Extracting low concentrations of bacteria from a sample

• US 9,562,896 B2
• Filed: 04/21/2011
• Issued: 02/07/2017
• 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: Esch, Victor C., Dryga, Sergey A., Clarizia, Lisa-Jo Ann
• Primary Examiner(s): Duffy, Patricia
• Application Number:
  US13/091,534
• Publication Number:
  US 20110262926A1
• Time in Patent Office: 2,119 Days
• Field of Search: None
• US Class Current: 1/1
• 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 bacterium from the sample, in which the assay isolates as low as about 1 CFU/ml of bacteria in the sample.

Claims

1. A method for isolating bacterium from a sample, the method comprising the steps of:

providing a vessel that contains a sample comprising a bacterium;

incubating said sample in a mixture comprising magnetic particles having attached thereto a binding moiety that specifically binds said bacterium and a cell lysis inhibitor;

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

separating the magnetic particles from other components in the sample by using the magnet to retain bacteria bound to magnetic particles while unbound sample components are not retained by the magnet;

washing the magnetic particles having bacteria bound thereto as well as the unbound sample components in a solution that imparts a negative charge to said magnetic particles; and

isolating as low as about 1 CFU of said bacteria from said mixture.

2. The method according to claim 1, wherein the sample is a human tissue or body fluid.

3. The method according to claim 2, wherein the body fluid is blood.

4. The method according to claim 3, wherein the bacterium is a blood borne bacterium.

5. The method according to claim 1, wherein the sample is a food sample.

6. The method according to claim 5, wherein the bacterium is a food borne bacterium.

7. The method according to claim 1, further comprising characterizing the bacterium.

8. The method according to claim 7, wherein said characterizing step comprises identifying the bacterium.

9. The method according to claim 1, wherein the bacterium is gram positive.

10. The method according to claim 1, wherein the bacterium is gram negative.

11. A method for isolating bacterium from a sample, the method comprising the steps of:

providing a vessel that contains a sample comprising a bacterium;

introducing magnetic particles comprising a bacterium-specific binding moiety to said sample;

incubating said sample in a solution that reduces particle aggregation and comprises a cell lysis inhibitor;

flowing said sample from the vessel through a channel in a microfluidic device and toward a magnet in the microfluidic device;

separating the magnetic particles from other components in the sample by using the magnet to retain bacteria bound to magnetic particles while unbound sample components are not retained by the magnet;

washing the magnetic particles; and

isolating as low as 1 CFU/ml of said bacterium.

12. The method according to claim 11, wherein said incubating step is conducted from about 10 seconds to about 2 hours.

13. The method according to claim 11, wherein the bacteria-specific binding moiety is an antibody.

14. The method according to claim 11, wherein the sample is a human tissue or body fluid.

15. The method according to claim 14, wherein the body fluid is blood.

16. The method according to claim 15, wherein the bacterium is a blood borne bacterium.

17. The method according to claim 11, wherein the sample is a food sample.

18. The method according to claim 17, wherein the bacterium is a food borne bacterium.

19. The method according to claim 11, further comprising characterizing the bacterium.

20. The method according to claim 19, wherein characterizing comprises identifying the bacterium.

21. The method according to claim 20, wherein identifying is selected from sequencing nucleic acid derived from the bacterium or amplifying nucleic acid derived from the bacterium.

22. The method according to claim 11, wherein the bacterium is gram positive.

23. The method according to claim 11, wherein the bacterium is gram negative.