초록 ▼

The present invention is directed to a method for separating, characterizing and/or identifying microorganisms in a test sample. The method of the invention comprises an optional lysis step for lysing non-microorganism cells that may be present in a test sample, followed by a subsequent separation s

The present invention is directed to a method for separating, characterizing and/or identifying microorganisms in a test sample. The method of the invention comprises an optional lysis step for lysing non-microorganism cells that may be present in a test sample, followed by a subsequent separation step. The method may be useful for the separation, characterization and/or identification of microorganisms from complex samples such as blood-containing culture media. The method may also be useful for the physical separation and/or enrichment of two or more different or individual microorganism species contained in a mixed test sample. The invention further provides for spectroscopic interrogation of the separated microorganism sample(s) to produce measurements of the microorganism and characterizing and/or identifying the microorganism(s) in the sample using said spectroscopic measurements.

대표청구항 ▼

1. A method of identifying two or more different and unknown microorganism species from a test sample, comprising: (a) obtaining a test sample known to contain or that may contain two or more different, unknown microorganism species;(b) optionally selectively lysing non-microorganism cells in said t

1. A method of identifying two or more different and unknown microorganism species from a test sample, comprising: (a) obtaining a test sample known to contain or that may contain two or more different, unknown microorganism species;(b) optionally selectively lysing non-microorganism cells in said test sample to produce a lysed sample;(c) layering said test or lysed sample on a density cushion in a container wherein said density cushion has a homogenous density of from about 1.025 to about 1.22 g/ml;(d) centrifuging the container to separate said two or more microorganism species from other components of said sample, said microorganism species passing through said density cushion to form a mixed pellet of said two or more microorganism species at the bottom of said container, wherein said two or more microorganism species form two or more enriched layers within said mixed pellet;(e) spectroscopically interrogating each of said enriched layers of said two or more microorganism species in said mixed pellet in situ to produce excitation-emission matrix (EEM) of said two or more unknown microorganism species, wherein said spectroscopic interrogation comprises intrinsic fluorescence, and wherein said intrinsic fluorescence is measured in front face mode; and(f) identifying each of said two or more unknown microorganism species in said enriched layers of said mixed pellet by comparison of the spectroscopic measurements with spectroscopic measurements taken, or spectroscopic properties predicted, of known microorganisms, wherein said two or more unknown microorganisms are identified to the genus level, species level, and/or strain level. 2. The method of claim 1, wherein said spectroscopic interrogation of said two or more enriched layers of microorganisms is carried out through an optical window located on the side of said separation container. 3. The method of claim 1, wherein steps (d) and (e) are carried out in a sealed container and wherein said interrogation step (e) is non-invasive. 4. The method of claim 1, wherein said spectroscopic interrogation further comprise Raman spectroscopy. 5. The method of claim 1, wherein said spectroscopic interrogation further comprises fluorescence spectroscopy, diffuse reflectance spectroscopy, infrared spectroscopy, terahertz spectroscopy, Raman spectroscopy, Surface Enhanced Raman Spectroscopy (SERS), Spatially Offset Raman spectroscopy (SORS), resonance Raman spectroscopy or any combination thereof. 6. The method of claim 1, wherein said EEM is compared to a database of EEMs of known microorganisms. 7. The method of claim 1, wherein said identification further comprises characterization of said microorganisms based on one or more phenotypic and/or morphologic characteristics. 8. The method of claim 1, wherein said identification further comprises characterization of said microorganisms based on one or more measurements of detection time, growth rate and microorganism pellet size, shape, color and/or density. 9. The method of claim 1, wherein said identification further comprises characterization of said microorganisms into on one or more classification models selected from the group consisting of Gram Groups, Clinical Gram Groups, Therapeutic Groups, Functional Groups, and Natural Intrinsic Fluorescence Groups. 10. The method of claim 1, wherein said selective lysis step (b) is performed by sonication, osmotic shock, chemical treatment, or a combination thereof. 11. The method of claim 1, wherein said selective lysis step (b) is performed using a lysis solution comprising one or more detergents. 12. The method of claim 11, wherein said one or more detergents is selected from the group consisting of octylphenol ethoxylate, NP-40, polyoxyethylene detergent, (Octylphenoxy)polyethoxyethanol, polyoxyethylene 10 oleoyl ether, CHAPS, octyl β-D-glucopyranoside, saponin, nonaethylene glycol monododecyl ether, sodium dodecyl sulfate, N-laurylsarcosine, sodium deoxycholate, bile salts, hexadecyltrimethylammonium bromide, SB3-10, SB3-12, amidosulfobetaine-14, C7BzO, polyoxyethylene (20) oleyl ether, polyoxyethylene 20 cetyl ether, polyoxyethylene (23) lauryl ether, polyoxyethylenesorbitan monooleate, polyoxyethylene sorbitol ester, polyoxyalkylene ether, non-detergent sulfobetaines, amphipols, and methyl-β-cyclodextrin. 13. The method of claim 11, wherein said detergent is a polyoxyethylene detergent comprising the structure C12-18/E9-10. 14. The method of claim 13, wherein said polyoxyethylene detergent is selected from the group consisting of polyoxyethylene 10 oleoyl ether polyoxyethylene detergent, and polidocenol. 15. The method of claim 11, wherein said lysis solution further comprises an enzyme composition comprising one or more proteinases and one or more nucleases. 16. The method of claim 11, wherein said lysis solution comprises one or more buffering agents. 17. The method of claim 1, wherein said density cushion comprises one or more of colloidal silica, iodinated contrast agents, sucrose, microscope immersion oil, mineral oil, silicone oil, fluorosilicone oil, silicone gel, diatrizoate-dextran, carboxymethyl cellulose, hydroxypropylmethyl cellulose, polyethylene oxide (high molecular weight), polyoxyalkylene ether, polyacrylic acid, cross-linked polyvinyl alcohol, cross-linked polyvinyl pyrrolidine, PEG methyl ether methacrylate, pectin, agarose, xanthan, gellan, Gellan Gum, sorbitol, a sucrose and epichlorohydrin copolymer, glycerol, dextran, glycogen, cesium chloride, perfluorocarbon fluids, and/or hydrofluorocarbon fluid. 18. The method of claim 1, wherein said test sample is a culture sample known to contain two or more microorganism species. 19. The method of claim 1, wherein visual determination of the presence of a mixed culture and/or of separate or distinct layers of a mixed culture, are aided with the use one or more selective dyes. 20. A method of identifying two or more different and unknown microorganism species from a blood culture, comprising: (a) obtaining a sample from a blood culture known to contain or that may contain two or more different, unknown microorganism species;(b) selectively lysing non-microorganism cells in said test sample to produce a lysed sample;(c) layering said lysed sample on a density cushion in a container wherein said density cushion comprises cesium chloride, iodinated contrast agents or colloidal silica;(d) centrifuging the container to separate said two or more microorganism species from other components of said sample, said microorganism species passing through said density cushion to form a mixed pellet of said two or more microorganism species at the bottom of said container, wherein said two or more microorganism species form two or more enriched layers within said pellet;(e) spectroscopically interrogating each of said enriched layers of said two or more microorganism species in said mixed pellet in situ to produce excitation-emission matrix (EEM) of said two or more microorganism species, wherein said spectroscopic interrogation comprises intrinsic fluorescence, and wherein said intrinsic fluorescence is measured in front face mode; and(f) identifying each of said two or more unknown microorganism species in said enriched layers of said mixed pellet by comparison of the spectroscopic measurements with spectroscopic measurements taken, or spectroscopic properties predicted, of known microorganisms, wherein said two or more unknown microorganisms are identified to the genus level, species level, and/or strain level.