US 12,221,647 B2
Methods for isolating microbial cells from a blood sample
Aryeh Gassel, Jerusalem (IL); Yaron Suissa, Jerusalem (IL); Raphael Gassel, Jerusalem (IL); Maoz Tal, Moshav Aviezer (IL); Iaacov Man, Neve Yaakov (IL); and Boaz Arieli, Mevaseret Tzion (IL)
Assigned to BACTERIA DETECTION LTD, Jerusalem (IL)
Filed by BACTERIA DETECTION LTD, Jerusalem (IL)
Filed on Nov. 15, 2021, as Appl. No. 17/526,048.
Application 17/526,048 is a continuation of application No. 15/759,217, abandoned, previously published as PCT/IL2016/051006, filed on Sep. 11, 2016.
Claims priority of provisional application 62/217,093, filed on Sep. 11, 2015.
Prior Publication US 2022/0064695 A1, Mar. 3, 2022
Int. Cl. C12Q 1/24 (2006.01); C12N 1/00 (2006.01); C12Q 1/04 (2006.01); C12Q 1/6806 (2018.01)
CPC C12Q 1/24 (2013.01) [C12N 1/00 (2013.01); C12Q 1/04 (2013.01); C12Q 1/6806 (2013.01); G01N 2800/26 (2013.01); G01N 2800/44 (2013.01)] 20 Claims
 
1. A method for isolating bacterial cells from a blood sample containing or suspected of containing one or more bacteria, the method comprising:
providing a blood sample from a mammal;
adding a first selective lysis reagent and a second selective lysis reagent comprising a non-ionic detergent to the blood sample and incubating the blood sample for a time period sufficient to lyse most of the erythrocytes and release hemoglobin therefrom, and for the non-ionic detergent to lyse leukocytes in the blood sample and release mammalian DNA therefrom, wherein most bacterial cells in the blood sample are preserved;
separating and removing from the blood sample at least a portion of the mammalian proteins, including hemoglobin, while preserving most or all of the bacterial cells;
incubating the sample with an endonuclease, for a time period sufficient to lyse mammalian DNA;
adding an insoluble solid surface, and at least one of a water soluble polymer and at least one inorganic salt in a quantity sufficient to cause blood plasma, blood cell components and bacteria to displace directly or indirectly onto the solid surface;
separating bacterial cells displaced onto the solid surface;
eluting bacterial cells from the solid surface by incubating the bacterial cells on the solid surface with a solution comprising at least one protease; and
isolating released bacterial cells from the solid surface.