Method for detecting bacteria according to the gram signal thereof in a complex sample
AssigneesMaat Pharma, Bioaster
InventorsAurore Duquenoy, Samuel Bellais, Vincent Thomas
Life of this patent
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Abstract
The invention relates to a method for detecting the proportion of Gram-positive and Gram-negative bacteria in a complex sample. The bacteria are detected according to the Gram signal thereof. The invention enables distinction between species of Gram-positive bacteria and species of Gram-negative bacteria in a complex sample. The invention also relates to a kit for marking Gram-positive and Gram-negative bacteria, particularly for use in flow cytometry, particularly for complex samples such as human or animal microbiota samples.
Claims17
A method for detecting, in a sample containing bacteria, the proportion of Gram-positive bacteria relative to the proportion of Gram-negative bacteria, comprising the steps of:
- a) incubating the sample, simultaneously or sequentially, with:
- wheat lectin (WGA) coupled to a first fluorochrome,
- vancomycin coupled to a second fluorochrome emitting a fluorescence at a different wavelength from the first fluorochrome coupled to WGA,
- optionally vancomycin that is not coupled to a fluorochrome,
- optionally potassium chloride,
- b) centrifuging the sample resulting from step a),
- c) optionally washing the sample resulting from step b), and
- d) measuring the fluorescence of the first and second fluorochromes of the sample resulting from step b) or c) using flow cytometry at different wavelengths to determine the proportion of Gram-positive bacteria relative to the proportion of Gram-negative bacteria in the sample.
A method for detecting, optionally distinguishing and identifying families, genera and/or species of Gram-positive bacteria and Gram-negative bacteria in a bacterial sample, comprising the steps of:
- a) incubating the sample, simultaneously or sequentially, with:
- wheat lectin (WGA) coupled to a first fluorochrome,
- vancomycin coupled to a second fluorochrome emitting a fluorescence at a different wavelength from the first fluorochrome coupled to WGA,
- optionally vancomycin,
- optionally potassium chloride,
- b) centrifuging the sample,
- c) optionally washing the sample,
- d) measuring the fluorescence of the sample using flow cytometry,
- e) analyzing the relative fluorescences of the first and second fluorochromes, and
- f) optionally distinguishing and identifying families, genera and/or species of Gram-positive and Gram-negative bacteria by comparing the relative fluorescences of the first and second fluorochromes at different wavelengths.
A kit for detecting the proportion of Gram-positive bacteria and the proportion of Gram-negative bacteria in a bacterial sample by flow cytometry, comprising:
- i. wheat lectin (WGA) coupled to a first fluorochrome,
- ii. vancomycin coupled to a second fluorochrome emitting a fluorescence at a different wavelength from the first fluorochrome coupled to WGA, and
- iii. optionally vancomycin,
- iv. optionally potassium chloride.
Dependencies are inferred from the claim text.