| CPC C12N 15/1017 (2013.01) [C12N 1/063 (2013.01); C12N 1/066 (2013.01); C12N 9/2462 (2013.01); C12Y 302/01017 (2013.01)] | 24 Claims |
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1. 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.
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21. A lysis 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), and
a buffer.
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