Selective lysis of cells
Methods and devices for the selective lysis of cells in a sample comprising micro-organisms such as bacteria are provided. The selective lysis is obtained by incubating the sample in a non-ionic detergent under alkaline conditions.
1. A method for detecting the presence of micro-organisms contained or suspected to be contained within a mammalian blood sample, the method comprising the steps of:
providing the mammalian blood sample containing or suspected to contain the micro-organisms, wherein:
said mammalian blood sample comprises whole blood that has not been cultivated, said whole blood including blood plasma and eukaryotic blood cells,
said mammalian blood sample has a volume between 1 ml and 20 ml,
said micro-organisms are selected from the group consisting of bacteria and fungi, and
there are less than 10,000 micro-organisms per milliliter of the mammalian blood sample,
adding a selective lysis buffer at a selected concentration to the mammalian blood sample to obtain a final solution having a pH level of around 9.5 or higher, said selective lysis buffer including (i) a non-ionic detergent and (ii) an alkaline buffer, said non-ionic detergent present in a concentration of between 0.1% and 5% (w/v % or v/v %),
wherein the selected concentration of the selective lysis buffer in the final solution is a concentration that achieves said pH level of around 9.5 or higher for the final solution, with a volume of the selective lysis buffer that is between 200% and 10% of a volume of the mammalian blood sample;
incubating the final solution in the presence of the selective lysis buffer for a time period sufficiently long to selectively lyse the blood cells in the mammalian blood sample while keeping the micro-organisms intact, thereby forming a lysed sample, wherein the percentage of micro-organism cells that remain intact in the lysed sample is at least two times higher than the percentage of blood cells that remain intact in the lysed sample; and
detecting the presence of the micro-organisms without cultivation of the micro-organisms within said mammalian blood sample.
2. The method according to claim 1 , wherein said time period for the incubating step is between 30 seconds and 10 minutes.
3. The method according to claim 1 , wherein the non-ionic detergent is selected from the group consisting of Nonidet P40, deoxycholate, lgepal CA 630, Triton-X 100, and any combinations thereof.
4. The method according to claim 1 , further comprising centrifuging the incubated final solution and isolating the micro-organisms.
5. The method according to claim 1 , further comprising filtering the incubated final solution on a filter with a pore size that retains micro-organisms on the filter.
6. The method according to claim 1 , further comprising lysing the micro-organisms.
7. The method according to claim 6 , further comprising using a nucleic acid based molecular assay on the lysed micro-organisms.
8. The method according to claim 1 , wherein the method further comprises:
adding an acid or acidic buffer to neutralize the lysed sample.
9. The method according to claim 8 , wherein the lysed sample is neutralized to a pH in a range between 7 and 9.