Method for isolating microbial DNA
The present invention relates to a new method for enriching and/or isolating nucleic acids from microbial cells which comprises filtering a liquid sample through a nucleic acid-binding matrix which has a pore size small enough to retain microbial cells, lysing the microbial cells on the matrix to release the nucleic acids from the microbial cells, binding the nucleic acids to the matrix and subsequently eluting the DNA. The invention also relates to a method for enriching and/or isolating nucleic acids from microbial cells which are present in a liquid sample that comprises microbial cells and higher eukaryotic cells and/or tissues. The invention also provides a cartridge for carrying out the methods of the invention. Finally, the invention relates to kits for carrying out the methods of the invention.
1. Method for enriching and/or isolating nucleic acids from microbial cells, comprising
(a) filtering a liquid sample comprising microbial cells through a nucleic acid-binding matrix, which has a pore size small enough to retain the microbial cells;
(b) lysing the microbial cells on the matrix to release the nucleic acids from the microbial cells, wherein said lysing does not involve precipitation of the nucleic acids released from the microbial cells;
(c) optionally, degrading proteins on the matrix by the addition of an enzyme having proteinase activity;
(d) binding the nucleic acids to the matrix by the addition of monovalent or multivalent cations;
(e) optionally, washing the matrix to remove any unbound material; and
(f) eluting the nucleic acids.
2. Method according to claim 1 , wherein the matrix has a pore size of less than 4 μm.
3. Method according to claim 1 , wherein the filtration in step (a) is carried out either by centrifugation or by use of a vacuum device.
4. Method according to claim 1 , wherein the cell wall degrading enzyme in step (b) is lysozyme, lysostaphin, or mutanolysin.
5. Method according to claim 1 , wherein the enzyme having proteinase activity in step (c) is proteinase K.
6. Method according to claim 1 , wherein said monovalent and/or multivalent cations in step (d) are selected from the group consisting of Na + , Ca 2+ , Mg 2+ , NH 4+ , and Al 2+ .
7. Method according to claim 6 , wherein said monovalent cations in step (d) are derived from a chaotropic salt.
8. Method according to claim 1 , wherein a washing buffer is used in step (e) which comprises at least 50% (vol/vol) alcohol.
9. The method of claim 1 , wherein said liquid sample in step (a) comprises both microbial cells and higher eukaryotic cells and/or tissues, said method further comprising:
lysing the higher eukaryotic cells and/or tissues in the sample under conditions which do not result in the lysis of the microbial cells;
degrading nucleic acids released from the eukaryotic cells and/or tissues by the addition of an enzyme having nuclease activity; and
optionally, degrading proteins released from the eukaryotic cells and/or tissues by the addition of an enzyme having proteinase activity;
before filtering said liquid sample through said nucleic-acid binding matrix in step (a).
10. The method of claim 9 , wherein the higher eukaryotic cells are blood cells or tissue cells of human or animal origin.
11. Method according to claim 9 , wherein said enzyme having nuclease activity is a DNA-degrading nuclease.
12. Method according to claim 7 , wherein the chaotropic salt is selected from the group consisting of guanidine hydrochloride, sodium iodide, guanidine isothiocyanate, and mixtures thereof.
13. Method according to claim 11 , wherein the DNA-degrading nuclease is active under chaotropic conditions.
14. Method according to claim 1 , wherein said nucleic acid-binding matrix is a silica or glass matrix.
15. Method according to claim 1 , wherein said microbial cells are bacterial cells.