Methods, systems, and compositions for isolating analytes

Abstract

Methods for isolating analytes from a sample are disclosed. In some embodiments, the methods are for selectively isolating microbial cell analytes, such as nucleic acids, from a sample containing or suspected of containing mammalian cells. The selective isolation method involves selective lysis of 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 analytes. The released analytes are then eluted from the filter. In other embodiments, 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 can be analyzed using an appropriate assay depending on the type of analyte molecule.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to European Patent Application No. 22306967.5, filed December 21, 2022, which is incorporated herein by reference in its entirety. [Background technology]

[0002] background Many molecular diagnostic applications require the highest possible sensitivity in order to be state-of-the-art and competitive with products from other molecular diagnostic manufacturers. High sensitivity is typically a distinct asset of all molecular diagnostic applications and is a characteristic that is typically valued by end users.

[0003] Furthermore, in certain cases, there are sensitivity requirements that cannot be achieved by any typical sample extraction method, regardless of their sensitivity, due to the small number of analyte molecules in the sample volume used for extraction. Some applications in this field include direct detection of blood sepsis pathogens, liquid biopsies for circulating tumor cells (CTCs), circulating tumor DNA, and exosomes (e.g., from blood / plasma and urine matrices), and water analysis and airborne pathogen detection, for example.

[0004] For example, direct microbial detection in blood samples is highly valuable for faster overall results in pathogen presence, identification, and, for example, antibiotic resistance characterization. The current gold standard method is based on pathogen enrichment by blood culture, which is time-consuming (e.g., 24–72 hours) and has low detection sensitivity even after culture. Worldwide, approximately 30 million sepsis cases occur annually, resulting in 7–9 million deaths (one death every 3.5 seconds). Published data show an 8% decrease in survival rate for every hour of delayed antibiotic administration in sepsis, and 15–40% of sepsis patients receive inappropriate empirical antibiotic treatment due to an inability to rapidly detect the causative pathogen, highlighting the importance of early pathogen detection, identification, and antibiotic resistance profiling.

[0005] In the field of direct blood sepsis testing, nucleic acid-based assays utilizing pathogen enrichment from blood samples have been developed by Qvell, T2 Biosystems, and DNAe. All of these assays are based on multiplex PCR, preceded by pathogen enrichment from blood samples. DNAe assays use magnetic particles coupled with pathogen-specific antibodies, while T2 Biosystems and Qvell assays use centrifugation for enrichment. The use of biomolecules such as antibodies for sample enrichment raises issues of storage / stability, lot-to-lot variability, and cost, in addition to incubation time. The use of centrifugation in a closed system results in large equipment and specific fluidics capable of retaining the correct fraction of the centrifugal product.

[0006] For example, there is a need for sample concentration methods in a variety of applications that enable analyte detection sensitivity beyond that achievable by conventional methods, as well as improved efficiency in cost, assay time, and instrumentation space. Summary of the Invention

[0007] Abstract This Summary is provided to introduce a selection of concepts in a simplified form that are further described below in the Detailed Description. This Summary is not intended to identify key features of the claimed subject matter, nor is it intended to be used as an aid in determining the scope of the claimed subject matter.

[0008] The systems and methods described herein are based on the use of selective filtration to concentrate analytes from larger original sample volumes. In this way, processing larger sample volumes can improve overall assay sensitivity. This concept is applicable to downstream nucleic acid (e.g., RNA / DNA) amplification and molecular diagnostics using qPCR and / or microarray detection, but also to other detection modalities, including next-generation sequencing, and protein / other biomolecule detection via, for example, ELISA, protein arrays, MALDI-TOF, nanoLC / UPLC-ESI-MS, and other related amplification / separation / detection modalities.

[0009] In one aspect, the present disclosure provides a method for selectively isolating microbial cell analytes (e.g., microbial cell nucleic acids) from a sample containing mammalian cells and potentially containing microbial cells. The method generally includes: (a) providing a sample containing mammalian (e.g., human) cells and potentially containing microbial cells; (b) mixing the sample with a first lysis solution that selectively lyses mammalian cells to obtain a first lysate containing lysed mammalian cells and, if present, intact microbial cells; (c) filtering the first lysate through a filter having a pore size that retains intact microbial cells; (d) contacting the filter containing the retained microbial cells with a second lysis solution effective to lyse the microbial cells and release analytes therefrom; (e) providing conditions for lysing the retained microbial cells in the second lysis solution, thereby lysing at least a portion of the microbial cells and releasing the analytes therefrom; and (f) eluting the analytes from the filter to obtain an eluate containing the isolated analyte. In some embodiments, the microbial cells are bacterial cells or yeast cells. In another non-mutually exclusive variation, the mammalian cells are blood cells.

[0010] Particularly suitable filters for use in the present methods include filters comprising polyethersulfone (PES), cellulose, nylon, polyvinylidene fluoride (poly(1,1-difluoroethylene), PVDF), polycarbonate, or glass (e.g., borosilicate glass) fibers. In certain variations, the filters have pore sizes of about 1 μm or less (e.g., pore sizes of about 0.1 μm to about 1 μm, pore sizes of about 0.2 to about 1 μm, or pore sizes of about 0.22 μm). In other, non-mutually exclusive variations, the filters comprise asymmetric structures. In certain variations, the pores on a first side of the asymmetric filter have a size of about 5 μm to about 20 μm, and the pores on a second side of the asymmetric filter have a size of up to about 0.2 μm.

[0011] In some embodiments of the above method, the first lysis solution comprises a chaotropic salt and a detergent. In some such embodiments, the chaotropic salt is guanidine hydrochloride and / or the detergent is saponin. In certain variations, the first lysis solution further comprises a second detergent, such as, for example, polysorbate 20.

[0012] In some embodiments of the methods described above, the first lysis solution comprises guanidine hydrochloride, polysorbate 20, saponin, and a buffer. In some such embodiments, the guanidine hydrochloride is present in the first lysis solution at a concentration of about 1 M to about 8 M; the polysorbate 20 is present in the first lysis solution at a concentration of 1% (v / v) to about 10% (v / v); the saponin is present in the first lysis solution at a concentration of about 1% (w / v) to about 10% (w / v); and / or the buffer in the first lysis solution is Tris and is present at a concentration of about 20 mM to about 200 mM. In more specific variations, guanidine hydrochloride is present in the first lysis solution at a concentration of about 4 M; polysorbate 20 is present in the first lysis solution at a concentration of 4% (v / v); saponin is present in the first lysis solution at a concentration of about 4% (w / v); and / or Tris is present in the first lysis solution at a concentration of about 35 mM to about 45 mM (e.g., about 40.5 mM).

[0013] In particular variations of the above method, the volume of the sample provided in step (a) is about 0.5 mL to about 5 mL. In more particular variations, the volume of the sample is about 1 mL to about 5 mL or about 2 mL to about 5 mL (e.g., about 4 mL).

[0014] In certain embodiments of the method as described above, during the mixing step (b), the ratio of first lysis solution to sample is about 1:1. In other, non-mutually exclusive variations, the concentration of guanidine hydrochloride in the first lysate is about 0.5 M to about 4 M; the concentration of polysorbate 20 in the first lysate is about 0.5% (v / v) to about 5% (v / v); the concentration of saponin in the first lysate is about 0.5% (w / v) to about 5% (w / v); and / or the buffer in the first lysis solution is Tris and the concentration of Tris in the first lysate is about 10 mM to about 100 mM. In some such variations, the concentration of guanidine hydrochloride in the first lysate is about 2 M; the concentration of polysorbate 20 in the first lysate is about 2% (v / v); the concentration of saponin in the first lysate is about 2% (w / v); and / or the buffer in the first lysate is Tris and the concentration of Tris in the first lysate is about 18 mM to about 23 mM (e.g., about 20.25 mM).

[0015] In some embodiments of the above-described method, the second lysis solution comprises sodium hydroxide, dimethyl sulfoxide (DMSO), glycerol, and a buffer. In some such embodiments, the sodium hydroxide is present in the second lysis solution at a concentration of about 20 mM to about 500 mM; the DMSO is present in the second lysis solution at a concentration of about 5% (v / v) to about 30% (v / v); the glycerol is present in the second lysis solution at a concentration of about 1% (v / v) to about 15% (v / v); and / or the buffer in the second lysis solution is Tris and is present at a concentration of about 1 mM to about 50 mM. In more specific variations, sodium hydroxide is present in the second lysis solution at a concentration of about 50 mM to about 100 mM; DMSO is present in the second lysis solution at a concentration of about 10% (v / v) to about 25% (v / v); glycerol is present in the second lysis solution at a concentration of about 3% (v / v) to about 10% (v / v); and / or the buffer in the second lysis solution is Tris and is present at a concentration of about 5 mM to about 10 mM. In even more specific variations, sodium hydroxide is present in the second lysis solution at a concentration of about 85 mM; DMSO is present in the second lysis solution at a concentration of about 18% (v / v); glycerol is present in the second lysis solution at a concentration of about 7% (v / v); and / or the buffer in the second lysis solution is Tris and is present at a concentration of about 5 mM to about 10 mM (e.g., about 7.5 mM). In some variations, the second lysis solution further comprises a chelating agent. A particularly suitable chelating agent is 2,2',2'',2''''-(ethane-1,2-diyldinitrilo)tetraacetic acid (EDTA), which may be present in the second lysis solution at a concentration of about 0.1 mM to about 4 mM, about 0.5 mM to about 2 mM, or about 1 mM. In some embodiments, the second lysis solution consists of, or consists essentially of, sodium hydroxide, DMSO, glycerol, a chelating agent, a buffer, and water.

[0016] In certain variations of the methods described above, the lysis conditions of step (e) comprise incubating the filter with a second lysis solution at a temperature of about 80° C. to about 150° C. (e.g., a temperature of about 90° C. to about 130° C.). In other, non-mutually exclusive, embodiments, the lysis conditions of step (e) comprise sonication.

[0017] In certain embodiments, the above method further comprises an enzyme treatment step between steps (c) and (d), which comprises contacting the filter containing the retained microbial cells with an enzyme effective to degrade one or more microbial cell wall components (e.g., peptidoglycan, chitin, protein). Particularly suitable enzymes are mutanolysin, lyticase, lysozyme, endoglucanase, protease, chitinase, and combinations thereof.

[0018] In some variations of the methods described above, the eluting step (f) comprises passing an immiscible liquid (e.g., an oil and / or hydrocarbon, such as mineral oil, silicone oil, or hexadecane) through the filter to force an eluate containing the analytes through the filter. In some such embodiments, the method further comprises separating the eluate from the immiscible liquid. In other variations, the eluting step (f) comprises using a second lysis solution as an elution buffer, and passing the elution buffer through the filter to obtain an eluate containing the nucleic acids. The direction of fluid flow through the filter in the elution step (f) can be the same as or opposite to the direction of fluid flow applied in the filtering step (c).

[0019] In certain embodiments, the above method further comprises (g) adding a neutralization buffer to the eluate obtained in step (f). In some such embodiments, the neutralization buffer may comprise hydrochloric acid (HCl) and Tris.

[0020] In some embodiments, the above method further comprises a washing step between steps (c) and (d), wherein the washing step comprises flowing a wash buffer through the filter. A particularly suitable washing buffer comprises polysorbate 20, EDTA, and Tris. In other embodiments, the wash buffer is the same as the second lysis solution or a diluted concentration of the second lysis solution. In an alternative variation, the method does not comprise a washing step between steps (c) and (d). In a particular variation that comprises a washing step between steps (c) and (d), in a method that further comprises an enzyme treatment step between steps (c) and (d), as described above, the washing step is performed before the enzyme treatment step.

[0021] The above method may further include analyzing the isolated analyte. For example, if the isolated analyte is a nucleic acid, the step for analyzing the isolated nucleic acid may include (i) performing an amplification reaction using the isolated nucleic acid as a template to generate an amplification product, and (ii) detecting the amplification product. In some such embodiments, the amplification reaction is PCR, e.g., quantitative PCR (qPCR). In other variations, the amplification is an isothermal amplification reaction, e.g., a transcription-mediated amplification reaction. The detection step (ii) may be performed in real time. The method may optionally include an analyte isolation / purification step to isolate the target analyte of interest from non-target analytes in the microbial analyte.

[0022] In some embodiments of the above methods, the method further comprises analyzing the isolated nucleic acid analyte, wherein the analyzing step comprises immobilizing the isolated nucleic acid or amplification product on a solid support. In some such embodiments, the isolated nucleic acid or amplification product is hybridized to an immobilized probe attached to the solid support, e.g., an immobilized probe contained in a nucleic acid array.

[0023] In another aspect, the present disclosure provides a fluidic system, which may be a microfluidic system, configured to selectively isolate microbial cell analytes from a sample according to the above-described method. The system generally includes: (1) an input port configured to receive the sample; (2) a first reservoir for a first lysis solution; (3) a lysis chamber for mixing the sample with the first lysis solution to obtain a first lysate; (4) a filter chamber containing a filter; (5) a second reservoir for a second lysis solution; (6) a third reservoir for an elution reagent effective to elute released analytes from the filter; (7) an elution chamber for receiving the eluate; and (8) a fluidic channel array configured to provide (a) fluid communication between the lysis chamber and each of the input port and the first reservoir, and (b) fluid communication between the filter chamber and each of the lysis chamber, the second and third reservoirs, and the elution chamber. In some embodiments, the fluidic system further includes a heater thermally coupled to the filter chamber, a sonotrode coupled to the filter chamber, or a conductive mesh structure disposed on one or both sides of the filter chamber. In other, non-mutually exclusive variations, the fluid system further includes one or both of: (i) a fourth reservoir for neutralization buffer, wherein the fluid channel array further provides fluid communication between the elution chamber and the fourth reservoir; and (ii) a fifth reservoir for wash buffer, wherein the fluid channel array further provides fluid communication between the filter chamber and the fifth reservoir.In yet other non-mutually exclusive embodiments, the fluid system further comprises one or both of a first lysis solution and a second lysis solution (e.g., a first lysis solution comprising guanidine hydrochloride at a concentration of about 1 M to about 8 M, polysorbate 20 at a concentration of about 0.5% (v / v) to about 5% (v / v), saponin at a concentration of about 0.5% (w / v) to about 5% (w / v), and Tris at a concentration of about 10 mM to about 100 mM; and / or a second lysis solution containing sodium hydroxide at a concentration of about 20 mM to about 500 mM, dimethyl sulfoxide (DMSO) at a concentration of about 5% (v / v) to about 30% (v / v), glycerol at a concentration of about 1% (v / v) to about 15% (v / v), 2,2',2'',2'''-(ethane-1,2-diyldinitrilo)tetraacetic acid (EDTA) at a concentration of about 0.1 mM to about 4 mM, and Tris at a concentration of about 1 mM to about 50 mM.

[0024] In another aspect, the present disclosure provides a method for isolating an analyte (e.g., a nucleic acid) from a sample using immiscible fluid elution. The method generally includes: (a) providing a sample potentially containing intact cells; (b) filtering the sample through a filter having a pore size that retains the intact cells; (c) contacting the filter containing the retained cells with a lysis solution effective to lyse the cells and release an analyte therefrom; (d) providing conditions for lysing the retained cells in the lysis solution, thereby lysing at least a portion of the cells and releasing the analyte therefrom; and (e) passing the immiscible liquid through the filter to force an analyte-containing eluate through the filter. The direction of fluid flow through the filter in elution step (f) can be the same as or opposite to the direction of fluid flow applied in filtration step (b). The method may further include (f) adding a neutralization buffer (e.g., a buffer containing hydrochloric acid (HCl) and Tris) to the eluate obtained in step (e). In some embodiments, the method further includes separating the eluate from the immiscible liquid. In other, non-mutually exclusive variations, the immiscible liquid is selected from the group consisting of oils and hydrocarbons, such as mineral oil, silicone oil, and hexadecane. Particularly suitable filters for use in the present method include filters comprising polyethersulfone (PES), cellulose, nylon, polyvinylidene fluoride (PVDF), polycarbonate, or glass (e.g., borosilicate glass) fibers. In certain variations, the filter has a pore size of about 1 μm or less (e.g., about 0.1 μm to about 1 μm, about 0.2 to about 1 μm, or about 0.22 μm). In other, non-mutually exclusive variations, the filter comprises an asymmetric structure. In certain variations, the pores on a first side of the asymmetric filter have a size of about 5 μm to about 20 μm, and the pores on a second side of the asymmetric filter have a size of up to about 0.2 μm.

[0025] In some embodiments of the method for isolating an analyte from a sample using immiscible fluid elution as described above, the cells are microbial cells, such as bacterial or yeast cells. In some embodiments where the cells are microbial cells, the lysis solution in step (c) comprises sodium hydroxide, dimethyl sulfoxide (DMSO), glycerol, and a buffer. In some such embodiments, the sodium hydroxide is present in the lysis solution at a concentration of about 20 mM to about 500 mM; the DMSO is present in the lysis solution at a concentration of about 5% (v / v) to about 30% (v / v); the glycerol is present in the lysis solution at a concentration of about 1% (v / v) to about 15% (v / v); and / or the buffer in the lysis solution is Tris, present at a concentration of about 1 mM to about 50 mM. In more specific variations, sodium hydroxide is present in the lysis solution at a concentration of about 50 mM to about 100 mM; DMSO is present in the lysis solution at a concentration of about 10% (v / v) to about 25% (v / v); glycerol is present in the lysis solution at a concentration of about 3% (v / v) to about 10% (v / v); and / or the buffer in the lysis solution is Tris and is present at a concentration of about 5 mM to about 10 mM. In even more specific variations, sodium hydroxide is present in the lysis solution at a concentration of about 85 mM; DMSO is present in the lysis solution at a concentration of about 18% (v / v); glycerol is present in the lysis solution at a concentration of about 7% (v / v); the chelating agent in the lysis solution is EDTA and is present at a concentration of about 1 mM; and / or the buffer in the lysis solution is Tris and is present at a concentration of about 5 mM to about 10 mM (e.g., about 7.5 mM). In some variations, the lysis solution further comprises a chelating agent. In some such variations, the chelating agent is 2,2',2'',2'''-(ethane-1,2-diyldinitrilo)tetraacetic acid (EDTA) and is present in the lysis solution at a concentration of about 0.1 mM to about 4 mM, about 0.5 mM to about 2 mM, or about 1 mM. In some embodiments, the lysis solution consists of, or consists essentially of, sodium hydroxide, DMSO, glycerol, a chelating agent, a buffer, and water.

[0026] In certain non-mutually exclusive variations, the lysis conditions of step (d) comprise incubating the filter with a lysis solution at a temperature of about 80° C. to about 150° C. (e.g., a temperature of about 90° C. to about 130° C.). In other non-mutually exclusive embodiments, the lysis conditions of step (d) comprise sonication.

[0027] In certain embodiments of the method of isolating an analyte from a sample using immiscible fluid elution as described above, the cells are microbial cells, and the method further comprises an enzyme treatment step between steps (b) and (c), which comprises contacting the filter containing the retained microbial cells with an enzyme effective to degrade one or more microbial cell wall components (e.g., peptidoglycan, chitin, protein). Particularly suitable enzymes are mutanolysin, lyticase, lysozyme, endoglucanase, protease, chitinase, and combinations thereof.

[0028] In some embodiments of the methods for isolating an analyte from a sample using immiscible fluid elution as described above, the cells are microbial cells, and the method further comprises a selective lysis step prior to step (b), which comprises selectively lysing any mammalian (e.g., human) cells in the sample while leaving the microbial cells intact. In some such variations, the mammalian cells in the sample are blood cells.

[0029] In some embodiments of the method for isolating an analyte from a sample using immiscible fluid elution as described above, the method further includes a washing step between steps (b) and (c), where the washing step includes flowing a wash buffer through the filter. A particularly suitable wash buffer includes EDTA and Tris, and may also include polysorbate 20. In other embodiments, the wash buffer is the same as the lysis solution or a dilute concentration of the lysis solution. In alternative variations, the method does not include a washing step between steps (b) and (c). In certain variations that include a washing step between steps (b) and (c), in methods that further include an enzyme treatment step between steps (b) and (c), as described above, the washing step occurs before the enzyme treatment step.

[0030] The method of isolating an analyte from a sample using immiscible fluid elution as described above may further include analyzing the isolated analyte. For example, if the isolated analyte is a nucleic acid, the steps for analyzing the isolated nucleic acid may include (i) performing an amplification reaction using the isolated nucleic acid as a template to generate an amplification product, and (ii) detecting the amplification product. In some such embodiments, the amplification reaction is PCR, e.g., quantitative PCR (qPCR). In other variations, the amplification is an isothermal amplification reaction, e.g., a transcription-mediated amplification reaction. The detection step (ii) may be performed in real time. In some embodiments, the analyzing step includes immobilizing the isolated nucleic acid or amplification product to a solid support; in some such embodiments, the isolated nucleic acid or amplification product is hybridized to an immobilized probe attached to the solid support, e.g., an immobilized probe contained in a nucleic acid array.

[0031] In another aspect, the present invention provides a lysis solution as described above. In some embodiments, the lysis solution is a buffered lysis solution effective for lysing microbial cells to release analytes therefrom, the lysis solution comprising sodium hydroxide at a concentration of about 20 mM to about 500 mM, dimethyl sulfoxide (DMSO) at a concentration of about 5% (v / v) to about 30% (v / v), glycerol at a concentration of about 1% (v / v) to about 15% (v / v), and a buffer. In more specific variations, the sodium hydroxide is present at a concentration of about 50 mM to about 100 mM (e.g., about 85 mM); the DMSO is present at a concentration of about 10% (v / v) to about 25% (v / v) (e.g., about 18% (v / v)); and / or the glycerol is present at a concentration of about 3% (v / v) to about 10% (v / v) (e.g., about 7% (v / v)). In even more specific variations, sodium hydroxide is present at a concentration of about 85 mM; DMSO is present at a concentration of about 18% (v / v); and / or glycerol is present at a concentration of about 7% (v / v). In certain embodiments, the buffer is Tris and is present at a concentration of about 1 mM to about 50 mM or about 5 mM to about 10 mM (e.g., about 7.5 mM). In some variations, the lysis solution further comprises a chelating agent. A particularly suitable chelating agent is 2,2',2'',2'''-(ethane-1,2-diyldinitrilo)tetraacetic acid (EDTA), which may be present at a concentration of about 0.1 mM to about 4 mM, about 0.5 mM to about 2 mM, or about 1 mM. In some embodiments, the lysis solution consists of, or consists essentially of, sodium hydroxide, DMSO, glycerol, chelating agent, buffer, and water.

[0032] These and other aspects of the present invention will become evident upon reference to the following detailed description. [Brief explanation of the drawings]

[0033] DESCRIPTION OF THE DRAWINGS The accompanying drawings, which are incorporated in and form a part of this specification, illustrate various embodiments of the presently disclosed subject matter, in which like reference numbers indicate identical or functionally similar elements.

[0034] The foregoing aspects and many of the attendant advantages of this invention will become more readily appreciated as the same become better understood by reference to the following detailed description, when taken in conjunction with the accompanying drawings, wherein:

[0035] [Figure 1] FIG. 1 is a schematic diagram of a fluidic system 100 for isolating an analyte from a sample using the methods described herein and analyzing the isolated analyte to detect whether the sample contains the analyte of interest.

[0036] [Figure 2] FIG. 2 is a flow chart illustrating a method for selectively isolating a microbial cell analyte according to the present disclosure.

[0037] [Figure 3] FIG. 3 is a flow chart illustrating a method for isolating an analyte from a sample using immiscible liquid elution according to the present disclosure.

[0038] [Figure 4] FIG. 4 is a flow chart illustrating a method for detecting a target nucleic acid within an isolated analyte. DETAILED DESCRIPTION OF THE INVENTION

[0039] Embodiment Embodiment 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 mammalian cells to obtain a first lysate containing lysed mammalian cells and, if present, intact microbial cells; (c) filtering the first lysate through a filter having a pore size that retains the intact microbial cells; (d) contacting the filter containing the retained microbial cells with a second lysis solution effective to lyse the microbial cells and release an analyte therefrom; (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 is released therefrom; and (f) eluting the analyte from the filter to obtain an eluate comprising the isolated analyte.

[0040] Embodiment 2: The method of embodiment 1, wherein the microbial cells are bacterial cells.

[0041] Embodiment 3: The method of embodiment 1, wherein the microbial cell is a yeast cell.

[0042] Embodiment 4: The method of any one of embodiments 1 to 3, wherein the filter has a pore size of about 1 μm or less.

[0043] Embodiment 5: The method of embodiment 4, wherein the filter has a pore size of about 0.1 μm to about 1 μm.

[0044] Embodiment 6: The method of embodiment 4, wherein the filter has a pore size of about 0.2 μm to about 1 μm.

[0045] Embodiment 7: The method of embodiment 6, wherein the filter has a pore size of about 0.22 μm.

[0046] Embodiment 8: The method of any one of embodiments 1 to 7, wherein the filter comprises polyethersulfone (PES), cellulose, nylon, polyvinylidene fluoride (PVDF), polycarbonate, or glass fiber.

[0047] Embodiment 9: The method of any one of embodiments 1 to 8, wherein the filter comprises an asymmetric structure, preferably the asymmetric filter comprises pores on a first side having a size of about 5 μm to about 20 μm and pores on a second side having a size of up to about 0.2 μm.

[0048] Embodiment 10: The method of any one of embodiments 1 to 9, wherein the first lysis solution comprises a chaotropic salt and a detergent.

[0049] Embodiment 11: The method of embodiment 10, wherein the chaotropic salt is guanidine hydrochloride.

[0050] Embodiment 12: The method of embodiment 10 or 11, wherein the detergent is saponin.

[0051] Embodiment 13: The method of embodiment 12, wherein the first lysis solution further comprises a second detergent.

[0052] Embodiment 14: The method of embodiment 13, wherein the second detergent is polysorbate 20.

[0053] Embodiment 15: The method of any one of embodiments 1 to 9, wherein the first lysis solution comprises guanidine hydrochloride, polysorbate 20, saponin and a buffer.

[0054] Embodiment 16: The method of embodiment 15, wherein guanidine hydrochloride is present in the first lysis solution at a concentration of about 1 M to about 8 M.

[0055] Embodiment 17: The method of embodiment 16, wherein guanidine hydrochloride is present in the first lysis solution at a concentration of about 4 M.

[0056] Embodiment 18: The method of any one of embodiments 15 to 17, wherein Polysorbate 20 is present in the first lysis solution at a concentration of about 1% (v / v) to about 10% (v / v).

[0057] Embodiment 19: The method of embodiment 18, wherein Polysorbate 20 is present in the first lysis solution at a concentration of 4% (v / v).

[0058] Embodiment 20: The method of any one of embodiments 15 to 19, wherein the saponin is present in the first lysis solution at a concentration of about 1% (w / v) to about 10% (w / v).

[0059] Embodiment 21: The method of any of embodiment 20, wherein the saponin is present in the first lysis solution at a concentration of about 4% (w / v).

[0060] Embodiment 22: The method of any one of embodiments 15 to 21, wherein the buffer in the first lysis solution is Tris and is present at a concentration of about 20 mM to about 200 mM.

[0061] Embodiment 23: The method of embodiment 22, wherein Tris is present in the first lysis solution at a concentration of about 35 mM to about 45 mM.

[0062] Embodiment 24: The method of any one of embodiments 1 to 23, wherein the volume of the sample provided in step (a) is from about 0.5 mL to about 5 mL.

[0063] Embodiment 25: The method of embodiment 24, wherein the volume of the sample provided in step (a) is from about 1 mL to about 5 mL.

[0064] Embodiment 26: The method of embodiment 24, wherein the volume of the sample provided in step (a) is about 4 mL.

[0065] Embodiment 27: The method of any one of embodiments 1 to 26, wherein during the mixing step (b), the ratio of first lysis solution:sample is about 1:1.

[0066] Embodiment 28: The method of embodiment 15, wherein the concentration of guanidine hydrochloride in the first lysate is from about 0.5 M to about 4 M.

[0067] Embodiment 29: The method of embodiment 28, wherein the concentration of guanidine hydrochloride in the first lysate is about 2 M.

[0068] Embodiment 30: The method of any one of embodiments 15, 28, and 29, wherein the concentration of polysorbate 20 in the first lysate is from about 0.5% (v / v) to about 5% (v / v).

[0069] Embodiment 31: The method of embodiment 30, wherein the concentration of polysorbate 20 in the first lysate is about 2% (v / v).

[0070] Embodiment 32: The method of any one of embodiments 15 and 28-31, wherein the concentration of saponin in the first lysate is from about 0.5% (w / v) to about 5% (w / v).

[0071] Embodiment 33: The method of embodiment 32, wherein the saponin concentration in the first lysate is about 2% (w / v).

[0072] Embodiment 34: The method of any one of embodiments 15 and 28 to 33, wherein the buffer in the first lysis solution is Tris, and the concentration of Tris in the first lysate is from about 10 mM to about 100 mM.

[0073] Embodiment 35: The method of embodiment 34, wherein the concentration of Tris in the first lysate is about 18 mM to about 23 mM.

[0074] Embodiment 36: The method of any one of embodiments 1 to 35, wherein the second lysis solution comprises sodium hydroxide, dimethyl sulfoxide (DMSO), glycerol and a buffer.

[0075] Embodiment 37: The method of embodiment 36, wherein sodium hydroxide is present in the second lysis solution at a concentration of about 20 mM to about 500 mM.

[0076] Embodiment 38: The method of embodiment 37, wherein sodium hydroxide is present in the second lysis solution at a concentration of about 50 mM to about 100 mM.

[0077] Embodiment 39: The method of embodiment 37, wherein sodium hydroxide is present in the second lysis solution at a concentration of about 85 mM.

[0078] Embodiment 40: The method of any one of embodiments 36 to 39, wherein DMSO is present in the second lysis solution at a concentration of about 5% (v / v) to about 30% (v / v).

[0079] Embodiment 41: The method of embodiment 40, wherein DMSO is present in the second lysis solution at a concentration of about 10% (v / v) to about 25% (v / v).

[0080] Embodiment 42: The method of embodiment 40, wherein DMSO is present in the second lysis solution at a concentration of about 18% (v / v).

[0081] Embodiment 43: The method of any one of embodiments 36 to 42, wherein glycerol is present in the second lysis solution at a concentration of about 1% (v / v) to about 15% (v / v).

[0082] Embodiment 44: The method of embodiment 43, wherein glycerol is present in the second lysis solution at a concentration of about 3% (v / v) to about 10% (v / v).

[0083] Embodiment 45: The method of any one of embodiments 43, wherein glycerol is present in the second lysis solution at a concentration of about 7% (v / v).

[0084] Embodiment 46: The method of any one of embodiments 36 to 45, wherein the buffer in the second lysis solution is Tris and is present at a concentration of about 1 mM to about 50 mM.

[0085] Embodiment 47: The method of embodiment 46, wherein Tris is present in the second lysis solution at a concentration of about 5 mM to about 10 mM.

[0086] Embodiment 48: The method of any one of embodiments 36 to 47, wherein the second lysis solution further comprises a chelating agent.

[0087] Embodiment 49: The method of embodiment 48, wherein the chelating agent in the second lysis solution is 2,2',2'',2'''-(ethane-1,2-diyldinitrilo)tetraacetic acid (EDTA) and is present at a concentration of about 0.1 mM to about 4 mM.

[0088] Embodiment 50: The method of embodiment 49, wherein EDTA is present in the second lysis solution at a concentration of about 0.5 mM to about 2 mM.

[0089] Embodiment 51: The method of embodiment 49, wherein EDTA is present in the second lysis solution at a concentration of about 1 mM.

[0090] Embodiment 52: The method of any one of embodiments 1 to 51, wherein the conditions for lysing in step (e) comprise incubating the filter with the second lysis solution at an incubation temperature of about 80°C to about 150°C.

[0091] Embodiment 53: The method of embodiment 52, wherein the incubation temperature is from about 90°C to about 130°C.

[0092] Embodiment 54: The method of any one of embodiments 1 to 53, wherein the dissolving conditions in step (e) comprise sonication and / or ultrasound and / or electrolysis.

[0093] Embodiment 55: The method of any one of embodiments 1 to 54, wherein the eluting step (f) comprises passing an immiscible liquid through the filter to force an eluate containing the analyte through the filter.

[0094] Embodiment 56: The method of embodiment 55, wherein the immiscible liquid is selected from the group consisting of oils and hydrocarbons.

[0095] Embodiment 57: The method of embodiment 55 or 56, further comprising separating the eluate from the immiscible liquid.

[0096] Embodiment 58: The method of any one of embodiments 1 to 54, wherein the eluting step (f) comprises using the second lysis solution as an elution buffer, and the elution buffer is passed through a filter to obtain an eluate comprising the nucleic acids.

[0097] Embodiment 59: The method according to any one of embodiments 1 to 58, wherein the direction of fluid flow through the filter in eluting step (f) is the same as the direction of fluid flow applied in filtering step (c).

[0098] Embodiment 60: The method according to any one of embodiments 1 to 58, wherein the direction of fluid flow through the filter in eluting step (f) is opposite to the direction of fluid flow applied in filtering step (c).

[0099] Embodiment 61: The method of any one of embodiments 1 to 60, further comprising: (g) adding a neutralization buffer to the eluate obtained in step (f).

[0100] Embodiment 62: The method of embodiment 61, wherein the neutralization buffer comprises hydrochloric acid (HCl) and Tris.

[0101] Embodiment 63: The method of any one of embodiments 1 to 62, further comprising a washing step between steps (c) and (d), wherein the washing step comprises flushing a wash buffer through the filter.

[0102] Embodiment 64: The method of embodiment 63, wherein the wash buffer comprises EDTA and Tris, and optionally polysorbate 20.

[0103] Embodiment 65: The method of embodiment 63, wherein the wash buffer is the same as or a diluted concentration of the second lysis solution.

[0104] Embodiment 66: The method of any one of embodiments 1 to 62, wherein the method does not include a washing step between steps (c) and (d).

[0105] Embodiment 67: The method of any one of embodiments 1 to 66, wherein the mammalian cell is a human cell.

[0106] Embodiment 68: The method of any one of embodiments 1 to 67, wherein the mammalian cells are blood cells.

[0107] Embodiment 69: The method of any one of embodiments 1 to 68, wherein the analyte is a nucleic acid.

[0108] Embodiment 70: The method of any one of embodiments 1 to 69, further comprising analyzing the isolated analyte.

[0109] Embodiment 71: The method of embodiment 69, further comprising analyzing the isolated nucleic acid.

[0110] Embodiment 72: The method of embodiment 71, wherein analyzing the isolated nucleic acid comprises (i) performing an amplification reaction using the isolated nucleic acid as a template to generate an amplification product, and (ii) detecting the amplification product.

[0111] Embodiment 73: The method of embodiment 72, wherein the amplification reaction is PCR.

[0112] Embodiment 74: The method of embodiment 72, wherein the amplification reaction is an isothermal amplification reaction.

[0113] Embodiment 75: The method of embodiment 74, wherein the isothermal amplification reaction is a transcription-mediated amplification reaction.

[0114] Embodiment 76: The method of any one of embodiments 72 to 75, wherein the detecting step (ii) is performed in real time.

[0115] Embodiment 77: The method of any one of embodiments 72 to 75, wherein analyzing the isolated nucleic acid comprises immobilizing the isolated nucleic acid or the amplification product on a solid support.

[0116] Embodiment 78: The method of embodiment 77, wherein the isolated nucleic acid or amplification product hybridizes to an immobilized probe attached to a solid support.

[0117] Embodiment 79: The method of embodiment 78, wherein the immobilized probes are contained in a nucleic acid array.

[0118] Embodiment 80: A fluidic system configured to selectively isolate microbial cell analytes from a sample according to the method of any one of embodiments 1 to 71, the system comprising: an input port configured to receive the sample; a first reservoir configured to contain a first lysis solution; a lysis chamber configured to mix the sample with the first lysis solution to obtain a first lysate; a filter chamber containing a filter; a second reservoir configured to contain a second lysis solution; a third reservoir configured to contain an elution reagent effective to elute released analytes from the filter; an elution chamber configured to receive an eluate; and a fluidic channel array configured to provide (a) fluid communication between the lysis chamber and each of the input port and the first reservoir, and (b) fluid communication between the filter chamber and each of the lysis chamber, the second and third reservoirs, and the elution chamber.

[0119] Embodiment 81: A fluid system as described in embodiment 80, wherein the fluid channel array can be configured to direct fluid flow in a first direction from the lysis chamber through the filter chamber to the elution chamber, and can be selectively configured to direct fluid flow in the first direction from the filter chamber through the third reservoir to the elution chamber, or in a second direction through the filter chamber opposite to the first direction from the third reservoir through the filter chamber to the elution chamber.

[0120] Embodiment 82: The fluid system described in embodiment 80, further comprising at least one of a heater thermally coupled to the filter chamber, a sonotrode coupled to the filter chamber, or a conductive mesh positioned within the filter chamber to apply an electrolytic field.

[0121] Embodiment 83: A fluid system described in any one of embodiments 80 to 82, further comprising one or both of a fourth reservoir for neutralization buffer, the fluid channel array further providing fluid communication between the elution chamber and the fourth reservoir, and a fifth reservoir for wash buffer, the fluid channel array further providing fluid communication between the filter chamber and the fifth reservoir.

[0122] Embodiment 84: A fluid system described in any one of embodiments 80 to 83, further comprising one or both of a first dissolution solution and a second dissolution solution.

[0123] Embodiment 85: The fluid system of embodiment 84, wherein the fluid system comprises a first dissolution solution comprising guanidine hydrochloride at a concentration of about 1 M to about 8 M, polysorbate 20 at a concentration of about 0.5% (v / v) to about 5% (v / v), saponin at a concentration of about 0.5% (w / v) to about 5% (w / v), and Tris at a concentration of about 10 mM to about 100 mM.

[0124] Embodiment 86: A fluid system according to embodiment 84 or 85, comprising a second dissolution solution, the second dissolution solution comprising sodium hydroxide at a concentration of about 20 mM to about 500 mM, dimethyl sulfoxide (DMSO) at a concentration of about 5% (v / v) to about 30% (v / v), glycerol at a concentration of about 1% (v / v) to about 15% (v / v), and a buffer.

[0125] Embodiment 87: The fluidic system of embodiment 86, wherein the buffer in the second lysis solution is Tris and is present at a concentration of about 1 mM to about 50 mM.

[0126] Embodiment 88: A fluid system described in embodiment 86 or 87, wherein the second dissolution solution further comprises 2,2',2'',2'''-(ethane-1,2-diyldinitrilo)tetraacetic acid (EDTA) at a concentration of about 0.1 mM to about 4 mM.

[0127] Embodiment 89: A fluid system described in any one of embodiments 80 to 88, further comprising an amplification chamber for containing a nucleic acid amplification reaction, the amplification chamber being directly or indirectly connected or connectable to the elution chamber by a fluid channel array.

[0128] Embodiment 90: The fluid system described in embodiment 89, further comprising an analysis chamber in which a detection process is carried out to detect an analyte, and wherein the amplification chamber is directly or indirectly connected or connectable to the amplification chamber by a fluid channel array.

[0129] Embodiment 91: A fluidic system as described in embodiment 90, wherein the analytical chamber comprises a biosensor for detecting the presence of a specific target molecule in the sample.

[0130] Embodiment 92: A method for isolating an analyte from a sample, the method comprising: (a) providing a sample potentially containing intact cells; (b) filtering the sample through a filter having a pore size that retains the intact cells; (c) contacting the filter containing the retained cells with a lysis solution effective to lyse the cells and release the analyte therefrom; (d) providing conditions for lysing the retained cells in the lysis solution, whereby at least a portion of the cells are lysed and the analyte is released therefrom; and (e) passing an immiscible liquid through the filter to force an eluate containing the analyte through the filter.

[0131] Embodiment 93: The method of embodiment 92, further comprising separating the eluate from the immiscible liquid.

[0132] Embodiment 94: The method of embodiment 92 or 93, wherein the immiscible liquid is selected from the group consisting of oils and hydrocarbons.

[0133] Embodiment 95: The method of any one of embodiments 92 to 94, wherein the cell is a microbial cell.

[0134] Embodiment 96: The method of embodiment 95, wherein the microbial cells are bacterial cells.

[0135] Embodiment 97: The method of embodiment 95, wherein the microbial cell is a yeast cell.

[0136] Embodiment 98: The method of any one of embodiments 95 to 97, further comprising a selective lysis step prior to step (b), wherein the selective lysis step comprises selectively lysing any mammalian cells in the sample while leaving microbial cells intact.

[0137] Embodiment 99: The method of embodiment 98, wherein the mammalian cell is a human cell.

[0138] Embodiment 100: The method of embodiment 98 or 99, wherein the mammalian cells are blood cells.

[0139] Embodiment 101: The method of any one of embodiments 95 to 100, wherein the lysis solution in step (c) comprises sodium hydroxide, dimethyl sulfoxide (DMSO), glycerol and a buffer.

[0140] Embodiment 102: The method of embodiment 101, wherein sodium hydroxide is present in the lysis reaction at a concentration of about 20 mM to about 500 mM.

[0141] Embodiment 103: The method of embodiment 102, wherein sodium hydroxide is present in the lysis solution at a concentration of about 50 mM to about 100 mM.

[0142] Embodiment 104: The method of embodiment 102, wherein sodium hydroxide is present in the lysis reaction at a concentration of about 85 mM.

[0143] Embodiment 105: The method according to any one of embodiments 101 to 104, wherein DMSO is present in the lysis solution at a concentration of about 5% (v / v) to about 30% (v / v).

[0144] Embodiment 106: The method of embodiment 105, wherein DMSO is present in the lysis solution at a concentration of about 10% (v / v) to about 25% (v / v).

[0145] Embodiment 107: The method of embodiment 105, wherein DMSO is present in the lysis solution at a concentration of about 18% (v / v).

[0146] Embodiment 108: The method according to any one of embodiments 101 to 107, wherein glycerol is present in the lysis solution at a concentration of about 1% (v / v) to about 15% (v / v).

[0147] Embodiment 109: The method of embodiment 108, wherein glycerol is present in the lysis solution at a concentration of about 3% (v / v) to about 10% (v / v).

[0148] Embodiment 110: The method of any one of embodiments 101 to 108, wherein glycerol is present in the lysis solution at a concentration of about 7% (v / v).

[0149] Embodiment 111: The method according to any one of embodiments 101 to 110, wherein the buffer in the lysis solution is Tris and is present at a concentration of about 1 mM to about 50 mM.

[0150] Embodiment 112: The method of embodiment 111, wherein Tris is present in the lysis solution at a concentration of about 5 mM to about 10 mM.

[0151] Embodiment 113: The method of any one of embodiments 101 to 111, wherein the lysis solution further comprises a chelating agent.

[0152] Embodiment 114: The method of embodiment 113, wherein the chelating agent in the lysis solution is 2,2',2'',2'''-(ethane-1,2-diyldinitrilo)tetraacetic acid (EDTA) and is present at a concentration of about 0.1 mM to about 4 mM.

[0153] Embodiment 115: The method of embodiment 114, wherein EDTA is present in the lysis solution at a concentration of about 0.5 mM to about 2 mM.

[0154] Embodiment 116: The method of embodiment 114, wherein EDTA is present in the lysis solution at a concentration of about 1 mM.

[0155] Embodiment 117: The method of any one of embodiments 95 to 116, wherein the conditions for lysing in step (d) comprise incubating the filter with the lysis solution at an incubation temperature of about 80°C to about 150°C.

[0156] Embodiment 118: The method of embodiment 117, wherein the incubation temperature is from about 90°C to about 130°C.

[0157] Embodiment 119: The method of any one of embodiments 95 to 118, wherein the conditions for dissolving in step (d) comprise sonication and / or ultrasound and / or electrolysis.

[0158] Embodiment 120: The method of any one of embodiments 92 to 119, wherein the filter has a pore size of about 1 μm or less.

[0159] Embodiment 121: The method of embodiment 120, wherein the filter has a pore size of about 0.1 μm to about 1 μm.

[0160] Embodiment 122: The method of embodiment 120, wherein the filter has a pore size of about 0.2 μm to about 1 μm.

[0161] Embodiment 123: The method of embodiment 122, wherein the filter has a pore size of about 0.22 μm.

[0162] Embodiment 124: The method of any one of embodiments 92 to 123, wherein the filter comprises polyethersulfone (PES), cellulose, nylon, polyvinylidene fluoride (PVDF), polycarbonate, or glass fiber.

[0163] Embodiment 125: The method according to any one of embodiments 92 to 124, wherein the direction of fluid flow through the filter in step (e) is the same as the direction of fluid flow applied in filtering step (b).

[0164] Embodiment 126: The method of any one of embodiments 92 to 124, wherein the direction of fluid flow through the filter in step (e) is opposite to the direction of fluid flow applied in filtering step (b).

[0165] Embodiment 127: The method of any one of embodiments 92 to 126, further comprising adding a neutralization buffer to the eluate obtained in step (f).

[0166] Embodiment 128: The method of embodiment 127, wherein the neutralization buffer comprises hydrochloric acid (HCl) and Tris.

[0167] Embodiment 129: The method of any one of embodiments 92 to 128, further comprising a washing step between steps (b) and (c), wherein the washing step comprises flushing a wash buffer through the filter.

[0168] Embodiment 130: The method of embodiment 129, wherein the wash buffer comprises polysorbate 20, EDTA and Tris.

[0169] Embodiment 131: The method of embodiment 129, wherein the wash buffer is the same as the lysis solution or a diluted concentration of the lysis solution.

[0170] Embodiment 132: The method of any one of embodiments 92 to 128, which does not include a washing step between steps (b) and (c).

[0171] Embodiment 133: The method of any one of embodiments 92 to 132, wherein the analyte is a nucleic acid.

[0172] Embodiment 134: The method of any one of embodiments 92 to 133, further comprising analyzing the isolated analyte.

[0173] Embodiment 135: The method of embodiments 92 to 133, further comprising analyzing the isolated nucleic acid.

[0174] Embodiment 136: The method of embodiment 135, wherein analyzing the isolated nucleic acid comprises (i) performing an amplification reaction using the isolated nucleic acid as a template to generate an amplification product, and (ii) detecting the amplification product.

[0175] Embodiment 137: The method of embodiment 136, wherein the amplification reaction is PCR.

[0176] Embodiment 138: The method of embodiment 136, wherein the amplification reaction is an isothermal amplification reaction.

[0177] Embodiment 139: The method of embodiment 138, wherein the isothermal amplification reaction is a transcription-mediated amplification reaction.

[0178] Embodiment 140: The method of any one of embodiments 136 to 139, wherein the detecting step (ii) is performed in real time.

[0179] Embodiment 141: The method of any one of embodiments 136 to 139, wherein analyzing the isolated nucleic acid comprises immobilizing the isolated nucleic acid or the amplification product on a solid support.

[0180] Embodiment 142: The method of embodiment 141, wherein the isolated nucleic acid or amplification product hybridizes to an immobilized probe attached to a solid support.

[0181] Embodiment 143: The method of embodiment 142, wherein the immobilized probes are contained in a nucleic acid array.

[0182] Embodiment 144: The method of any one of embodiments 1 to 79, further comprising an enzyme treatment step between steps (c) and (d), wherein the enzyme treatment step comprises contacting the filter containing the retained microbial cells with an enzyme effective to degrade one or more microbial cell wall components.

[0183] Embodiment 145: The method of embodiment 144, wherein the enzyme is mutanolysin, lyticase, and / or lysozyme.

[0184] Embodiment 146: The method of any one of embodiments 92 to 143, further comprising an enzyme treatment step between steps (b) and (c), wherein the enzyme treatment step comprises contacting the filter containing the retained microbial cells with an enzyme effective to degrade one or more microbial cell wall components.

[0185] Embodiment 147: The method of embodiment 146, wherein the enzyme is mutanolysin, lyticase, and / or lysozyme.

[0186] Embodiment 148: The method of any one of embodiments 1 to 79, further comprising an enzyme treatment step between steps (c) and (d), wherein the enzyme treatment step comprises contacting the filter containing the retained microbial cells with an enzyme solution effective to degrade one or more microbial cell wall components.

[0187] Embodiment 149: The method of embodiment 148, wherein the enzyme is mutanolysin, lyticase, and / or lysozyme.

[0188] Embodiment 150: The method of any one of embodiments 92 to 143, further comprising an enzyme treatment step between steps (b) and (c), wherein the enzyme treatment step comprises contacting the filter containing the retained microbial cells with an enzyme solution effective to degrade one or more microbial cell wall components.

[0189] Embodiment 151: The method of embodiment 150, wherein the enzyme is mutanolysin, lyticase, and / or lysozyme.

[0190] Embodiment 152: The method of any one of embodiments 1 to 79, further comprising an enzyme treatment step between steps (c) and (d), wherein the enzyme treatment step comprises contacting the filter containing the retained microbial cells with an enzyme solution containing at least two enzymes, each effective to degrade one or more microbial cell wall components.

[0191] Embodiment 153: The method described in embodiment 152, wherein the at least two enzymes contained in the enzyme solution are mutanolysin at a concentration of about 0 U / mL to about 1,000 U / mL, lyticase at a concentration of about 0 U / mL to about 1,000 U / mL, and lysozyme at a concentration of about 0 U / mL to about 1,000 U / mL.

[0192] Embodiment 154: The method of any one of embodiments 92 to 143, further comprising an enzyme treatment step between steps (b) and (c), wherein the enzyme treatment step comprises contacting the filter containing the retained microbial cells with an enzyme solution containing at least two enzymes, each effective to degrade one or more microbial cell wall components.

[0193] Embodiment 155: The method described in embodiment 154, wherein the at least two enzymes contained in the enzyme solution are mutanolysin at a concentration of about 0 U / mL to about 1,000 U / mL, lyticase at a concentration of about 0 U / mL to about 1,000 U / mL, and lysozyme at a concentration of about 0 U / mL to about 1,000 U / mL.

[0194] Embodiment 156: A fluidic system configured for selectively isolating a microbial cell analyte from a sample, the system comprising: an input port configured to receive a sample containing mammalian cells and potentially containing microbial cells; a first reservoir configured to contain a first lysis solution; a lysis chamber configured to mix the sample with the first lysis solution to obtain a first lysate containing lysed mammalian cells and, if present, intact microbial cells, the lysis chamber being in fluid communication with the input port and the first reservoir; a filter chamber containing a filter having a pore size of about 0.1 μm to about 1 μm, and / or an asymmetric filter comprising first side pores having a size of about 5 μm to about 20 μm and second side pores having a size of up to about 0.2 μm. a filter chamber in fluid communication with a lysis chamber and a waste chamber configured to receive a lysate; a second reservoir configured to contain a second lysis solution, the second reservoir in fluid communication with the filter chamber; a third reservoir configured to contain an elution reagent, the third reservoir in fluid communication with the filter chamber; an elution chamber configured to receive an eluate, the elution chamber in fluid communication with the filter chamber; and a fluid channel array configured to provide (a) fluid communication between the lysis chamber and each of the inlet port and the first reservoir, and (b) fluid communication between the filter chamber and each of the lysis chamber, the second and third reservoirs, and the elution chamber.

[0195] Embodiment 157: A dissolution solution comprising sodium hydroxide at a concentration of about 20 mM to about 500 mM, dimethyl sulfoxide (DMSO) at a concentration of about 5% (v / v) to about 30% (v / v), glycerol at a concentration of about 1% (v / v) to about 5% (v / v), a chelating agent, and a buffer.

[0196] Embodiment 158: The lysis buffer of embodiment 157, wherein sodium hydroxide is present in the second lysis solution at a concentration of about 20 mM or about 85 mM.

[0197] Embodiment 159: The lysis solution of embodiment 157 or 158, wherein DMSO is present in the second lysis solution at a concentration of about 18% (v / v).

[0198] Embodiment 160: A lysis solution according to any one of embodiments 157 to 159, wherein glycerol is present in the second lysis solution at a concentration of about 7% (v / v).

[0199] Embodiment 161: The lysis solution of any one of embodiments 157 to 160, wherein EDTA is present in the second lysis solution at a concentration of about 1 mM.

[0200] Embodiment 162: A lysis solution according to any one of embodiments 157 to 161, wherein Tris is present in the second lysis solution at a concentration of about 5 mM to about 10 mM.

[0201] definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art related to the methods and compositions being described. As used herein, the following terms and phrases have the meanings ascribed to them unless specified otherwise.

[0202] The terms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0203] As used herein, the terms "microbe," "microorganism," and "microbial" refer to bacteria, archaea, fungi, and protists. In some embodiments, the microbe is a prokaryote (i.e., a species of bacteria or archaea). In other embodiments, the microbe is a microbial species having a cell wall, such as any species of bacteria, archaea, or fungi, or some protist species.

[0204] As used herein with respect to cells (e.g., microbial cells), the term "intact" means a cell that has a substantially undisrupted cell membrane and / or cell wall such that intracellular components are generally retained within the cell.

[0205] As used herein, "analyte" refers to a substance or one or more components thereof for identification and / or characterization, such as, for example, detection via a probe or sequencing. Examples of analytes include, but are not limited to, DNA, RNA, and protein. In the context of the present disclosure, an analyte is a component of a cell (e.g., a microbial cell).

[0206] A "sample" includes any specimen that may contain an analyte. A sample includes a "biological sample," which includes any tissue or material derived from a living or dead mammal (e.g., a human). A sample can also include processed samples, such as samples obtained from passing a sample over or through a filtration device, or after centrifugation, or by attachment to a medium, matrix, or support.

[0207] "Detergent" refers to a substance that can disperse hydrophobic substances (e.g., lipids) in water by emulsification and can be used to dissolve or solubilize biological samples for subsequent analysis. Detergents can be ionic or non-ionic.

[0208] A buffer refers to a weak acid or base used to maintain the pH of a solution.

[0209] As used herein, the term "immiscible liquid" refers to any liquid that is not miscible with water, such that the immiscible liquid and the aqueous phase solution do not mix to achieve homogeneity. Immiscible liquids typically have a high carbon content and may include, for example, silicone oil, mineral oil, fluorocarbon oil, vegetable oil, or combinations thereof, among others.

[0210] As used herein with respect to filters, the term "asymmetric structure" refers to a filter having pores with variable pore sizes, where the pores on one side of the filter are generally larger than the pores on the other side of the filter.

[0211] "Nucleic acid" refers to a polymeric compound containing nucleotides or analogs having nitrogenous heterocyclic bases or base analogs linked together to form polymers, including conventional RNA, DNA, mixed RNA-DNA, and their analogs.

[0212] The term "microfluidic device" or "microfluidic cartridge" refers to a device through which fluid can flow and in which at least a portion of any fluid passage, chamber, etc. in which the fluid flows or is held is geometrically constrained to a small scale (e.g., sub-millimeter) such that surface forces acting on the fluid meet or exceed volumetric forces.

[0213] As used herein, the term "biosensor" means a device that measures a biological or chemical reaction by producing a signal that is proportional to the concentration of an analyte in the reaction.

[0214] As used herein, the term "nucleic acid array" refers to a solid support on which a collection of target-specific nucleic acids is arranged at predetermined locations, either by spotted or directed synthesis.

[0215] As used herein, a "nucleotide" is a subunit of a nucleic acid consisting of a phosphate group, a five-carbon sugar, and a nitrogenous base (also referred to herein as a "nucleobase"). The five-carbon sugar found in RNA is ribose. In DNA, the five-carbon sugar is 2'-deoxyribose.

[0216] "Oligomer," "oligonucleotide," or "oligo" generally refers to a nucleic acid less than 1,000 nucleotides (nt), including those with a size range having a lower limit of about 5 nt and an upper limit of about 900 nt. The term oligonucleotide does not indicate any specific function for the reagent, but is used generically to encompass all such reagents described herein. Although oligomers can be referred to by functional names (e.g., capture probe, detection probe, primer, or promoter primer), those skilled in the art will understand that these terms refer to oligomers.

[0217] Any reference to "pore diameter" or pore size refers to a measure of each pore that characterizes the size of the largest particle that can pass through the pore. For example, in the context of pores having a circular or nearly circular cross-sectional shape, the pore diameter may refer to the diameter of the shape. In the context of pores having a more rectangular or elliptical shape, the pore diameter may refer to the smaller width dimension of the shape, corresponding to the width of the largest particle that can pass through the pore.

[0218] As used herein, the term "target sequence" or "target nucleic acid sequence" refers to a specific nucleotide sequence of a nucleic acid analyte to be amplified and / or detected. A "target sequence" includes a complexing sequence to which an oligonucleotide (e.g., a priming oligonucleotide and / or a promoter oligonucleotide) complexes during an amplification process (e.g., PCR, TMA). Unless the context clearly indicates otherwise, if the nucleic acid analyte is originally single-stranded, the term "target sequence" also refers to a sequence complementary to the "target sequence" present in the nucleic acid analyte; if the nucleic acid analyte is originally double-stranded, the term "target sequence" refers to both the sense (+) strand and the antisense (-) strand.

[0219] "Nucleic acid amplification" refers to any well-known in vitro procedure that produces multiple copies of a target nucleic acid sequence. Examples of such procedures include transcription-related methods such as transcription-mediated amplification (TMA), nucleic acid sequence-based amplification (NASBA), etc. (e.g., U.S. Pat. Nos. 5,399,491, 5,554,516, 5,437,990, 5,130,238, 4,868,105, and 5,124,246), replicase-mediated amplification (e.g., U.S. Pat. No. 4,786,600), polymerase chain reaction (PCR) (e.g., U.S. Pat. Nos. 4,683,195, 4,683,202, and 4,800,159), ligase chain reaction (LCR) (e.g., European Patent No. 0320308), and strand displacement amplification (SDA) (e.g., U.S. Pat. No. 5,422,252).

[0220] "Amplicon" or "amplification product" refers to a nucleic acid molecule produced in a nucleic acid amplification reaction and derived from a nucleic acid analyte. The amplicon or amplification product contains a target nucleic acid sequence that can be of the same or opposite sense as the nucleic acid analyte.

[0221] An "amplification oligonucleotide" or "amplification oligomer" is an oligonucleotide that hybridizes to a nucleic acid analyte and participates in a nucleic acid amplification reaction, e.g., serves as a primer. An amplification oligomer can have a 3' end that is extended by polymerization as part of a nucleic acid amplification reaction. Alternatively, an amplification oligomer can have a 3' end that is not extended by polymerization but provides a moiety that facilitates nucleic acid amplification, such as a promoter sequence attached 5' to the target-specific sequence of the amplification oligomer. Such an amplification oligomer is referred to as a promoter-provider. An amplification oligomer that provides both a 3' target-specific sequence and a 5' promoter sequence that can be extended by polymerization is referred to as a promoter-primer. Amplification oligomers may be optionally modified to include 5' non-target-specific sequences, such as tags, (referred to as) promoters, or other sequences used or useful for manipulating or amplifying the primer or target oligonucleotide.

[0222] "Detection probe oligomer," "detection probe," or "probe" refers to an oligomer that specifically hybridizes to a target sequence, including an amplification product, under conditions that promote nucleic acid hybridization for the detection of a nucleic acid analyte. Detection can be either direct (i.e., a probe hybridized directly to the target) or indirect (i.e., a probe hybridized to an intermediate structure that links the probe to the target). The target-specific sequence of a probe generally refers to the specific sequence within a larger sequence to which the probe specifically hybridizes. Detection probes can include target-specific and non-target-specific sequences. Such non-target-specific sequences can include sequences that impart desired secondary or tertiary structures, such as hairpin structures, that can be used to facilitate detection and / or amplification.

[0223] A "label" or "detectable label" refers to a moiety or compound directly or indirectly attached to a probe that is detected or provides a detectable signal. Direct attachment may use covalent or non-covalent interactions (e.g., hydrogen bonding, hydrophobic or ionic interactions, and the formation of chelate or coordination complexes), while indirect attachment may use a bridging moiety or linker (e.g., via an antibody or additional oligonucleotide that amplifies the detectable signal). Any detectable moiety may be used, such as radionuclides, ligands such as biotin or avidin, enzymes, enzyme substrates, reactive groups, chromophores such as dyes or particles (e.g., latex or metal beads) that impart a detectable color, luminescent compounds (e.g., bioluminescent, phosphorescent, or chemiluminescent compounds such as acridinium ester ("AE") compounds), and fluorescent compounds (i.e., fluorophores). Fluorophores may be used in combination with a quencher molecule that absorbs light when in close proximity to the fluorophore, thereby reducing background fluorescence. Detectably labeled probes include, for example, hydrolysis (eg, TaqMan™) probes, AE-labeled probes, molecular torches, and molecular beacons.

[0224] References herein to numerical ranges (eg, "X to Y" or "X to Y") include the endpoints defining the range, and all values ​​subsumed within the range.

[0225] As used herein, the terms "first" and "second" preceding the name of an element (e.g., a component, device, position, feature or portion thereof, or direction of movement, force, or other dynamic action) are used for distinguishing purposes to distinguish between similar elements and are not necessarily intended to denote order, nor are the terms "first" and "second" intended to exclude the inclusion of additional similar elements. Furthermore, the use of the term "first" preceding the name of an element (e.g., a component, device, position, feature or portion thereof, or direction of movement, force, or other dynamic action) does not necessarily imply or require the presence of more such elements, e.g., a "second," a "third," etc.

[0226] The description may use various terms that describe the relative spatial arrangement and / or orientation or direction of a component, device, location, feature or portion thereof, or when describing a direction of movement, force or other dynamic action. Unless otherwise stated or dictated by the context of the description, such terms, including but not limited to top, bottom, above, below, on top of, upper, lower, left, right, front of, behind, below, next to, adjacent, between, horizontal, vertical, diagonal, longitudinal, transverse, radial, axial, clockwise, counter-clockwise, etc., are used for convenience in referring to such components, devices, locations, features or portions thereof, or to motions, forces, or other dynamic actions depicted in the drawings, and are not intended to be limiting.

[0227] Unless otherwise indicated or suggested otherwise by context, terms used herein to describe the physical and / or spatial relationship between a first component, structure, or portion thereof and a second component, structure, or portion thereof, such as attached, connected, fixed, joined, coupled, connected, or similar terms or variations of such terms, are intended to encompass both a direct relationship in which the first component, structure, or portion thereof is in direct contact with the second component, structure, or portion thereof, or a relationship in which there are one or more intervening components, structures, or portions thereof between the first component, structure, or portion thereof and the second component, structure, or portion thereof.

[0228] The term "fluid communication" means either direct fluid communication, e.g., two regions can be in fluid communication with each other through an unobstructed fluid passageway connecting the two regions, or can be in fluid communication, e.g., two regions can be in fluid communication with each other when connected through a fluid passageway that may include a valve installed therein, such that opening the valve can establish fluid communication between the two regions, e.g., by melting a dissolvable valve, rupturing a rupturable seal, or actuating a mechanical valve installed in the fluid passageway.

[0229] As used herein, the term "about" or "approximately" applies to all numerical values ​​and terms indicating a particular physical orientation or relationship, such as horizontal, vertical, parallel, perpendicular, concentric, or similar terms, as specified herein, whether explicitly stated or not. This term generally refers to a range of numbers, orientations, and relationships that, in the context of this disclosure, one of ordinary skill in the art would consider a reasonable amount of deviation from the recited numerical values, orientations, and relationships (i.e., having an equivalent function or result). For example, and without intending to be limiting, this term can be interpreted to include a ±10 percent deviation from a given numerical value, orientation, or relationship, provided that such deviation does not alter the ultimate function or result of the stated value, orientation, or relationship. Thus, in some circumstances, a value of about or approximately 1% can be interpreted as being in the range of 0.9% to 1.1%, as would be understood by one of ordinary skill in the art.

[0230] Detailed Description The present disclosure provides methods and systems for isolating analytes from samples, including methods and systems for selectively isolating microbial cell analytes from samples containing mammalian cells. The disclosed methods and systems are particularly useful, for example, for increasing the sensitivity of downstream molecular diagnostic assays targeting the isolated analytes. For example, detection sensitivity in analytical applications such as nucleic acid amplification and detection generally depends on the amount of analyte introduced into the detection reaction. While typical molecular diagnostic applications are considered highly sensitive, typical analyte extraction procedures for such applications (e.g., DNA or RNA extraction for downstream use in amplification) do not provide the necessary sensitivity. In certain aspects, the present disclosure addresses this challenge by providing a method for concentrating microbial cell analytes that may be present in relatively small amounts in a larger volume sample containing mammalian cells, using selective lysis to lyse mammalian cells and filtering the lysate to capture and isolate microbial cells, while allowing the mammalian DNA released by the selective lysis process to pass through the filter. In this way, overall assay sensitivity can be improved. In other aspects, the present disclosure provides methods for isolating analytes from a sample using on-filter lysis of intact cells, followed by elution using an immiscible liquid to force the eluate containing the released analytes through the filter. The methods and corresponding systems are applicable, for example, to molecular diagnostics for nucleic acid detection (e.g., by quantitative PCR, nucleic acid arrays and / or other microarrays, and other nucleic acid detection modalities such as, for example, next-generation sequencing), as well as detection of other biomolecules such as proteins (e.g., ELISA, protein arrays, MALDI-TOF, nanoLC / UPLC-ESI-MS, and other related detection modalities).

[0231] One concept of this disclosure is the use of size-selective filtering to concentrate microorganisms from vast background matrix volumes (e.g., blood or other biological matrices), thus both concentrating the analyte concentration in the sample and replacing the sample matrix with another compatible downstream analytical procedure (e.g., PCR or other detection methods, including immunological detection). Therefore, large sample volumes (e.g., up to approximately 5 mL) can be processed to detect analytes that may be present at very low concentrations. For example, the initial concentration of microorganisms in the blood of patients with bloodstream infections (BSIs) can be very low (e.g., 1 or 2 microorganisms / mL). In such cases, it may be advantageous to filter a larger sample volume so that the concentrated eluate contains enough target to be detected. At the same time, maximizing the volume can be difficult due to the potential for clogging the filter: for example, blood samples can vary significantly in patients with infections, with different levels of white blood cells, proteins, infection markers, hematocrit, clotting factors, etc. In certain aspects of the present disclosure, this problem is addressed by formulating the dissolution solution for selective dissolution (the "first dissolution solution" disclosed herein; e.g., a dissolution solution comprising guanidine hydrochloride, saponin, and polysorbate 20 as described herein) and / or by using a filter comprising an advantageous membrane type (e.g., polyethersulfone (PES)).

[0232] Size selection is achieved by using specific filters and filter materials with selected pore sizes, optionally with an asymmetric structure so that pore size decreases with downstream movement through the filter membrane. In some examples, prokaryotic and eukaryotic target organisms (bacteria and yeast) are captured with filters with pore sizes of, for example, 0.22 μm and constructed of, for example, polyethersulfone (PES) or some other applicable membrane material. The selected membrane size and material enable efficient capture of organisms containing the target analytes, and furthermore, the membrane type does not easily clog, even with larger sample volumes or by excessive washing of the membrane for particles smaller than the pore size. On-filter lysis is used to transfer analytes to downstream analytical processes (e.g., PCR, isothermal RNA / DNA amplification, protein, or other biomolecular analyte detection). Microorganisms / pathogenic organisms captured by the filter are lysed / disintegrated by chemical and / or physical methods, which release the analyte molecules from the intact organisms and allow the analytes to be released from the capture filter. Another possibility is to employ reverse elution flow on the filter by flowing the sample solution through the filter membrane in one direction and the elution reagent through the filter membrane in the opposite direction. Lysing intact analyte-containing organisms is not necessary, as the organisms are released from the capture filter surface / matrix by reversing the flow. Another option is to use a combination of the two previous options.

[0233] By way of example, dissolution / disintegration methods described herein may consist of, for example, (a) chemical dissolution using detergents and / or chaotropic salts and / or extreme pH values, glycerol / other alcohols; (b) enzymatic dissolution on the filter; (c) physical dissolution using an externally controlled heat source embedded in the filter or induction-mediated heating where a conductive or semi-conductive material such as brass, copper, steel, iron, aluminum, graphite, carbon or silicon is embedded on / in the filter matrix, for example by lamination of a porous mesh or co-molding with the actual filter material; (d) physical dissolution by direct electricity (electrolysis), for example where a conductive mesh structure is placed on both sides of the filter by lamination or similar process, or sonication / ultrasonication using a sonotrode in contact with the filter, or any combination of chemical / enzymatic / physical dissolution / disintegration.

[0234] Fluid Systems As shown in FIG. 1 , the present disclosure provides a fluidic system 100 for isolating analytes from a sample and analyzing (e.g., detecting) the isolated analytes using the methods described herein. System 100 may include a fluidic cartridge (e.g., a microfluidic cartridge) configured to be operably coupled to a processing device or docking station (not shown), which includes a substrate that may include a first substrate portion 102a containing components of a first fluidic system 100a for isolating analytes from a sample, such as chambers interconnected by microchannels that may include valves for controlling fluid flow through the microchannels, and a second substrate portion 102b containing components of a second fluidic system 100b for analyzing the isolated analytes, such as additional chambers interconnected by microchannels that may include valves for controlling fluid flow through the microchannels. Dashed line 102 separates first substrate portion 102a and first fluidic system 100a from second substrate portion 102b and second fluidic system 100b. The dashed line 102 signifies that the first substrate portion 102a and the second substrate portion 102b may comprise a single continuous substrate, or that the first substrate portion 102a and the second substrate portion 102b may be separate, distinct substrates (e.g., separate cartridges) that may be fluidly coupled to one another.

[0235] The first and second substrate portions 102a and 102b may be made from suitable materials that can be molded and / or machined to form the features of the first and second fluidic systems 100a and 100b and that do not adversely react with the sample or reaction process materials. Suitable materials include thermoplastic polymer materials such as cyclic olefin copolymer (COC) or cyclic olefin polymer (COP), or any thermoplastic polymer suitable for injection molding. In one embodiment, the first and second substrate portions 102a and 102b may be formed from polypropylene (PP). Other exemplary materials may be selected from the group including polycarbonate, polyacrylamide, polyethylene, polymethyl methacrylate (PMMA), polydimethylsiloxane (PDMS), and polyvinyl chloride (PVC).

[0236] The microchannels may be defined by grooves formed in the outer surface of the first substrate portion 102a or the second substrate portion 102b and covered with a membrane secured to the outer surface of the substrate portion. The chambers may be defined by recessed cavities formed in the outer surface of the first substrate portion 102a or the second substrate portion 102b and covered with a film secured to the outer surface of the substrate portion. The valves may include occlusions in the microchannels that are changeable from a closed state, which prevents fluid flow through the associated microchannel, to an open state, which allows fluid flow through the associated microchannel. The valves may include dissolvable or rupturable occlusions or flexible seals that can be operated between closed and open states, for example, by an external actuator. In one embodiment, the valves are changeable between closed and open states, and vice versa, to enable selective fluid flow control through the associated microchannel. Exemplary cartridge designs and associated processing device elements that may be applicable to the methods and systems described herein are described in U.S. Pat. No. 10,654,039.

[0237] In some embodiments, the first fluidic system 100a provides a platform for selectively isolating microbial cell analytes from a sample using selective lysis and filtration methods or using immiscible liquid elution as described herein. The first fluidic system 100a generally includes an input port or sample chamber or reservoir 104 (which may include an input port) configured to receive a sample, a first reservoir 106 that may contain a first process fluid, such as a first lysis solution; a lysis chamber 108 for mixing the sample with the first lysis solution to obtain a first lysate; a filter chamber 120 that includes a filter and may also include components 122 for performing physical lysis on the filter; a second reservoir 110 that may contain a second process fluid, such as a second lysis solution; and a second reservoir 110 for discharging the sample. a third reservoir 116 that may contain a third process fluid, such as an elution reagent effective to elute the analytes from the filter; an elution chamber 124 for receiving an eluate; and a fluid channel array (described in more detail below) configured to provide (a) fluid communication between the lysis chamber 108 and each of the inlet port / sample chamber 104 and the first reservoir 106, and (b) fluid communication between the filter chamber 120 and each of the lysis chamber 108, the second reservoir 110 and the third reservoir 116, and the elution chamber 124.

[0238] Components 122 of filter chamber 120 may include a heat source thermally coupled to the filter chamber (e.g., an induction-mediated heater embedded in the filter to heat the filter with the retained microbial cells in the presence of the second lysis solution), a sonotrode coupled to the filter chamber (e.g., to apply sonication or ultrasound with the retained microbial cells in the presence of the second lysis solution), or a conductive mesh structure disposed in the filter chamber (e.g., to apply electrolysis with the retained microbial cells in the presence of the second lysis solution).

[0239] The first fluid system 100a may also include a fourth reservoir 114 that may contain a fourth process fluid such as a neutralization buffer, and the fluid channel array further provides fluid communication between the elution chamber 124 and the fourth reservoir 114 and / or a fifth reservoir 112 that may contain a fifth process fluid such as a wash buffer, and the fluid channel array further provides fluid communication between the filter chamber 120 and the fifth reservoir 112.

[0240] In the schematic diagram of FIG. 1, the elution chamber 124 and the fourth reservoir 114 are shown as being located within the second substrate portion 102b, but the elution chamber 124 and the fourth reservoir 114 may be located within the first substrate portion 102a, or, as described below, the elution chamber 124 may be omitted.

[0241] Solutions and other fluid materials contained in reservoirs of the first fluid system 100a may initially be stored as dry, reconstitutable materials, and such dry materials may initially be stored in corresponding reservoirs, where a reconstitution reagent is combined with the dry material to form a solution within the reservoir. The reconstitution reagent may initially be stored in a reservoir external to the first fluid system 100a or in one or more additional reservoirs that are part of the first fluid system 100a. The fluid channel array of the first fluid system 100a may also include microchannels and valves for transport of the reconstitution reagent to the reservoirs containing the dry materials.

[0242] The first fluid system 100 a may also include a waste chamber 118 , with the fluid channel array further providing fluid communication between the filter chamber 120 and the waste chamber 118 .

[0243] First fluid system 100a includes a first fluid channel array that provides selective fluid connections between various reservoirs, chambers, and other components of system 100. The first fluid channel array may include several fluid channels or conduits through which fluid may flow in a first or second direction, and valves for controlling fluid flow through particular channels (e.g., controlling fluid flow by allowing or preventing fluid flow through the channels). For example, in system 100 shown in FIG. 1, sample reservoir 104 may be connected by channel 10 to valve 50 that is configured to be selectively operated (e.g., by an actuator of a processing device to which first fluid system 100a is coupled) to (a) prevent fluid flow from sample reservoir 104, (b) direct fluid flow from sample reservoir 104 to lysis chamber 108 via channel 10a, or (c) direct fluid flow from sample reservoir 104 to filter chamber 120 via channel 10b.

[0244] The first reservoir 106 may be connected to the lysis chamber 108 by channel 12 via a valve 52 configured to be operated (e.g., by an actuator of a processing device to which the first fluid system 100a is coupled) to selectively prevent fluid flow from the first reservoir 106 or to direct fluid flow from the first reservoir 106 to the lysis chamber 108 via channel 12.

[0245] The lysis chamber 108 may be connected to the filter chamber 20 by the channel 14 via a valve 54 configured to be operated (e.g., by an actuator of a processing device to which the first fluid system 100a is coupled) to selectively prevent fluid flow from the lysis chamber 108 or to direct fluid flow from the lysis chamber 108 to the filter chamber 120 via the channel 14.

[0246] The second reservoir 110 may be connected to the filter chamber 120 by channel 16 via a valve 56 configured to be operated (e.g., by an actuator of a processing device to which the first fluid system 100a is coupled) to selectively prevent fluid flow from the second reservoir 110 or to direct fluid flow from the second reservoir 110 to the filter chamber 120 via channel 16.

[0247] The third reservoir 116 may be connected to a valve 60 by a channel 20 (e.g., by an actuator of a processing device to which the first fluid system 100a is coupled), and the valve is configured to be selectively operated to (a) prevent fluid flow from the third reservoir 116, (b) direct fluid flow from the third reservoir 116 to the filter chamber 120 via channel 20a in direction "A" (from right to left in FIG. 1), or (c) direct fluid flow from the third reservoir 116 to the filter chamber 120 via channel 20b in direction "B" (from left to right in FIG. 1).

[0248] The fifth reservoir 112 may be connected to the filter chamber 120 by channel 18 via a valve 58 configured to be operated (e.g., by an actuator of a processing device to which the first fluid system 100a is coupled) to selectively prevent fluid flow from the fifth reservoir 112 or to direct fluid flow from the fifth reservoir 112 to the filter chamber 120 via channel 18.

[0249] The filter chamber 120 may be connected to the waste chamber 118 by the channel 22 via a valve 64 configured to be operated (e.g., by an actuator of a processing device to which the first fluid system 100a is coupled) to selectively prevent fluid flow through the channel 22 or to direct fluid flow from the filter chamber 120 to the waste chamber 118 via the channel 22.

[0250] Filter chamber 120 may be connected by channels 24a and 24b to a valve 62 configured to be operated (e.g., by an actuator of a processing device to which first fluid system 100a is coupled) to (a) prevent fluid flow through channels 24a and 24b, (b) direct fluid flow from filter chamber 120 to elution chamber 124 in direction "A" via channels 24a and 24b, or (c) direct fluid flow from filter chamber 120 to elution chamber 124 in direction "B" via channels 24b and 24b.

[0251] The fourth reservoir 114 may be connected to the elution chamber 124 by the channel 26 via a valve 66 configured to be operated (e.g., by an actuator of a processing device to which the first fluid system 100a or the second fluid system 100b is coupled) to selectively prevent fluid flow from the fourth reservoir 114 or to direct fluid flow from the fourth reservoir 114 to the elution chamber 124 via the channel 26.

[0252] Fluid flow through the various channels may be achieved by any known means, including one or more sources of negative and / or positive pressure (e.g., a syringe pump (not shown) drawing fluid from one reservoir, such as sample reservoir 104, and then expelling fluid into another reservoir, such as lysis chamber 108), or capillary action.

[0253] In some variations, the fluid system 100 further includes one or both of a first lysis solution contained in the first reservoir 106 and a second lysis solution contained in the second reservoir 110, as described herein. For example, in certain embodiments, the system includes a first lysis solution containing guanidine hydrochloride at a concentration of about 1 M to about 8 M, polysorbate 20 at a concentration of about 0.5% (v / v) to about 5% (v / v), saponin at a concentration of about 0.5% (w / v) to about 5% (w / v), and a buffer (e.g., Tris) at a concentration of about 10 mM to about 100 mM (e.g., a first lysis solution containing guanidine hydrochloride at a concentration of about 4 M, polysorbate 20 at a concentration of about 4% (v / v), saponin at a concentration of about 4% (w / v), and Tris at a concentration of about 40.5 mM). In other, non-mutually exclusive embodiments, the system includes a second lysis solution containing sodium hydroxide at a concentration of about 20 mM to about 500 mM, dimethyl sulfoxide (DMSO) at a concentration of about 5% (v / v) to about 30% (v / v), glycerol at a concentration of about 1% (v / v) to about 15% (v / v), and a buffer such as Tris at a concentration of about 1 mM to about 50 mM (e.g., a second lysis solution containing sodium hydroxide at a concentration of about 85 mM, DMSO at a concentration of about 18% (v / v), glycerol at a concentration of about 7% (v / v), EDTA at a concentration of about 1 mM, and Tris at a concentration of about 7.5 mM). In some variations involving the second lysis solution described above, the second lysis solution further comprises a chelating agent, such as 2,2',2'',2''-(ethane-1,2-diyldinitrilo)tetraacetic acid (EDTA), for example, at a concentration of about 0.1 mM to about 4 mM (e.g., about 1 mM). In some embodiments, the second lysis solution described above consists of or consists essentially of sodium hydroxide, DMSO, glycerol, a chelating agent, a buffer, and water.

[0254] In other embodiments, the fluidic system is for isolating an analyte from a sample using the immiscible liquid elution methods described herein. In some such embodiments, the fluidic system is the system described above for selective lysis and filtration, and the elution reagent is an immiscible liquid. In some variations, the system further comprises an immiscible liquid.

[0255] In some embodiments, the second fluidic system 100b provides a platform for performing one or more processes related to the analysis of analytes, including, for example, target analyte isolation (target capture), target analyte amplification, and target analyte detection. The second fluidic system 100b may include a sample preparation chamber (e.g., a target isolation chamber or target capture chamber) 126, an amplification chamber 128, a hybridization / detection (or analysis) chamber 130, a hybridization buffer reservoir 138, a hybridization wash buffer reservoir 140, a wash buffer reservoir 134, an elution buffer / amplification reagent reservoir 136, and a waste chamber 132. In an alternative embodiment, the first fluidic system 100a and the second fluidic system 100b may share a common waste chamber.

[0256] Solutions and other fluid materials contained in reservoirs of second fluid system 100b may initially be stored as dry, reconstitutable materials, and such dry materials may initially be stored in corresponding reservoirs where a reconstitution reagent is combined with the dry materials to form a solution within the reservoir. Reconstitution agents may initially be stored in reservoirs external to second fluid system 100b or in one or more additional reservoirs that are part of second fluid system 100b. The fluid channel array of second fluid system 100b may also be microchannels and valves for transporting reconstitution agents to reservoirs containing dry materials.

[0257] The sample preparation chamber may include a target isolation chamber 126 (also referred to as "isolation chamber 126") for isolating or capturing one or more specific analytes. The target isolation chamber 126 may contain a silica-like membrane for DNA binding. According to various embodiments, the target isolation chamber 126 may contain, for example, a gel, beads, or a paper filter for DNA binding and concentration. By way of example, agarose gel, silica beads, and filter paper such as cellulose, base purification may also be used.

[0258] The second fluidic system 100b includes a second fluidic channel array that provides selective fluid connections between various reservoirs, chambers, and other components of the system 100. The second fluidic channel array may include several fluidic channels or conduits through which fluid may flow in a first or second direction, and valves for controlling the flow of fluid through particular conduits. For example, in the system 100 shown in FIG. 1, the elution chamber 124 may be connected to the isolation chamber 126 by channel 28 via a valve 68 configured to be operated (e.g., by an actuator of a processing device to which the second fluidic system 100b is coupled) to selectively prevent fluid flow from the elution chamber 124 or direct fluid flow from the elution chamber 124 to the target isolation chamber 126 via channel 28. In an alternative arrangement, the target isolation chamber 126 may be connected to the filter chamber 120 of the first fluidic system 100a via channels 24, 24a, 24b controlled by valve 62, and the elution chamber 124 and the fourth reservoir 114 may be omitted. In another alternative embodiment, the elution chamber 124 may be omitted and the fourth reservoir 114 may be connected directly to the target isolation chamber 126 without an intervening elution chamber.

[0259] The wash buffer reservoir 134 may be connected to the target isolation chamber 126 by channel 36 via a valve 76 configured to be operated (e.g., by an actuator of a processing device to which the second fluid system 100b is coupled) to selectively prevent fluid flow from the wash buffer reservoir 134 or to direct fluid flow from the wash buffer reservoir 134 to the target isolation chamber 126 via channel 36.

[0260] The elution buffer / amplification reagent reservoir 136 may be connected to the isolation chamber 126 by channel 38 via a valve 78 configured to be operated (e.g., by an actuator of a processing device to which the second fluid system 100b is coupled) to selectively prevent fluid flow from the elution buffer / amplification reagent reservoir 136 or to direct fluid flow from the elution buffer / amplification reagent reservoir 136 to the isolation chamber 126 via channel 38.

[0261] The target isolation chamber 126 may be connected to the waste chamber 132 by the channel 30 via a valve 70 configured to be operated (e.g., by an actuator of a processing device to which the second fluid system 100b is coupled) to selectively prevent fluid flow through the channel 30 or to direct fluid flow from the target isolation chamber 126 to the waste chamber 132 via the channel 30.

[0262] The target isolation chamber 126 may be connected to the amplification chamber 128 by channel 32 via a valve 72 configured to be operated (e.g., by an actuator of a processing device to which the second fluid system 100b is coupled) to selectively prevent fluid flow through channel 32 or to direct fluid flow from the target isolation chamber 126 to the amplification chamber 128 via channel 32.

[0263] In embodiments in which the protocol for analyte detection does not include a process for isolating and purifying microbial nucleic acids, target isolation chamber 126, wash buffer reservoir 134, elution buffer / amplification reagent reservoir 136, and waste chamber 132, as well as channels 30, 32, 36, and 38 and valves 70, 72, 76, and 78, may be omitted from second fluidic system 100b. If target isolation chamber 126 is omitted, elution chamber 124 may be connected to amplification chamber 128, or in an alternative arrangement, amplification chamber 128 may be connected to filter chamber 120 of first fluidic system 100a via channels 24, 24a, and 24b controlled by valve 62, and elution chamber 124 and fourth reservoir 114 may be omitted.

[0264] The amplification chamber 128 may be connected to the hybridization / detection chamber 130 by channel 34 via a valve 74 configured to be operated (e.g., by an actuator of a processing device to which the second fluid system 100b is coupled) to selectively prevent fluid flow through channel 34 or to direct fluid flow from the amplification chamber 128 to the hybridization / detection chamber 130 via channel 34.

[0265] The hybridization wash buffer reservoir 140 may be connected to the hybridization / detection chamber 130 by the channel 40 via a valve 80 configured to be operated (e.g., by an actuator of a processing device to which the second fluidic system 100b is coupled) to selectively prevent fluid flow from the hybridization wash buffer reservoir 140 or to direct fluid flow from the hybridization wash buffer reservoir 140 through the channel 40 to the hybridization / detection chamber 130.

[0266] The hybridization buffer reservoir 138 may be connected to the hybridization / detection chamber 130 by channel 42 via a valve 82 configured to be operated (e.g., by an actuator of a processing device to which the second fluid system 100b is coupled) to selectively prevent fluid flow from the hybridization buffer reservoir 138 or to direct fluid flow from the hybridization buffer reservoir 138 to the hybridization / detection chamber 130 via channel 42.

[0267] In an embodiment, the hybridization chamber 130 contains a biosensor for detecting the presence of a specific target molecule in a sample. The biosensor interacts with the target molecule through ligation. Capturing amplification products or amplicons among multiple candidates on a surface is a well-known technique for multiplexed detection. One example of a biosensor is a microarray (e.g., a nucleic acid array). In such a microarray, the identity and quantity of a target nucleic acid in a sample are detected by measuring the level of association between the target sequence and a probe provided specifically for that sequence. In nucleic acid biochip technology, a set of probe nucleic acids, each with a defined sequence, is immobilized on a solid support or substrate, such as a nucleic acid array, with each probe occupying a predetermined position. The processing device to which the second fluidic system 100b is coupled may include means for optically exciting the array of the second fluidic system 100b and means for optically detecting an optical signal representative of the nucleic acids in the sample analyzed by the second fluidic system 100b.

[0268] In other embodiments, amplification and detection may occur in the same chamber.

[0269] Microbial enrichment using selective lysis and filtration 2 is a flowchart illustrating a method 10 for selectively isolating a microbial cell analyte according to the present disclosure. The method 10 generally includes the following steps: (a) providing a sample containing or potentially containing mammalian cells and microbial cells; (b) combining the sample with a first lysis solution that selectively lyses the mammalian cells, if present, to obtain a first lysate containing lysed mammalian cells and, if present, intact microbial cells; (c) filtering the first lysate through a filter having a pore size that retains the intact microbial cells but passes the lysed mammalian cells; (d) providing conditions for lysing the retained microbial cells, thereby lysing at least a portion of the microbial cells and releasing the analyte therefrom; and (e) eluting the analyte from the filter to obtain an eluate containing the analyte released from the lysed microbial cells. In some embodiments, the conditions for lysis in step (d) include contacting the filter containing the retained microbial cells with a second lysis solution effective to lyse the microbial cells and release the analyte therefrom. In other non-mutually exclusive embodiments, the method 10 is performed using a first fluid system 100a.

[0270] In step S12, a sample containing or potentially containing mammalian cells and microbial cells is provided, for example, by dispensing the sample into a sample reservoir 104 or by connecting a sample container (e.g., a BD Vacutainer® (Becton Dickinson, Franklin Lakes, NJ)) to the sample inlet port of the first fluid system 100a.

[0271] A range of samples containing or potentially containing mammalian cells and microbial cells can be analyzed according to the present disclosure. In typical variations, the sample is known to contain mammalian cells and is at least suspected to contain one or more microorganisms. In some such variations, the sample is isolated from a mammal. For example, a sample particularly suitable for analysis using the methods disclosed herein is a mammalian (e.g., human) blood sample, e.g., for blood sepsis pathogen detection. In other embodiments, the sample is a sample not known to contain mammalian cells but suspected to do so, e.g., a sample not isolated from a mammal but derived from another location (e.g., an environmental sample), etc. Microbial cells can be any bacterial, archaeal, fungal, or protist species, particularly species characterized by a cell wall. In typical variations, the microbial cells are pathogenic microorganisms or microorganisms that cause infection in the body, although microbial cells can be pathogenic or non-pathogenic. Furthermore, the step of providing a sample need not involve isolation of the sample from a mammal or other source, but may be performed by retrieving the sample from a container in which it is stored, or by initiating contact of the sample with the first lysis solution immediately prior to the mixing step (b).

[0272] In certain embodiments, the volume of the sample provided in step S12 is up to about 6 mL, up to about 5 mL, or up to about 4 mL. For example, in some variations, the sample volume is about 0.5 mL to about 6 mL, about 0.5 mL to about 5 mL, about 1 mL to about 5 mL, about 2 mL to about 5 mL, about 2 mL to about 4 mL, about 3 mL to about 5 mL, about 3 mL to about 4 mL, or about 4 mL. In some embodiments involving the above-described sample volumes, the sample is a blood sample (e.g., a blood sample for blood sepsis pathogen detection). In other, non-mutually exclusive embodiments involving the above-described sample volumes, the first lysis solution is a lysis solution comprising a chaotropic salt (e.g., guanidine hydrochloride), saponin, and a polyoxyethylene surfactant (e.g., polysorbate 20) as described herein. In yet other, non-mutually exclusive embodiments involving the above-described sample volumes, the filter is composed of polyethersulfone (PES) (e.g., a PES filter having a pore size of about 0.22 μm).

[0273] Non-limiting examples of microorganisms that can be targets for analyte isolation according to the present disclosure include Gram-negative bacteria, such as Acinetobacter baumannii, Bacteroides fragilis, Enterobacterales (e.g., E. cloacae, E. coli, K. aerogenes, K. oxytoca, K. pneumoniae, Proteus, Salmonella, S. marcescens), Haemophilus influenzae, Neisseria meningitidis, Pseudomonas aeruginosa, and Stenotrophomonas maltophilia; Gram-positive bacteria, such as Enterococcus faecalis, Enterococcus faecium, Listeria monocytogenes, Staphylococcus (e.g., S. aureus, S. epidermis, S. lugdunensis), and Streptococcus (e.g., S. agalactiae, S. pneumoniae, S. pyogenes); and Candida. Examples of yeasts include Candida albicans, Candida auris, Candida glabrata, Candida krusei, Candida parapsilosis, Candida tropicalis, Cryptococcus neoformans, and Cryptococcus gatti.

[0274] In step S14, the sample is combined with the first lysis solution. In one example, step S14 is accomplished by (a) opening valve 50 to channel 10a, closing valves 52 and 54, and transporting a certain amount of sample from sample reservoir 104 to lysis chamber 108 via channels 10 and 10a, and then (b) opening valves 52, 50, and 54 and transporting the first lysis solution from first reservoir 106 to lysis chamber 108 via channel 12, thereby combining the sample and the first lysis solution in lysis chamber 108. As described above, the first lysis solution can lyse mammalian cells while leaving any microbial cells, including microbial cells of interest, intact. The lysis solution can be mixed with the sample in any appropriate ratio based on the lysis solution components and their respective concentrations to achieve effective concentrations of the lysis solution components in the final mixture for selective lysis. Exemplary ratios of first lysis solution:sample include ratios of about 3:1 to about 1:3, about 2:1 to about 1:2, about 1.5:1 to about 1:1.5, or about 1:1.

[0275] Typically, the first lysis solution contains a chaotropic salt and a detergent. A particularly suitable chaotropic salt is guanidine hydrochloride, which may be present in the first lysis solution at a concentration of, for example, about 1 M to about 8 M, about 1 M to about 6 M, about 1 M to about 5 M, about 2 M to about 8 M, about 2 M to about 6 M, about 2 M to about 5 M, about 3 M to about 8 M, about 3 M to about 6 M, about 3 M to about 5 M, or about 4 M. Suitable effective concentrations of guanidine hydrochloride (i.e., the concentration in the initial lysis reaction after mixing with the sample, or the concentration in the final lysate) include, for example, about 0.5 M to about 4 M, about 0.5 M to about 3 M, about 0.5 M to about 2.5 M, about 1 M to about 4 M, about 1 M to about 3 M, about 1 M to about 2.5 M, about 1.5 M to about 4 M, about 1.5 M to about 3 M, about 1.5 M to about 2.5 M, or about 2 M. Other suitable chaotropic salts include, for example, guanidinium thiocyanate, urea, and lithium perchlorate.

[0276] The detergent used in the first lysis solution can be a non-ionic detergent, an ionic detergent, or a zwitterionic detergent. To keep the microbial cells intact, a mild detergent and / or a low detergent concentration is typically used. For example, non-ionic detergents such as saponin or polyoxyethylene surfactants are preferred. Saponin is particularly preferred and may be present in the first lysis solution at a concentration of, for example, about 1% (w / v) to about 10% (w / v), about 2% (w / v) to about 8% (w / v), about 2% (w / v) to about 6% (w / v), about 3% (w / v) to about 5% (w / v), or about 4% (w / v), and saponin may be used at an effective concentration (i.e., the concentration in the initial lysis reaction after mixing with the sample, or the concentration in the final lysate) of about 0.5% (v / v) to about 5% (v / v), about 1% (v / v) to about 4% (v / v), about 1% (v / v) to about 3% (v / v), about 1.5% (v / v) to about 2.5% (v / v), or about 2% (v / v). A particularly suitable polyoxyethylene surfactant is polysorbate 20 (Tween®-20), which may be present in the first lysis solution at a concentration of, for example, about 1% (v / v) to about 10% (v / v), about 2% (v / v) to about 8% (v / v), about 2% (v / v) to about 6% (v / v), about 3% (v / v) to about 5% (v / v), or about 4% (v / v), and polysorbate 20 may be used at an effective concentration of about 0.5% (v / v) to about 5% (v / v), about 1% (v / v) to about 4% (v / v), about 1% (v / v) to about 3% (v / v), about 1.5% (v / v) to about 2.5% (v / v), or about 2% (v / v). Other suitable detergents include, for example, nonylphenoxypolyethoxyethanol (NP-40), or polyethylene oxide surfactants such as Triton® X-100. In some embodiments of the method, the first lysis solution comprises at least two detergents. For example, in some variations where the first lysis solution comprises saponin, the lysis solution further contains polysorbate 20 as a second detergent.

[0277] In a typical variation, the first lysis solution further contains a buffer, typically present at a concentration sufficient to maintain a pH of, for example, about 6.0 to about 10.0, about 6.5 to about 9.0, about 7.0 to about 8.0, or about 7.2 to about 7.6. Suitable buffers include Tris (2-amino-2-(hydroxymethyl)-1,3-propanediol), PIPES (piperazine-N,N'-bis(2-ethanesulfonic acid)), HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), phosphate, citrate, succinate, and histidine. In certain embodiments, the buffer is Tris, which may be present in the first lysis solution at a concentration of, for example, about 20 mM to about 200 mM, about 30 mM to about 100 mM, about 30 mM to about 50 mM, about 35 mM to about 45 mM, or about 40.5 mM. Tris may be used at an effective concentration (i.e., in the initial lysis reaction after mixing with the sample, or in the final lysate) of about 10 mM to about 100 mM, about 15 mM to about 50 mM, about 15 mM to about 25 mM, about 18 mM to about 23 mM, or about 20.25 mM. Other appropriate concentrations of buffer for formulations according to the present disclosure can be readily determined by one of skill in the art.

[0278] The first lysis reagent and the sample are mixed in step S14 to induce sufficient mammalian cell lysis to allow the resulting first lysate to pass through the filter, while leaving microbial cells, if present, sufficiently intact and captured on the filter surface. Preferably, all or substantially all (e.g., at least 80%) of the microbial cells present in the sample remain intact after selective lysis of the mammalian cells. In some variations, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the microbial cells in the sample remain intact. In typical variations, a separate incubation period before passing the lysate through the filter is not required, and sufficient lysis is achieved upon mixing the first lysis reagent and the sample. In other embodiments, a short incubation period before passing the lysate through the filter may be used (e.g., an incubation period of 10 minutes or less, 5 minutes or less, or 1 minute or less). Incubation may occur in the lysis chamber 108, with temperature ranges for the initial lysis reaction including 5-50° C., 10-45° C., 15-37° C., 20-30° C., 22-27° C., or 25° C. Ambient temperature (room temperature) is particularly suitable.

[0279] In some variations involving lysis of a blood sample with a first lysis solution, the first lysis solution comprises guanidine hydrochloride, saponin, and polysorbate 20, wherein (i) the effective concentration of guanidine hydrochloride in the first lysis reaction is at least about 1.5 M, at least 1.8 M, or at least about 2 M; (ii) the effective concentration of saponin in the first lysis reaction is at least about 1.5% (w / v), at least about 1.8% (w / v), or at least about 2% (w / v); and (iii) the effective concentration of polysorbate 20 in the first lysis reaction is at least about 0.5% (v / v), at least about 1% (v / v), at least about 1.5% (v / v), or at least about 2% (v / v). Such a variation is particularly suitable for lysis of blood samples with high white blood cell and platelet counts, inducing sufficient lysis to allow the resulting first lysate to be passed through a filter (e.g., for lysis of laboratory-generated buffy coat samples that may have a normal white blood cell (WBC) count in the range of approximately 17,000-44,000 / μl, a red blood cell (RBC) count of approximately 4 x 10 / μl, and a platelet (PLT) count in the range of approximately 1.2-2.1 x 10 / μl).

[0280] In step S16, the first lysate obtained in the selective lysis of mammalian cells (step S14) is passed through a filter having a pore size that retains intact microbial cells while allowing the lysed mammalian material to pass through, thereby separating the intact microbial cells from the lysed mammalian cells. The portion of the first lysate that flows through the filter, including the lysed mammalian cells, can be collected, for example, in a waste chamber. In one example, step S16 is accomplished by opening valves 54 and 64, closing all other valves, and transporting the first lysate from lysis chamber 108 through channel 14 to filter chamber 120 in direction "A," and from filter chamber 120 to waste chamber 118 through channel 22.

[0281] Filtration is performed using a filter with a pore size suitable for capturing the target microbial cells. The filter may have a pore size of about 1 μm or less, preferably about 0.5 μm or less, and more preferably 0.25 μm or less. In some variations, the filter has a pore size of about 0.1 μm to about 1 μm, about 0.1 μm to about 0.5 μm, about 0.2 μm to about 1 μm, or about 0.2 μm to about 0.5 μm. In some embodiments, the filter has a pore size of about 0.22 μm. Suitable filter materials include, for example, polyethersulfone (PES), cellulose, nylon, polyvinylidene fluoride (PVDF), polycarbonate, and glass fiber (e.g., borosilicate glass fiber).

[0282] In some embodiments, the filter includes an asymmetric structure. This provides a kind of "built-in" prefilter and a larger effective membrane area: larger pores on the upstream side of the membrane capture larger particles at the top of the membrane, while smaller particles can continue downstream, depending on their size, into or through the membrane. The pore size gradually decreases from the upstream side of the membrane to the downstream side, so particles are captured in different parts of the membrane depending on their size. In contrast, track-edged membranes collect all particles larger than the pore size to the same level, making the membrane more susceptible to clogging. Asymmetric filters may have pore sizes of about 5 μm to about 20 μm on the first side of the asymmetric filter and up to about 0.2 μm on the second side of the asymmetric filter.

[0283] Following the filtration step, the filter may optionally be washed in step S18 to remove residual lysis solution and mammalian cell debris prior to lysis of the intact microbial cells. Washing is typically accomplished by passing a wash buffer through the filter. In one example, optional step S18 is accomplished by opening valves 58 and 64, closing all other valves, and transporting wash buffer from fifth reservoir 112 via channel 18 through filter chamber 120 in direction "A," and from filter chamber 120 via channel 22 to waste chamber 118.

[0284] Suitable wash buffers may include a detergent and may further include a chelating agent (e.g., 2,2',2'',2'''-(ethane-1,2-diyldinitrilo)tetraacetic acid (EDTA)). For example, in some variations, the wash buffer includes a non-ionic detergent, such as polysorbate 20, and in some such embodiments, the wash buffer further includes Tris and / or EDTA. In other variations, the wash buffer does not contain a detergent. For example, in some such variations, the wash buffer contains sodium hydroxide, dimethyl sulfoxide (DMSO), glycerol, a chelating agent (e.g., EDTA), and a buffer (e.g., Tris). In more specific variations of a detergent-free wash buffer, the wash buffer is the same as or a diluted concentration of the second lysis solution described herein. In still other embodiments, the wash buffer is a buffer solution selected from TE buffer, Tris-buffered saline (TBS), and phosphate-buffered saline (PBS).

[0285] After filtering step S16 and optional washing step S18, the retained intact microbial cells are lysed using an on-filter lysis step S20. The intact microbial cells retained on the filter can be lysed by chemical and / or physical lysis methods to release the microbial cell analytes from the intact organisms, allowing the analytes to be released from the filter in an elution step. For example, lysis of intact microbial cells can include (a) chemical lysis using detergents, chaotropic salts, alcohols (e.g., glycerol), and / or extreme pH values; (b) enzymatic lysis on the filter; (c) physical lysis using an externally controlled heat source embedded in the filter (e.g., induction-mediated heating, in which a conductive or semiconductive material such as brass, copper, steel, iron, aluminum, graphite, carbon, or silicon is embedded on / in the filter matrix, e.g., by laminating a porous mesh or co-molding with the actual filter material); (d) physical lysis by direct electricity (electrolysis), e.g., by placing a conductive mesh structure on both sides of the filter; (e) sonication / ultrasonication with a sonotrode in contact with the filter; or (f) any combination of chemical, enzymatic, and / or physical lysis. In one example, after a filter washing step, an enzyme is applied to the filter, followed by a time / temperature incubation, followed by the addition of a small amount of a second lysis solution, followed by induction heating or sonication. In certain embodiments, on-filter lysis step S20 involves contacting the filter containing the retained microbial cells with a second lysis solution effective to lyse the microbial cells. In one example, step S20 is accomplished by opening valves 56 and 64, closing all other valves, and transporting the second lysis solution from second reservoir 110 via channel 16 through filter chamber 120 in direction "A," and from filter chamber 120 via channel 22 to waste chamber 118. The filter is incubated with the second lysis solution under conditions sufficient to induce lysis of intact microbial cells and release analytes therefrom.In some embodiments involving contacting the filter with a second lysis solution, the on-filter lysis step S20 further comprises inductive heating and / or sonication.

[0286] In some preferred variations, the second lysis solution comprises sodium hydroxide and a buffer. In some such embodiments, sodium hydroxide is present in the second lysis solution at a concentration of about 20 mM to about 500 mM, about 35 mM to about 400 mM, about 45 mM to about 300 mM, about 50 mM to about 250 mM, about 50 mM to about 150 mM, about 50 mM to about 100 mM, about 60 mM to about 150 mM, about 60 mM to about 100 mM, or about 85 mM. Buffers suitable for use in the second lysis solution can include those buffers described above for the first lysis solution; for example, in some variations, the buffer is Tris and is present in the second lysis solution at a concentration of about 1 mM to about 50 mM, about 2 mM to about 25 mM, about 5 mM to about 10 mM, or about 7.5 mM. In some embodiments, the second dissolution solution (e.g., the second dissolution solution containing sodium hydroxide described above) is dimethyl sulfoxide (DMSO) (e.g., about 5% (v / v) to about 30% (v / v), about 5% (v / v) to about 25% (v / v), about 10% (v / v) to about 30% (v / v), about 10% (v / v) to about 25% (v / v), about 10% (v / v) to about 20% (v / v)). % (v / v), about 15% (v / v) to about 20% (v / v), or about 18% (v / v)) and / or glycerol (e.g., about 1% (v / v) to about 15% (v / v), about 2% (v / v) to about 12% (v / v), about 3% (v / v) to about 10% (v / v), about 5% (v / v) to about 10% (v / v), or about 7% (v / v)). In certain variations, the second lysis solution (e.g., the second lysis solution containing sodium hydroxide and further containing one or both of DMSO and glycerol described above) further comprises a chelating agent; in some such variations, the chelating agent is EDTA, present in the second lysis solution at a concentration of about 0.1 mM to about 4 mM, about 0.25 mM to about 3 mM, about 0.5 mM to about 2 mM, or about 1 mM. In some embodiments, the second lysis solution consists of, or consists essentially of, sodium hydroxide, DMSO, glycerol, the chelating agent, buffer, and water.

[0287] In some variations in which the on-filter lysis step comprises contacting the filter with a second lysis solution as described above, the on-filter lysis step further comprises sonication. In some such embodiments, sodium hydroxide is present in the second lysis solution at a concentration of about 20 mM to about 85 mM, about 35 mM to about 85 mM, about 20 mM to about 70 mM, about 35 mM to about 70 mM, about 20 mM to about 60 mM, about 35 mM to about 60 mM, about 20 mM to about 50 mM, or about 35 mM to about 50 mM. In other non-mutually exclusive embodiments involving sonication, DMSO is present in the second lysis solution at a concentration of about 5% (v / v) to about 18% (v / v), about 5% (v / v) to about 15% (v / v), about 5% (v / v) to about 10% (v / v), about 10% (v / v) to about 18% (v / v), or about 10% (v / v) to about 15% (v / v).

[0288] In some variations of the method involving the use of a second lysis solution as described above to lyse intact microbial cells, the on-filter lysis step S20 may also include incubating the filter with the second lysis solution at a temperature of, for example, about 80°C to about 150°C or about 90°C to about 130°C. Exemplary times for incubating the filter with the second lysis solution, optionally in the presence of heat and / or sonication, include 1 to 30 minutes, 2 to 20 minutes, 3 to 15 minutes, 4 to 12 minutes, 5 to 10 minutes, or 5 to 8 minutes. In other, non-mutually exclusive variations, the second lysis step includes sonication and / or electrolysis.

[0289] In certain variations involving lysis with a second lysis solution as described above, the second lysis solution contains DMSO and glycerol. Such embodiments are particularly preferred for improving the lysis efficiency of certain microorganisms, such as Gram-positive bacteria (e.g., S. aureus) and yeast species (e.g., Candida, e.g., C. albicans). Embodiments including DMSO and glycerol are also particularly suitable for co-directional elution of released analytes from the filter after lysis.

[0290] In some embodiments involving lysis with a second lysis solution as described above, lysis of microbial cells further comprises an enzyme treatment step prior to contacting the filter with the second lysis solution. The enzyme treatment step typically involves contacting the filter containing retained microbial cells with an enzyme solution effective to degrade one or more microbial cell wall components (e.g., peptidoglycan, chitin, protein). Particularly suitable enzymes include mutanolysin, lyticase, lysozyme, endoglucanase, protease, chitinase, and combinations thereof, to name a few. In some particular variations of the enzyme solution, mutanolysin is used at a concentration of about 1 U / mL to about 1,000 U / mL, or about 1 U / mL to about 500 U / mL. In some particular variations of the enzyme solution, lyticase is used at a concentration of about 1 U / mL to about 1,000 U / mL, or about 1 U / mL to about 500 U / mL. In some specific variations of the enzyme solution, lysozyme is used at a concentration of about 1 U / mL to about 1,000 U / mL, or about 1 U / mL to about 500 U / mL. In some specific variations, the enzyme solution contains at least two enzymes: mutanolysin is used at a concentration of about 0 U / mL to about 1,000 U / mL, lyticase is used at a concentration of about 0 U / mL to about 1,000 U / mL, and lysozyme is used at a concentration of about 0 U / mL to about 1,000 U / mL. Typically, once the enzyme solution is added to the filter, the filter is incubated with the enzyme solution at an appropriate temperature (e.g., 20°C to 50°C). Exemplary times for incubating the filter with the enzyme solution include 5 to 30 minutes, 5 to 25 minutes, 5 to 20 minutes, or 10 to 20 minutes (e.g., 15 minutes). In variations further including a washing step (step S18) after filtration of the first lysate as described above, the enzyme treatment of the filter typically occurs after the washing step. Mutanolysis (available, for example, from Sigma-Aldrich as catalog number SRE0007) has a stock specific activity of approximately 4,000 U / mg protein (one [o]ne unit generates a ΔA600 of 0.01 per minute at 37°C, pH 6.0 in a 1 mL volume using a suspension of Streptococcus faecalis cell walls as substrate).Lysozyme (available, for example, from Sigma-Aldrich as catalog number L4919) has a stock specific activity of approximately 40,000 U / mg protein (one [o]ne unit generates a ΔA450 of 0.001 per minute at pH 6.24 at 25°C in a 2.6 mL reaction mixture (1 cm light path) using a suspension of Micrococcus lysodeikticus as substrate). Lyticase (available, for example, from A&A Biotechnology, μl. Strzelca 40, 80-299 Gdańsk, Poland (catalog number 1018)) has a stock concentration of 10 U / μL (one [o]ne unit generates a ΔA800 of 0.001 per minute at pH 7.5 at 25°C using a suspension of S. cerevisiae as substrate in a 3 mL reaction mixture).

[0291] Following on-filter lysis of the intact microbial cells (step S20), the released analytes are recovered from the filter in step S22. The recovered analytes may be collected in a downstream container or reservoir for further processing (e.g., performing an assay). An exemplary analyte recovery method involves eluting the analytes from the filter by passing an immiscible liquid through the filter, forcing the analyte-containing eluate out of the filter. Elution with an immiscible liquid is particularly suitable for recovering hydrophilic analytes, such as nucleic acids, because hydrophilic analytes are retained with the aqueous liquid contents forced out of the filter. Furthermore, using an immiscible liquid for the elution step is advantageous because the aqueous phase recovered from the filter is not diluted with the eluate, thereby avoiding dilution of low-copy-number analytes. Because the immiscible liquid does not mix with the aqueous phase from the filter, an excess of the immiscible liquid may be used, and the aqueous eluate can be easily separated from the immiscible liquid for use in downstream assays. Suitable immiscible liquids include oils and hydrocarbons, such as mineral oil, silicone oil, essential oil, hexadecane, or a combination of two or more oils, to name a few. In alternative variations, the aqueous elution buffer may be passed through a filter for the elution step, and in some such embodiments, the elution buffer is the same as the second lysis solution described herein. In other variations, the aqueous elution buffer is selected from TE buffer, TBS, and PBS.

[0292] When passing the elution fluid through the filter, the direction of fluid flow may be the same as the direction of fluid flow applied in the filtration step (also referred to herein as "co-current elution"). Alternatively, the direction of fluid flow during elution may be opposite to the direction of fluid flow applied in the filtration step (also referred to herein as "countercurrent elution"). Countercurrent elution may be advantageous in certain variations, including, for example, the use of asymmetric filter membranes in which intact microorganisms as a sample are captured on the side of the membrane with the largest pores, while the first lysis solution passes through the filter. Using countercurrent elution in such variations can facilitate the elution of partially lysed microorganisms, since they do not have to pass through the filter membrane surface with the smallest pores. Countercurrent elution may increase downstream assay sensitivity in certain embodiments.

[0293] In one example, the co-directional elution of step S22 is achieved by opening valve 60 to channel 20a, opening valve 62 to channel 24a, closing all other valves, and transporting elution reagent from third reservoir 116 through filter chamber 120 via channels 20 and 20a in direction "A," and from filter chamber 120 through channels 24a and 24b to elution chamber 124. Conversely, the countercurrent elution of step S22 is achieved by opening valve 60 to channel 20b, opening valve 62 to channel 24b, closing all other valves, and transporting elution reagent from third reservoir 116 through filter chamber 120 via channels 20 and 20b in direction "B," and from filter chamber 120 through channels 24b and 24b to elution chamber 124.

[0294] After collection (or, if an immiscible liquid is used, after separating the eluate from the immiscible liquid), in optional step S24, the eluate may be neutralized by adding an appropriate neutralization buffer. In one example, step S24 is accomplished by opening valve 66, closing all other valves, and transporting neutralization buffer from fourth reservoir 114 to elution chamber 124 via channel 26.

[0295] For example, in certain embodiments involving the use of sodium hydroxide in the second lysis solution described herein, the neutralization buffer contains hydrochloric acid and Tris (e.g., a 20x neutralization buffer comprising HCl at a concentration of about 500 mM and Tris at a concentration of about 50 mM).

[0296] Analyte isolation using immiscible liquid elution 3 is a flow chart illustrating a method 30 for isolating an analyte from a sample using immiscible liquid elution according to the present disclosure. In one embodiment, method 30 may generally include the following steps: (a) providing a sample containing or potentially containing intact cells; (b) filtering the sample through a filter having a pore size that retains the intact cells; (c) providing conditions for lysing the cells retained on the filter, thereby lysing at least a portion of the cells and providing conditions for the analyte to be released from the filter; and (d) passing an immiscible liquid through the filter to force an eluate containing the analyte through the filter. In some embodiments, the conditions of step (c) include contacting the filter containing the retained cells with a lysis solution effective to lyse the cells, optionally in combination with one or more heat or sonication treatments, to release the analyte therefrom.

[0297] In step S32, a sample containing or potentially containing intact mammalian cells and microbial cells is provided, for example, by dispensing the sample into a sample reservoir 104 or by connecting a sample container (e.g., a BD Vacutainer® (Becton Dickinson, Franklin Lakes, NJ)) to the sample inlet port of the first fluid system 100a.

[0298] In some variations where the intact cells of interest are microbial cells, the method further includes a selective lysis step S34 prior to filtering the sample, which involves selectively lysing any mammalian cells in the sample while leaving the microbial cells intact. In certain embodiments, the selective lysis step is performed as described above in connection with the method for microbial enrichment using selective lysis. In one example, step S34 is accomplished by (a) opening valve 50 to channel 10a, closing valves 52 and 54, and transporting a volume of sample from sample reservoir 104 to lysis chamber 108 via channels 10 and 10a, and then (b) opening valves 52, 50, and 54, and transporting a first lysis solution from first reservoir 106 to lysis chamber 108 via channel 12, thereby combining the sample and the first lysis solution in lysis chamber 108.

[0299] After step S32 or step S34, filtration is performed in step S36 using a filter having a pore size suitable for capturing the cells of interest. In some variations, such as for capturing microbial cells, the filter has a pore size of about 1 μm or less, about 0.5 μm or less, or about 0.25 μm or less (e.g., pore sizes of about 0.1 μm to about 1 μm, about 0.1 μm to about 0.5 μm, about 0.2 μm to about 1 μm, about 0.2 μm to about 0.5 μm, or about 0.22 μm). Suitable filter materials include, for example, polyethersulfone (PES), cellulose, nylon, polyvinylidene fluoride (PVDF), polycarbonate, and glass fiber (e.g., borosilicate glass fiber). In some embodiments, the filter comprises an asymmetric structure. In certain variations, the pores on the first side of the asymmetric filter have a size of about 5 μm to about 20 μm, and the pores on the second side of the asymmetric filter have a size of up to about 0.2 μm.

[0300] In one example, if step S36 follows step S32 and step S34 is omitted, step S36 is accomplished by opening valve 50 to channel 10b, opening valve 64, closing all other valves, and transporting the sample from sample reservoir 104 via channels 10 and 10b through filter chamber 120 in the direction "A", and from filter chamber 120 via channel 22 to waste chamber 118.

[0301] In another example, when step S36 follows step S34, step S36 is accomplished by opening valves 54 and 64, closing all other valves, and transporting the first lysate from lysis chamber 108 via channel 14 through filter chamber 120 in direction "A," and from filter chamber 120 via channel 22 to waste chamber 118.

[0302] After the filtration step, the filter may be washed, if necessary, in step S38 before lysis of the intact cells. A washing step may be desirable, for example, if the filter contains substances that are inhibitory to downstream processing. Washing is typically performed by passing a wash buffer through the filter. Suitable wash buffers include those described above in connection with the method for microbial enrichment using selective lysis. In one example, optional step S38 is accomplished by opening valves 58 and 64, closing all other valves, and transporting the wash buffer from fifth reservoir 112 through channel 18 through filter chamber 120 in the direction "A," and from filter chamber 120 through channel 22 to waste chamber 118.

[0303] After the filtration step S36 and optional washing step S38, the retained intact cells are lysed using an on-filter lysis step S40. The intact cells can be lysed by chemical and / or physical lysis methods to release the analytes, which can then be released from the filter in an elution step. For example, lysis of intact cells can include chemical lysis using detergents, chaotropic salts, alcohols (e.g., glycerol), and / or extreme pH values; on-filter enzymatic lysis; physical lysis using an externally controlled heat source embedded in the filter (e.g., induction-mediated heating in which a conductive or semiconductive material such as brass, copper, steel, iron, aluminum, graphite, carbon, or silicon is embedded on / in the filter matrix (e.g., lamination of a porous mesh or co-molded with the actual filter material); physical lysis by direct electricity (electrolysis), for example, in which a conductive mesh structure is laminated and placed on both sides of the filter; ultrasonic treatment / ultrasonication with a sonotrode in contact with the filter; or any combination of chemical, enzymatic, and / or physical lysis.

[0304] In some embodiments, the on-filter lysis step S40 involves contacting the filter containing the retained cells with a lysis solution effective to lyse the cells, and the filter can be incubated with the lysis solution under conditions sufficient to induce lysis of intact cells and release analytes therefrom. For example, a lysis solution particularly suitable for lysing microbial cells is the "second" lysis solution described above in connection with the method for microbial concentration. In one example, step S40 is accomplished by opening valves 56 and 64, closing all other valves, and transporting the second lysis solution from second reservoir 110 through channel 16 through filter chamber 120 in direction "A," and from filter chamber 120 through channel 22 to waste chamber 118.

[0305] In some variations involving lysis of intact microbial cells, the on-filter lysis step S40 involves incubating the filter with the lysis solution, e.g., at a temperature of about 80°C to about 130°C or about 90°C to about 130°C. Exemplary times for incubating the filter with the lysis solution, optionally in the presence of heat and / or sonication, include 1 to 30 minutes, 2 to 20 minutes, 3 to 15 minutes, 4 to 12 minutes, 5 to 10 minutes, or 5 to 8 minutes. In other, non-mutually exclusive variations involving lysis of intact microbial cells, the lysis step involves sonication and / or electrolysis.

[0306] In some embodiments involving on-filter lysis of microbial cells with a lysis solution as described above, lysis of the microbial cells further comprises an enzyme treatment step prior to contacting the filter with the lysis solution. The enzyme treatment step typically involves contacting the filter containing retained microbial cells with an enzyme solution effective to degrade one or more microbial cell wall components (e.g., peptidoglycan, chitin, protein). Particularly suitable enzymes include mutanolysin, lyticase, lysozyme, endoglucanase, protease, chitinase, and combinations thereof, to name a few. In some particular variations of the enzyme solution, mutanolysin is used at a concentration of about 1 U / mL to about 1,000 U / mL, or about 1 U / mL to about 500 U / mL. In some particular variations of the enzyme solution, lyticase is used at a concentration of about 1 U / mL to about 1,000 U / mL, or about 1 U / mL to about 500 U / mL. In some specific variations of the enzyme solution, lysozyme is used at a concentration of about 1 U / mL to about 1,000 U / mL, or about 1 U / mL to about 500 U / mL. In some specific variations, the enzyme solution uses at least two enzymes: mutanolysin is used at a concentration of about 0 U / mL to about 1,000 U / mL, lyticase is used at a concentration of about 0 U / mL to about 1,000 U / mL, and lysozyme is used at a concentration of about 0 U / mL to about 1,000 U / mL. Typically, once the enzyme solution is added to the filter, the filter is incubated with the enzyme solution at an appropriate temperature (e.g., 20°C to 50°C). Exemplary times for incubating the filter with the enzyme solution include 5 to 30 minutes, 5 to 25 minutes, 5 to 20 minutes, or 10 to 20 minutes (e.g., 15 minutes). In variations further including a washing step (step S38) after filtration of the first lysate as described above, the enzyme treatment of the filter is performed after the washing step.

[0307] Following on-filter lysis of intact cells (step S40), the released analytes are recovered from the filter in step S42. The recovered analytes may be collected in a downstream container or reservoir for further processing (e.g., performing an assay). An exemplary analyte recovery method involves eluting the analytes from the filter by passing an immiscible liquid through the filter, forcing the analyte-containing eluate out of the filter. As previously mentioned, elution with an immiscible liquid is particularly suitable for recovering hydrophilic analytes, such as nucleic acids, because hydrophilic analytes are retained in the aqueous liquid contents pushed out of the filter. Furthermore, using an immiscible liquid for the elution step is advantageous because the aqueous phase recovered from the filter is not diluted with the eluate, thereby avoiding dilution of low-copy-number analytes. Because the immiscible liquid does not mix with the aqueous phase from the filter, an excess of the immiscible liquid may be used, and the aqueous eluate can be easily separated from the immiscible liquid for use in downstream assays.

[0308] In some embodiments, the immiscible liquid has a lower density than the aqueous phase being forced through the filter, thereby allowing the immiscible liquid to remain above the aqueous phase (e.g., in embodiments involving the use of a dissolution solution in step (c), the immiscible liquid may have a lower density than the dissolution solution). Suitable immiscible liquids include oils and hydrocarbons, such as mineral oil, silicone oil, and hexadecane, to name a few. Typically, enough immiscible liquid is used so that some of the immiscible liquid is also forced through the filter. When passing the immiscible liquid through the filter, either co-current or countercurrent elution may be used.

[0309] In one example, the co-directional elution of step S42 is achieved by opening valve 60 to channel 20a, opening valve 62 to channel 24a, closing all other valves, and transporting elution reagent from third reservoir 116 through filter chamber 120 via channels 20 and 20a in direction "A," and from filter chamber 120 through channels 24a and 24b to elution chamber 124. Conversely, the countercurrent elution of step S42 is achieved by opening valve 60 to channel 20b, opening valve 62 to channel 24b, closing all other valves, and transporting elution reagent from third reservoir 116 through filter chamber 120 via channels 20 and 20b in direction "B," and from filter chamber 120 through channels 24b and 24b to elution chamber 124.

[0310] Following elution separation of the eluate from the immiscible liquid, in optional step S44, the eluate may be neutralized by adding an appropriate neutralization buffer (e.g., a neutralization buffer as described above in connection with the method for microbial enrichment using selective lysis). In one example, step S44 is accomplished by opening valve 66, closing all other valves, and transporting neutralization buffer from fourth reservoir 114 to elution chamber 124 via channel 26.

[0311] Analyte isolation / detection Following release and recovery of the cellular analyte, the above-described methods of isolating an analyte can further include analyzing the isolated analyte. The type of assay will depend on the analyte.

[0312] For example, in some variations where the analyte is a nucleic acid, analyzing the isolated nucleic acid involves amplification. In such embodiments, the isolated nucleic acid analyte is used as a template in an in vitro nucleic acid amplification reaction, which utilizes at least two amplification oligomers flanking a target sequence within the nucleic acid analyte to generate an amplification product corresponding to the target sequence. The nucleic acid analyte can be amplified using methods such as isothermal amplification reactions (e.g., transcription-modified amplification (TMA), nucleic acid sequence-based amplification (NASBA), loop-mediated isothermal amplification (LAMP), polymerase chain reaction (PSR (Liu et al., Sci. Rep. 5:12723, 2015), ligase chain reaction (LCR), and other isothermal amplification methods), or temperature cycling amplification reactions (e.g., polymerase chain reaction (PCR) or other temperature cycling amplification methods), or other amplification methods. In certain embodiments involving PCR, the PCR is selected from quantitative PCR (qPCR) and real-time PCR (rt-PCR).

[0313] Amplification can be performed with or without prior capture of the nucleic acid analyte. In some variations involving a capture step, the isolated nucleic acid analyte is captured prior to the amplification step by hybridizing the nucleic acid to an immobilized capture probe attached to a solid support.

[0314] Detection of amplified nucleic acid analyte products can be carried out using any known method during amplification (real-time) or after amplification (endpoint). Amplified nucleic acids can be detected in solution phase or by immobilizing them on a solid support (e.g., a nucleic acid array) and detecting their associated labels (e.g., intercalating agents, such as ethidium bromide). Some detection methods use detection probes complementary to sequences in the amplification products to detect the presence of probe:product complexes (e.g., by detecting the labels bound to the probes), or use probe complexes to amplify the signals detected from the amplification products (e.g., U.S. Patent Nos. 5,424,413, 5,451,503, and 5,849,481). Other detection methods use probes in which signal generation is related to the presence of the target sequence, such as in molecular beacons, molecular torches, or hybridization switch probes, where a signal change occurs only when the labeled probe binds to the amplification product (e.g., U.S. Pat. Nos. 5,118,801, 5,210,015, 5,312,728, 5,538,848, 5,541,308, 5,656,207, 5,658,737, 5,925,517, 6,150,097, 6,361,945, 6,534,274, 6,835,542, and 6,849,412; and U.S. Patent Application Publication No. 2006 / 0194240 A1). Such probes typically use a label (e.g., a fluorophore) attached to one end of the probe and an interacting compound (e.g., a quencher) attached to another location on the probe to inhibit signal generation from the label when the probe is in one conformation ("closed"), indicating that the probe is not hybridized to an amplification product, but a detectable signal is generated when the probe hybridizes to an amplification product that changes its conformation ("open"). Detection of a signal from a directly or indirectly labeled probe that specifically associates with an amplification product indicates the presence of amplified target nucleic acid.

[0315] In a specific variation, the amplification and detection assay for analyzing isolated nucleic acids is a qPCR assay. Such an assay includes forward and reverse primers for target amplification and a target-specific detection probe (also called a hydrolysis probe or TaqMan probe) labeled with a fluorophore at the 5' end and a quencher at the 3' end. In this format, the quencher neutralizes the fluorophore emission. If the target nucleic acid is present in the sample, the probe binds to a complementary sequence within the amplification target region. When the polymerase extends the 3' end of the primer and synthesizes the nascent complementary strand, the 5' to 3' exonuclease activity of the polymerase degrades the bound probe, thus neutralizing the effect of the quencher on the fluorophore. This is seen as an increase in fluorescence intensity depending on the amplification rate.

[0316] FIG. 4 is a flow chart illustrating a method 50 of analyzing analytes (e.g., nucleic acids) received from a lysis process, for example, by isolating, amplifying, and detecting target nucleic acids, as described, for example, in U.S. Pat. No. 10,654,039.

[0317] In processes where nucleic acid capture occurs prior to amplification, target capture or isolation occurs in step S52. In one example, step S52 is accomplished by selectively connecting elution chamber 124 to target isolation chamber 126 (e.g., by opening valve 70 while valves 68, 72, 76, and 78 are closed) and transporting eluate from elution chamber 124 to target isolation chamber 126 via channel 28, where the eluate is typically contacted with the target capture reagent present in the target isolation chamber. In an alternative embodiment, the eluate is transported directly from filter chamber 120 to target isolation chamber 126 without first receiving it in an intermediate elution chamber. Target isolation chamber 126 may contain a binding substrate or membrane, such as a silica-like membrane, for nucleic acid binding. According to various embodiments, target isolation chamber 126 may contain, for example, a gel, bead, or paper filter for nucleic acid binding and concentration. Examples include agarose gel, silica beads, and / or filter paper, such as cellulose.

[0318] Once binding to the binding substrate or membrane is complete, waste chamber 132 is selectively connected to target isolation chamber 126 (e.g., by opening valve 70 and closing valves 68, 72, 76, and 78), and a portion of the sample containing unbound non-target analytes is transported from target isolation chamber 126 to waste chamber 132 via channel 30, while the captured target nucleic acid is retained within isolation chamber 126.

[0319] In step S54, the bound target nucleic acid in the target isolation chamber 126 is recovered by washing to remove inhibitors and purify the nucleic acid. In one example, step S54 is accomplished by selectively connecting the wash buffer reservoir 134 to the target isolation chamber 126 (e.g., by opening valve 76 and closing valves 68, 70, 72, and 78), transporting the wash buffer from the wash buffer reservoir 134 to the target isolation chamber 126 via channel 36, and washing the binding membrane in the target isolation chamber 126 with the wash buffer. In one embodiment, the same operation may be repeated using a second DNA wash buffer contained in another wash buffer reservoir (not shown). The used wash buffer may be directed to the waste chamber 132 by selectively connecting the waste chamber 132 to the isolation chamber 126 (e.g., by opening valve 68 while valves 70, 72, 76, and 78 are closed) and transporting the used wash buffer from the target isolation chamber 126 to the waste chamber 132 via channel 30.

[0320] In step S56, the nucleic acids bound to the binding membrane are eluted with an elution buffer, which may contain amplification reagents. In one embodiment, elution buffer / amplification reagent reservoir 136 is selectively connected to target isolation chamber 126 (e.g., by opening valve 78 and closing valves 68, 70, 72, and 76), and elution buffer is transported from elution buffer / amplification reagent reservoir 136 through channel 38 to target isolation chamber 126 to elute the nucleic acids bound to the binding membrane. The amplification mix solution contained in elution buffer / amplification reagent reservoir 136 can be used as the elution buffer.

[0321] At the end of step S56, an isolated nucleic acid sample remains.

[0322] In step S58, after elution, the amplification chamber 128 is selectively connected to the target isolation chamber 126 (e.g., by opening valve 68 while valves 72, 70, 76, and 78 are closed), and the isolated nucleic acids are transported from the target isolation chamber 126 to the amplification chamber 128 via channel 32 for amplification. As mentioned above, the steps of isolating and purifying the analyte of interest in the second fluidic system 100b may be omitted; therefore, steps S52, S54, and S56 may be considered optional, depending on the protocol used. In such an embodiment, the lysed microbial analyte from the first fluidic system 100a is delivered to the amplification chamber 128 directly from the filter chamber 120 or from the elution chamber 124. In such an arrangement, amplification reagents may be pre-stored in the amplification chamber 128 or may be transferred to the amplification chamber 128 from a separate amplification reagent reservoir before or after the sample material is transferred to the amplification chamber 128.

[0323] In some embodiments, to calibrate the appropriate volume to be injected into the amplification chamber, the isolated nucleic acid sample amplification mix is ​​transferred to a metering chamber before being transferred to the amplification chamber 128. In some embodiments, in step S60, a nucleic acid amplification reaction can be performed in the amplification chamber 128.

[0324] In step S60, nucleic acid amplification in the amplification chamber 128 can be performed by a standard amplification protocol (typically any amplification method, including but not limited to polymerase chain reaction (PCR), reverse transcriptase PCR, isothermal amplification, and other amplification protocols, including those mentioned above) that achieves very good sensitivity and specificity for up to 20 markers. In one embodiment, a set of primers may be immobilized (e.g., lyophilized) in the amplification chamber 128 during the cartridge manufacturing process. These primers can be resuspended when the amplification mix is ​​transferred to the amplification chamber 128. The amplification mix may contain polymerase, nucleotides, and a reaction buffer at concentrations appropriate for efficient amplification of the nucleic acid template. At the end of step S60, if the target nucleic acid was originally present in the sample, an amplified nucleic acid sample is obtained.

[0325] In step S62, hybridization buffer reservoir 138 is selectively connected to hybridization / detection chamber 130 (e.g., by opening valve 74 while valves 82 and 80 are closed), and hybridization buffer is transported from hybridization buffer reservoir 138 to hybridization / detection chamber 130 via channel 42.

[0326] Next, in step S64, amplification chamber 128 is selectively connected to hybridization / detection chamber 130 (e.g., by opening valve 72 while valves 74, 80, and 82 are closed), and amplification solution is transported from amplification chamber 128 to hybridization / detection chamber 130 via channel 34, thereby contacting the amplified nucleic acid sample with the hybridization buffer transferred in step S62.

[0327] In step S66, the amplified DNA sample may be placed in contact with an affinity biosensor (e.g., a biochip) in hybridization / detection chamber 130 so that complementary sequences can combine with the immobilized probes, for example, by hybridization, association, or ligation to the probes. After removal of unrelated material, the related sequences are ready for detection and measurement.

[0328] In one embodiment, in step S66, the amplified nucleic acid sample is hybridized in the hybridization / detection chamber 130 for several minutes, for example, about 30 minutes.

[0329] In step S68, hybridization wash buffer reservoir 140 is selectively connected to hybridization / detection chamber 130 (e.g., by opening valve 74 while valves 80 and 82 are closed), and hybridization wash buffer is transported from hybridization wash buffer reservoir 140 to hybridization / detection chamber 130 via channel 40 to recover the hybridized nucleic acids, thus obtaining a hybridized nucleic acid sample.

[0330] In a variation of the analytical method, a DNA melting step can be added at the end of hybridization, allowing for increased detection specificity.

[0331] In step S70, the array image is acquired and analyzed. The interaction between the target nucleic acid and the biosensor probe is detected by an optical detection device. Localized hybridization can be detected, for example, by the emission of a colorimetric signal. Herein, "colorimetric signal" should be understood as any optical signal emitted directly or indirectly after excitation by an appropriate light source or after chemical or enzymatic conversion. Colorimetric signals include colorimetric, photoluminescent, fluorescent, chemiluminescent, and bioluminescent signals. Such signals are emitted directly by the molecule of interest or by a detectable element (tag) attached and / or grafted thereto.

[0332] The processing device to which the second fluidic system 100b is coupled may include an optical detector, such as a fluorescence reader, configured to acquire a fluorescence image of the biosensor surface. To this end, the biosensor is illuminated (excited) by a light source at the excitation wavelength of the fluorophore that labels the target molecule, and an adapted optical system forms an image of the biosensor's fluorescence at the emission wavelength of the fluorophore. The light intensity at each point of this image is related to the amount of fluorophore present at the corresponding point of the affinity biosensor, which is itself proportional to the number of target molecules selectively attached to this location during the hybridization step, making it possible to collect information (often quantitative) about the nucleic acid content of the sample. [Example]

[0333] The following examples are provided to illustrate certain disclosed embodiments and should not be construed in any way as limiting the scope of the disclosure.

[0334] Example 1 In this study, pathogen enrichment protocols using set concentrations of NaOH, glycerol, and EDTA were evaluated with different elution techniques.

[0335] Previous studies examining oil elution initially used glycerol and increased EDTA. Zhang et al. (Lett. Appl. Microbiol. 51:114-118, 2010) reported that a sodium phosphate / EDTA / glycerol solution can lyse yeast cells, but when this lysis solution was studied using on-filter lysis, the detection power for C. albicans and S. aureus was low. The addition of glycerol and increased EDTA to the NaOH solution subsequently provided promising improvements for detection by co-directional elution.

[0336] In this study, the following elution methods were used to evaluate lysis solutions containing 85 mM NaOH, 1 mM EDTA, and 7% (v / v) glycerol: 1) co-elution with oil after heating the filters in an oven (98°C for 30 minutes); 2) co-elution with oil after heating the filters in a specific filter holder where the heating element is in direct contact with the filter; 3) co-elution with lysis buffer after heating the filters in an oven at 98°C for 30 minutes; and 4) counter-elution with oil after heating the filters in an oven at 98°C for 30 minutes.

[0337] The protocol used in this study is summarized below. 1) Mix the blood sample thoroughly by inverting the tube more than 30 times. 2) Take 4 ml of blood sample into a 50 ml Falcon tube. Spike centrifuge (9500 rpm / min, 10 min) + wash pathogen to 50 cfu / ml (20 μl from 10^4 cfu / ml stock) and mix gently. 3) Add 4 ml of hemolysis solution (4 M Gu-HCl, 4% Tween®-20 (v / v), 4% saponin, 40.5 mM Tris-HCl) and mix by gently pipetting back and forth 25 times. 4) Using a needle, draw the lysed blood sample into a 10 ml syringe. Remove all air. Attach the syringe directly to the filter at a 90° angle. Filter the blood. 5) Wash the filter by filtering through 15 ml of GENT solution at a rate of approximately 20 ml / min. 6) Cap the filter and place it in the filter heating holder. The following induction heating protocol was performed: heat the filter to 50°C and hold for 40 seconds; increase the heat to 70°C and hold for 20 seconds; increase the heat to 90°C and hold for 5 minutes. Allow to cool for a few minutes, then remove from the heating holder. 7) Connect a 1.2 ml syringe with mineral oil (1, 2, and 4) or 300 µl of lysis buffer (3) to the filter inlet (1, 2, 3) or outlet (4) and elute by steadily pressurizing the oil or lysis buffer through the filter. Collect the resulting liquid in a tube. 8) For oil elution (1, 2, and 4), the miscible liquid lower phase is separated from the oil. 9) Neutralize the eluate by adding 20x Neutralizer (500 mM HCl, 50 mM Tris) and checking the pH with pH paper. Aim for a pH of 8.5. (Approximately 40 μl of 20x Neutralizer was required for 250 μl of eluate.)

[0338] The results are shown in Table 1 below. [Table 1]

[0339] S. aureus detection performed well using co-directional elution with oil compared to the positive control. S. pneumoniae performed poorly, suggesting that the cell wall structure of Streptococcus is more resistant than Staphylococcus to lysis by this chemistry.

[0340] The Candida results clearly demonstrate that countercurrent oil elution very efficiently collected yeast cells located on the membrane (4). With countercurrent elution, cells trapped on the top of the membrane readily emerged. With co-directional elution (1, 2, 3), non-lysed cells could not emerge because they would need to pass through the membrane. Therefore, these data suggest that this chemistry plus heat does not efficiently lyse cells and release nucleic acids for Candida (as well as S. pneumoniae), resulting in poor results after co-directional elution. However, countercurrent elution can be used to collect non-lysed cells. Therefore, detection is improved because PCR thermal cycling imposes additional stress on sensitized cells, releasing DNA for amplification.

[0341] Because reverse flow elution did not produce as good results as Candida, lysis was not optimal for Gram-positive bacteria (especially S. pneumoniae). The size of Gram-positive bacteria allows them to enter the pores of the asymmetric membrane structure (gradually decreasing to 0.2 μm). When reverse flow elution is performed, the microorganisms either cannot exit the membrane pores, or they exit but remain so intact that further stress in the PCR thermal cycles cannot destroy them. Candida cells are larger, therefore more likely to be located on the upper (not the interior) membrane. Therefore, with reverse flow elution, Candida cells exit and are detected by PCR. The results demonstrate the benefits of reverse flow for Candida and its ability to be destroyed by further stress in PCR. An optimal protocol providing good detection for all tested microorganisms has not yet been found, so further chemical refinements or lysis stresses are needed to allow either complete lysis in the filter with unidirectional elution or partial lysis with reverse flow before further stress for complete lysis in downstream processes.

[0342] Example 2 Microbial suspensions derived from Staphylococcus aureus, Candida albicans, and Haemophilus influenzae were prepared from colonies of pure microbial cultures. The colonies were suspended in PBS and diluted to an OD of 0.5 McF. A 104 cfu / ml suspension was prepared and centrifuged to remove any potential free DNA. After dissolving the pellet, a 1000 cfu / ml suspension was prepared and used for spiking blood samples. The suspension was then plated for culture to determine the exact final concentration.

[0343] Four milliliters of blood was spiked with the test microorganism to a concentration of 10 cfu / ml. The blood was mixed with an equal volume of lysis solution containing 4 M guanidine hydrochloride, 4% (v / v) Tween® 20, 4% (w / v) saponin, and 40.5 mM Tris. The resulting lysate was filtered through a 0.22 μm pore size syringe filter using a syringe.

[0344] The filter was washed by filtering 15 ml of the solution using a second lysis solution containing 85 mM sodium hydroxide, 18% (v / v) DMSO, 7% (v / v) glycerol, 1 mM EDTA, and 7.5 mM Tris. The solution was left on the filter, and the filter was incubated at 92°C for 5 minutes in a filter heating block.

[0345] After incubation, the filters were cooled for 3 minutes, and then 2 ml of mineral oil was passed through the filters, removing the second lysis solution containing the free DNA as eluate.

[0346] Approximately 200 μl of eluate was separated from the immiscible liquid, neutralized with 20x neutralization solution (500 mM HCl, 50 mM Tris), and run in quintuplicate qPCR using the target-specific assay. 4 / 5 replicates tested positive for C. albicans with an average Cq value of 40.2, 4 / 5 for S. aureus with an average Cq value of 38.1, and 5 / 5 for H. influenzae with an average Cq value of 37.6.

[0347] Example 3 Microbial suspensions derived from Staphylococcus aureus, Candida albicans, and Haemophilus influenzae were prepared from colonies of pure microbial cultures. The colonies were suspended in PBS and diluted to an OD of 0.5 McF. A 104 cfu / ml suspension was prepared and centrifuged to remove any potential free DNA. After dissolving the pellet, a 1000 cfu / ml suspension was prepared and used for spiking blood samples. The suspension was then plated for culture to determine the exact final concentration.

[0348] Four ml of blood was spiked with test microorganisms to concentrations of 30 and 15 cfu / ml. The blood was mixed with an equal volume of lysis solution containing 4 M guanidine hydrochloride, 4% (v / v) Tween® 20, 4% (w / v) saponin, and 40.5 mM Tris. The resulting lysate was filtered through a syringe filter with a pore size of 0.22 μm using a syringe. Experiments were performed in duplicate.

[0349] A second lysis solution containing 85 mM sodium hydroxide, 7% (v / v) glycerol, 1 mM EDTA, and 7.5 mM Tris was tested both without DMSO and with the addition of 20% (v / v) DMSO. The second lysis solution was used to wash the filter by filtering 15 ml of the solution. The solution was left on the filter, and the filter was incubated in an oven at 90°C for 20 minutes.

[0350] After incubation, the filters were allowed to cool for several minutes, and then 2 ml of mineral oil was forced through the filters in the same direction, removing the second lysis solution containing the free DNA from the filters as eluate.

[0351] Approximately 200 μl of the eluate was separated from the immiscible liquid, neutralized with 20x neutralization solution (500 mM HCl, 50 mM Tris), and run in 10 replicate qPCR using the target-specific assay. The results are shown in Table 2 below. [Table 2]

[0352] The results show that the addition of DMSO had a clear beneficial effect on both C. albicans and S. aureus. C. albicans Cq values ​​and PCR replicate positivity (PCR replicates from the same eluate) were improved using DMSO (comparing conditions 1 & 2 to 3 & 4 (with 30 cfu / mL spike) and conditions 5 & 6 to 7 & 8 (with 15 cfu / mL spike)). Some improvement was also observed for S. aureus (especially when comparing conditions 1 & 2 to 3 & 4). These results for Candida and S. aureus were unexpected. On the other hand, H. influenzae showed no improvement in Cq values, indicating that more sensitive microorganisms for lysis were already efficiently lysed without the aid of DMSO.

[0353] Example 4 During the development of the enrichment protocol, we found that some Gram-positive bacteria were more resistant to lysis, requiring optimization of the protocol. Therefore, we included an enzymatic treatment in the enrichment protocol for better degradation of cell wall components (e.g., peptidoglycan).

[0354] Microbial suspensions of (A) Haemophilus influenzae, (B) Listeriae monocytogenes, (C) Candida albicans, (D) Streptococcus pneumoniae, (E) Streptococcus agalactiae, (F) Stenotrophomonas maltophilia, (G) Enterococcus faecium, and (H) Staphylococcus aureus were prepared from colonies of pure cultures. Colonies were suspended in PBS to a high concentration, centrifuged to remove free DNA, and the pellet was resuspended in fresh PBS and adjusted to an OD of 0.5 McF. A 104 cfu / ml suspension was prepared and used to spike blood samples.

[0355] Four milliliters of blood was spiked with the test microorganism to a concentration of 20 cfu / ml. The blood was mixed with an equal volume of lysis solution containing 4 M guanidine hydrochloride, 4% (v / v) Tween® 20, 4% (w / v) saponin, and 40.5 mM Tris. The resulting lysate was filtered through a 0.22 μm pore size syringe filter using a syringe.

[0356] The filter was washed by filtering 15 ml of the solution using diluted second lysis solution (3.5% (v / v) glycerol, 0.5 mM EDTA, 42.5 mM sodium hydroxide, 10 mM Tris, and 9% (v / v) DMSO).

[0357] 1 ml of 500 U / ml mutanolysin solution was added through a syringe filter.

[0358] The filters were incubated with the mutanolysin solution for 15 minutes at room temperature.

[0359] The concentrated second lysis solution was added to the filter in such a volume (100 μl) that it was likely unable to push the liberated analyte out of the filter, and the lysis solution concentration in the filter was diluted to 20 mM sodium hydroxide, 18% (v / v) DMSO, 7% (v / v) glycerol, 1 mM EDTA, 7.5 mM Tris during the reaction.

[0360] The filters were placed on an induction heater and incubated at a maximum temperature of 110°C for 8 minutes.

[0361] After incubation, the filters were cooled for 3 minutes, and then 2 ml of mineral oil was passed through the filters, removing the second lysis solution containing the free DNA as eluate.

[0362] Approximately 200 μl of eluate was separated from the immiscible liquid and run in duplicate in qPCR using target-specific assays.

[0363] A positive control was prepared at the concentration expected to be present in the eluate after successful enrichment (i.e., approximately 10-fold enrichment from the initial sample) using a MagNA Lyser instrument (Roche Diagnostics), which automatically disrupts cells by bead beating. The results are shown in Table 3 below. [Table 3]

[0364] The results show that after enrichment, most of the tested targets reach Cq values ​​similar to that of the 200 cfu / ml positive control. C. albicans, which is not susceptible to mutanolysin, remains approximately 3 Cq behind the positive control result. Nevertheless, the PCR positivity rate for yeast from the initial spiked sample at 20 cfu / ml is 100%. Of the tested targets, E. faecium, which should be susceptible to mutanolysin, requires further protocol optimization for optimal lysis. Overall, the addition of the mutanolysin step improved the detection of Gram-positive bacteria to Cq levels similar to the positive control.

[0365] Example 5 The pathogen enrichment protocol was evaluated using microbial cell lysis under three separate conditions: (1) DMSO alone (in water); (2) DMSO in 85 mM NaOH; and (3) DMSO in "GENT" lysis solution (85 mM NaOH, 7% glycerol, 1 mM EDTA, 7.5 mM Tris). DMSO was tested at 20% (v / v) and 40% (v / v) for each of conditions (1)-(3) (for a total of six different test solutions).

[0366] The protocol used in this study is summarized below. 1) Take a 4ml PBS sample into a 50ml Falcon tube. (Spike centrifuge (9500 rpm / min, 10 min) + wash pathogen to 30 cfu / ml (12 μl from 10^4 cfu / ml stock) and mix gently. 2) Using a needle, collect a PBS sample into a 10 ml syringe. Attach the syringe to the filter inlet. Filter the sample. 3) Wash the filter by filtering 13 ml of the GENT dissolution solution or solutions under study at a rate of approximately 20 ml / min. 4) Cap the filter and place in a 90°C oven for 20 minutes. 5) Elute by connecting a 1 ml syringe filled with mineral oil directly to the top inlet (for co-directional elution) and pushing steadily through at a slow speed. 6) Use a needle attached to a 1 ml syringe to separate the lower liquid phase (containing the lysis solution and microbial DNA) and transfer it to a new tube. 7) Neutralize the eluate by adding 20x Neutralizer (500 mM HCl, 50 mM Tris) and checking the pH with pH paper. Aim for a pH of 8.5. (Approximately 50 μl of 20x Neutralizer was required for 250 μl of eluate.)

[0367] The results are shown in Table 4 below. [Table 4]

[0368] The results show that a 40% DMSO concentration was unable to lyse C. albicans alone in water or 85 mM NaOH. H. influenzae was always lysed in the presence of NaOH. Some detection power was also seen for Candida when 40% DMSO was in a solution containing both glycerol and NaOH (GENT).

[0369] In 20% DMSO in water, cells were not lysed, but in 20% DMSO containing 85 mM NaOH, detection was observed. Detection was significantly improved for Candida when 20% DMSO was in a solution containing glycerol and NaOH (GENT).

[0370] 40% DMSO in GENT appears to be inhibitory in PCR (or requires PCR optimization) compared to 20% DMSO in GENT.

[0371] Example 6 The induction heating pathogen enrichment protocol was evaluated using a lysis solution with or without DMSO. The basic lysis solution (GENT) contained 85 mM NaOH, 7% glycerol, 1 mM EDTA, and 7.5 mM Tris. DMSO was used at a concentration of 18% (v / v).

[0372] The protocol used in this study is summarized below. 1) Mix the blood sample thoroughly by inverting the tube more than 30 times. 2) Take a 4ml blood sample into a 50ml Falcon tube. (Spike centrifuge (9500 rpm / min, 10 min) + wash pathogen to 50cfu / ml (20µl from 10^4cfu / ml stock) and mix gently. 3) Add 4 ml of hemolysis solution (4 M Gu-HCl, 4% Tween®-20 (v / v), 4% saponin, 40.5 mM Tris-HCl) and mix by gently pipetting back and forth 25 times. 4) Using a needle, draw the lysed blood sample into a 10 ml syringe. Remove all air. Attach the syringe directly to the filter at a 90° angle. Filter the blood. 5) Wash the filter by filtering through 15 ml of GENT solution at a rate of approximately 20 ml / min. 6) Cap the filter and place it in the filter heating holder. The following induction heating protocol was performed: heat the filter to 50°C and hold for 40 seconds; increase the heat to 70°C and hold for 20 seconds; increase the heat to 90°C and hold for 5 minutes. Allow to cool for a few minutes, then remove from the heating holder. 7) Attach a 1.2 ml syringe containing mineral oil to the filter inlet and elute by steadily pushing the oil through the filter. 8) Separating the miscible liquid lower phase from the oil. 9) Neutralize the eluate by adding 20x Neutralizer (500 mM HCl, 50 mM Tris) and checking the pH with pH paper. Aim for a pH of 8.5. (Approximately 40 μl of 20x Neutralizer was required for 250 μl of eluate.)

[0373] The results are shown in Table 5 below. [Table 5]

[0374] The results show a clear improvement in Candida albicans detection (3 Cq) when 18% DMSO was added to the GENT solution (compare #1 and #2).

[0375] Example 7 The sonication pathogen enrichment protocol was evaluated using a lysis solution containing DMSO either with or without NaOH. The lysis solution with NaOH (GENTD) contained 85 mM NaOH, 18% DMSO, 7% glycerol, 1 mM EDTA, and 7.5 mM Tris.

[0376] The protocol used in this study is summarized below. 1) Take 4 ml of blood in a 50 ml Falcon tube. Add pathogen to 50 cfu / ml (20 μl from 10^4 cfu / ml stock) and mix gently. 2) Using a needle, collect a sample into a 10 ml syringe. Attach the syringe to the filter inlet. Filter the blood. 3) Wash the filter by passing 15 ml of the relevant GENTD solution or TE buffer (10 mM Tris, 1 mM EDTA) through it at a rate of approximately 20 ml / min. 4) Cap the filter and sonicate using the sonotrode for the filter: ensure the same tight connection of the filter to the sonotrode. 5) Elute by connecting a 2 ml syringe filled with silicone oil (polydimethylsiloxane, trimethylsiloxy terminated) directly to the top inlet (for co-directional elution) and pushing it steadily through at a slow speed. 6) Use a needle attached to a 1 ml syringe to separate the lower liquid phase (containing the lysis solution and microbial DNA) and transfer it to a new tube. 7) Neutralize the eluate by adding 20x Neutralizer (500 mM HCl, 50 mM Tris) and checking the pH with pH paper. Aim for a pH of 8.5. (Approximately 50 μl of 20x Neutralizer was required for 250 μl of eluate.)

[0377] The results are shown in Table 6 below. [Table 6]

[0378] The results demonstrate the importance of NaOH in the lysis solution, even when using more aggressive mechanical lysis methods: readily lysed H. influenzae lysed poorly or inefficiently without NaOH (#3 and #6), or not at all (#4 and #5).

[0379] From the foregoing, it will be understood that, although specific embodiments of the invention have been described herein for purposes of illustration, various modifications may be made without departing from the spirit and scope of the invention. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.

[0380] The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

Claims

1. 1. A method for selectively isolating a microbial cell analyte from a sample, said 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, if present, intact microbial cells, wherein the first lysis solution is 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, the pore size being between about 0.1 μm and about 1 μm; (d) contacting the filter containing the retained microbial cells with a second lysis solution effective to lyse the microbial cells to release analytes therefrom, the second lysis solution being a buffer solution comprising sodium hydroxide at a concentration of about 20 mM to about 500 mM, dimethyl sulfoxide (DMSO) at a concentration of about 5% (v / v) to about 30% (v / v), and glycerol at a concentration of about 1% (v / v) to about 15% (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 is released therefrom; and (f) eluting the analyte from the filter to obtain an eluate comprising the isolated analyte.

2. sodium hydroxide is present in said second lysis solution at a concentration of about 50 mM to about 100 mM; DMSO is present in the second lysis solution at a concentration of about 10% (v / v) to about 25% (v / v); 10. The method of claim 1, wherein glycerol is present in the second lysis solution at a concentration of about 3% (v / v) to about 10% (v / v).

3. The method according to claim 1 or 2, wherein the microbial cells are bacterial cells and / or yeast cells.

4. 4. The method of claim 1, wherein the filter comprises polyethersulfone (PES), cellulose, nylon, polyvinylidene fluoride (PVDF), polycarbonate, or glass fiber, and / or the filter comprises an asymmetric structure, and / or the pores on a first side of the asymmetric filter have a size of about 5 μm to about 20 μm, and the pores on a second side of the asymmetric filter have a size of up to about 0.2 μm.

5. The method of claim 4 , wherein the filter comprises polyethersulfone (PES).

6. 6. The method according to any one of claims 1 to 5, wherein between steps (b) and (c) or between steps (c) and (d), the method further comprises an enzyme treatment step comprising contacting the filter containing the retained microbial cells with an enzyme solution containing at least one enzyme effective to degrade one or more microbial cell wall components, wherein the at least one enzyme is preferably mutanolysin, lyticase, or lysozyme.

7. the first lysis solution further comprises a second detergent which is polysorbate 20; 7. The method of any one of claims 1 to 6, wherein optionally, polysorbate 20 is present in the first lysis solution at a concentration of about 1% (v / v) to about 10% (v / v).

8. guanidine hydrochloride is present in said first lysis solution at a concentration of about 1 M to about 8 M; and / or saponin is present in said first lysis solution at a concentration of about 1% (w / v) to about 10% (w / v); and / or the first lysis solution is buffered with about 20 mM to about 200 mM Tris buffer; and / or 8. The method of claim 1, wherein the second lysis solution is buffered with about 1 mM to about 50 mM Tris buffer.

9. the second dissolution solution further comprises a chelating agent; 9. The method of any one of claims 1 to 8, wherein optionally the chelating agent in the second lysis solution is 2,2',2'',2'''-(ethane-1,2-diyldinitrilo)tetraacetic acid (EDTA) and is present at a concentration of about 0.1 mM to about 4 mM or about 0.5 mM to about 2 mM.

10. 10. The method of any one of claims 1 to 9, wherein the volume of the sample provided in step (a) is from about 0.5 mL to about 5 mL.

11. The method according to any one of claims 1 to 10, wherein the sample is a blood sample.

12. 10. The method of any one of claims 1 to 9, wherein during said mixing step (b), the ratio of said first lysis solution to said sample is about 1:

1.

13. 13. The method of any one of claims 1 to 12, wherein the conditions for lysing in step (e) comprise incubating the filter with the second lysis solution at an incubation temperature of from about 80°C to about 150°C, more preferably from about 90°C to about 130°C.

14. 14. The method of any one of claims 1 to 13, wherein the conditions for lysing in step (e) comprise sonication.

15. 15. The method of any one of claims 1 to 14, wherein the eluting step (f) comprises passing an immiscible liquid through the filter to force the eluate containing the analyte through the filter, wherein the immiscible liquid is selected from the group consisting of oils and hydrocarbons.

16. 16. The method of any one of claims 1 to 15, wherein the direction of fluid flow through the filter in the eluting step (f) is the same as the direction of fluid flow applied in the filtering step (c).

17. 16. The method of any one of claims 1 to 15, wherein the direction of fluid flow through the filter in the eluting step (f) is opposite to the direction of fluid flow applied in the filtering step (c).

18. 18. The method of any one of claims 1 to 17, further comprising: (g) adding a neutralization buffer to the eluate obtained in step (f), wherein the neutralization buffer comprises hydrochloric acid (HCl) and Tris; and / or further comprising a washing step between steps (c) and (d), wherein the washing step comprises flowing a washing buffer through the filter.

19. The method of any one of claims 1 to 18, wherein the analyte is a nucleic acid.

20. 20. The method of any one of claims 1 to 19, further comprising analyzing the isolated analyte, where optionally the analyte is a nucleic acid, and analyzing the isolated nucleic acid analyte, the method comprising: (i) performing a nucleic acid amplification reaction using the isolated nucleic acid as a template to generate an amplification product; and (ii) detecting the amplification product.

21. 21. The method of claim 20, wherein analyzing the isolated nucleic acid comprises immobilizing the isolated nucleic acid or the amplification product on a solid support.

22. A dissolution solution comprising: sodium hydroxide at a concentration of about 20 mM to about 500 mM; dimethyl sulfoxide (DMSO) at a concentration of about 5% (v / v) to about 30% (v / v); glycerol at a concentration of about 1% (v / v) to about 15% (v / v); a lysis solution comprising: a buffer;

23. sodium hydroxide is present in said second lysis solution at a concentration of about 50 mM to about 100 mM; DMSO is present in the second lysis solution at a concentration of about 10% (v / v) to about 25% (v / v); 10. The lysis solution of claim 1, wherein glycerol is present in the second lysis solution at a concentration of about 3% (v / v) to about 10% (v / v).

24. 24. The lysis solution of claim 22 or 23, further comprising a chelating agent.

25. the chelating agent is 2,2',2'',2'''-(ethane-1,2-diyldinitrilo)tetraacetic acid (EDTA) and is present at a concentration of about 0.1 mM to about 4 mM; 25. The lysis solution of claim 24, wherein optionally the EDTA is present at a concentration of about 0.5 mM to about 2 mM.

26. the buffer is Tris and is present at a concentration of about 1 mM to about 50 mM; 26. The lysis solution of any one of claims 22 to 25, optionally wherein Tris is present at a concentration of about 5 mM to about 10 mM.

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