Rapid FRET-based diagnosis of bacterial pathogens
Substrates for detecting microorganisms are provided, wherein the substrate comprises a set of molecular markers linked, optionally with linker molecules or moieties, to a di-, or tripeptide consisting of amino acids X1 and X2, or X1, X2 and X3, in which one of them, for example, X1, is a D-amino acid and the others, for example, X2 and X3, may be any D- or L-amino acid. The substrate preferably is used for the detection of Bacillus anthracis . Also provided are substrates for detecting Pseudomonas aeruginosa , wherein the substrate comprises a set of molecular markers linked, optionally with linker molecules or moieties to a tri-, tetra-, or pentapeptide consisting of glycine amino acids. The invention further comprises methods for detecting microorganisms, specifically B. anthracis and P. aeruginosa , with the substrates of the invention and use of the substrate(s) in such a method.
1. A substrate for detection of Bacillus anthracis , wherein the substrate comprises FITC-Ahx-X1-X2-X3-Lys-DABCYL, wherein X1 is L-Leu, D-Leu, or Gly; X2 is L-Leu, D-Leu, or D-Val; X3 is L-Leu, D-Leu, or absent, Lys stands for Lysine, Ahx stands for aminohexanoic acid, FITC is fluorescein-5-isothiocyanate, DABCYL is 4-((-4-dimethylamino)-phenyl)-azo-) benzoic acid, and one of the amino acids is a D-amino acid and the others are D- or L-amino acids.
2. A substrate for detection of Bacillus anthracis , wherein the substrate comprises AcNH-Cys(S-Ac)-X1-X2-X3-Cys(S-Ac)-OH, wherein X1 is L-Leu, D-Leu, or Gly; X2 is L-Leu, D-Leu, or D-Val; X3 is L-Leu, D-Leu, or absent, Cys(S-Ac) stands for thiol acetylated cysteine and AcNH for an acetylated amino terminus, and one of the amino acids is a D-amino acid and the others are D- or L-amino acids.
3. The substrate of claim 1 , wherein the substrate is selected from the group consisting of:
FITC-Ahx-Leu-DLeu-Lys-DABCYL,
FITC-Ahx-DLeu-Leu-Lys-DABCYL,
FITC-Ahx-DLeu-DLeu-Lys-DABCYL,
FITC-Ahx-Leu-DLeu-Leu-Lys-DABCYL,
FITC-Ahx-Leu-DVal-Lys-DABCYL,
and
FITC-Ahx-Gly-DLeu-Lys-DABCYL.
4. The substrate of claim 1 or claim 2 , wherein the L-amino acid or one of the two L-amino acids is Leu.
5. The substrate of claim 2 , wherein said substrate is:
AcNH-Cys(S-Ac)-X1-X2-X3-Cys(S-Ac)-OH,
wherein the L-amino acid or one of the two L-amino acids is Leu, wherein X3 may be absent, and wherein Cys(S-Ac) stands for thiol acetylated cysteine and AcNH for an acetylated amino terminus.
6. The substrate of claim 2 , wherein the substrate is selected from the group consisting of:
AcNH-Cys(S-Ac)-Leu-DLeu-Cys(S-Ac)-OH,
AcNH-Cys(S-Ac)-DLeu-Leu-Cys(S-Ac)-OH,
AcNH-Cys(S-Ac)-DLeu-DLeu-Cys(S-Ac)-OH,
AcNH-Cys(S-Ac)-Leu-DLeu-Leu-Cys(S-Ac)-OH,
AcNH-Cys(S-Ac)-Leu-DVal-Cys(S-Ac)-OH,
and
AcNH-Cys(S-Ac)-Gly-DLeu-Cys(S-Ac)-OH.
7. The substrate of claim 1 or claim 2 , wherein the D-amino acid is DLeu or DVal.
8. A method for the detection of Bacillus anthracis in a sample comprising adding the substrate of claim 1 or claim 2 to the sample and detecting a fluorescent signal or a shift in color.
9. The method of claim 8 wherein the fluorescent signal is located intracellularly in the microbial cells.
10. The method of claim 8 , wherein the sample is selected from a body fluid, powder, water, food, medium, or any other biological matrix.
11. A method for the identification of Bacillus anthracis PXO1+ in a sample comprising adding the substrate of claim 1 or claim 2 to the sample and detecting a fluorescent signal, a shift in color, a specific mass spectrum, or detection by capillary electrophoresis.