpha.-amino group and an .alpha.-phenyl group or a derivative of an .alpha.-phenyl group with either an organism thought to produce Beta-lactamase, or a Beta-lactamase preparation;
- (2) incubating the substrate and the organism or preparation in step 1 under conditions and for a period of time sufficient to effect hydrolysis of the substrate;
- (3) determining whether the reaction product between the penicillinase substrate and either the organism or the preparation fluoresces as a result of Beta-lactamase hydrolysis of said substrate;
- (4) contacting a non-fluorescing cephalosporinase substrate with an acyl side chain containing an .alpha.-amino group and an .alpha.-phenyl group or a derivative of an .alpha.-phenyl group with the same organism or preparation used in step 1;
- (5) incubating the substrate and the organism or preparation in step 4 under conditions and for a period of time sufficient to effect hydrolysis of the substrate;
- and
- (6) determining whether the reaction product between the cephalosporinase substrate and either the organism or the preparation fluoresces as a result of Beta-lactamase hydrolysis of said substrate.
A method of detecting the fluorescent end-product initiated by the presence of Beta-lactamase activity within a culture medium without removal of the bacteria from the milieu, comprising:
- impregnating an interface layer with a non-fluorescing Beta-lactam ring containing substrate with an acyl side chain containing an .alpha.-amino group and an .alpha.-phenyl group or a derivative of an .alpha.-phenyl group, whose amide bond is hydrolyzed in the presence of Beta-lactamase;
- placing the interface layer containing said substrate in contact with the bacteria on the culture medium;
- and
- determining whether the reaction product between the substrate and the bacteria fluoresces as a result of Beta-lactamase hydrolysis of said substrate.
Dependencies are inferred from the claim text.