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Publication Number
US-10450558-B2
Publication Date
2019-10-22
Expiration Date
Abstract
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.
Core Innovation
A method for enriching and/or isolating nucleic acids from microbial cells is provided by filtering a liquid sample comprising microbial cells through a nucleic acid-binding matrix. The matrix has a pore size small enough to retain the microbial cells during filtration. The microbial cells are then lysed on the matrix to release the nucleic acids, and the lysing does not involve precipitation of the nucleic acids released from the microbial cells.
Released nucleic acids are subsequently bound to the matrix by the addition of monovalent or multivalent cations. The matrix can be optionally washed to remove any unbound material before eluting the nucleic acids. Optionally, proteins on the matrix are degraded by adding an enzyme having proteinase activity, in particular proteinase K.
For mixed samples comprising microbial cells together with higher eukaryotic cells and/or tissues, the higher eukaryotic cells and/or tissues are lysed without lysing the microbial cells. Released eukaryotic nucleic acids are degraded by enzyme having nuclease activity, while proteins can be degraded by an enzyme having proteinase activity, followed by filtering the sample through the nucleic-acid-binding matrix.
Claims Coverage
The document identifies one independent claim with multiple dependent claims. Across the claim set, the inventive subject matter centers on filtration through a nucleic-acid-binding matrix followed by on-matrix lysis without nucleic-acid precipitation, cation-mediated binding, optional washing, and elution, with selective handling of mixed microbial and higher eukaryotic samples.
Filtering through a nucleic acid-binding matrix retaining microbial cells
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.
On-matrix lysis without nucleic-acid precipitation
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.
Protein degradation on the matrix using a proteinase
Optionally, degrading proteins on the matrix by the addition of an enzyme having proteinase activity.
Cation-mediated binding of nucleic acids to the matrix
Binding the nucleic acids to the matrix by the addition of monovalent or multivalent cations.
Optional washing of the matrix and elution of nucleic acids
Optionally, washing the matrix to remove any unbound material; and eluting the nucleic acids.
Matrix pore size threshold
The nucleic acid-binding matrix has a pore size of less than 4 μm.
Silica or glass nucleic-acid-binding matrix
The nucleic acid-binding matrix is either a silica matrix or a glass matrix.
Filtration performed by centrifugation or vacuum device
The filtration in step (a) is performed either by centrifugation or by using a vacuum device.
Proteinase activity provided by proteinase K
In step (c), the proteinase enzyme is proteinase K.
Selective handling of mixed microbial and higher eukaryotic samples
For a liquid sample containing both microbial cells and higher eukaryotic cells and/or tissues: lysing the higher eukaryotic cells/tissues without lysing microbial cells; degrading released eukaryotic nucleic acids with a nuclease enzyme; optionally degrading released proteins with a proteinase enzyme; and then filtering the sample through said nucleic-acid binding matrix in step (a).
Overall, claim coverage emphasizes a filtration-based enrichment/isolation approach using a nucleic-acid-binding matrix that retains microbial cells, with on-matrix lysis performed without nucleic-acid precipitation. Nucleic acids are then bound via monovalent or multivalent cations, optionally washed, and eluted, with dependent claims specifying pore-size and material constraints, filtration mode, proteinase choice, and selective processing of mixed microbial/eukaryotic samples.
Stated Advantages
Not explicitly described in patent.
Documented Applications
Not explicitly described in patent.
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