Method for genetic identification of unknown organisms

• US 20100055677A1
• Filed: 06/15/2007
• Published: 03/04/2010
• Est. Priority Date: 01/04/2007
• Status: Active Grant

• Current Assignee: Lawrence Livermore National Security LLC (Government of the United States of America)
• Original Assignee: Regents of the University of California (University of California)
• Inventors: Fitch, Joseph P., Carter, J. Chance, Hindson, Benjamin J., Colston, Billy W. JR.
• Granted Patent: US 9,422,586 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: B01L 2200/0673 Handling of plugs of fluid surrounded by immiscible fluid
• CPC Class Codes: B01L 2200/10 Integrating sample preparation and analysis in single entity, e.g. lab-on-a-chip concept
• CPC Class Codes: B01L 2200/12 Specific details about manufacturing devices
• CPC Class Codes: B01L 2300/0816 Cards, e.g. flat sample carriers usually with flow in two horizontal directions
• CPC Class Codes: B01L 2300/0864 comprising only one inlet and multiple receiving wells, e.g. for separation, splitting
• CPC Class Codes: B01L 2300/0867 Multiple inlets and one sample wells, e.g. mixing, dilution
• CPC Class Codes: B01L 2300/087 Multiple sequential chambers
• CPC Class Codes: B01L 2300/0887 Laminated structure
• CPC Class Codes: B01L 2300/1822 using Peltier elements
• CPC Class Codes: B01L 2300/1827 using resistive heater
• CPC Class Codes: B01L 2300/1861 using radiation
• CPC Class Codes: B01L 2400/0421 electrophoretic flow
• CPC Class Codes: B01L 2400/0487 fluid pressure, pneumatics
• CPC Class Codes: B01L 3/502753 characterised by bulk separation arrangements on lab-on-a-chip devices, e.g. for filtration or centrifugation separation in general B01D; microapparatus for analysis using electrophoresis G01N27/44791; sample preparation G01N1/28
• CPC Class Codes: B01L 3/502761 specially adapted for handling suspended solids or molecules independently from the bulk fluid flow, e.g. for trapping or sorting beads, for physically stretching molecules investigating characteristics of particles G01N15/00
• CPC Class Codes: B01L 3/502784 specially adapted for droplet or plug flow, e.g. digital microfluidics automatic analysis using a stream of discrete samples in a tube system G01N35/08
• CPC Class Codes: B01L 7/52 with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples amplification or hybridisation processes per se C12Q1/68; controlling sequential reactions for synthesis B01J19/0046
• CPC Class Codes: B01L 7/525 with physical movement of samples between temperature zones
• CPC Class Codes: C12Q 1/04 Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor C12Q1/6897 takes precedence
• CPC Class Codes: C12Q 1/6888 for detection or identification of organisms
• CPC Class Codes: G01N 33/5432 : Liposomes or microcapsules
• CPC Class Codes: G01N 33/569 : for microorganisms, e.g. protozoa, bacteria, viruses
• CPC Class Codes: G01N 33/587 : Nanoparticles

Abstract

A method of rapid, genome and proteome based identification of unknown pathogenic or non-pathogenic organisms in a complex sample. The entire sample is analyzed by creating millions of emulsion encapsulated microdroplets, each containing a single pathogenic or non-pathogenic organism sized particle and appropriate reagents for amplification. Following amplification, the amplified product is analyzed.

Claims

1. A method of identifying pathogenic and non-pathogenic organisms in a sample, comprising:

preparing the sample,

isolating the organisms from the sample into microdroplets,

amplifying the organisms,

sorting said microdroplets,

analyzing said microdroplets, and

classifying and identifying the organisms.

2. The method of identifying pathogenic and non-pathogenic organisms in a sample of claim 1 wherein said step of preparing the sample comprises chemical treatment, dilution, buffer exchange, separation, cytometry, filtration, or concentration or any combination of chemical treatment, dilution, buffer exchange, separation, cytometry, filtration, or concentration.

3. The method of identifying pathogenic and non-pathogenic organisms in a sample of claim 1 wherein said step of isolating the organisms from the sample into microdroplets comprises creating sub-nanoliter volume reactor droplets containing the organisms.

4. The method of identifying pathogenic and non-pathogenic organisms in a sample of claim 1 wherein said step of isolating the organisms from the sample into microdroplets includes genomic analysis, proteomic analysis, or physical analysis or any combination of genomic analysis, proteomic analysis, or physical analysis.

5. The method of identifying pathogenic and non-pathogenic organisms in a sample of claim 1 wherein said step of isolating the organisms from the sample into microdroplets comprises forcing the sample through an orifice.

6. The method of identifying pathogenic and non-pathogenic organisms in a sample of claim 5 wherein said step of isolating the organisms from the sample into microdroplets comprises forcing the sample through an orifice and merging it with an immiscible fluid.

7. The method of identifying pathogenic and non-pathogenic organisms in a sample of claim 5 wherein said step of forcing the sample through an orifice comprises a microfluidic, microjet, or inkjet, pin system of forcing the sample through an orifice.

8. The method of identifying pathogenic and non-pathogenic organisms in a sample of claim 1 wherein said step of isolating the organisms from the sample into microdroplets includes incorporating unique identifiers into said microdroplets.

9. The method of identifying pathogenic and non-pathogenic organisms in a sample of claim 1 wherein said step of isolating the organisms from the sample into microdroplets includes incorporating barcodes into said microdroplets.

10. The method of identifying pathogenic and non-pathogenic organisms in a sample of claim 1 wherein said step of isolating the organisms from the sample into microdroplets includes forming a protective coating around said microdroplets.

11. The method of identifying pathogenic and non-pathogenic organisms in a sample of claim 1 wherein said step of isolating the organisms from the sample into microdroplets includes forming a protective coating around said microdroplets by in-situ polymerization.

12. The method of identifying pathogenic and non-pathogenic organisms in a sample of claim 1 wherein said step of isolating the organisms from the sample into microdroplets includes lysis.

13. The method of identifying pathogenic and non-pathogenic organisms in a sample of claim 1 wherein said step of isolating the organisms from the sample into microdroplets includes lysis, isolating a single organism from the organisms, creating a microdroplet from said microdroplets, and encapsulating said single organism in said microdroplet.

14. The method of identifying pathogenic and non-pathogenic organisms in a sample of claim 1 wherein said step of sorting said microdroplets comprises optical sorting, hydrodynamic sorting, or magnetic sorting or any combination of optical sorting, hydrodynamic sorting, or magnetic sorting.

15. The method of identifying pathogenic and non-pathogenic organisms in a sample of claim 1 wherein said step of sorting said microdroplets comprises measuring the spectroscopic properties of said microdroplets.

16. The method of identifying pathogenic and non-pathogenic organisms in a sample of claim 1 wherein said step of sorting said microdroplets comprises incorporation of inert or labeled paramagnetic nano-particles to said microdroplets and application of a magnetic field to said microdroplets.

17. The method of identifying pathogenic and non-pathogenic organisms in a sample of claim 1 wherein said step of sorting said microdroplets comprises cytometry sorting, diffusion sorting, or focusing sorting or any combination of cytometry sorting, diffusion sorting, or focusing sorting.

18. The method of identifying pathogenic and non-pathogenic organisms in a sample of claim 1 wherein said step of analyzing said microdroplets comprises separation, sequencing, mass spectrometry, spectroscopic, fluorescence, Raman, NMR, X-ray crystallography, SEM, AFM, ligand binding, or electrochemical analysis or any combination of separation, sequencing, mass spectrometry, spectroscopic, fluorescence, Raman, NMR, X-ray crystallography, SEM, AFM, ligand binding, or electrochemical analysis.

19. The method of identifying pathogenic and non-pathogenic organisms in a sample of claim 1 wherein said step of classifying and identifying the organisms comprises performing informatics analysis to generate genomic and proteomic profiles for the organisms in the sample.

20. The method of identifying pathogenic and non-pathogenic organisms in a sample of claim 1 wherein said step of classifying and identifying the organisms includes physical analysis.

21. The method of identifying pathogenic and non-pathogenic organisms in a sample of claim 1 wherein said step of classifying and identifying the organisms includes size, shape, pH, mobility, charge, viscosity, refractive index, or kinetics analysis or any combination of size, shape, pH, mobility, charge, viscosity, refractive index, or kinetics analysis.

34. The method of identifying organisms in a sample of claim 5 wherein said step of isolating the organisms from the sample into microdroplets comprises forcing the sample through an orifice and merging it with an immiscible fluid.

35. The method of identifying organisms in a sample of claim 5 wherein said step of forcing the sample through an orifice comprises a microfluidic, microjet, or inkjet, pin system of forcing the sample through an orifice.

22. An apparatus for identifying pathogenic and non-pathogenic organisms in a sample, comprising:

a sample preparation system for preparing the sample,

an isolating system for isolating the organisms from the sample into microdroplets,

an amplifier for amplifying the organisms,

a sorter for sorting said microdroplets,

an analyzer for analyzing said microdroplets, and

a system for classifying and identifying the organisms.

23. The apparatus for identifying pathogenic and non-pathogenic organisms in a sample of claim 22 wherein said sample preparation system for preparing the sample comprises a chemical treatment system, a dilution system, a buffer exchange system, a separation system, a cytometry system, a filtration system, or a concentration system or any combination of said systems.

24. The apparatus for identifying pathogenic and non-pathogenic organisms in a sample of claim 22 wherein said sorter comprises an optical sorter, a hydrodynamic sorter, or a magnetic sorter or any combination of an optical sorter, a hydrodynamic sorter, or a magnetic sorter.

25. The apparatus for identifying pathogenic and non-pathogenic organisms in a sample of claim 22 wherein said system for classifying and identifying the organisms comprises a separator, a sequencer, a mass spectrometer, a spectroscope, a fluorescence system, a Raman system, a NMR system, an X-ray crystallograph, a SEM system, an AFM system, a ligand binding system, or electrochemical analyzer or any combination thereof.

26. The apparatus for identifying pathogenic and non-pathogenic organisms in a sample of claim 22 wherein said analyzer for analyzing said microdroplets is a parallel analyzer.

27. A method of identifying organisms in a sample, wherein said organisms may be pathogenic organisms or non-pathogenic organisms, comprising the steps of:

preparing the sample containing the organisms,

isolating the organisms from the sample into microdroplets,

amplifying the organisms,

sorting said microdroplets,

analyzing said microdroplets, and

classifying and identifying the organisms.

28. The method of identifying organisms in a sample of claim 27 wherein said steps of preparing the sample, isolating the organisms from the sample into microdroplets, amplifying the organisms, sorting said microdroplets, analyzing said microdroplets, and classifying and identifying the organisms are performed in series.

29. The method of identifying organisms in a sample of claim 27 wherein said step of analyzing said microdroplets is performed by a parallel analyzer.

30. The method of identifying organisms in a sample of claim 27 wherein said step of preparing the sample comprises chemical treatment, dilution, buffer exchange, separation, cytometry, filtration, or concentration or any combination of chemical treatment, dilution, buffer exchange, separation, cytometry, filtration, or concentration.

31. The method of identifying organisms in a sample of claim 27 wherein said step of isolating the organisms from the sample into microdroplets comprises creating sub-nanoliter volume reactor droplets containing the organisms.

32. The method of identifying organisms in a sample of claim 27 wherein said step of isolating the organisms from the sample into microdroplets includes genomic analysis, proteomic analysis, or physical analysis or any combination of genomic analysis, proteomic analysis, or physical analysis.

33. The method of identifying organisms in a sample of claim 27 wherein said step of isolating the organisms from the sample into microdroplets comprises forcing the sample through an orifice.

36. The method of identifying organisms in a sample of claim 27 wherein said step of isolating the organisms from the sample into microdroplets includes incorporating unique identifiers into said microdroplets.

37. The method of identifying organisms in a sample of claim 27 wherein said step of isolating the organisms from the sample into microdroplets includes incorporating barcodes into said microdroplets.

38. The method of identifying organisms in a sample of claim 27 wherein said step of isolating the organisms from the sample into microdroplets includes forming a protective coating around said microdroplets.

39. The method of identifying organisms in a sample of claim 27 wherein said step of isolating the organisms from the sample into microdroplets includes forming a protective coating around said microdroplets by in-situ polymerization.

40. The method of identifying organisms in a sample of claim 27 wherein said step of isolating the organisms from the sample into microdroplets includes lysis.

41. The method of identifying organisms in a sample of claim 27 wherein said step of isolating the organisms from the sample into microdroplets includes lysis, isolating a single organism from the organisms, creating a microdroplet from said microdroplets, and encapsulating said single organism in said microdroplet.

42. The method of identifying organisms in a sample of claim 27 wherein said step of sorting said microdroplets comprises optical sorting, hydrodynamic sorting, or magnetic sorting or any combination of optical sorting, hydrodynamic sorting, or magnetic sorting.

43. The method of identifying organisms in a sample of claim 27 wherein said step of sorting said microdroplets comprises measuring the spectroscopic properties of said microdroplets.

44. The method of identifying organisms in a sample of claim 27 wherein said step of sorting said microdroplets comprises incorporation of inert or labeled paramagnetic nano-particles to said microdroplets and application of a magnetic field to said microdroplets.

45. The method of identifying organisms in a sample of claim 27 wherein said step of sorting said microdroplets comprises cytometry sorting, diffusion sorting, or focusing sorting or any combination of cytometry sorting, diffusion sorting, or focusing sorting.

46. The method of identifying organisms in a sample of claim 27 wherein said step of analyzing said microdroplets comprises separation, sequencing, mass spectrometry, spectroscopic, fluorescence, Raman, NMR, X-ray crystallography, SEM, AFM, ligand binding, or electrochemical analysis or any combination of separation, sequencing, mass spectrometry, spectroscopic, fluorescence, Raman, NMR, X-ray crystallography, SEM, AFM, ligand binding, or electrochemical analysis.

47. The method of identifying organisms in a sample of claim 27 wherein said step of classifying and identifying the organisms comprises performing informatics analysis to generate genomic and proteomic profiles for the organisms in the sample.

48. The method of identifying organisms in a sample of claim 27 wherein said step of classifying and identifying the organisms includes physical analysis.

49. The method of identifying organisms in a sample of claim 27 wherein said step of classifying and identifying the organisms includes size, shape, pH, mobility, charge, viscosity, refractive index, or kinetics analysis or any combination of size, shape, pH, mobility, charge, viscosity, refractive index, or kinetics analysis.