Analyzing bacteria without culturing

• US 10,677,789 B2
• Filed: 01/10/2018
• Issued: 06/09/2020
• 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: Dryga, Sergey A., Clarizia, Lisa-Jo Ann, Adams, Eddie W., Esch, Victor C.
• Primary Examiner(s): Hines, Jana A
• Application Number:
  US15/866,628
• Publication Number:
  US 20180136205A1
• Time in Patent Office: 881 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 pathogen in a sample, the method comprising:

providing a vessel that contains an unwashed body fluid sample;

introducing to the vessel;

magnetic particles comprising a pathogen-binding moiety, and

a buffer that promotes binding of the particles to pathogen;

binding the magnetic particles to a pathogen in the sample;

flowing the pathogen-bound magnetic particles and unwashed sample from the vessel into a fluidic device and through a channel in the fluidic device comprising a magnet;

applying a magnetic field to isolate the pathogen from the sample; and

analyzing the pathogen,

wherein the pathogen is selected from the group consisting of a bacterium, a fungus, and a virus.

2. The method according to claim 1, further comprising washing the magnetic particles 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 comprise at least about 65% magnetic material by weight.

6. The method according to claim 1, wherein the pathogen-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 pathogen is a blood-borne pathogen.

10. The method according to claim 1, wherein the identifying step comprises one selected from the group consisting of sequencing nucleic acid derived from the pathogen and amplifying nucleic acid derived from the pathogen.

11. The method of claim 1, wherein the buffer comprises sodium chloride.

12. The method of claim 11, wherein said buffer further comprises tris(hydroxymethyl)aminomethane hydrochloride and polysorbate 20.

13. The method of claim 12, wherein the buffer comprises tris(hydroxymethyl)aminomethane hydrochloride at a concentration of about 75 mM.

14. The method of claim 1, wherein the introducing step produces a mixture of sample:

buffer at a ratio of about 1;

1.

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

16. The method of claim 1, wherein the flowing step comprises flowing the sample toward a magnet.

17. A method for detecting a pathogen in a sample, the method comprising:

providing a vessel that contains a body fluid sample;

introducing to the sample in the vessel;

magnetic particles comprising a pathogen-binding moiety, and

a buffer that promotes binding of the particles to the pathogen;

binding the magnetic particles to a pathogen in the sample;

flowing the pathogen-bound magnetic particles and unwashed sample from the vessel into a fluidic device and through a channel in the fluidic device comprising a magnet;

applying a magnetic field to isolate the pathogen from the sample; and

detecting the pathogen,

wherein the pathogen is selected from the group consisting of a bacterium, a fungus, and a virus.

18. The method according to claim 17, wherein the body fluid is blood.

19. The method according to claim 18, wherein the pathogen is a blood-borne pathogen.