Analyzing bacteria without culturing

• US 9,671,395 B2
• Filed: 04/21/2011
• Issued: 06/06/2017
• Est. Priority Date: 04/21/2010
• Status: Active Grant

• Current Assignee: DNAE Group Holdings Limited (Genting Bhd.)
• Original Assignee: DNAE Group Holdings Limited (Genting Bhd.)
• Inventors: Clarizia, Lisa-Jo Ann, Adams, Eddie W., Esch, Victor C., Dryga, Sergey A.
• Primary Examiner(s): Hines, Ja'Na
• Application Number:
  US13/091,548
• Publication Number:
  US 20110262927A1
• Time in Patent Office: 2,238 Days
• CPC Class Codes: B01L 2200/0647 Handling flowable solids, e.g. microscopic beads, cells, particles
• CPC Class Codes: B01L 2400/043 magnetic forces
• CPC Class Codes: C07K 1/22 Affinity chromatography or related techniques based upon selective absorption processes
• CPC Class Codes: C07K 16/1267 from Gram-positive bacteria
• CPC Class Codes: C12N 13/00 Treatment of microorganisms or enzymes with electrical or wave energy, e.g. magnetism, sonic waves
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/689 for bacteria
• CPC Class Codes: C12Q 2563/143 Magnetism, e.g. magnetic label
• CPC Class Codes: C12Q 2565/629 being a microfluidic device
• CPC Class Codes: G01N 2333/195 from bacteria
• CPC Class Codes: G01N 2446/20 the magnetic material being present in the particle core
• CPC Class Codes: G01N 33/54326 Magnetic particles
• CPC Class Codes: G01N 33/54333 Modification of conditions of immunological binding reaction, e.g. use of more than one type of particle, use of chemical agents to improve binding, choice of incubation time or application of magnetic field during binding reaction
• CPC Class Codes: G01N 33/56911 Bacteria
• CPC Class Codes: Y10T 428/2982 Particulate matter [e.g., sphere, flake, etc.]

Abstract

The invention generally relates to conducting an assay on a sample that isolates a pathogen from the sample and allows for analysis of the pathogen with minimal (i.e., at most 24 hrs of culturing) or no culturing of the pathogen. In certain embodiments, the invention provides methods for identifying a pathogen from a sample that involve obtaining a sample including a pathogen, conducting an assay that isolates the pathogen from the sample, culturing the isolated pathogen for at most about 24 hrs, and analyzing the pathogen.

Claims

1. A method for analyzing a bacterium from a sample, the method comprising:

providing a vessel that contains a body fluid sample comprising a bacterium;

introducing to the sample, magnetic particles comprising a bacterium-specific binding moiety and a buffer that promotes binding of the particles to the bacterium in order to create a mixture;

incubating the mixture in the vessel to allow the particles to bind to the bacterium in the sample;

flowing the mixture from the vessel through a channel in a fluidic device and toward a magnet;

applying a magnetic field to isolate bacterium/magnetic particle complexes from the sample;

culturing the isolated bacterium for at most about 24 hrs; and

analyzing the bacterium.

2. The method according to claim 1, further comprising washing with a wash solution that reduces particle aggregation.

3. The method according to claim 1, wherein the particles are superparamagnetic particles.

4. The method according to claim 3, wherein the particles have a diameter from about 100 nm to about 250 nm.

5. The method according to claim 4, wherein the particles are at least about 65% magnetic material by weight.

6. The method according to claim 1, wherein the bacterium-specific binding moiety is an antibody.

7. The method according to claim 6, wherein the antibody comprises a detectable label.

8. The method according to claim 1, wherein the body fluid is blood.

9. The method according to claim 8, wherein the bacterium is a blood borne bacterium.

10. The method according to claim 1, wherein identifying step comprises sequencing nucleic acid derived from the bacterium or amplifying nucleic acid derived from the bacterium.

11. The method according to claim 1, wherein culturing is for at most about 12 hrs.

12. The method according to claim 1, wherein culturing is for at most about 6 hrs.

13. The method according to claim 1, wherein culturing is for at most about 3 hrs.

14. The method according to claim 1, wherein culturing is for at most about 1 hr.

15. The method of claim 1, wherein said buffer comprises sodium chloride.

16. The method of claim 15, wherein said buffer further comprises tri(hydroxymethyl)aminomethane-hydrochloric acid (Tris-HCl) and polysorbate 20.

17. The method of claim 16, wherein said Tris-HCl is concentration of about 75 mM.

18. The method of claim 1, wherein said buffer is present in said mixture at a ratio of about 1:

1 with said sample.

20. The method of claim 1, wherein the fluidic device is a microfluidic device.

19. A method for detecting a bacterium from a sample, the method comprising:

providing a vessel that contains a body fluid sample comprising a bacterium;

introducing to the sample, magnetic particles comprising a bacterium-specific binding moiety and a buffer that promotes binding of the particles to the bacterium in order to create a mixture;

incubating the mixture in the vessel to allow the particles to bind to the bacterium in the sample;

flowing the mixture from the vessel through a channel and toward a magnet;

applying a magnetic field to isolate bacterium/magnetic particle complexes from the sample;

culturing the isolated bacterium for at most about 24 hrs; and

detecting the bacterium.