US 12,460,197 B2Granted

Methods and compositions for isolating an analyte

AssigneeGen-Probe Incorporated

InventorsAnne H. Niinivaara, Juha M. N. Saharinen, Katri M. Holappa

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Abstract

Disclosed are methods for isolating an analyte from a sample. In some aspects, the methods are for selectively isolating a microbial cell analyte, such as a nucleic acid, from a sample containing or suspected of containing mammalian cells. The selective isolation method includes selective lysis of the mammalian cells and filtration of the resulting lysate through a filter that retains intact microbial cells, followed by on-filter lysis of the retained microbial cells to release the microbial cell analyte. The released analyte is then eluted from the filter. In other aspects, the methods utilize on-filter lysis of a sample containing intact cells (e.g., microbial cells) to release the analytes, followed by elution of the released analytes from the filter by passing an immiscible liquid through the filter. The isolated analytes may be analyzed using a suitable assay depending on the type of analyte molecule. Also disclosed are fluidic systems and lysis solutions that may be used for isolating an analyte according to the disclosed methods.

Claims24

Dependent claims

A method for selectively isolating a microbial cell analyte from a sample, the method comprising:

(a) providing a sample containing mammalian cells and potentially containing microbial cells;
(b) mixing the sample with a first lysis solution that selectively lyses the mammalian cells to obtain a first lysate containing lysed mammalian cells and intact microbial cells, if present, wherein the first lysis solution a buffered solution comprising a chaotropic salt that is guanidine hydrochloride, and a detergent that is saponin;
(c) filtering the first lysate through a filter having a pore size that retains the intact microbial cells, wherein the pore size from about 0.1 μm to about 1 μm;
(d) contacting the filter containing the retained microbial cells with a second lysis solution that is effective to lyse the microbial cells and release therefrom an analyte, wherein the second lysis solution is a buffered solution comprising sodium hydroxide at a concentration of from about 50 mM to about 100 mM, dimethyl sulfoxide (DMSO) at a concentration of from about 10% (v/v) to about 25% (v/v), glycerol at a concentration of from about 3% (v/v) to about 10% (v/v);
(e) providing conditions for lysing the retained microbial cells in the second lysis solution whereby at least a portion of the microbial cells are lysed and the analyte released therefrom;
and
(f) eluting the analyte from the filter to obtain an eluate comprising isolated analyte.

4 more elements

Dependencies are inferred from the claim text.

Facts

Application: 19/240,880Filed 17 Jun 2025 · continuation
Priority: EP 2230696721 Dec 2022
Published: US 2025/0313826 A19 Oct 2025
Granted: 4 Nov 2025
Term ends: Not estimatedThe source records don’t support an estimate.

Classification9 codes

C12N 15/1017
Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses…
C12Q 1/6806
Involving nucleic acids: preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
C12N 1/063
Lysis of microorganisms: of yeast
C12N 1/066
Lysis of microorganisms: by physical processes