Methods for isolating microbial analytes

Abstract

A method for selectively isolating microbial cell analytes (e.g., nucleic acids) from a sample containing or suspected of containing mammalian cells is disclosed. The selective isolation method comprises selective lysis of mammalian cells, filtration of the lysate obtained by passing it through a filter that retains intact microbial cells, and subsequent release of the retained microbial cells from the filter. The microbial cells are then collected from the filter using backflow elution or aspiration, and the collected microbial cells are lysed to release the analytes from the intact cells. The isolated analytes can be analyzed using appropriate assays depending on the type of analyte molecule.

Description

Background Art

[0001] (Background) Many molecular diagnostic applications are at the forefront and require the best possible sensitivity to compete with products from other molecular diagnostic manufacturers. High sensitivity is typically a clear advantage for all molecular diagnostic applications and is a feature typically evaluated by end-users.

[0002] Furthermore, in certain cases, due to the low number of analyte molecules in the volume of the sample used for extraction, sensitivity requirements are raised that cannot be achieved by any typical sample extraction method, regardless of its sensitivity. Some applications in this field include direct blood sepsis pathogen detection, liquid biopsy for circulating tumor cells (CTCs), liquid biopsy for circulating tumor DNA and exosomes (e.g., derived from blood / plasma and urine matrices), and, for example, water quality analysis and airborne pathogen detection.

[0003] For example, direct blood sample microbial detection would have great value due to the more rapid overall results regarding the presence of the pathogen, pathogen identification, and, for example, antibiotic resistance characteristics. The current gold standard method is based on pathogen enrichment by blood culture, which is time-consuming (e.g., 24 hours to 72 hours) and has low detection sensitivity even after culturing. Worldwide, approximately 30 million cases of sepsis occur each year, resulting in 7 million to 9 million deaths (one death every 3.5 seconds). Published data show an 8% decrease in survival rate per hour due to the delay in antibiotic administration in sepsis, and published data show that 15% to 40% of sepsis patients receive inappropriate empirical antibiotic treatment due to the inability to rapidly detect the causative pathogen, highlighting the importance of early pathogen detection, early pathogen identification, and early antibiotic resistance profiling.

[0004] In the field of direct hematological 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, with pathogen enrichment from the blood sample performed first. DNAe's assay uses magnetic particles combined with pathogen-specific antibodies, while T2 Biosystems' and Qvell's assays use centrifugation for enrichment. The use of biomolecules (e.g., antibodies) for sample enrichment raises issues regarding storage / stability, lot-to-lot variability, and cost, in addition to incubation time. The use of centrifugation in a closed system results in bulky equipment and specific fluidics that can retain precise fractions of the centrifugated material. European Patent No. EP2510123 discloses a method and device for using selective lysis to isolate a microbial cell analyte from a sample, the method comprising the steps of: providing a sample comprising eukaryotic cells (particularly animal cells) containing microorganisms or eukaryotic cells (particularly animal cells) suspected to contain microorganisms; adding a nonionic washing agent and buffer to the sample to obtain a solution having a pH of about 9.5 or greater; incubating the solution for a period of time long enough to lyse the animal cells; filtering the first lysate through a filter having a pore size that retains intact microbial cells, the pore size being about 0.1 μm to about 1 μm; and lysing the microbial cells to obtain a second lysate containing the analyte. In EP2510123, it is further disclosed that the filter retaining intact microbial cells is incubated at 95°C for 10 minutes with a lysis buffer containing 200 mM sodium hydroxide (NaOH) and 0.5% SDS. [Prior art documents] [Patent Documents]

[0005] [Patent Document 1] European Patent No. EP251012 [Overview of the project] [Problems that the invention aims to solve]

[0006] In various application fields, there is a need for sample enrichment methods that enable analyte detection sensitivity beyond what is achievable by conventional methods, as well as increased efficiency in terms of cost, assay time, and instrument space. [Means for solving the problem]

[0007] (overview) The methods described herein are based on enriching microbial cell analytes from larger volume samples containing mammalian cells by using selective filtration in combination with the collection of backflow filter contents and off-filter lysis of microbial cells downstream. In this manner, the overall assay sensitivity can be improved by processing larger sample volumes. Furthermore, since microorganisms may differ in their tolerance to lysis treatments, releasing and collecting microbial cells from the filter allows for the use of more potent lysis methods that would be impossible or extremely difficult to perform inside the filter. This concept is applicable not only to molecular diagnostics involving downstream amplification of nucleic acids (e.g., RNA / DNA) and detection by qPCR and / or microarrays, but also to the detection of proteins / other biomolecules by other detection methods (next-generation sequencing being one example), as well as by other suitable amplification / separation / detection methods, such as ELISA, protein arrays, MALDI-TOF, nanoLC / UPLC-ESI-MS, and other appropriate amplification / separation / detection methods.

[0008] A method for selectively isolating microbial cell analytes from a sample in accordance with this disclosure generally comprises: (a) providing a sample containing mammalian cells and possibly microbial cells; (b) mixing the sample with a first solution that selectively lyses the 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 solution effective in enabling the release of the microbial cells from the filter; and (e) incubating the filter together with the second solution under conditions that promote the release of the microbial cells from the filter. The process includes: (f) collecting the microbial cells from the filter, wherein the collection step includes (i) using an elution buffer to elute the microbial cells from the filter, wherein the direction of the fluid flow through the filter is opposite to the direction of the fluid flow applied in the filtration step (c); or (ii) aspirating the microbial cells from the filter, wherein the microbial cells are aspirated from the same side of the filter that was first contacted by the first lysate in step (c); and (g) lysing the microbial cells collected in step (f) to obtain a second lysate containing an analyte released from the microbial cells, thereby selectively isolating the microbial cell analyte from the sample. In some embodiments, the analyte to be isolated is nucleic acid. In other non-mutually exclusive embodiments, the microbial cells are bacterial cells or yeast cells. In other non-mutually exclusive variants, the mammalian cells are blood cells. In yet another non-mutually exclusive embodiment, the mammalian cells are human cells.

[0009] Filters particularly suitable for use in the above method include those containing polyethersulfone (PES), cellulose, nylon, polyvinylidene fluoride (poly(1,1-difluoroethylene), PVDF), polycarbonate, or glass (e.g., borosilicate glass) fibers. In certain variants, the filter has a pore size of about 1 μm or less (e.g., pore size of about 0.1 μm to about 1 μm, pore size of about 0.2 μm to about 1 μm, or pore size of about 0.22 μm). In other non-mutually exclusive variants, the filter includes an asymmetric structure. In certain variants, 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.

[0010] In some embodiments of the method described above, the first solution comprises a chaotropic salt and a detergent. In some such embodiments, the chaotropic salt is guanidine hydrochloride, and / or the detergent is a saponin. In certain variants, the first solution further comprises a second detergent, such as polysorbate 20.

[0011] In some embodiments of the method described above, the first solution comprises guanidine hydrochloride, polysorbate 20, saponin, and a buffer. In some such embodiments, guanidine hydrochloride is present in the first solution at a concentration of about 1 M to about 8 M; polysorbate 20 is present in the first solution at a concentration of 1% (v / v) to about 10% (v / v); saponin is present in the first solution at a concentration of about 1% (w / v) to about 10% (w / v); and / or the buffer in the first solution is Tris, present at a concentration of about 20 mM to about 200 mM. In a more specific variant, guanidine hydrochloride is present in the first solution at a concentration of about 4 M; polysorbate 20 is present in the first solution at a concentration of 4% (v / v); saponin is present in the first solution at a concentration of about 4% (w / v); and / or tris is present in the first solution at a concentration of about 35 mM to about 45 mM (e.g., about 40.5 mM).

[0012] In a particular embodiment of the method described above, the ratio of the first solution to the sample during the mixing step (b) is approximately 1:1. In other non-mutually exclusive variants, the concentration of guanidine hydrochloride in the first solution is approximately 0.5 M to approximately 4 M; the concentration of polysorbate 20 in the first solution is approximately 0.5% (v / v) to approximately 5% (v / v); the concentration of saponin in the first solution is approximately 0.5% (w / v) to approximately 5% (w / v); and / or the buffer in the first solution is Tris, and the concentration of Tris in the first solution is approximately 10 mM to approximately 100 mM. In some such variant forms, the concentration of guanidine hydrochloride in the first solution is approximately 2 M; the concentration of polysorbate 20 in the first solution is approximately 2% (v / v); the concentration of saponin in the first solution is approximately 2% (w / v); and / or the buffer in the first solution is Tris, and the concentration of Tris in the first solution is approximately 18 mM to approximately 23 mM (e.g., approximately 20.25 mM).

[0013] In some embodiments of the method described above, the second solution comprises sodium hydroxide, dimethyl sulfoxide (DMSO), glycerol, a chelating agent, and a buffer. In some such embodiments, sodium hydroxide is present in the second solution at a concentration of about 10 mM to about 100 mM; DMSO is present in the second solution at a concentration of about 2% (v / v) to about 20% (v / v); glycerol is present in the second solution at a concentration of about 1% (v / v) to about 15% (v / v); the chelating agent in the second solution is 2,2',2'',2'''-(ethane-1,2-diyldinitro)tetraacetic acid (EDTA) at a concentration of about 0.1 mM to about 4 mM; and / or the buffer in the second solution is Tris at a concentration of about 1 mM to about 50 mM. In a more specific variant, sodium hydroxide is present in the second solution at a concentration of about 30 mM; DMSO is present in the second solution at a concentration of about 10% (v / v); glycerol is present in the second solution at a concentration of about 7% (v / v); the chelating agent in the second dissolution is EDTA, present at a concentration of about 1 mM; and / or the buffer in the second solution is Tris, present at a concentration of about 5 mM to about 10 mM (e.g., about 7.5 mM).

[0014] In a particular variation of the method described above, the conditions of step (e) include incubating the filter with the second solution at a temperature of about 70°C to about 99°C (for example, a temperature of about 90°C to about 95°C).

[0015] In certain embodiments, the method described above further includes the step of adding a neutralizing buffer to the eluate obtained in step (g) (h). In some such embodiments, the neutralizing buffer may include hydrochloric acid (HCl) and Tris. Alternatively, in certain embodiments, the method includes the step of analyzing an isolated target, wherein the analysis step includes the step of carrying out an amplification reaction (see below), the amplification reaction mixture is buffered to neutralize the pH of the eluate / amplification reaction mixture (for example, to make the pH of the eluate / amplification reaction mixture less alkaline).

[0016] In some embodiments, the method described above further includes a washing step between step (c) and step (d), the washing step including passing the washing buffer through the filter. In some such embodiments, the washing step is performed once after step (c) and before step (d). In other embodiments, the filtration step (c) and the washing step are repeated sequentially multiple times before step (d), each iteration of step (c) including filtering a portion of the first dissolution through the filter. Particularly suitable washing buffers include EDTA and Tris; in some such variants, the washing buffer further includes polysorbate 20, which may be present in the washing buffer at a concentration of about 0.5% (v / v) to about 5% (v / v). In other embodiments, the washing buffer is the same as the second solution or the second dissolution at a diluted concentration. In an alternative modified form, the above method does not include a washing step between step (c) and step (d).

[0017] In some variations of the above-described method, step (f) includes the step of using an elution buffer to elute microbial cells from a filter, wherein the elution buffer comprises EDTA and Tris. In some such embodiments, the elution buffer further comprises polysorbate 20, which may be present in the elution buffer at a concentration of about 0.5% (v / v) to about 5% (v / v).

[0018] In certain embodiments of the methods described above, the lysis of the microbial cells in step (g) includes physical lysis. Particularly suitable physical lysis methods include one or more of sonic treatment, ultrasound, bead beating, radiolysis, and electrolysis. In some variations, the physical lysis includes electromagnetic lysis.

[0019] The above method may further include a step of analyzing the isolated analyte. For example, if the isolated analyte is nucleic acid, the step of analyzing the isolated nucleic acid may include (i) carrying out an amplification reaction using the isolated nucleic acid as a template to produce 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 variants, the amplification is an isothermal amplification reaction, e.g., a transcription-mediated amplification reaction. The detection step (ii) may be carried out in real time. The method may optionally include an analyte isolation / purification step to isolate the target analyte of interest from the non-target analyte in the microbial analyte.

[0020] In some embodiments of the above-described method, the method further includes the step of analyzing an isolated nucleic acid analyte, the analysis step including 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 within a nucleic acid array).

[0021] A typical embodiment of this method is further described below. Embodiment

[0022] Embodiment 1. A method for selectively isolating a microbial cell analyte from a sample, comprising: (a) providing a sample containing mammalian cells and possibly containing microbial cells; (b) mixing the sample with a first solution that selectively lyses the mammalian cells to obtain a first lysate containing the 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 solution effective to allow release of the microbial cells from the filter; (e) incubating the filter with the second solution under conditions that promote release of the microbial cells from the filter; (f) collecting the microbial cells from the filter, wherein the collecting step comprises: (i) using an elution buffer to elute the microbial cells from the filter, wherein the direction of fluid flow through the filter is opposite to the direction of fluid flow applied in the filtering step (c); or (ii) aspirating the microbial cells from the filter, wherein the microbial cells are aspirated from the same side of the filter that was first contacted by the first lysate in step (c); and (g) lysing the microbial cells collected in step (f) to obtain a second lysate containing the analyte released from the microbial cells, thereby selectively isolating the microbial cell analyte from the sample.

[0023] Embodiment 2. The method of Embodiment 1, wherein the microbial cells are bacterial cells.

[0024] Embodiment 3. The method of Embodiment 2, wherein the bacterial cells are Gram-positive bacterial cells.

[0025] Embodiment 4. The method of Embodiment 2, wherein the bacterial cells are Gram-negative bacterial cells.

[0026] Embodiment 5. The method of Embodiment 2, wherein the microbial cells are yeast cells.

[0027] Embodiment 6. Any one of Embodiments 1 to 5, wherein the filter has a pore size of about 1 μm or less.

[0028] Embodiment 7. The method of Embodiment 6, wherein the filter has a pore size of approximately 0.1 μm to approximately 1 μm.

[0029] Embodiment 8. The method according to Embodiment 7, wherein the filter has a pore size of approximately 0.22 μm.

[0030] Embodiment 9. Any one of Embodiments 1 to 8, wherein the filter comprises polyethersulfone (PES), cellulose, nylon, polyvinylidene fluoride (PVDF), polycarbonate, or glass fiber.

[0031] Embodiment 10. The method according to any one of Embodiments 1 to 9, wherein the filter includes an asymmetric structure, preferably the asymmetric filter includes 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.

[0032] Embodiment 11. Any one of Embodiments 1 to 10, wherein the first solution comprises a chaotropic salt and a detergent.

[0033] Embodiment 12. The method of Embodiment 11, wherein the chaotropic salt is guanidine hydrochloride.

[0034] Embodiment 13. The method of Embodiment 11 or 12, wherein the cleaning agent is a saponin.

[0035] Embodiment 14. The method of Embodiment 13, wherein the first solution further comprises a second cleaning agent.

[0036] Embodiment 15. The method of Embodiment 14, wherein the second cleaning agent is polysorbate 20.

[0037] Embodiment 16. Any one of Embodiments 1 to 10, wherein the first solution comprises guanidine hydrochloride, polysorbate 20, saponin, and buffer.

[0038] Embodiment 17. The method of Embodiment 16, wherein guanidine hydrochloride is present in the first solution at a concentration of about 1 M to about 8 M.

[0039] Embodiment 18. The method of Embodiment 17, wherein guanidine hydrochloride is present in the first solution at a concentration of about 4 M.

[0040] Embodiment 19. Any one of Embodiments 16 to 18, wherein polysorbate 20 is present in the first dissolution solution at a concentration of about 1% (v / v) to about 10% (v / v).

[0041] Embodiment 20. The method of Embodiment 19, wherein polysorbate 20 is present in the first dissolution solution at a concentration of 4% (v / v).

[0042] Embodiment 21. Any one of Embodiments 16 to 20, wherein saponin is present in the first solution at a concentration of about 1% (w / v) to about 10% (w / v).

[0043] Embodiment 22. The method of Embodiment 21, wherein saponin is present in the first solution at a concentration of approximately 4% (w / v).

[0044] Embodiment 23. Any one of Embodiments 16 to 22, wherein the buffer solution in the first solution is Tris, present at a concentration of about 20 mM to about 200 mM.

[0045] Embodiment 24. The method of Embodiment 23, wherein Tris is present in the first solution at a concentration of about 35 mM to about 45 mM.

[0046] Embodiment 25. The method according to any one of Embodiments 1 to 24, wherein during the mixing step (b), the ratio of the first solution to the sample is approximately 1:1.

[0047] Embodiment 26. The method of Embodiment 16, wherein the concentration of guanidine hydrochloride in the first solution is about 0.5 M to about 4 M.

[0048] Embodiment 27. The method of Embodiment 26, wherein the concentration of guanidine hydrochloride in the first solution is approximately 2 M.

[0049] Embodiment 28. Any one of Embodiments 16, 26, and 27, wherein the concentration of polysorbate 20 in the first solution is about 0.5% (v / v) to about 5% (v / v).

[0050] Embodiment 29. The method of Embodiment 28, wherein the concentration of polysorbate 20 in the first solution is approximately 2% (v / v).

[0051] Embodiment 30. Any one of Embodiments 16 and 26-29, wherein the concentration of saponin in the first solution is about 0.5% (w / v) to about 5% (w / v).

[0052] Embodiment 31. The method of Embodiment 30, wherein the concentration of saponin in the first solution is approximately 2% (w / v).

[0053] Embodiment 32. Any one of Embodiments 16 and 26-31, wherein the buffer in the first solution is Tris, and the concentration of Tris in the first dissolve is about 10 mM to about 100 mM.

[0054] Embodiment 33. The method of Embodiment 32, wherein the concentration of Tris in the first solution is approximately 18 mM to approximately 23 mM.

[0055] Embodiment 34. Any one of Embodiments 1 to 33, wherein the second solution comprises sodium hydroxide, dimethyl sulfoxide (DMSO), glycerol, a chelating agent, and a buffer.

[0056] Embodiment 35. The method of Embodiment 34, wherein sodium hydroxide is present in the second solution at a concentration of about 10 mM to about 100 mM.

[0057] Embodiment 36. The method of Embodiment 35, wherein sodium hydroxide is present in the second solution at a concentration of about 30 mM.

[0058] Embodiment 37. Any one of Embodiments 34 to 36, wherein DMSO is present in the second solution at a concentration of about 2% (v / v) to about 20% (v / v).

[0059] Embodiment 38. The method of Embodiment 37, wherein DMSO is present in the second solution at a concentration of approximately 10% (v / v).

[0060] Embodiment 39. Any one of Embodiments 34 to 38, wherein glycerol is present in the second solution at a concentration of about 1% (v / v) to about 15% (v / v).

[0061] Embodiment 40. The method of Embodiment 39, wherein glycerol is present in the second solution at a concentration of approximately 7% (v / v).

[0062] Embodiment 41. Any one of Embodiments 34 to 40, wherein the chelating agent in the second solution is 2,2',2'',2'''-(ethane-1,2-diyldinitrilo)tetraacetic acid (EDTA), present at a concentration of about 0.1 mM to about 4 mM.

[0063] Embodiment 42. The method of Embodiment 41, wherein EDTA is present in the second solution at a concentration of approximately 1 mM.

[0064] Embodiment 43. Any one of Embodiments 34 to 42, wherein the buffer solution in the second solution is Tris, present at a concentration of about 1 mM to about 50 mM.

[0065] Embodiment 44. The method of Embodiment 43, wherein Tris is present in the second solution at a concentration of about 5 mM to about 10 mM.

[0066] Embodiment 45. The method according to any one of Embodiments 1 to 44, wherein the conditions for promoting the release of the microbial cells from the filter in step (e) include incubating the filter with the second solution at an incubation temperature of about 70°C to about 99°C.

[0067] Embodiment 46. The method of Embodiment 45, wherein the incubation temperature is approximately 90°C to approximately 95°C.

[0068] Embodiment 47. Any one of Embodiments 1 to 46, further comprising a washing step between step (c) and step (d), wherein the washing step comprises a step of passing a washing buffer through the filter.

[0069] Embodiment 48. The method of Embodiment 47, wherein the filtration step (c) and the washing step are carried out sequentially multiple times before step (d), and each iteration of step (c) includes a step of filtering a portion of the first dissolved substance through the filter.

[0070] Embodiment 49. The method of Embodiment 47 or 48, wherein the washing buffer comprises EDTA and Tris.

[0071] Embodiment 50. The method of Embodiment 49, wherein the washing buffer further comprises polysorbate 20.

[0072] Embodiment 51. The method of Embodiment 50, wherein the polysorbate 20 is present in the washing buffer at a concentration of about 0.5% (v / v) to about 5% (v / v).

[0073] Embodiment 52. Step (f) is, (i) Steps of using the elution buffer to elute the microbial cells from the filter. A method according to any one of Embodiments 1 to 51, wherein the direction of the fluid flow through the filter is opposite to the direction of the fluid flow applied in the filtration step (c) above.

[0074] Embodiment 53. The method of Embodiment 52, wherein the elution buffer contains EDTA and Tris.

[0075] Embodiment 54. The method of Embodiment 53, wherein the elution buffer further comprises polysorbate 20.

[0076] Embodiment 55. The method of Embodiment 54, wherein the polysorbate 20 is present in the elution buffer at a concentration of approximately 0.5% (v / v) to approximately 5% (v / v).

[0077] Embodiment 56. Step (f) is, (ii) Step of aspirating the microbial cells from the filter mentioned above. The method according to any one of Embodiments 1 to 51, wherein the microbial cells are aspirated from the same side of the filter that was first contacted by the first lysate in step (c).

[0078] Embodiment 57. Any one of Embodiments 1 to 56, wherein the lysis of the microbial cells in step (g) includes physical lysis.

[0079] Embodiment 58. The method according to Embodiment 57, wherein the physical dissolution includes sonication, ultrasound, bead beating, radiolysis, and / or electrolysis.

[0080] Embodiment 59. The method of Embodiment 57 or 58, wherein the physical dissolution includes electromagnetic dissolution.

[0081] Embodiment 60.(h) Step of adding a neutralizing buffer to the second solution obtained in step (g). The method according to any one of embodiments 1 to 59, further including the above.

[0082] Embodiment 61. The method according to Embodiment 60, wherein the neutralization buffer comprises hydrochloric acid (HCl) and Tris.

[0083] Embodiment 62. Any one of Embodiments 1 to 61, wherein the mammalian cells are human cells.

[0084] Embodiment 63. Any one of Embodiments 1 to 62, wherein the mammalian cells are blood cells.

[0085] Embodiment 64. Any one of Embodiments 1 to 63, wherein the analyte is nucleic acid.

[0086] Embodiment 65. Any one of Embodiments 1 to 64, further comprising the step of analyzing the isolated analyte.

[0087] Embodiment 66. The method of Embodiment 64, further comprising the step of analyzing the isolated nucleic acid.

[0088] Embodiment 67. The method of Embodiment 66, wherein the step of analyzing the isolated nucleic acid includes (i) a step of carrying out an amplification reaction using the isolated nucleic acid as a template to produce an amplification product; and (ii) a step of detecting the amplification product.

[0089] Embodiment 68. The method of Embodiment 67, wherein the amplification reaction is PCR.

[0090] Embodiment 69. The method of Embodiment 67, wherein the amplification reaction is an isothermal amplification reaction.

[0091] Embodiment 70. The method of Embodiment 69, wherein the isothermal amplification reaction is a transcription-mediated amplification reaction.

[0092] Embodiment 71. The method according to any one of Embodiments 67 to 70, wherein the detection step (ii) described above is performed in real time.

[0093] Embodiment 72. Any one of Embodiments 67 to 70, wherein the step of analyzing the isolated nucleic acid includes the step of immobilizing the isolated nucleic acid or amplification product on a solid support.

[0094] Embodiment 73. The method according to Embodiment 72, wherein the isolated nucleic acid or amplification product is hybridized to an immobilized probe attached to the solid support.

[0095] Embodiment 74. The method of Embodiment 73, wherein the fixed probe is included in a nucleic acid array. [Modes for carrying out the invention]

[0096] These and other aspects of the present invention will become apparent from the following detailed description.

[0097] (definition) Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art with respect to the methods and compositions described herein. Where used herein, the following terms and phrases have the same meanings unless otherwise specified.

[0098] The terms “a,” “an,” and “the” refer to multiple objects unless the context explicitly states otherwise.

[0099] As used herein, the terms “microbe,” “microorganism,” and “microbial” refer to bacteria, archaea, fungi, and protists. In some embodiments, a microbe is a prokaryote (i.e., certain types of bacteria or archaea). In other embodiments, a microbe is a microbial species having a bacterial wall (e.g., any species of bacteria, archaea, or fungi, or certain protist species).

[0100] As used herein with respect to microbial cells, the term “intact” means a cell having a cell membrane and / or cell wall that is substantially undamaged, such that the intracellular components are retained as a whole within the cell.

[0101] As used herein with respect to microbial cells or other contents captured on a filter, “release” means the detachment of the captured contents from the membrane. The release of captured filter contents may include partial lysis of microbial cells. For example, in certain embodiments, the release process (e.g., chemical incubation with or without heating) may lyse certain types of microorganisms, while Gram-positive microorganisms and yeasts that are difficult to lyse will remain intact.

[0102] As used herein, “analyte” refers to a substance or one or more components thereof intended for identification and / or characterization, such as by detection by probe or sequencing. Examples of analytes include, but are not limited to, DNA, RNA, and proteins. In the context of this disclosure, an analyte is a component of a microbial cell.

[0103] "Sample" includes any specimen that may contain an analyte. "Sample" includes "biological samples" that contain any tissue or material derived from a living mammal (e.g., human) or a dead mammal (e.g., human). "Sample" may also include processed samples, such as samples obtained by passing a sample over or through a filtration device, or samples after centrifugation, or samples obtained by adhesion to a medium, a matrix, or a support.

[0104] A "cleansing agent" 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. Cleansing agents can be ionic or nonionic.

[0105] A buffer refers to a weak acid or weak base used to maintain the pH of a solution.

[0106] As used herein with respect to filters, the term “asymmetric structure” refers to a filter having pores of varying pore diameters, where the pores on one side of the filter are generally larger than those on the opposite side. In some embodiments, the asymmetric structure comprises a single filter membrane. In other variations, the asymmetric structure comprises multiple filter membranes, each having a different pore diameter (e.g., a first filter membrane with a larger pore diameter as a pre-filtration membrane, followed by another additional membrane, the additional membrane furthest distal to the first membrane having the smallest pore diameter). Unless otherwise explicitly stated in the context, “one (a) membrane” or “the membrane” includes reference to multiple membranes that may be present within a filter (e.g., a filter having an asymmetric structure).

[0107] "Nucleic acids" refer to polymeric compounds containing nucleotides or analogues of nitrogen-containing heterocyclic bases or nitrogen-containing heterocyclic base analogues that are linked together to form polymers (such as conventional RNA, DNA, mixed RNA-DNA, and their analogues).

[0108] As used herein, the term "nucleic acid array" refers to a solid support on which a group of target-specific nucleic acids are positioned at predetermined locations, either by spotting or direct synthesis.

[0109] As used herein, "nucleotide" refers to a nucleic acid subunit consisting of a phosphate group, a pentose sugar, and a nitrogen-containing base (also referred to herein as a "nucleic acid base"). In RNA, the pentose sugar is ribose. In DNA, the pentose sugar is 2'-deoxyribose.

[0110] The terms "oligomer," "oligonucleotide," or "oligo" generally refer to nucleic acids with fewer than 1,000 nucleotides (nt), including nucleic acids with a size range of approximately 5 nt to a lower limit and approximately 900 nt to an upper limit. The term oligonucleotide does not represent any specific function of a reagent; rather, the term is used generally to encompass all such reagents described herein. Oligomers may be referred to by their functional names (e.g., capture probe, detection probe, primer, or promoter primer), but those skilled in the art will understand that such terms refer to oligomers.

[0111] 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 that pore. For example, in the context of a pore with an annular or nearly annular cross-section, the pore diameter may refer to the diameter of that shape. In the context of a pore with a more rectangular or elliptical shape, the pore diameter may refer to the dimension of the smaller of the shapes, which corresponds to the width of the largest particle that can pass through that pore.

[0112] As used herein, the terms “target sequence” or “target nucleic acid sequence” refer to a specific nucleotide sequence in a nucleic acid analyte that is to be amplified and / or detected. The “target sequence” includes a complex-forming sequence in which an oligonucleotide (e.g., priming oligonucleotide and / or promoter oligonucleotide) forms a complex during the amplification process (e.g., PCR, TMA). Unless otherwise explicitly stated in the context, 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 (+) and antisense (-) strands.

[0113] "Nucleic acid amplification" refers to any well-known in vitro procedure that generates 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) (e.g., U.S. Patents 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. Patent 4,786,600), polymerase chain reaction (PCR) (e.g., U.S. Patents 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. Patent 5,422,252).

[0114] The term "amplicon" or "amplification product" refers to the nucleic acid molecule produced in a nucleic acid amplification reaction, which originates from the nucleic acid analyte. The amplicon or amplification product contains a target nucleic acid sequence that may be in the same sense or the opposite sense of its nucleic acid analyte.

[0115] An "amplifying oligonucleotide" or "amplifying oligomer" is an oligonucleotide that hybridizes to a nucleic acid analyte and participates in a nucleic acid amplification reaction (e.g., functions as a primer). An amplifying oligomer may have a 3' end that is extended by polymerization as part of its nucleic acid amplification reaction. An amplifying oligomer may also have an alternative 3' end that is not extended by polymerization but provides a component that promotes nucleic acid amplification (e.g., a promoter sequence that is bound to the 5' side of the target-specific sequence of the amplifying oligomer). Such an amplifying oligomer is called a promoter provider. An amplifying oligomer that provides both a 3' target-specific sequence that can be extended by polymerization and a 5' promoter sequence is called a promoter primer. An amplifying oligomer may be modified as needed to include a 5' non-target-specific sequence (e.g., a tag, promoter (as described above), or other sequence) that is used or useful to manipulate or amplify its primer or target oligonucleotide.

[0116] A “detection probe oligomer,” “detection probe,” or “probe” refers to an oligomer for the detection of a nucleic acid analyte that specifically hybridizes to a target sequence (including the amplified product) under conditions that facilitate nucleic acid hybridization. Detection can be either direct (i.e., a probe that hybridizes directly to the target) or indirect (i.e., a probe that hybridizes to an intermediate structure that binds the probe to the target). The target-specific sequence of a probe generally refers to a specific sequence within a larger sequence into which the probe specifically hybridizes. A detection probe may include target-specific sequences and non-target-specific sequences. Such non-target-specific sequences may include sequences that confer a desired secondary or tertiary structure (e.g., a hairpin structure, which may be used to facilitate detection and / or amplification).

[0117] "Label" or "detectable label" refers to a moiety or compound that is directly or indirectly bound to a probe to be detected or a probe that gives rise to a detectable signal. Direct binding may use covalent or non-covalent interactions (e.g., hydrogen bonds, hydrophobic or ionic interactions, and chelate or coordination complex formation), while indirect binding may use a bridging moiety or linker (e.g., by an antibody or additional oligonucleotide(s) (which amplify the detectable signal)). Any detectable moiety may be used, which include, for example: radionuclides, ligands (e.g., biotin or avidin), enzymes, enzyme substrates, reactive groups, chromophores (e.g., dyes, or particles that impart a detectable color (e.g., latex or metal beads)), luminescent compounds (e.g., bioluminescent compounds, phosphorescent compounds, or chemiluminescent compounds (e.g., acridinium ester ("AE") compounds)), and fluorescent compounds (i.e., fluorophores). Fluorophores can be used in combination with quencher molecules that absorb light when in proximity to the fluorophore to reduce background fluorescence. Examples of detectably labeled probes include hydrolysis (e.g., TaqMan®) probes, AE-labeled probes, molecular torches, and molecular beacons.

[0118] In this specification, references to numerical ranges (e.g., "X to Y" or "from X to Y") include the endpoint defining the range and all values ​​that fall within that range.

[0119] Unless it is clear from the context otherwise, when a value is expressed as "about" X or "approximately" X, the stated value X is understood to be accurate to within ±10%. In particular, with regard to temperature values, the terms "about" or "approximately" mean ±1°C.

[0120] In the extent used herein, the terms “first” and “second” preceding the names of elements (e.g., solutions or their components) are used for specific purposes to distinguish similar elements and are not necessarily intended to imply 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 does not necessarily imply or require the existence of additional such elements (e.g., “second,” “third,” etc.).

[0121] (explanation) This disclosure provides a method for selectively isolating microbial cell analytes from a sample containing mammalian cells. The disclosed method is particularly useful, for example, to increase the sensitivity of downstream molecular diagnostic assays that target the isolated analytes. For example, detection sensitivity in analytical applications (e.g., nucleic acid amplification and detection) generally depends on the amount of analyte introduced into the detection reaction. Typical molecular diagnostic applications are expected to be highly sensitive, but typical analyte extraction procedures for such applications (e.g., extraction of DNA or RNA for use in downstream amplification) do not provide the required sensitivity. In certain embodiments, this disclosure addresses this challenge by providing a method for enriching microbial cell analytes (which may be present in relatively small amounts in a larger volume sample containing mammalian cells), the method comprising the steps of lysing mammalian cells using selective lysis, filtering the lysate to capture and isolate intact microbial cells (while simultaneously allowing mammalian cell components released by the selective lysis step to pass through the filter), releasing and collecting the captured filter contents using backflow elution or aspiration, and lysing the microbial cells collected from the filter. In this manner, the overall assay sensitivity can be improved. For example, in addition to enrichment of microbial cells achieved using selective lysis and filtration, lysis of the microbial cells away from the filter allows for a wider range of lysis methods. This is because microorganisms have very different tolerances to various (e.g., chemical or enzymatic) lysis techniques, which can make on-filter lysis very difficult or even impossible. Using the method of this disclosure, intact microbial cells containing the analyte are not necessarily lysed inside the filter, but are lysed downstream of the collection process using an appropriate lysis treatment.This method and the corresponding system are applicable to molecular diagnostics, for example, for nucleic acid detection (e.g., by quantitative PCR, nucleic acid arrays, and / or other microarrays, as well as other nucleic acid detection methods (e.g., next-generation sequencing)) and detection of other biomolecules (e.g., proteins) (e.g., by ELISA, protein arrays, MALDI-TOF, nanoLC / UPLC-ESI-MS, and other suitable detection methods).

[0122] One of the concepts of this disclosure is the use of size-selective filtration for enriching microorganisms from a large background matrix volume (e.g., blood or other biological matrix), thereby resulting in enrichment of the analyte concentration in the sample, and both replacing the sample matrix with one or more suitable downstream analytical procedures (e.g., PCR or other detection methods, including immunological detection). Thus, large sample volumes can be processed to detect analytes that may be present at very low concentrations. The size selection is achieved by using specific filters having selected pore sizes and filter materials, and having an asymmetric structure such that the pore size decreases as it moves downstream through one or more filter membranes, if necessary. In some examples, target prokaryotes and target eukaryotes (bacteria and yeast) are captured by filters having, for example, a pore size of 0.22 μm and composed of, for example, polyethersulfone (PES) or any other applicable membrane material. The selected membrane size and material allow for sufficient capture of organisms, including the target analyte, and furthermore, the membrane type does not easily clog, even by using a larger sample volume or by excessive washing of the membrane with particles smaller than its pore size.

[0123] To guide the above analytes to downstream analytical processes (e.g., PCR, isothermal RNA / DNA amplification, detection of proteins or other biomolecular analytes), lysis away from the filter is used. Microorganisms captured by the filter are released from the capture filter by chemical and / or physical methods, and the microbial cells are collected from the filter for downstream lysis. The collection of the microbial cells may utilize a 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. Alternatively, the collection step may include aspirating the microbial cells from the filter, and the cells are collected from the same side of the filter that the sample first came into contact with.

[0124] The backflow method of this disclosure, combined with the lysis of collected microorganisms away from the filter, is particularly advantageous for the isolation of microbial cell analytes. For example, methods utilizing elution through a membrane after "in-filter lysis" carry a greater risk of failing to achieve efficient enrichment of microbial analytes than methods using lysis performed outside the filter, as in this method. Alternatively, in-filter lysis is possible for microorganisms that are more easily soluble (ultrasonic treatment can also destroy microorganisms that are difficult to lyse, releasing their analytes), but these methods are more difficult to design the instruments for. The backflow method of this disclosure can be easily implemented in instruments and exhibits very good results (see, for example, Example 3 below).

[0125] A method for selectively isolating microbial cell analytes from a sample generally involves the following steps: (a) providing a sample containing or possibly containing mammalian cells and microbial cells; (b) mixing the sample with a first 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 allows the lysed mammalian cells to pass through; (d) contacting the filter containing the retained microbial cells with a second solution effective in enabling the release of the microbial cells from the filter; and (e) contacting the filter with the second solution to enable the release of the microbial cells from the filter. The steps include: (f) incubating under conditions that promote separation; (i) collecting the microbial cells from the filter, wherein the collection step includes (i) using an elution buffer to elute the microbial cells from the filter, wherein the direction of the fluid flow through the filter is opposite to the direction of the fluid flow applied in the filtration step (c); or (ii) aspirating the microbial cells from the filter, wherein the microbial cells are aspirated from the same side of the filter that was first contacted by the first lysate in step (c); and (g) lysing the microbial cells collected in step (f) to obtain a second lysate containing the analyte released from the microbial cells, thereby selectively isolating the microbial cell analyte from the sample.

[0126] A range of samples containing or potentially containing mammalian cells and microbial cells may be analyzed in accordance with this disclosure. In a typical variant, the sample is known to contain mammalian cells and is suspected to contain at least one or more microorganisms. In some such variants, the sample is isolated from a mammal. For example, a sample particularly suitable for analysis using the methods disclosed herein is, for example, a mammalian (e.g., human) blood sample for the detection of septicemic pathogens in blood. In other embodiments, the sample is suspected to contain mammalian cells but is not known to contain them (e.g., a sample not isolated from a mammal but originating elsewhere (e.g., an environmental sample)). The microbial cells may be any bacterial, archaeal, fungal, or protist species, in particular a species characterized by a bacterial wall. In a typical variant, the microbial cells may be pathogenic microorganisms or microorganisms that cause infection in the body, but the microbial cells may be pathogenic or non-pathogenic. Furthermore, the step of providing the sample does not necessarily involve isolating the sample from a mammal or other source, but can be carried out, for example, by retrieving the sample from the container in which it is stored, or by initiating contact between the sample and the first dissolving solution immediately before the mixing step (b).

[0127] Non-limiting examples of microorganisms that may be targeted for analyte isolation as relating to this disclosure include: Gram-negative bacteria (e.g., 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, Pseudomanas aeruginosa, and Stenotrophomonas maltophilia, etc.); Gram-positive bacteria (e.g., 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, etc.); and yeast (e.g., Candida). Examples include Candida albicans, Candida auris, Candida glabrata, Candida krusei, Candida parapsilosis, Candida tropicalis, Cryptococcus neogormans, and Cryptococcus gatti.

[0128] As shown above, the first solution is capable of lysing mammalian cells while leaving all microbial cells (including microbial cells of interest) intact. The first solution can be mixed with the sample in any appropriate ratio, based on its solution components and individual concentrations, to achieve effective concentrations of the solution components in the final mixture for selective lysis. Exemplary ratios of the first solution to the 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.

[0129] Typically, the first solution described above comprises a chaotropic salt and a detergent. A particularly suitable chaotropic salt is guanidine hydrochloride, which may be present in the first solution at concentrations 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 first dissolution reaction after mixing with the sample, or the concentration in the final solution) include, for example, concentrations of about 0.5 M to 4 M, about 0.5 M to 3 M, about 0.5 M to 2.5 M, about 1 M to 4 M, about 1 M to 3 M, about 1 M to 2.5 M, about 1.5 M to 4 M, about 1.5 M to 3 M, about 1.5 M to 2.5 M, or about 2 M. Other suitable chaotropic salts include, for example, guanidine thiocyanate, urea, and lithium perchlorate.

[0130] The detergent for use in the first solution described above may be a nonionic detergent, an ionic detergent, or a zwitterionic detergent. Mild detergents and / or low detergent concentrations may typically be used to maintain microbial cells intact. Nonionic detergents (e.g., saponins or polyoxyethylene surfactants) are preferred. Saponins are particularly preferred and may be present in the first dissolution solution at concentrations 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 saponins may be used at effective concentrations 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) (i.e., the concentration in the first dissolution reaction after mixing with the sample, or the concentration in the final solution). A particularly suitable polyoxyethylene surfactant is polysorbate 20 (Tween®-20), which may be present in the above first dissolution solution at concentrations 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 effective concentrations 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, nonylphenoxypolyethoxylethanol (NP-40) or polyethylene oxide surfactants (e.g., Triton® X-100). In some embodiments of the above method, the first solution comprises at least two detergents. For example, in some variants in which the first solution comprises a saponin, the dissolution further comprises polysorbate 20 as a second detergent.

[0131] In a typical variant, the first solution further contains a buffer, typically present in a concentration sufficient to maintain a pH of approximately 6.0–10.0, 6.5–9.0, 7.0–8.0, or 7.2–7.6. 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 buffer, citrate buffer, succinate buffer, and histidine. In certain embodiments, the buffer is Tris and may be present in the first solution at concentrations, 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; and Tris may be used at effective concentrations 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 (i.e., the concentration in the first dissolution reaction after mixing with the sample, or the concentration in the final dissolution). Other suitable buffer concentrations for the formulations according to this disclosure can be readily determined by those skilled in the art.

[0132] The first lysis reagent and the sample are mixed to induce sufficient lysis of the mammalian cells, allowing the resulting first lysate to pass through the filter, while simultaneously leaving microbial cells, if present, sufficiently intact to remain trapped 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 the selective lysis of the mammalian cells. In some variant forms, 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 a typical variant form, a separate incubation time before passing the lysate through the filter is unnecessary, as sufficient lysis is achieved by mixing the first lysis reagent and the sample. In other embodiments, a short incubation time before passing the lysate through the filter may be used (e.g., an incubation time of 10 minutes or less, 5 minutes or less, or 1 minute or less). Examples of the temperature ranges for the first dissolution reaction described above include 5-50°C, 10-45°C, 15-37°C, 20-30°C, 22-27°C, or 25°C. Ambient (room) temperature is particularly suitable.

[0133] In some modified forms, which involve dissolving a blood sample in the first dissolving solution, the first dissolving solution comprises guanidine hydrochloride, saponin, and polysorbate 20, wherein (i) the effective concentration of guanidine hydrochloride in the first dissolution reaction is at least about 1.5 M, at least about 1.8 M, or at least about 2 M; (ii) the effective concentration of saponin in the first dissolution 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 dissolution 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 modified form is particularly suitable for lysing blood samples with high white blood cell and platelet counts, inducing sufficient lysis to allow the resulting first lysate to pass through the filter (for example, laboratory-made buffy coat samples (which have a normal white blood cell (WBC) count in the range of approximately 17,000 / μl to approximately 44,000 / μl, approximately 4 × 10⁶) 6 Red blood cell (RBC) count per μl, and approximately 1.2 × 10⁻⁶ 6 / μl ~ approx. 2.1×10 6 For the dissolution of platelets (PLTs) that may have a platelet count within the range of / μl.

[0134] The first lysate obtained in the selective lysis of mammalian cells (step (b)) is passed through a filter having a pore size that allows intact microbial cells to pass through while also 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 (containing the lysed mammalian cells) that flows through the filter can be collected, for example, in a waste chamber.

[0135] Filtration is carried out using a filter having a pore size suitable for capturing microbial cells of interest. 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 modified forms, the filter has 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, 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 fibers (e.g., borosilicate glass fibers).

[0136] In some embodiments, the filter includes an asymmetric structure. This asymmetric structure provides a kind of "built-in" prefilter and a larger effective membrane area: larger pores on the upstream side of the membrane trap larger particles on the membrane, while smaller particles, depending on their size, can proceed downstream to the interior of the membrane or pass through it. The pore size gradually decreases from the upstream side to the downstream side of the membrane, thereby trapping particles in various parts of the membrane according to their size. In contrast, a track-edged membrane collects all particles, from those larger than its pore size to those of the same size, making it easier to clog the membrane. The asymmetric filter may have pore sizes of about 5 μm to about 20 μm on the first side of the asymmetric filter and pore sizes up to about 0.2 μm on the second side of the asymmetric filter.

[0137] After the filtration step described above, the filter may be washed as necessary to remove any remaining first solution and mammalian cell debris before step (d) (i.e., before the filter containing the retained microbial cells comes into contact with the second solution). Washing is typically carried out by passing a washing buffer through the filter. In some embodiments, step (c) (i.e., the step of filtering the first lysate through the filter) and the washing step are repeated sequentially multiple times before step (d), each iteration of step (c) includes filtering a portion of the first lysate through the filter, and after each iteration of step (c), the washing buffer is passed through the filter.

[0138] A suitable washing buffer may contain a detergent, and a suitable washing buffer may further contain a chelating agent (e.g., 2,2',2'',2'''-(ethane-1,2-diyldinitro)tetraacetic acid (EDTA)). For example, in some variants, the washing buffer contains a nonionic detergent (e.g., polysorbate 20); in some such embodiments, the washing buffer further contains Tris and / or EDTA. Suitable concentrations of polysorbate 20 in the washing buffer include concentrations of about 0.5% (v / v) to about 5% (v / v). In other variants, the washing buffer does not contain a specific detergent. For example, in some such variants, the washing buffer contains sodium hydroxide, dimethyl sulfoxide (DMSO), glycerol, a chelating agent (e.g., EDTA), and a buffer (e.g., Tris). In a more specific variant of a washing buffer that does not contain a detergent, the washing buffer is the same as the second solution described herein, or a diluted concentration of that washing buffer. In yet another embodiment, the washing buffer is a buffer solution selected from TE buffer (i.e., a buffer containing Tris and EDTA), Tris-buffered saline (TBS), and phosphate-buffered saline (PBS).

[0139] Following filtration of the first dissolution and washing of the filter as necessary, the filter containing the retained intact microbial cells is incubated in contact with the second solution under conditions sufficient to promote the release of the microbial cells from the filter. In some preferred variants, the second dissolution comprises sodium hydroxide, a chelating agent, and a buffer. In some such embodiments, sodium hydroxide is present in the second dissolution at concentrations of about 10 mM to about 100 mM, about 10 mM to about 80 mM, about 20 mM to about 60 mM, about 20 mM to about 50 mM, about 20 mM to about 40 mM, or about 30 mM; and / or the chelating agent is EDTA, present in the second dissolution at concentrations 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. Suitable buffers for use in the second dissolution solution include the buffers described above for the first solution; for example, in some modified forms, the buffer is Tris and is present in the second dissolution solution at concentrations of approximately 1 mM to 50 mM, approximately 2 mM to 25 mM, approximately 5 mM to 10 mM, or approximately 7.5 mM. In some embodiments, the second solution (for example, the second dissolution solution containing sodium hydroxide and a chelating agent as described above) contains dimethyl sulfoxide (DMSO) (for example, at concentrations of about 2% (v / v) to about 20% (v / v), about 2% (v / v) to about 15% (v / v), about 5% (v / v) to about 20% (v / v), about 5% (v / v) to about 15% (v / v), or about 10% (v / v)) and / or glycerol (for example, at concentrations of about 1% (v / v) to about 15% (v / v), about 2% (v / v) to about 12% (v / v), about 5% (v / v) to about 10% (v / v), or about 7% (v / v)).

[0140] The action of the second solution in step (d) is not necessarily limited to the successful release of the filter contents, but can also dissolve, for example, any minute or large coagulations that may form in the sample. For example, the contents of a blood sample may vary depending on the person and the patient's condition, and chemical treatment with the second solution may be used to ensure that no interfering aggregates or coagulations are present in the downstream process.

[0141] In some variations of the above-described method, conditions for promoting the release of microbial cells include incubating the filter with the second solution at a temperature of, for example, about 70°C to about 99°C, about 80°C to about 99°C, about 80°C to about 95°C, or about 90°C to about 95°C. The filter may be heated in the presence of the second solution using an externally controlled heat source embedded in or in contact with the filter, or using inductive mediated heating (a conductive or semiconducting material (such as brass, copper, steel, iron, aluminum, graphite, carbon, or silicon) embedded on / in the filter matrix, for example, by lamination of porous mesh or by co-molding with the actual filter material). In a typical variation using an external heating element, the heating element is located on the same side of the filter that was first contacted by the first solution in step (c) (also referred to herein as the "inlet side"). Exemplary incubation times for the above-mentioned filter with the second solution include, if necessary, 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 under heating.

[0142] After the release of microbial cells, the released microbial cells are collected from the filter in step (f). In this step, the filter contents are not collected through the filter membrane (i.e., by "co-directional elution"), but instead are collected from the same side that was first contacted by the first lysate, i.e., using a "backflow" collection method. The use of an asymmetric filter in certain embodiments is particularly suited to backflow collection of its filter contents because its contents (packed inside the small membrane pores and immobile due to the decreasing pore size) can be released from the side of the filter with the larger pore size. Furthermore, with backflow collection, the contents of most interest (contents from the side that was first contacted by the first lysate (i.e., the "inlet" side)) are released from the filter first. Backflow elution can increase the sensitivity of downstream assays in certain embodiments.

[0143] In some embodiments, microbial cells are collected using an elution buffer to elute them from a filter, and the direction of the fluid flow through the filter is opposite to the direction of the fluid flow applied during the filtration step (c). A suitable elution buffer may contain a detergent, and a suitable elution buffer may further contain a chelating agent (e.g., EDTA). For example, in some variants, the elution buffer contains a nonionic detergent (e.g., polysorbate 20); in some such embodiments, the elution buffer further contains Tris and / or EDTA. Polysorbate 20 may be present in the elution buffer at a concentration, for example, about 0.5% (v / v) to about 5% (v / v). In other variants, the elution buffer does not contain a detergent. For example, in some such variants, the elution buffer contains sodium hydroxide, dimethyl sulfoxide (DMSO), glycerol, a chelating agent (e.g., EDTA), and a buffer (e.g., Tris). In a more specific variant of the elution buffer that does not contain a detergent, the elution buffer is the same as the second solution described herein, or a diluted concentration thereof. In yet another embodiment, the elution buffer is a buffer solution selected from TE buffer (i.e., a buffer containing Tris and EDTA), Tris-buffered saline (TBS), and phosphate-buffered saline (PBS). The fluid flow rate in the backflow elution step may be, for example, up to about 20 ml / min (e.g., about 10 ml / min to about 20 ml / min, or about 15 ml / min).

[0144] In an alternative variant, microbial cells are collected by aspirating them from a filter. In such an embodiment, the microbial cells are aspirated from the same side of the filter that was initially contacted by the first lysate in step (c). Aspiration is particularly advantageous for promoting enrichment of the microbial cell contents because this collection method can be achieved without the use of any elution buffer and therefore without any dilution. Aspiration may also be easier to perform in an automated system.

[0145] After collecting microbial cells from the filter, the cells are lysed away from the filter to obtain a second lysate containing microbial cell analytes released from intact organisms. The collected microbial cells may be lysed by chemical, enzymatic, and / or physical lysis methods. For example, the lysis of the microbial cells may include: (a) chemical lysis using detergents, chaotropic salts, alcohols (e.g., glycerol), and / or extreme pH values; (b) enzymatic lysis using enzymes effective in breaking down one or more microbial cell wall components (e.g., peptidoglycans, chitins, proteins) (e.g., enzymes selected from mutanolisins, lithicases, lysozyme, endoglucanases, proteases, chitinases, and combinations thereof); (c) physical lysis using heat; (d) physical lysis using direct electricity (electrolysis); (e) sonotropic / ultrasonic treatment using sonotrodes; (f) physical lysis using bead beating (e.g., using a MagNA Lyser bead beating instrument); (g) physical lysis using radiolysis; or (h) any combination of chemical lysis, enzymatic lysis, and / or physical lysis. In some variant forms, the microbial cells are lysed using electromagnetic lysis.

[0146] Following microbial cell lysis, the second lysate may be neutralized by adding a suitable neutralizing buffer. For example, in certain embodiments, including the use of sodium hydroxide in the second solution and / or elution buffer described herein, the neutralizing buffer comprises hydrochloric acid and Tris (e.g., a 20× neutralizing buffer comprising HCl at a concentration of about 500 mM and Tris at a concentration of about 50 mM). In other embodiments, neutralization is not performed separately but is instead achieved during the subsequent processing of the sample for analysis of the isolated target analyte. For example, in certain embodiments, including an analytical step involving an in vitro nucleic acid amplification reaction (e.g., see the discussion of target nucleic acid amplification below), the amplification (e.g., PCR) reaction mixture is buffered such that the mixture of amplification reaction components and eluate is less alkaline than can be tolerated in the final reaction mixture.

[0147] The above method for isolating microbial cell analytes may further include a step of analyzing the isolated nucleic acids. The type of assay depends on the analyte.

[0148] For example, in some variant forms in which the analyte is a nucleic acid, the step of analyzing the isolated nucleic acid includes amplification. In such embodiments, the isolated nucleic acid analyte is used as a template in an in vitro nucleic acid amplification reaction, utilizing at least two amplification oligomers adjacent to the target sequence in the nucleic acid analyte to generate an amplification product corresponding to the target sequence. The nucleic acid analyte may be amplified using methods such as isothermal amplification reactions (e.g., transcription-mediated amplification (TMA), nucleic acid sequence-based amplification (NASBA), loop-mediated isothermal amplification (LAMP), polymerase spiral reaction (PSR) (Liu et al., Sci. Rep. 5:12723, 2015), ligase chain reaction (LCR), and other isothermal amplification methods), or temperature-cycle amplification reactions (e.g., polymerase chain reaction (PCR) or other temperature-cycle amplification methods), or other amplification methods. In certain embodiments including PCR, the PCR is selected from quantitative PCR (qPCR) and real-time PCR (rt-PCR).

[0149] Amplification may be carried out with or without prior capture of the nucleic acid analyte. In some modified forms that include a capture step, the isolated nucleic acid analyte is captured before the amplification step by hybridizing its nucleic acid to an immobilized capture probe attached to a solid support.

[0150] Detection of amplified nucleic acid analytes can be performed during amplification (in real time) or after amplification (at an endpoint) using any known method. Amplified nucleic acids can be detected in a solution phase, or by immobilizing them on a solid support (e.g., a nucleic acid array) and detecting labels associated with those amplified nucleic acids (e.g., insertors (e.g., ethidium bromide)). Some detection methods use a detection probe complementary to the sequence in the amplified product and detect the presence of the probe:product complex (e.g., by detecting the label bound to the probe), or use the probe complex to amplify the signal detected from the amplified product (see, for example, U.S. Patents 5,424,413, 5,451,503, and 5,849,481). Other detection methods use probes whose signal generation is associated with the presence of the target sequence, because the signal change occurs only when the labeled probe (e.g., in molecular beacons, molecular torches, or hybridization switch probes) binds to the amplified product (e.g., U.S. Patents 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. Public Appeal No. 2006 / 0194240). A1). Such a probe typically uses a label (e.g., a fluorophore) attached to one end of the probe and an interacting compound (e.g., a quencher) attached to the other end of the probe to inhibit signal generation from the label when the probe is in a conformation that indicates it is not hybridized to the amplified product ("closed"), but generates a detectable signal when the probe hybridizes to the amplified product and changes its conformation ("open").Detection of a signal from a directly or indirectly labeled probe that specifically associates with the amplified product indicates the presence of the amplified target nucleic acid.

[0151] In a specific variant form, the amplification and detection assay for analyzing isolated nucleic acids is the qPCR assay. Such an assay includes forward and reverse primers for targeted amplification, as well as a target-specific detection probe (labeled with a fluorophore at its 5' end and a quencher at its 3' end) (also called a hydrolysis probe or TaqMan probe). In this form, the quencher prevents the fluorescein of its fluorophore. If the target nucleic acid is present in the sample, the probe binds to a complementary sequence within the amplification target region. As the polymerase extends the 3' end of its primer to synthesize a nascent complementary chain, the polymerase's 5'→3' exonuclease activity degrades the bound probe, thereby neutralizing the quencher's action on the fluorophore. This is evident by an increase in fluorescence intensity depending on the amplification factor. [Examples]

[0152] The following embodiments are provided to illustrate specific embodiments disclosed and should not be construed as limiting the scope of this disclosure.

[0153] (Example 1) The protocol for enriching microbial cell analytes from larger sample volumes containing mammalian cells was performed manually as described below. 1) Collect a 4 ml donor blood sample and place it in a 50 ml Falcon tube. 2) Add the microorganism under study to the blood sample to a concentration of 5 cfu / ml. 3) Add 4 ml of hemolysis solution (4M guanidine hydrochloride, 4% (v / v) Tween® 20, 4% (w / v) saponin, 40.5 mM Tris) to the blood sample and mix by pipetting up and down 20 times (this is for selective lysis, i.e., to lyse human cells). 4) Collect the hemolyzed blood (8 ml in this case) and put it into a 10 ml syringe. 5) Connect the syringe (perpendicularly) to the syringe filter (25 mm diameter filter, PES membrane, 0.2 / 0.22 μm pore size). 6) The hemolyzed blood is filtered through the filter (intact microorganisms are captured by the size-selective membrane, while most of the human cell debris and human DNA pass through). 7) Remove the empty blood syringe from the filter. 8) Attach a 20 ml syringe containing 15 ml of washing solution ("GENTD"; 30 mM sodium hydroxide, 10% (v / v) DMSO, 7% (v / v) glycerol, 1 mM EDTA, 7.5 mM Tris) to the filter. 9) Inject the cleaning solution. 10) Cover the inlet and outlet of the filter (this includes the GENTD filter), rotate the filter, and place it on a heater set to 95°C with the inlet facing downwards (the material trapped in the membrane with intact microorganisms is located on the inlet side of the membrane, and therefore heating is more efficient when applied to the inlet side). Incubate for 15 minutes.

[0154] 11) Option A: a. After incubation, the container was allowed to cool for 3 minutes, the lid was removed, the filter outlet was attached to a three-way connector, and a syringe filled with TE buffer was connected to the infusion pump. b. Attach an empty 1 ml syringe to the filter inlet and perform automated infusion of 350 μl of TE buffer at a rate of 15 ml / min (= physical cleaning of the filter, i.e., removal of contents containing microorganisms from the filter). c. Remove the 1 ml syringe containing the eluted material (including the filter contents with intact microorganisms / microbial analytes), add it to the MagNA Lyser bead beating tube, and perform mechanical dissolution.

[0155] Option B: d. After incubation, let it cool for 3 minutes, remove the lid, and attach the filter inlet to an empty 20ml syringe. e. Filter - Draw the contents into the syringe (the membrane is airtight, so it is vacuum-free). f. Remove the syringe (by actively holding the plunger in the 20 ml syringe against vacuum) and transfer the eluate to a preferred tube for further processing in the dissolution method (MagNA Lyser bead beating).

[0156] 12) The eluted material obtained by bead beating is analyzed by PCR.

[0157] In this protocol, the following two steps enable the collection of microorganisms from the filter: chemical cleansing (GENTD incubation, step 10) and physical cleansing (backflow, step 11).

[0158] After step 9, the contents of the captured blood residue, along with intact microorganisms (which could not be removed by the washing step), are packed into the membrane and into the membrane pores. The membrane is incubated with GENTD to release the captured contents from the membrane in a physical cleansing step (step 11). Heating accelerates the reaction induced by GENTD (i.e., denaturation and lysis of its contents). This chemical incubation will likely lyse certain types of microorganisms (brought about by heating and reagents in GENTD), but Gram-positive microorganisms and yeasts that are difficult to lyse remain intact. Lysis inside the filter is neither necessary nor important, because the backflow elution / aspiration step collects the contents of interest (possibly including released DNA) from the filter.

[0159] In option A of step 11, TE buffer was used to push out the contents of the filter in the backflow elution step. In this otherwise manual protocol, the step was performed using an injection pump to determine the rate for physical cleaning (15 ml / min was tested and it was shown to be efficient for this physical cleaning step). The internal volume of the filter was approximately 250 μl, meaning that 250 μl of GENTD was present inside the filter during the incubation step. When 350 μl of liquid was pushed into the filter, approximately 250 μl came out (this was due to the extremely large inlet and outlet portions of the commercially available filter (which take part of the eluate)). The eluate (which could also be another buffer or even GENTD) did not completely push out the contents when mixed with them. However, the contents from the inlet side came out first, with the captured material present on the inlet side, and as a result, the contents of interest came out first. These results indicated that this protocol was effective in collecting even low concentrations of analytes, meaning that the most concentrated portion of its contents was present within its 250 μl eluate volume.

[0160] Alternative collection step 11, option B, was also used. Filter contents were collected using aspiration to replace backflow elution. By using a large syringe (10 ml or 20 ml), the pulling force of the plunger was able to draw out a similar volume from the filter as that eluted by the push / backflow step above, without any dilution as no elution buffer was used. Similar to backflow elution, this alternative aspiration step was used to first remove the contents of most interest (contents from the inlet side).

[0161] (Example 2) A microbial cell enrichment protocol similar to that described in Example 1 was performed manually, utilizing multiple repetitions of the selective filtration step followed by a washing (rinsing) step. This protocol is summarized below.

[0162] 1) Mix 4 ml of blood sample with 4 ml of hemolysis solution (same as in Example 1). 2) Perform filtration using a syringe filter (the syringe filter is the same as in Example 1): a. First, "prime" the filter with 2 ml of rinse solution (TE + 2% (v / v) Tween(registered trademark)-20); b. Filter 2 ml of hemolyzed blood; c. 2.5 ml of rinse solution; d. 2 ml of hemolyzed blood; e. 2.5 ml of rinse solution; f. 2 ml of hemolyzed blood; g. 2.5 ml of rinse solution; h.2 ml of hemolyzed blood; i. 2.5 ml of rinse solution; 3) Filter through 10 ml of washing solution (GENTD; see Example 1). 4) Incubation of the filter by external heating (90°C, 15 minutes). 5) Collect the contents of the filter by aspirating them (see Example 1). 6) Mechanical dissolution using MagNA Lyser bead beating.

[0163] 7) The eluted material obtained by bead beating is analyzed by PCR.

[0164] (Example 3) This example describes the isolation and detection of microbial cell analytes from samples with low microbial concentrations, using a microbial cell enrichment protocol similar to that described in Example 2. This study included: 1) Spiked samples with 5 CFU / ml (23 different microorganisms or targets); 2) Multiple rinse / washing steps during filtration of hemolyzed blood (TE+2% Tween®-20: 1ml, 2.5ml, 2.5ml, 2.5ml, 3.5ml); 3) Incubate with GENTD containing 30 mM NaOH, 10% DMSO, and 7% glycerol; 4) Suction; 5) Addition of 0.5% foam-BAN to the aspirated material to prevent foaming during dissolution; 6) Bead beating of MagNA Lyser for dissolution; 7) Neutralization; 8) 12 replicates on a PCR plate; 9) A 50 CFU / ml positive control was prepared by directly spiking microorganisms in GENTD containing 60 mM NaOH, 10% DMSO, and 7% glycerol, lysed in MagNA Lyser, neutralized, and analyzed by PCR; this represents the concentration to be achieved when enriching a 5 CFU / ml spiked sample); 10) Plate the microbial stock used for the spike and investigate the actual cell concentration used.

[0165] Table 1 below shows the actual spiked cell concentrations (target 5 CFU / ml) according to the microbial strains and number of plates tested. [Table 1]

[0166] Since Cryptococcus is known to be a difficult target pathogen to lyse, we also considered the tendency of most Cryptococcus species to form a polysaccharide capsule in response to environmental stimuli. This capsule is a major virulence factor for these species, and cells with this capsule are larger in size. In this study, we tested two different C. neoformans strains because the ATCC 208821 strain is known to form its capsule more readily than the ATCC MYA-4567 strain. We tested capsule-inducible and non-capsule-inducible C. neoformans cells and compared their results. The induction protocol was as follows: Cryptococcus cells grown overnight at 30°C on Sabouraud dextrose agar plates under aerobic conditions were transferred to a broth containing DMEM + 2 mM MgCl2 + 10% FBS and incubated at 37°C with 10% CO2 for 24 to 48 hours. After incubation, the formation of polysaccharide capsules was observed under a microscope using nigrosine staining.

[0167] The eluted substances were analyzed using Biorad CFX PCR with an in-house PCR assay.

[0168] The results of this study are shown in Tables 2 and 3 below. [Table 2]

[0169] [Table 3]

[0170] Overall, these results demonstrate that the developed protocol can efficiently enrich the microorganisms tested. A small number of these microorganisms presented challenges in cultivation and stock preparation, as discussed below.

[0171] S. pneumoniae: The enrichment protocol for this microorganism was repeated several times because the PCR positivity rate in enriched samples was low. However, positive controls prepared by bead beating in GENTD solution were 100% positive. Streptococcus pneumoniae has a tendency towards autolysis, which is induced when the growth of this microorganism reaches a stationary phase. Modeling the actual physiological growth conditions for this microorganism can be difficult, and the performance of actual protocols can only be studied if positive clinical blood samples are available.

[0172] C. difficile: C. difficile also tends to initiate the destruction of its own DNA. In this study, feasible detection was achieved. The cell count on the plate was unreliable (only 0-2 CFUs were produced out of 100 CFUs), which was most likely due to suboptimal plating conditions (possibly not fully anaerobic).

[0173] C. neoformans: Some differences were observed in the detection of capsular-inducible cells and non-capsular-inducible cells, indicating variations in lysis efficiency. However, their positive rates were still similar to, or even better than, those observed with positive controls. The capsular structure alters the size of this microorganism, making stock preparation difficult, but for all microorganisms tested, the plate counts indicated true spike concentrations. The spike concentration according to plate counts for the inducer strain ATCC 208821, which had the highest inducer-inducible capsular count, was 4 CFU / ml. For this low cell concentration and for partially capsular cells, the detection rate was still 75%, and the mean Cq value was 38.3.

[0174] (Conclusion): All tested target microorganisms could be detected with at least an acceptable positive rate using this microbial cell enrichment protocol, and many of the tested microbial targets showed a high positive rate of 100% or near 100%.

[0175] From the foregoing, it will be understood that while specific embodiments of the present invention have been described herein for illustrative purposes, various modifications can be made without departing from the spirit and scope of the invention. All publications, patents, and patent applications cited herein are incorporated herein by reference in their entirety for all purposes.

Claims

1. A method for selectively isolating microbial cell analytes from a sample, (a) A step of providing a sample that contains mammalian cells and possibly microbial cells; (b) A step of mixing the sample with a first 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 solution is a buffer solution containing a chaotropic salt which is guanidine hydrochloride and a washing agent which is saponin; (c) A step of filtering the first lysate through a filter having a pore size that retains intact microbial cells, wherein the pore size is approximately 0.1 μm to approximately 1 μm; (d) A step of contacting the filter containing the retained microbial cells with a second solution effective in enabling the release of the microbial cells from the filter, wherein the second solution is a buffer solution containing sodium hydroxide, dimethyl sulfoxide (DMSO), glycerol, and a chelating agent; (e) Incubating the filter with the second solution at an incubation temperature of approximately 70°C to approximately 99°C to promote the release of the microbial cells from the filter; (f) A step of collecting the microbial cells from the filter, wherein the collection step is (i) A step of using an elution buffer to elute the microbial cells from the filter, wherein the direction of the fluid flow through the filter is opposite to the direction of the fluid flow applied in the filtration step (c); or (ii) A step of aspirating the microbial cells from the filter, wherein the microbial cells are aspirated from the same side of the filter that was first contacted by the first lysate in step (c), Processes including; and (g) A step of lysing the microbial cells collected in step (f) to obtain a second lysate containing the analyte released from the microbial cells, thereby selectively isolating the microbial cell analyte from the sample. Methods that include...

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

3. The method according to claim 2, wherein the microbial cells are Gram-positive bacterial cells.

4. The filter comprises polyethersulfone (PES), cellulose, nylon, polyvinylidene fluoridene (PVDF), polycarbonate, or glass fiber; and / or The filter includes an asymmetric structure, and optionally, the pores on the first side of the asymmetric filter have a size of approximately 5 μm to approximately 20 μm, and the pores on the second side of the asymmetric filter have a size of up to approximately 0.2 μm. The method according to any one of claims 1 to 3.

5. (A) The guanidine hydrochloride is present in the first solution at a concentration of about 1 M to about 8 M, and / or the saponin is present in the first solution at a concentration of about 1% (w / v) to about 10% (w / v); and / or (B) The sodium hydroxide is present in the second solution at a concentration of about 10 mM to about 100 mM, and / or the DMSO is present in the second solution at a concentration of about 2% (v / v) to about 20% (v / v), and / or the glycerol is present in the second solution at a concentration of about 1% (v / v) to about 15% (v / v), and / or the chelating agent in the second solution is 2,2',2'',2''''-(ethane-1,2-diyldinitrilo)tetraacetic acid (EDTA) at a concentration of about 0.1 mM to about 4 mM; and / or (C) The first solution is buffered with approximately 20 mM to approximately 200 mM Tris buffer: and / or (D) The second solution is buffered with approximately 1 mM to approximately 50 mM Tris buffer. The method according to any one of claims 1 to 4.

6. The method according to any one of claims 1 to 5, wherein the first solution further comprises a second cleaning agent which is polysorbate 20.

7. The method according to claim 6, wherein the polysorbate 20 is present in the first solution at a concentration of about 1% (v / v) to about 10% (v / v).

8. The method according to any one of claims 1 to 7, wherein during the mixing step (b), the ratio of the first dissolving solution to the sample is approximately 1:

1.

9. The method according to any one of claims 1 to 8, further comprising a washing step between step (c) and step (d), wherein the washing step comprises passing a washing buffer through the filter.

10. The method according to claim 9, wherein the filtration step (c) and the washing step are carried out sequentially multiple times before step (d), and each iteration of step (c) includes a step of filtering a portion of the first dissolved substance through the filter.

11. The method according to any one of claims 1 to 10, wherein the lysis of the microbial cells in step (g) includes physical lysis.

12. The method according to claim 11, wherein the physical dissolution includes electromagnetic dissolution.

13. The method according to any one of claims 1 to 12, wherein the analyte is nucleic acid.

14. A step of analyzing an isolated analyte, wherein the analyte is a nucleic acid, and the step of analyzing an isolated nucleic acid analyte. It further includes, The analysis method is, (i) A step of carrying out a nucleic acid amplification reaction using the isolated nucleic acid as a template to produce an amplification product; and (ii) Step of detecting the amplified product A method according to any one of claims 1 to 13, including the method described in any one of claims 1 to 13.

15. The method according to claim 14, wherein the step of analyzing the isolated nucleic acid includes the step of immobilizing the isolated nucleic acid or amplification product on a solid support.

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