Rapid colorimetric diagnostic test for C. difficile
Inventors
Rammos, Danae • Baskaran, Madhumita • Dorbala, Pranav • Wong, Allison Julie
Assignees
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Publication Number
US-11597961-B2
Publication Date
2023-03-07
Expiration Date
Abstract
In certain aspects, provided herein are methods and kits for detecting the presence of C. difficile in a biological sample. Also provided herein are methods of selecting of identifying a subject that would benefit from C. difficile treatment.
Core Innovation
The invention provides a point-of-care paper-based colorimetric diagnostic for detecting Clostridioides difficile in fecal samples by measuring glutamate dehydrogenase activity. A filter-paper medium is loaded with glutamate and an oxidizing agent selected from nicotinamide-adenine dinucleotide and nicotinamide-adenine dinucleotide phosphate, and the filter-paper medium includes a window configured for direct application of the fecal sample.
After the fecal sample is applied, a solution comprising an electron transfer agent and a tetrazolium dye precursor is applied to the filter-paper medium to generate a visible color change. The colorimetric detection is based on the activity of glutamate dehydrogenase enzyme of C. difficile present in the fecal sample. The chemistry supports visible color darkening as a readout associated with glutamate dehydrogenase activity.
The invention further includes kits with the paper medium and the solution components arranged for detection of C. difficile colorimetrically based on glutamate dehydrogenase activity. The kits include sample application features such as windows on the paper medium and include positive and negative control windows as described. The document also describes use for diagnosis, treatment selection, and post-treatment evaluation, with a rapid turnaround after developer addition and prototype testing showing color response across glutamate dehydrogenase concentrations.
Claims Coverage
The document provides two independent claims covering a method and a kit for detecting C. difficile in a fecal sample colorimetrically based on glutamate dehydrogenase activity. Across the independent claims and their dependent refinements, the inventive features include specific paper-medium loading chemistry, a defined two-step reagent application, and an integrated fecal-sample application window; additional dependent features specify particular reagent identities and control/window configurations.
Filter paper medium loaded with glutamate and an oxidizing agent
The method includes applying a fecal sample to a filter paper medium loaded with glutamate and an oxidizing agent selected from nicotinamide-adenine dinucleotide and nicotinamide-adenine dinucleotide phosphate, wherein the filter paper medium comprises a window for direct application of the fecal sample.
Electron transfer agent and tetrazolium dye precursor on the filter paper medium
A solution comprising an electron transfer agent and a tetrazolium dye precursor is applied to the filter paper medium.
Colorimetric detection based on glutamate dehydrogenase activity of C. difficile
C. difficile in the fecal sample is detected colorimetrically based on the activity of glutamate dehydrogenase enzyme of the C. difficile present in the fecal sample.
Kit with glutamate-loaded filter paper medium and oxidizing agent
The kit comprises a filter paper medium loaded with glutamate and an oxidizing agent selected from nicotinamide-adenine dinucleotide and nicotinamide-adenine dinucleotide phosphate, wherein the filter paper medium comprises a window for direct application of a fecal sample.
Kit solution with electron transfer agent and tetrazolium dye precursor
The kit comprises a solution comprising an electron transfer agent and a tetrazolium dye precursor.
Kit configured for colorimetric detection based on glutamate dehydrogenase activity
The kit is configured to detect the presence of C. difficile in the fecal sample colorimetrically based on the activity of glutamate dehydrogenase enzyme of the C. difficile present in the fecal sample.
Overall, the claims cover a glutamate- and oxidizing-agent-loaded filter-paper format with a fecal-sample window, followed by application of an electron transfer agent and tetrazolium dye precursor to enable colorimetric detection based on C. difficile glutamate dehydrogenase activity, implemented as either a method or a kit. Dependent claim refinements specify particular reagent identities, glutamate dehydrogenase content constraints in fecal samples, restriction to C. difficile ribotype O27, and paper layout features such as positive and negative control windows.
Stated Advantages
Rapid turnaround as described in the document.
Detectable visible color change as a colorimetric readout based on glutamate dehydrogenase activity.
Detection capability across a range of glutamate dehydrogenase concentrations as described in prototype testing.
Provides positive and negative control windows for the paper medium as described.
Supports diagnosis, treatment selection, and post-treatment evaluation as described in the document.
Documented Applications
Diagnosis of C. difficile infection using the described point-of-care paper-based colorimetric detection.
Treatment selection based on the detected C. difficile result as described in the document.
Post-treatment evaluation using the described detection approach as described in the document.
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