Methods and apparatus for pathogen detection, identification and/or quantification
First Claim
1. A method comprising:
- a) obtaining at least one sample suspected of containing one or more target cells or pathogens;
b) using the target cell or pathogen to generate pyrophosphate (PPi) by an enzyme-catalyzed reaction;
c) using the PPi to produce light by a luciferase dependent process;
d) accumulating the number of photons produced over a time interval; and
e) using the accumulated photons to detect the target cells or pathogens.
1 Assignment
0 Petitions
Accused Products
Abstract
The present invention concerns methods, compositions and apparatus for detecting, identifying and/or quantifying target cells or pathogens. In certain embodiments of the invention, the cells or pathogens may be detected by detection of a specific nucleic acid. In other embodiments, the cells or pathogens may be detected by use of an aptamer or a tagged protein that binds to the cells or pathogens. Alternatively, the cells or pathogens may be immobilized on a solid surface and endogenous ATP and/or PPi detected. In preferred embodiments of the invention, the ATP and/or PPi are detected by a process utilizing luciferase mediated bioluminescence, such as BRC. In other preferred embodiments, thermostable enzymes may be used in either isothermal or cyclic thermal reactions to generate PPi. Apparatus and compositions for cell or pathogen analysis are also disclosed.
206 Citations
48 Claims
-
1. A method comprising:
-
a) obtaining at least one sample suspected of containing one or more target cells or pathogens;
b) using the target cell or pathogen to generate pyrophosphate (PPi) by an enzyme-catalyzed reaction;
c) using the PPi to produce light by a luciferase dependent process;
d) accumulating the number of photons produced over a time interval; and
e) using the accumulated photons to detect the target cells or pathogens. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24)
-
-
25. A method comprising:
-
a) obtaining at least one sample suspected of containing one or more target cells or pathogens;
b) binding the target cells or pathogens to a solid surface;
c) removing unbound cells or pathogens;
d) lysing the bound cells or pathogens; and
e) detecting endogenous ATP and/or PPi from the lysed cells or pathogens by a luciferase dependent process. - View Dependent Claims (26, 27, 28, 29, 30, 31, 32, 33)
-
-
34. A method for cell or pathogen detection comprising:
-
a) generating pyrophosphate in a cell or pathogen dependent process;
b) using thermostable ATP sulfurylase and luciferase to produce light from the pyrophosphate; and
c) measuring the light output to detect the cell or pathogen. - View Dependent Claims (35, 36, 37, 38, 39, 40, 41)
-
-
42. A method comprising:
-
a) obtaining at least one sample suspected of containing one or more pathogen nucleic acids;
b) adding labeled nucleotides to the one or more pathogen nucleic acids with a thermostable terminal transferase; and
c) detecting the labeled nucleic acids. - View Dependent Claims (43)
-
-
44. A method of cell or pathogen detection comprising:
-
a) attaching a target cell or pathogen to a substrate;
b) binding a first binding moiety to the target cell or pathogen;
c) binding a second binding moiety to the first binding moiety, wherein the second binding moiety is attached to a dextran or dendromer molecule labeled with oligonucleotides;
d) generating pyrophosphate by terminal transferase mediated addition of nucleotides to the oligonucleotides; and
e) detecting the pyrophosphate. - View Dependent Claims (45)
-
-
46. A system comprising:
-
a) one or more reaction chambers;
b) a microfluidic system;
c) one or more photodectectors d) a thermostable luciferase; and
e) a thermostable ATP sulfurylase. - View Dependent Claims (47, 48)
-
Specification