Analyzing bacteria without culturing
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.
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 in the vessel, 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 containing bacterium/magnetic particle complexes;
flowing the mixture from the vessel into a fluidic device through a channel in the fluidic device and toward a magnet;
applying a magnetic field to isolate the bacterium/magnetic particle complexes from the sample; 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 of claim 1 , wherein said buffer comprises sodium chloride.
12. The method of claim 11 , wherein said buffer further comprises tri(hydroxymethyl)aminomethane-hydrochloric acid (Tris-HCl) and polysorbate 20.
13. The method of claim 12 , wherein said Tris-HCl is concentration of about 75 mM.
14. The method of claim 1 , wherein said buffer is present in said mixture at a ratio of about 1:1 with said sample.
15. The method of claim 1 , wherein the fluidic device is a microfluidic device.
16. The method of claim 1 , further comprising incubating the mixture in the vessel to allow the magnetic particles to bind to the bacterium and form bacterium/magnetic particle complexes.
17. 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 in the vessel, 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 and form bacterium/magnetic particle complexes;
flowing the mixture from the vessel into a fluidic device through a channel and toward a magnet;
applying a magnetic field to isolate bacterium/magnetic particle complexes from the sample; and
detecting the bacterium.