Methods for isolating microbial cells from a blood sample

Abstract

This disclosure relates to methods for isolating bacterial cells, fungal cells, and single-celled parasites present in a blood sample containing higher eukaryotic cells; particularly wherein the microorganisms are present at a concentration significantly lower than the eukaryotic cells in the sample.

1 . A method for isolating microbial cells from a blood sample containing or suspected of containing one or more microorganisms, the method comprising:
 providing an aqueous solution comprising a blood sample from a higher eukaryotic organism;   adding a first selective lysis reagent to the aqueous solution for a time period sufficient to lyse most of the erythrocytes and release hemoglobin therefrom, wherein the integrity of at least most microbial cells and leukocytes in the blood sample are preserved;   separating and removing from the aqueous solution at least a portion of the higher eukaryotic proteins, including hemoglobin, while preserving most or all of the microbial cells;   adding a second selective lysis reagent to the aqueous solution;   incubating the aqueous solution for a time period sufficient to lyse leukocytes in the blood sample and release therefrom higher eukaryotic DNA, wherein the integrity of at least most microbial cells in the blood sample is preserved;   providing an endonuclease to the aqueous solution, and incubating the aqueous solution for a time period sufficient to lyse higher eukaryotic DNA;   adding to the aqueous solution an insoluble solid surface, and at least one of a water soluble polymer and at least one inorganic salt in a quantity sufficient to cause blood plasma, blood cell components and microorganisms to displace directly or indirectly onto the solid surface;   separating microbial cells displaced onto the solid surface from the aqueous solution;   eluting microbial cells from the solid surface by incubating the sample with a solution comprising at least one protease; and   isolating released microbial cells from the solid surface and from at least a portion of the residual blood components in the solution.   
     
     
         2 . The method of  claim 1 , wherein the aqueous solution comprises at least one anticoagulant agent. 
     
     
         3 . The method of  claim 1 , wherein the aqueous solution is a volume up to 5 ml, or is an up to 5 ml aliquot that is subdivided from an undiluted (100%) or diluted (5%-95%) larger blood sample volume. 
     
     
         4 . The method of  claim 1 , wherein, subsequent to adding the first lysis reagent to the aqueous solution, the aqueous solution is centrifuged for a time period sufficient to precipitate a majority of the microbial cells to facilitate removing at least a portion of the supernatant comprising higher eukaryotic proteins, including hemoglobin, while preserving most or all of the microbial cells within the aqueous solution. 
     
     
         5 . The method of  claim 4 , wherein subsequent to removing at least a portion of the supernatant from the aqueous solution and either (a) prior to adding to the aqueous solution a second lysis reagent, (b) in conjunction with adding to the aqueous solution a second lysis reagent, or (c) subsequent to adding to the aqueous solution a second lysis reagent, the method further comprises adding an additional quantity of first lysis reagent to the aqueous solution, centrifuging the sample a second time and removing at least a portion of the supernatant from the aqueous solution while preserving most or all of the microbial cells within the aqueous solution. 
     
     
         6 . The method of  claim 1 , wherein, subsequent to adding to the aqueous solution a first lysis reagent, the aqueous solution is separated so as to cause at least a portion of the higher eukaryotic proteins, including hemoglobin, to be removed from the aqueous solution while most or all of the microbial cells are preserved in the aqueous solution. 
     
     
         7 . The method of  claim 1 , wherein subsequent to adding to the aqueous solution the insoluble solid surface, the separated microbial cells are washed one or more times with a wash solution. 
     
     
         8 . The method of  claim 1 , wherein the microorganism is a bacterium or a fungus. 
     
     
         9 . The method of  claim 1 , wherein the second selective lysis reagent comprises a non-ionic detergent. 
     
     
         10 . The method of  claim 1 , wherein the second selective lysis reagent further comprises an agent capable of reducing viscosity, and wherein incubating the aqueous solution containing said agent comprises a time period sufficient to reduce viscosity in the aqueous solution. 
     
     
         11 . The method of  claim 10 , wherein the agent capable of reducing viscosity is a deoxyribonuclease. 
     
     
         12 . The method of  claim 1 , wherein the water soluble polymer is a dextran, PolyVinylPyrrolidone (PVP), or polyethylene glycol (PEG). 
     
     
         13 . The method of  claim 12 , wherein the final concentration of PEG is 2%-20% w/v. 
     
     
         14 . The method of  claim 1 , wherein the solid surface is magnetic, ferromagnetic or para-magnetic, and wherein separating the microbial cells comprises immobilizing the solid surface with a magnet, and removing substantially all of the aqueous solution. 
     
     
         15 . The method of  claim 7 , wherein the wash solution comprises at least one of a water soluble polymer and an inorganic salt. 
     
     
         16 . The method of  claim 15 , wherein the inorganic salt in the wash solution is at least one salt selected from the group consisting of: sodium chloride, magnesium chloride, calcium chloride, potassium chloride, lithium chloride, barium chloride, and cesium chloride. 
     
     
         17 . The method of  claim 16 , wherein the water soluble polymer in the wash solution is Dextran, PolyVinylPyrrolidone (PVP), or polyethylene glycol (PEG). 
     
     
         18 . The method of  claim 1 , wherein the protease is Proteinase K. 
     
     
         19 . The method of  claim 1 , wherein the incubation in a protease is combined with, preceded and/or followed by agitation in a buffering solution at a pH above 8. 
     
     
         20 . The method of  claim 1 , wherein the ratio of the microbial cells to higher eukaryotic cells is at least 1:8000. 
     
     
         21 . The method of  claim 1 , wherein the second selective lysis reagent is added simultaneous with, or prior to, the first selective lysis reagent.