Systems, methods, and apparatus for the concentration and identification of microorganisms from blood.

Abstract

The present invention relates to systems, methods and devices for isolating and identifying microorganisms from samples known to or capable of containing microorganisms.

Description

Technical Field

[0001] Related Applications This application claims the benefit and priority of U.S.A Provisional Patent Application No.63 / 126,041, filed on December 16, 2020, the entire content of which is incorporated herein by reference.

[0002] 1. Technical Field Embodiments of the present disclosure generally relate to systems, methods, and devices for direct sepsis diagnosis from blood.

Background Art

[0003] 2. Background In the United States, Canada, and Western Europe, infectious diseases account for approximately 7% of human mortality, while in developing regions, infectious diseases account for more than 40% of human mortality. Infectious diseases cause various clinical symptoms. Common overt symptoms include fever, pneumonia, meningitis, diarrhea, and diarrhea including blood. Physical symptoms suggest some pathogens while excluding others as pathogens, leaving the possibility of various causative agents, and a clear diagnosis often requires the implementation of various assays.

[0004] In the United States, bloodstream infections (BSIs) and the resulting septic shock (also known as sepsis, septicemia, bacteremia, fungal infection, candidiasis, candidiasis, bloodborne infection, and other related terms) are a leading cause of death. For example, bacterial BSI is the 11th leading cause of death in adults and the 7th in infants. Candida spp. and other fungi can also cause BSIs. The high mortality rate (40%) associated with Candida spp. bloodstream infections is mainly due to the long diagnostic time required for blood cultures. Studies have shown that initiating appropriate antimicrobial or antifungal treatment can reduce mortality, and that each delay in antibiotic administration is associated with a significant increase in mortality. Therefore, early detection and definitive diagnosis, as well as rapid treatment with appropriate antibiotics or antifungal agents, are desirable for improving outcomes in patients suspected of having a BSI.

[0005] The current diagnostic gold standard for BSI requires the growth of the organism in culture, followed by microscopic observation, subculturing, and phenotypic identification of purified isolates. As a result, reporting times range from 36–72 hours for Gram-positive bacteria, 48–96 hours for Gram-negative bacteria, and 48–120 hours for fungal infections. Surprisingly, about one-third of patients treated for fungal BSI never show growth on positive blood cultures, and many positive cases of fungal BSI are definitively diagnosed simply through post-mortem analysis. Consequently, it is typical for physicians to initiate treatment with broad-spectrum antibiotics or antifungal regimens immediately after blood collection for culture in patients suspected of having BSI. This is not ideal. Studies have shown that the administration of inappropriate or ineffective antimicrobial agents does not help improve patient outcomes and, troublingly, leads to an increase in drug-resistant organisms, independently of overall patient outcomes and public health.

[0006] One alternative to the diagnostic gold standard (i.e., classical microbiological methods) involves the molecular identification of infectious bacteria and fungi from blood. However, the number of infectious organisms found in whole blood in BSI is usually low (approximately 1–100 colony-forming units (cfu / ml) per mL of blood, and about 1–10 cfu / ml is typical in most individuals with sepsis confirmed by culture). Furthermore, blood contains several inhibitors of polymerase chain reaction (PCR) (e.g., hemoglobin and other blood proteins (e.g., human serum albumin) and genomic DNA: derived from leukocytes, which can be co-purified with microorganisms and interfere with both nucleic acid recovery from target microorganisms and downstream PCR). Due to the low number of organisms in whole blood and the presence of PCR inhibitors, concentration from larger volumes of whole blood (e.g., 1–20 mL) is required to obtain the quality and quantity of DNA templates desired to achieve susceptibility at clinically relevant microbial levels.

[0007] There is an urgent need for faster and more accurate molecular-based diagnostics to reduce the number of ineffective or unnecessary broad-spectrum antibiotic administrations received by non-infected patients. Rapid diagnosis could enable the timely administration of more tailored and effective antimicrobial agents to patients who definitely have BSI. Despite these many potential benefits, many of the rapid BSI diagnostic solutions being attempted have not been widely adopted. This is due to a variety of reasons, including cumbersome workflows, time to results, and cost.

[0008] One commercially available product, called MolYsis®, offers the prospect of selective isolation of bacterial DNA from intact organisms in whole blood. The MolYsis® Complete 5 DNA Extraction Kit (catalog number D-321-100; Molzym GmbH & Co. KG, Bremen, Germany) contains chaotropic lysis buffer for selective lysis of blood factors (red blood cells, white blood cells, etc.) and DNase for degrading genomic DNA. Microbial cells are recovered by centrifugation, the supernatant is discarded, the cells are resuspended several times in different buffers, repelled, chemical lysis of the microbial cells is performed, and finally the microbial nucleic acids are recovered. All in all, the MolYsis® kit involves a cumbersome workflow that takes approximately 45 minutes for sample preparation, plus another 45 minutes for purification and lysis of microbial cells. Microbial identification requires inputting the nucleic acids recovered using the MolYsis® kit into another assay, which takes further time and involves further expenditure. Furthermore, successful use of MolYsis® kits requires skilled professionals. This reliance on operator skill carries the risk of variations in yield and quality from operator to operator. The numerous buffers and manual pipetting steps increase the risk of sample cross-contamination.

[0009] Another product intended for use in identifying BSI from whole blood is T2MR® from T2 Biosystems. The T2MR® system includes automated sample preparation, including selective lysis of blood factors, microbial recovery, microbial lysis, recovery of microbial nucleic acids, and PCR amplification. Nuclear magnetic resonance (NMR) is used in the T2MR® system for microbial identification. Superparamagnetic NMR nanoprobes in solution bind to DNA specific to the microorganism and form aggregates detectable by NMR. Nanoprobes that aggregate in the presence of microbial nucleic acids produce a stronger NMR signal compared to signals from non-aggregated nanoprobes. However, the T2MR® system requires 4-6 hours to acquire NMR data to obtain data of sufficient quality for use in microbial identification. Furthermore, the T2MR® system is expensive (the device costs approximately $150,000), and the device's throughput is significantly limited due to the time required for data acquisition. In addition, bacteria and fungi associated with BSI are tested against separate T2MR® panels. This means that patients presenting with sepsis will require testing against bacterial and fungal panels to include / exclude bacterial and fungal causes. Furthermore, the number of organisms tested against the bacterial and fungal panels is limited (approximately 6 organisms are tested for each), and the tests do not provide information on drug susceptibility / resistance.

[0010] This invention addresses various improvements in the direct identification of BSI-related microorganisms from blood with a simplified workflow and faster sample-to-response answers. [Overview of the project] [Means for solving the problem]

[0011] The present invention provides methods, systems, and apparatus for concentrating, characterizing, and / or identifying microorganisms from a sample. In one embodiment, the microorganism is a bacterium. In another embodiment, the microorganism is a fungus (e.g., yeast or mold). In a further embodiment, the microorganism is a parasite. The methods, systems, and apparatus may be particularly useful in the isolation, concentration, characterization, and / or identification of microorganisms from blood or a composite sample such as urine or cerebrospinal fluid. In a preferred embodiment, the methods, systems, and apparatus of the present invention may be used to concentrate, characterize, and / or identify microorganisms directly from whole blood in order to rapidly determine that a patient has sepsis. In typical sepsis, the concentration of microorganisms in the bloodstream is low, for example, < about 1 to 100 cfu / ml, and < about 1 to 10 cfu / ml is typical. In one or more patients suspected of having sepsis, microorganisms in the blood, if present, are very dilute and can be identified directly from a blood sample without using the methods described herein. Furthermore, the blood preferably contains several PCR inhibitors (e.g., hemoglobin, human serum albumin, and genomic DNA) that may be removed to ensure that the identification and analysis of microorganisms from whole blood and other complex matrices (e.g., urine and CSF) are always successful. The present invention provides methods, systems, and apparatus for the selective lysis of non-microbial cells in a sample and the concentration of microorganisms from relatively large volumes (e.g., 10-20 ml) of a sample.In preferred embodiments, the methods, systems, and apparatus described herein, without limitation, do not include the use of apparatus or steps such as mixing a blood sample and differential lysis buffer in a first vessel, then transferring the lysate to a centrifugal concentrator containing components other than the blood sample and differential lysis buffer in a centrifugal concentrator, opening the centrifugal concentrator after centrifugation and decanting the supernatant fraction, recovering microorganisms by centrifugation with a high-density cushion or physical separator, pre-treating the blood sample (other than mixing the blood sample with differential lysis buffer and treating it in the concentration and identification steps described herein), preliminary analysis and culture steps, subculturing the sample and identifying microorganisms present in the sample, or DNase steps for selectively digesting non-microbial DNA from non-microbial cells.

[0012] The inventions described herein may preferably include a method for isolating and identifying the described microorganisms. The method may preferably include the steps of: (a) providing a certain volume of blood sample suspected to contain microorganisms; (b) mixing the blood sample with a differential lysis buffer to obtain a lysate containing lysed blood cells and unlysed microorganisms; (c) concentrating the microorganisms from the lysate; (d) adding the microorganisms to an apparatus containing one or more reagents required to identify the microorganisms; and (e) identifying the microorganisms present in the blood sample. In the method, the microorganisms, if present, are concentrated to a range of 25 to 100 times the volume of the provided blood sample, and the microorganisms, if present, have a concentration in the provided blood sample in the range of < about 1 CFU / ml (but greater than 0) to about 100 CFU / ml (e.g., < 1 CFU / ml to about 10 CFU / ml).

[0013] Steps (a) to (c) of the method may preferably be completed within a time range of about 10 to 20 minutes. Steps (d) and (e) of the method may preferably be completed within a time range of less than 4 hours, less than 3 hours, less than 2 hours, or less than 1 hour.

[0014] The microorganisms in this method may preferably include one or more bacteria or fungi associated with bloodborne infections.

[0015] The identification in the method may preferably include one or more of the following: molecular testing, phenotypic testing, proteomics testing, optical testing, or culture-based testing. The identification may preferably include the steps of isolating one or more nucleic acids having microbial characteristics from a microorganism, and analyzing one or more nucleic acids to identify the microorganism present in the blood sample. In one embodiment of the method, the identification further includes amplifying one or more nucleic acids and then detecting one or more amplified nucleic acids. The detection of one or more amplified nucleic acids may preferably include the use of one or more dsDNA-binding dyes, real-time PCR, a post-amplification nucleic acid lysis step, a nucleic acid sequencing step, a labeled DNA-binding probe, or an unlabeled probe. The identification step may preferably be completed within a time range of about 5 to 75 minutes.

[0016] The method may preferably further include a culturing step of concentrated microorganisms in a culture medium to increase the concentration of microorganisms, and then an identification step, where the culturing step is carried out for 4 hours or less, 3 hours or less, or 2 hours or less, 1 hour or less, 30 minutes or less, 20 minutes or less, or 10 minutes or less, preferably 3 hours or less.

[0017] The differential lysis buffer used in the enumerated methods may preferably include a buffering agent, a nonionic surfactant, a salt, and a pH range of about 10–11 before mixing the blood sample with the differential lysis buffer. The differential lysis buffer may preferably have a pH of about 7.0–8.0 after mixing the blood sample with the differential lysis buffer. The buffering agent used in the differential lysis buffer may preferably be selected from the group consisting of CABS, CAPS, CAPS, CHES, and combinations thereof. The buffering agent used in the differential lysis buffer may preferably be CAPS. The pH of the differential lysis buffer mixed with the blood sample may preferably be in the pH range of about 1.5–2.5, below the pH buffering range of the buffering agent. The nonionic surfactant used in the differential lysis buffer is preferably one or more of polyoxyethylene (POE) ethers, preferably Arlasolve200 (also known as poly(oxy-1,2-ethanediyl)), BrijO10, and nonaethylene glycol monododecyl ether (also known as Brij35). The nonionic surfactant used in the differential lysis buffer is preferably selected from the group consisting of Triton X-114, NP-40, Arlasolve200, BrijO10 (also known as Brij96 / 97), octyl β-D-glucopyranoside, saponin, nonaethylene glycol monododecyl ether (also known as Brij35), and combinations thereof. In the differential lysis buffer combined with the blood sample, the detergent concentration (e.g., in the range of 0.1% to 0.5%) and pH (e.g., in the range of 7 to 11) may be preferably adjusted to minimize the pellet volume while maximizing the differential lysis of blood cells in the sample. Preferably, the pellet volume may be about 500 μL or less, about 400 μL or less, about 300 μL or less, about 200 μL or less, or about 100 μL or less. Preferably, up to 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, and 99% of non-microbial cells in the sample can be lysed within 2 to 5 minutes of combining the sample with the differential lysis buffer.

[0018] Microbial concentration from lysate preferably includes centrifugation, and the concentration further includes recovering a pellet fraction containing microorganisms from a supernatant fraction containing a lysed blood fraction. Microbial concentration from lysate preferably includes placing a blood sample mixed with differential lysis buffer into a centrifugal concentrator; centrifugating the centrifugal concentrator to concentrate microorganisms from the blood sample placed in the chamber; and generating the concentrated microorganisms from a second opening at the second end of the chamber, where the centrifugal concentrator is a chamber having an opening at the first end and a seal at the second end, the seal being designed to seal the second opening at the second end of the chamber; and a plunger at least partially movable inside the chamber, designed to be operated to open the seal. The generation of concentrated microorganisms from the second opening at the second end of the chamber preferably includes aseptically generating the pellet from the second end of the centrifugal concentrator into a vial or assay device. The centrifugal concentrator may preferably not include a high-density cushion or physical separator for separating microorganisms from the lysate.

[0019] The method preferably involves mixing a blood sample and differential lysis buffer in a first container, then transferring the lysate to a centrifugal concentrator containing components other than the blood sample and differential lysis buffer, opening the centrifugal concentrator after centrifugation and decanting the supernatant fraction, and may omit one or more of the following steps: a culture step before mixing the blood sample with the differential lysis buffer, or a DNase step for digesting genomic DNA in the lysate. The method preferably preferably involves mixing a blood sample with the differential lysis buffer, or may omit one or more of the following steps: a culture step before mixing the blood sample with the differential lysis buffer, or a DNase step for digesting genomic DNA in the lysate.

[0020] The microorganisms may preferably be concentrated from the lysate by filtration techniques. The method may preferably further include adding a filter having the concentrated microorganisms on top to one or more culture or assay devices designed to identify microorganisms present in a blood sample.

[0021] Preferably, the steps of mixing a blood sample with a differential lysis buffer to obtain a lysate and separating microorganisms from the lysate may be performed in a single tube. Preferably, the differential lysis buffer used in the method may be a single buffer provided in a single tube. Preferably, the differential lysis buffer used in the method may not contain DNase or protease, and the method may preferably not include the step of adding exogenous DNase or protease to a single tube. The differential lysis buffer used in the method may preferably be compatible with an anticoagulant selected from the group consisting of EDTA, citrate, dextrose citrate (ACD), sodium polyanethole sulfate (SPS), heparan, sodium fluoride / oxalate, and combinations thereof.

[0022] The inventions described herein may preferably include a method for concentrating and identifying microorganisms from blood. The method may preferably include the steps of: (a) providing a blood sample known to contain or capable of containing microorganisms; (b) mixing the blood sample with a differential lysis buffer comprising a buffer, a nonionic surfactant, and a salt to obtain a lysate containing lysed blood cells and unlysed microorganisms; (c) concentrating microorganisms from the lysate; and (d) identifying the microorganisms present in the blood sample, wherein the blood sample mixed with the differential lysis buffer has a pH of about 7.0 to 8.0, and the buffer has a useful pH buffering range of about 8.6 to 11.4, wherein the microorganisms are concentrated to a range of 25 to 100 times the initial volume of the provided blood sample, wherein the identification is achieved in 4 hours or less, 3 hours or less, 2 hours or less, or 1 hour or less.

[0023] The identification may preferably include one or more of molecular testing, phenotypic testing, proteomics testing, optical testing, or culture-based testing. The identification step of the method may preferably include the step of isolating one or more nucleic acids having the characteristics of the microorganism from the microorganism, and the step of analyzing the one or more nucleic acids to identify the microorganism present in the blood sample.

[0024] The nonionic surfactant listed in the method may preferably be one or more of polyoxyethylene (POE) ether, preferably Arlasolve200 (also known as poly(oxy-1,2-ethanediyl)), BrijO10, and nonaethylene glycol monododecyl ether (also known as Brij35). The nonionic surfactant listed in the method may preferably be selected from the group consisting of Triton X-114, NP-40, Arlasolve200, BrijO10 (also known as Brij96 / 97), octyl β-D-glucopyranoside, saponin, nonaethylene glycol monododecyl ether (also known as Brij35), and combinations thereof.

[0025] The buffer substance listed in the method may preferably be selected from the group consisting of CABS, CAPS, CAPSO, CHES, and combinations thereof. The buffer substance listed in the method may preferably be CAPS, and CAPS has a pH buffering range of about 9.7 to 11.1 and a pKa of about 10.4 at 25°C.

[0026] (For example, within the range of 0.1% to 0.5) the concentration of the detergent and (for example, within the range of 7 to 11) the pH may be preferably adjusted to minimize the pellet volume while maximizing the differential lysis of blood cells in the sample. Preferably, the pellet volume may be about 500 μL or less, about 400 μL or less, about 300 μL or less, about 200 μL or less, or about 100 μL or less. Preferably, up to 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99% of the non-microbial cells in the sample can be lysed within 2 to 5 minutes when the sample is combined with the differential lysis buffer.

[0027] The salts listed in the method may preferably be sodium chloride.

[0028] The method may preferably not include a blood culture step before concentration and / or a DNase step for digesting genomic DNA in the lysate.

[0029] Steps (a) to (c) of the method may preferably be completed within a time range of about 10 to 20 minutes. The steps of isolating and analyzing the method may preferably be completed within a time range of about 5 to 75 minutes. The time for obtaining the lysate may preferably be in the range of about 2 to 10 minutes, preferably about 3 to 5 minutes. The generation of the lysate may preferably not include additional steps other than combination (i.e., incubating the blood / buffer mixture for a time sufficient to lyse blood cells in the sample (about 2 to 10 minutes, preferably about 3 to 5 minutes) by combining the blood sample with the differential lysis buffer).

[0030] The following is the content to be described.

[0031] A1. A method for isolating and identifying microorganisms, comprising: (a) providing a blood sample known or likely to contain microorganisms; (b) Mixing a blood sample with a differential lysis buffer having a certain pH to obtain a lysate containing lysed blood cells and non-lysed microorganisms; (c) Isolating microorganisms from the lysate; (d) Adding the microorganism to an assay apparatus containing one or more reagents required to identify the microorganism; (e) Identifying microorganisms present in the blood sample, A method comprising, wherein identification comprises the steps of: isolating one or more nucleic acids having the characteristics of a microorganism from a microorganism; and analyzing one or more nucleic acids to identify the microorganism present in a blood sample.

[0032] A2. The method of Clause A1, wherein the microorganism is one or more bacteria or yeasts associated with bloodborne infections.

[0033] A3. The method of Clause A1 and / or Clause A2, which first includes identifying one or more symptoms in the patient, such as sepsis, septic infection, septic shock, or septicemia, and further including determining that the patient has a bloodborne infection.

[0034] A4. One or more methods of clauses A1 to A3, wherein the differential lysis buffer comprises a buffer, a nonionic surfactant, and a pH range of approximately 10–11 before mixing the blood sample with the differential lysis buffer and a pH of approximately 7.0–8.0 after mixing the blood sample with the differential lysis buffer.

[0035] A5. One or more methods of clauses A1 to A4, wherein the nonionic surfactant is polyoxyethylene (POE) ether.

[0036] A6. One or more methods of clauses A1 to A5, wherein the nonionic surfactant is selected from the group consisting of Triton X-114, NP-40, Arlasolve200, BrijO10 (also known as Brij96 / 97), octyl β-D-glucopyranoside, saponin, nonaethylene glycol monododecyl ether (C12E9, polidocenol), and combinations thereof.

[0037] A7. One or more methods of clauses A1 to A6, wherein the isolation of microorganisms from lysate comprises a centrifugation step, and the isolation further comprises recovering a pellet fraction containing microorganisms from a supernatant fraction containing a lysed blood fraction.

[0038] A8. The blood sample mixed with differential lysis buffer is placed in a centrifugal concentrator, The process involves centrifuging a centrifugal concentrator to pelletize microorganisms from a blood sample placed in the chamber; The plunger is pushed in, the seal is released, and pellets are generated from the opening at the second end of the chamber. It further includes, here a centrifugal concentrator, A chamber having an open portion at a first end and a sealing portion at a second end, wherein the sealing portion is designed to seal the second open portion at the second end of the chamber; and A plunger positioned at least partially movable inside a chamber, and designed to act to release a seal. One or more methods of clauses A1 to A7, including

[0039] A9. Pushing in the plunger, releasing the seal, and generating pellets, including pushing the plunger until it is tightly engaged with a portion of the body, and discharging the pellets from the second end by releasing the seal under pressure, one or more methods of clauses A1 to A8.

[0040] A10. One or more methods of clauses A1 to A9, further comprising generating a pellet from the second end of a centrifugal concentrator into a vial or self-contained assay device.

[0041] A11. One or more methods of clauses A1 to A10, wherein the centrifugal concentrator and the vial are designed such that the second end of the centrifugal concentrator is conjugated to the vial.

[0042] A12. The vial is designed to deliver the pellet to a self-contained molecular analyzer, one or more of the methods described in clauses A1 to A11.

[0043] A13. One or more methods of clauses A1 to A12, wherein the vial is designed to deliver the pellet to a self-contained molecular analyzer without decoupling the vial from the second end of the centrifugal concentrator.

[0044] A14. One or more methods of clauses A1-A13, including partitions, similar functional structures, etc., where the first end opening of the centrifugal concentrator is designed to aseptically fill the centrifugal concentrator with a sample mixed with differential lysis buffer.

[0045] A15. One or more methods of clauses A1 to A14, wherein the centrifugal concentrator does not include a high-density cushion or physical separator.

[0046] A16. One or more methods of clauses A1 to A15, which do not include one or more of the following steps: mixing the blood sample and differential lysis buffer in a first container, then transferring the lysate to a centrifugal concentrator containing components other than the blood sample and differential lysis buffer in a centrifugal concentrator, opening the centrifugal concentrator after centrifugation and decanting the supernatant fraction, pre-treating the blood sample, a blood culture step, a step of subculturing the blood sample to identify microorganisms present in the blood sample, or a DNase step for digesting genomic DNA in the lysate.

[0047] A17. One or more methods of clauses A1 to A16, which do not include one or more of the following steps: pre-treating a blood sample, a blood culture step, subculturing a blood sample and identifying microorganisms present in the blood sample, or a DNase step for digesting genomic DNA in a lysate.

[0048] A18. Steps (a) to (c) are completed within a time range of approximately 10 to 20 minutes, using one or more of the methods described in clauses A1 to A17.

[0049] A19. Steps (d) and (e) are completed within a time range of approximately 15 to 75 minutes, using one or more of the methods specified in clauses A1 to A18.

[0050] A20. Steps (a) to (e) are completed within a time range of approximately 25 to 95 minutes. One or more methods of clauses A1 to A19

[0051] A21. Microorganisms are isolated from the lysate by filtering, using one or more of the methods described in clauses A1 to A20.

[0052] A22. One or more methods of clauses A1 to A21, further comprising adding a filter to a self-contained assay device.

[0053] A23. One or more methods of clauses A1 to A22, wherein the differential lysis buffer contains a buffer having a certain pH buffer range, and the pH of the differential lysis buffer mixed with the blood sample is outside the pH buffer range of the buffer.

[0054] A23.1 One or more methods of clauses A1 to A23, wherein the buffering material is selected from the group consisting of CABS, CAPS, CAPS, CHES, and combinations thereof.

[0055] A23.2 The buffer material is CAPS, one or more of the methods of clauses A1 to A23.1

[0056] A24. The pH of the differential lysis buffer mixed with the blood sample is below the pH buffering range of the buffering agent, one or more methods according to clauses A1 to A23.2.

[0057] A25. The pH of the differential lysis buffer mixed with the blood sample is approximately 1.5 to 2.5 pH units below the pH buffering range of the buffering agent, according to one or more methods of clauses A1 to A24.

[0058] A26. One or more methods of clauses A1 to A25, wherein the blood sample mixed with differential lysis buffer has a pH of approximately 7.0 to 8.0, and the buffer has a useful pH buffering range of approximately 8.6 to 11.4 and a pKa in the range of approximately 9.5 to approximately 10.7 at 25°C.

[0059] A27. Identification further comprises amplifying one or more nucleic acids and then detecting one or more amplified nucleic acids, one or more of the methods of clauses A1 to A26.

[0060] A28. Detection of one or more amplified nucleic acids by one or more methods of clauses A1 to A27, including a nucleic acid lysis step.

[0061] A29. One or more methods of clauses A1 to A28, further comprising: performing first-stage multiplex amplification on one or more nucleic acids to obtain a first-stage amplification product; separating the diluted first-stage amplification product in a set of second-stage amplification wells, each having a set of amplification primers designed to further amplify specific nucleic acids that may be present in the sample; performing second-stage amplification in the second-stage amplification wells; and performing post-amplification nucleic acid lysis and lysis curve analysis to identify microorganisms present in the blood sample.

[0062] A30. One or more methods of clauses A1 to A29, wherein the analysis includes a nucleic acid sequencing step to produce sequencing data containing sequence information obtained from one or more nucleic acids sufficient to identify microorganisms present in a blood sample.

[0063] A31. The nucleic acid sequencing step includes one or more methods from clauses A1 to A30, including large-scale parallel or next-generation sequencing technology.

[0064] A32. One or more methods of clauses A1 to A31, in which the steps of mixing a blood sample with differential lysis buffer to obtain a lysate and separating microorganisms from the lysate are achieved in a single tube.

[0065] A33. The differential lysis buffer is a single buffer provided in a single tube, one or more of the methods in clauses A1 to A32.

[0066] A34. One or more methods of clauses A1 to A33, wherein the differential lysis buffer does not contain DNase or protease, and the method does not involve the step of adding exogenous DNase or protease to a single tube.

[0067] A35. One or more methods of clauses A1 to A34, wherein the differential lysis buffer conforms to a standard anticoagulant, for example, selected from the group consisting of, but not limited to, EDTA, citrate, polyanethole sulfate sodium (SPS), heparan, sodium fluoride / oxalate, and combinations thereof.

[0068] B1. A method for isolating and identifying microorganisms, (a) To provide a blood sample that is known to contain or may contain microorganisms; (b) Mixing the blood sample with a differential lysis buffer containing a buffering agent and a nonionic surfactant to obtain a lysate containing lysed blood cells and non-lysed microorganisms; (c) Isolating microorganisms from lysate; (d) Adding microorganisms to a self-contained assay device designed to perform an assay including amplification of one or more nucleic acids having microbial characteristics and analysis of one or more amplified nucleic acids, thereby identifying microorganisms present in the blood sample, The method includes a blood sample mixed with differential lysis buffer having a pH of approximately 7.0 to 8.0, and the buffer having a useful pH buffering range of approximately 8.6 to 11.4.

[0069] B2. Prior to amplification, the assay further comprises lysing of microorganisms and recovery of nucleic acids from the microorganisms, the recovered nucleic acids being amplified for the identification of microorganisms present in the blood sample, according to the method of clause B1.

[0070] B3. One or more methods of clause B1 or B2, wherein the nonionic surfactant is polyoxyethylene (POE) ether.

[0071] B4. One or more methods of clauses B1 to B3, wherein the nonionic surfactant is selected from the group consisting of Triton X-114, NP-40, Arlasolve200, BrijO10 (also known as Brij96 / 97), octyl β-D-glucopyranoside, saponin, nonaethylene glycol monododecyl ether (C12E9, polidocenol), and combinations thereof.

[0072] B5. One or more methods of clauses B1 to B4, wherein the buffering material is selected from the group consisting of CABS, CAPS, CAPSO, CHES, and combinations thereof.

[0073] B6. One or more methods of clauses B1 to B5, wherein the buffering material is CAPS, and CAPS has a pH buffering range of approximately 9.7 to 11.1 and a pKa of approximately 10.4 at 25°C.

[0074] B7. Combining a blood sample with differential lysis buffer in a centrifugal concentrator, A blood sample is combined with differential lysis buffer over a first time to obtain a lysate; The process involves centrifuging a centrifugal concentrator to pelletize microorganisms from a blood sample placed in the chamber; The plunger is pushed in, the seal is released, and pellets are generated from the opening at the second end of the chamber. It further includes, here a centrifugal concentrator, A chamber having an open portion at a first end and a sealing portion at a second end, wherein the sealing portion is designed to seal the second open portion at the second end of the chamber; and A plunger positioned at least partially movable inside a chamber, and designed to act to release a seal. One or more methods of clauses B1 to B6, including

[0075] B8. Pushing in the plunger, releasing the seal, and generating pellets, includes pushing the plunger until it is tightly engaged with a portion of the body, and discharging the pellets from the second end by releasing the seal under pressure, one or more methods of clauses B1 to B7.

[0076] B9. Further comprising optionally generating pellets in a vial having an internal volume containing a sample buffer disposed within it, and a cannula processing vial including a cannula extending from the bottom surface of the vial, A cannula that does not extend into the vial has a first end and a second end adjacent to the bottom surface of the vial. The vial further includes a filter located near the bottom surface of the vial, designed to filter the body fluid before it is introduced into the cannula, wherein the filter has a diameter large enough to allow fungi, viruses, protozoa, and / or bacterial organisms to pass through and enter the cannula, but has a pore size small enough to capture larger particulate matter. One or more methods of clauses B1 to B8

[0077] B10. One or more methods of clauses B1 to B9, further comprising positioning the second end of the cannula into the first port of a self-contained assay device, wherein the first port of the self-contained assay device is provided under vacuum to guide the volume of body fluid from the vial through the cannula to the self-contained assay device.

[0078] B11. Self-contained assay device, A cell lysis zone designed for dissolving microorganisms, with fluid connected to the first port; A nucleic acid preparation zone, designed for purifying nucleic acids from microorganisms, is fluid-connected to the cell lysis zone; A first-stage reaction zone, including a first-stage reaction chamber designed for the first-stage amplification of nucleic acids purified from microorganisms, which is fluid-connected to the nucleic acid preparation zone; and A second-stage reaction zone, fluidly connected to a first-stage reaction zone, comprising a plurality of second-stage reaction chambers, each containing a primer pair designed for further amplification of biospecific nucleic acids purified from microorganisms, and the second-stage reaction zone is designed for simultaneous heat circulation in all of the plurality of second-stage reaction chambers, and for performing post-amplified nucleic acid fusion and fusion curve analysis to identify microorganisms present in blood samples. Furthermore, one or more methods of clauses B1 to B10, including

[0079] B12. One or more methods of clauses B1 to B11, wherein the centrifugal concentrator and the vial of the cannula processing vial are designed to engage with each other in order to conjugate the centrifugal concentrator with the cannula processing vial, wherein the vial of the cannula processing vial is designed to surround the second end, and therefore the vial of the cannula processing vial is designed to collect the pellet expressed from the second end of the chamber.

[0080] B13. One or more methods of clauses B1 to B12, wherein the second end of the centrifugal concentrator includes a first engaging portion, and the vial body of the cannula processing vial includes a second complementary engaging portion for fixedly conjugating the centrifugal concentrator with the cannula processing vial.

[0081] B14. One or more methods of clauses B1 to B13, wherein the first and second engaging portions include threads for threadably conjugating the centrifugal concentrator to a cannula processing vial.

[0082] B15. One or more methods of clauses B1 to B14, further comprising engaging a centrifugal concentrator and a cannula processing vial to guide the volume of body fluid from the vial through the cannula to a self-contained assay device, removing the cannula from the cannula processing vial from the first port of the self-contained assay device, and disposing of the centrifugal concentrator and the cannula processing vial.

[0083] B16. One or more methods of clauses B1 to B15, including a partition designed for aseptically loading a sample mixed with differential lysis buffer into the centrifugal concentrator, such as the first end opening of the centrifugal concentrator.

[0084] B17. A centrifugal concentrator that does not contain a high-density cushion, one or more of the methods of clauses B1 to B16.

[0085] B18. One or more methods of clauses B1 to B17, which do not include one or more of the following steps: pre-treating a blood sample, a blood culture step, a step of subculturing a blood sample to identify microorganisms present in the blood sample, or a DNase step for digesting genomic DNA in a lysate.

[0086] B19. Steps (a) to (c) are completed within a time range of approximately 10 to 20 minutes, using one or more of the methods specified in clauses B1 to B18.

[0087] B20. Steps (d) and (e) are completed within a time range of approximately 15 to 75 minutes, using one or more of the methods specified in clauses B1 to B19.

[0088] B21. Steps (a) to (e) are completed within a time range of approximately 25 to 95 minutes, using one or more of the methods specified in clauses B1 to B20.

[0089] B22. One or more methods of clauses B1 to B21, wherein the first time to obtain the lysate is in the range of about 2 to 10 minutes, preferably about 5 minutes.

[0090] B23. Obtaining a license is done by one or more methods of clauses B1-B22, which do not involve any additional steps other than the combination.

[0091] C1. Blood samples known to contain or potentially containing microorganisms; and Differential lysis buffer to be combined with blood samples, comprising an aqueous medium, a buffering agent, and a nonionic surfactant. A composition comprising a buffer having a pH of approximately 7.0 to 8.0, where the buffer has a useful pH buffering range of approximately 8.6 to 11.4 and a pKa in the range of approximately 9.5 to approximately 10.7 at 25°C.

[0092] C2. The composition of clause C1, wherein the nonionic surfactant is polyoxyethylene (POE) ether.

[0093] C3. One or more compositions of clause C1 or C2 in which the nonionic surfactant is selected from the group consisting of Triton X-114, NP-40, Arlasolve200, BrijO10 (also known as Brij96 / 97), octyl β-D-glucopyranoside, saponin, nonaethylene glycol monododecyl ether (C12E9, polidocenol), and combinations thereof.

[0094] C4. The buffering material is selected from the group consisting of CABS, CAPS, CAPSO, CHES, and combinations thereof, one or more compositions of clauses C1 to C3.

[0095] C5. One or more compositions of clauses C1 to C4, wherein the buffering material is CAPS having a pH buffering range of approximately 9.7 to 11.1 and a pKa of approximately 10.4 at 25°C.

[0096] C6. One or more compositions of clauses C1 to C5, wherein the buffering material is substantially positively charged at a pH of approximately 7.0 to 8.0.

[0097] One or more compositions of clauses C1 to C6 that do not contain C7.DNase

[0098] C8. Blood samples known to contain or potentially containing microorganisms; and Differential lysis buffer containing buffering agent and nonionic surfactant Essentially consisting of one or more compositions of clauses C1 to C7, having a pH of about 7.0 to 8.0, where the buffering material is CAPS having a useful pH buffering range of about 9.7 to 11.1 and a pKa of about 10.4 at 25°C.

[0099] D1. Blood samples known to contain or potentially containing microorganisms; and Differential lysis buffer containing buffering agent and nonionic surfactant A composition comprising a buffer having a pH of about 7.0 to 8.0, wherein the buffer has a useful pH buffering range of about 8.6 to 11.4 and a pKa in the range of about 9.5 to about 10.7 at 25°C. A composition comprising a lysate containing lysed blood cells and, if present, non-lysed microorganisms; A centrifugal concentrator designed for pelletizing insoluble microorganisms in lysate, A chamber having an open portion at a first end and a sealing portion at a second end, wherein the sealing portion is designed to seal the second open portion at the second end of the chamber; and A plunger positioned at least partially movable inside a chamber, and designed to act to release a seal. A centrifugal concentrator, including; and A cannula processing vial designed to be conjugated to the second end of a centrifugal concentrator in order to receive microbial pellets from the centrifugal concentrator, Optionally, a vial having an internal volume containing a sample buffer, and a cannula extending from the bottom surface of the vial, having a first end adjacent to the bottom surface of the vial and a second end, and including a cannula that does not extend from the vial; and The vial further includes a filter located near the bottom surface of the vial, designed to filter body fluids before they enter the cannula, where the filter has a diameter large enough to allow fungi, viruses, protozoa, and / or bacterial organisms to pass through and enter the cannula, but has a pore size small enough to capture larger particulate matter. vials for cannula processing systems including

[0100] D2. The centrifugal concentrator does not include a high-density cushion, as per clause D1.

[0101] D3. A self-contained assay device further comprising one or more systems of clauses D1 and D2, having a first port designed to receive a second end of a cannula for introducing a sample into the self-contained assay device, wherein the first port of the self-contained assay device is provided under vacuum to guide the volume of the sample from the vial through the cannula into the self-contained assay device.

[0102] D4. Self-contained assay device, A cell lysis zone fluidly connected to a first port, a cell lysis zone designed for lysing microorganisms; A nucleic acid preparation zone fluidly connected to a cell lysis zone, designed for purifying nucleic acids from microorganisms; A first-stage reaction zone fluidly connected to a nucleic acid preparation zone, comprising a first-stage reaction chamber designed for first-stage amplification of nucleic acids purified from microorganisms; and A second-stage reaction zone is fluidly connected to a first-stage reaction zone, the second-stage reaction zone comprises a plurality of second-stage reaction chambers, each second-stage reaction chamber containing a primer pair designed for further amplification of biospecific nucleic acids purified from microorganisms, the second-stage reaction zone is designed for simultaneous heat circulation in all of the plurality of second-stage reaction chambers, and for performing post-amplified nucleic acid fusion and fusion curve analysis to identify microorganisms present in blood samples. Furthermore, one or more systems of clauses D1 to D3, including

[0103] D5. One or more systems of clauses D1-D4, wherein the centrifugal concentrator and the vial of the cannula processing vial are designed to engage with each other in order to conjugate the centrifugal concentrator with the cannula processing vial, and the vial of the cannula processing vial is designed to surround the second end, and therefore the vial of the cannula processing vial is designed to collect the pellet expressed from the second end of the chamber.

[0104] D6. One or more systems of clauses D1 to D5, wherein the second end of the centrifugal concentrator includes a first engagement portion, and the vial body of the cannula processing vial includes a complementary second engagement portion for fixedly conjugating the centrifugal concentrator with the cannula processing vial.

[0105] D7. One or more systems of clauses D1 to D6, wherein the first and second engaging portions include threads for threadably conjugating the centrifugal concentrator to a cannula processing vial.

[0106] D8. One or more systems of clauses D1-D7, including a partition designed to aseptically fill the centrifugal concentrator with a sample mixed with differential lysis buffer, such as the first end opening of the centrifugal concentrator.

[0107] D9. Centrifugal concentrator does not include high-density cushioning in one or more systems of clauses D1-D8.

[0108] D10. One or more systems of clauses D1-D9 in which the nonionic surfactant of the differential lysis buffer is polyoxyethylene (POE) ether.

[0109] D11. One or more systems of clauses D1 to D10 in which the nonionic surfactant of the differential lysis buffer is selected from the group consisting of Triton X-114, NP-40, Arlasolve200, BrijO10 (also known as Brij96 / 97), octyl β-D-glucopyranoside, saponin, nonaethylene glycol monododecyl ether (C12E9, polidocenol), and combinations thereof.

[0110] D12. The buffering agent of the differential lysis buffer is selected from the group consisting of CABS, CAPS, CAPSO, CHES, and combinations thereof, in one or more systems of clauses D1 to D11.

[0111] D13. The buffering agent of the differential lysis buffer is CAPS, and CAPS has a useful pH buffering range of approximately 9.7 to 11.1 and a pKa of approximately 10.4 at 25°C, in one or more systems of clauses D1 to D12.

[0112] D14. The buffering material in the differential lysis buffer is substantially positively charged at a pH of approximately 7.0–8.0 in one or more systems of clauses D1–D13.

[0113] D15. Differential lysis buffer is DNase-free, one or more systems of clauses D1-D14.

[0114] E1. A method for isolating and identifying microorganisms, (a) To provide a blood sample that is known to contain or may contain microorganisms; (b) Mixing a blood sample with a differential lysis buffer having a certain pH to obtain a lysate containing lysed blood cells and non-lysed microorganisms; (c) Placing the blood sample mixed with differential lysis buffer into a centrifugal concentrator; (d) Centrifugation of the concentrator to pelletize microorganisms from the blood sample placed in the chamber; (e) The step of adding microorganisms to a self-contained assay device containing one or more reagents required to identify the microorganisms; (f) Identifying microorganisms present in the blood sample, The process includes, where the addition of microorganisms to a self-contained assay apparatus involves pushing in a plunger, releasing the seal portion, and generating a pellet from the opening at the second end of the chamber, and the identification process includes the steps of isolating one or more nucleic acids having microbial characteristics from the microorganisms, performing nucleic acid amplification, and performing post-amplified nucleic acid lysis and lysis curve analysis to identify the microorganisms present in the blood sample. Here, the centrifugal concentrator is used. A chamber having an open portion at a first end and a sealing portion at a second end, wherein the sealing portion is designed to seal the second open portion at the second end of the chamber; and A plunger positioned at least partially movable inside a chamber, and designed to act to release a seal. Methods including

[0115] E2. Self-contained assay device, A first port, provided under vacuum, allows a certain amount of pellets to be introduced into a self-contained assay device; A cell lysis zone fluidly connected to a first port, a cell lysis zone designed for lysing microorganisms; A nucleic acid preparation zone fluidly connected to a cell lysis zone, designed for purifying nucleic acids from microorganisms; A first-stage reaction zone fluidly connected to a nucleic acid preparation zone, comprising a first-stage reaction chamber designed for performing first-stage multiplex amplification of one or more nucleic acids; A second-stage reaction zone fluidly connected to a first-stage reaction zone, the second-stage reaction zone comprising a plurality of second-stage reaction chambers, each second-stage reaction chamber comprising a primer pair designed for further amplification of a specific nucleic acid purified from one of the microorganisms, and the second-stage reaction zone being designed for simultaneous heat circulation in all of the plurality of second-stage reaction chambers. It further includes, and The process involves performing first-stage multiplex amplification in the first-stage reaction zone to obtain the first-stage amplification product, diluting the first-stage amplification product, separating the diluted first-stage amplification product in multiple second-stage reaction chambers, performing second-stage amplification in the second-stage amplification chambers, and performing nucleic acid lysis after amplification and lysis curve analysis after second-stage amplification to identify microorganisms present in the blood sample. The method of clause E1, which further includes

[0116] E3. One or more methods of clauses E1 and E2, which include pushing the plunger in until the plunger is tightly engaged with a portion of the body, and releasing the pellet from the second end under pressure by releasing the seal.

[0117] E4. The pellet is optionally generated in a cannula processing vial containing a sample buffer, and further includes a vial body having an internal volume and a cannula extending from the bottom surface of the vial body. A cannula that does not extend into the vial has a first end and a second end adjacent to the bottom surface of the vial. The vial further includes a filter located near the bottom surface of the vial, designed to filter the body fluid before it enters the cannula, wherein the filter has a diameter large enough to allow fungi, viruses, protozoa, and / or bacterial organisms to pass through and enter the cannula, but has a pore size small enough to capture larger particulate matter. One or more methods of clauses E1 to E3

[0118] E5. One or more methods of clauses E1 to E4, further comprising positioning the second end of the cannula into the first port of a self-contained assay device, wherein the first port of the self-contained assay device is provided under vacuum to guide a certain amount of body fluid from a vial through the cannula into the self-contained assay device.

[0119] E6. Steps (b) to (d) are performed within a single tube, using one or more methods of clauses E1 to E5.

[0120] E7. The differential lysis buffer is a single buffer provided in a single tube, one or more of the methods of clauses E1 to E6.

[0121] E8. One or more methods of clauses E1 to E7, wherein the differential lysis buffer does not contain DNase or protease, and the method does not involve the step of adding exogenous DNase or protease to a single tube.

[0122] E9. One or more methods of clauses E1 to E8, which do not include one or more of the following steps: pretreatment of a blood sample, a blood culture step, a step of subculturing a blood sample to identify microorganisms present in the blood sample, or a DNase step for digesting genomic DNA in a lysate.

[0123] E10. Steps (a) to (d) are completed within a time range of approximately 10 to 20 minutes, using one or more of the methods specified in clauses E1 to E9.

[0124] E11. Steps (e) and (f) are completed within a time range of approximately 15 to 75 minutes, using one or more of the methods of clauses E1 to E10.

[0125] E12. Steps (a) to (f) are completed within a time range of approximately 25 to 95 minutes, using one or more of the methods specified in clauses E1 to E11.

[0126] This summary is provided to introduce the selection of concepts in a simplified form, which will be further explained later in a more detailed description. This summary is not intended to reveal the main or essential features of the claimed subject matter, nor is it intended to be used to help determine the scope of the claimed subject matter.

[0127] Further features and advantages will be described in the following description, or will be evident in part from the description, or may be recognized through the practice of the present invention. These features and advantages may be realized and obtained, in particular, using the apparatus and combinations shown in the appended claims. These and other features will be more clearly evident from the following description and the appended claims, or may be recognized through the practice of the present invention, as described later. [Brief explanation of the drawing]

[0128] [Figure 1] This shows a portable pouch useful for self-contained PCR. [Figure 2] This is an exploded perspective view of the device for use with the pouch shown in Figure 1, including the pouch shown in Figure 1. [Figure 3] The pouch in Figure 1 is shown together with the bladder components in Figure 2. [Figure 4] Figure 2 shows a motor used in an exemplary embodiment of the apparatus. [Figure 5] This is a schematic diagram of one embodiment of a differential dissolution and centrifugation method using the system and apparatus described herein. [Figure 6A] This is an isometric view of a centrifugal concentrator according to one embodiment of the present invention. [Figure 6B]Figure 6A is a side view of the centrifugal concentrator. [Figure 6C] The diagram shows the same centrifugal concentrator as in Figure 6B, with the cap removed. [Figure 6D] This is another isometric view of a centrifugal concentrator. [Figure 6E] This is a detailed diagram of one end of a centrifugal concentrator. [Figure 6F] Figure 6E is a cross-sectional view of the end of the centrifugal concentrator shown. [Figure 6G] Figures 6A to 6F are isometric views of the plunger of the centrifugal concentrator. [Figure 7] This is an example of a workflow using differential lysis buffer. [Figure 8] This is a bar graph comparing differential lysis buffers with several other protocols. [Figure 9] This bar graph illustrates cell retrieval using differential lysis buffer. [Figure 10] The effect of removing human genomic DNA in an exemplary differential dissolution and centrifugation method is illustrated. [Figure 11] This data demonstrates that differential lysis buffers can selectively dissolve eukaryotic host cells while leaving microbial cells intact. [Figure 12] The workflow for microbial recovery rate and detection efficiency from whole blood is illustrated. [Figure 13] Figure 12 illustrates the average recovery rate of microorganisms in the test workflow shown. [Figure 14] Figure 12 illustrates the average inoculation and recovery of microorganisms in the test workflow shown. [Figure 15A-C] Flow-through methods for animal cell lysis, culture, and microbial enrichment are illustrated. [Figure 16] A filtration method for isolating and concentrating microorganisms is illustrated. [Figure 17A-C] Different filtration structures that can be used to separate cells by size are schematically illustrated. [Figure 18] Different types of pillar filters, such as (18A) polygonal, (18B) U-shaped, and (18C) butterfly-shaped micro-pillar shapes, are schematically illustrated. [Figure 19] The separation of large and small cells within a series of micropillars and crossflow structures in buffer and cell suspensions is schematically illustrated. [Figure 20] The concentration of large and small cells by movement along the oval filter unit is schematically illustrated. [Figure 21] This graph illustrates the solubility of blood samples over time using various differential lysis buffer formulations, showing the relationship between absorbance and incubation time. [Figure 22] This is a bar graph illustrating the increase in biological concentrations for several types of organisms and blood anticoagulants. [Modes for carrying out the invention]

[0129] Examples of embodiments are described below with reference to the accompanying drawings. Since many forms and embodiments are possible without departing from the spirit and teachings of this disclosure, this disclosure should not be construed as limiting to the examples of embodiments described herein. Rather, these examples of embodiments are provided to make this disclosure thorough and complete and to convey the scope of this disclosure to those skilled in the art. In the drawings, the sizes and relative sizes of layers and areas may be exaggerated for clarity. Similarity reference numbers refer to similar elements throughout this description.

[0130] Unless otherwise specified, all terms used herein (including technical and scientific terms) have the same meaning as those commonly understood by those skilled in the art to whom this disclosure relates. Terms as defined in commonly used dictionaries should be interpreted as having the same meaning as those in relation to the present application and related art, and should not be interpreted in an idealized or overly formal sense unless specifically defined herein. The terms used in the description of the invention herein are intended solely to describe specific embodiments and are not intended to limit the invention. While several methods and materials similar or equivalent to those described herein may be used in the practice of this disclosure, only specific representative materials and methods are described herein.

[0131] All publications, patent applications, patents, or other references referenced herein are incorporated by reference in their entirety. In the event of any inconsistency in the use of terms, this specification shall prevail.

[0132] Various aspects of this disclosure, including apparatus, systems, methods, etc., may be illustrated in relation to one or more representative practices. As used herein, the terms “representative” and “exemplary” mean “serving as an example, illustration, or demonstration,” and should not necessarily be construed as being preferable or advantageous to other practices disclosed herein. Furthermore, “practices” or “embodiments” of this disclosure or the present invention include specific references to one or more embodiments thereof, and vice versa, and are intended to provide examples without limiting the scope of the invention as set forth by the appended claims rather than by the following description.

[0133] As used herein and in the appended claims, the singular forms "a," "an," and "the" will be accepted to include multiple references unless otherwise explicitly indicated in the context. For example, "a tile" may include one, two, or more tiles. Similarly, multiple references should be interpreted to include a single reference and / or multiple references unless otherwise indicated or explicitly stated in the context. Therefore, "tiles" does not necessarily require multiple such tiles. Instead, it will be understood that, apart from combinations, one or more tiles are contemplated herein.

[0134] As used throughout this application, the terms “can” and “may” are used in a permissive sense (i.e., meaning “to have the ability to do something”) rather than a compulsory sense (i.e., meaning “must”). In addition, the terms “including,” “having,” “involving,” “containing,” and “characterized by,” their variations (e.g., “includes,” “has,” “involves,” “contains,” etc.), and similar terms, when used herein, including in the claims, are inclusive and / or unrestrictive, and are to have the same meaning as the term “comprising” and its variations (e.g., “comprise” and “comprises”), and do not exclude, by example, additional unlisted elements or method steps.

[0135] As used herein, terms of directionality and / or subjectivity such as “top,” “bottom,” “left,” “right,” “up,” “down,” “upper,” “lower,” “inside,” “outside,” “internal,” “external,” “interior,” “exterior,” “proximal,” “distal,” “forward,” and “reverse” may be used alone to indicate relative direction and / or orientation and may not be intended to particularly limit the scope of this disclosure, including the specification, invention, and / or claims.

[0136] When one element is referred to as being “conjugated,” “connected,” or “responsive” to another element, or as “relating to” another element, it can be directly conjugated, connected, or responsive to or with respect to the other element, or there may be an intervening element. In contrast, when one element is referred to as being “directly conjugated,” “directly connected,” or “directly responsive” to another element, or as “directly toward” another element, it will be understood that there is no intervening element.

[0137] Examples of conceptual embodiments of the present invention are described herein with reference to schematic cross-sectional views of idealized embodiments (and intermediate-stage structures) of the examples of embodiments. Thus, variations from the shapes of the drawings, for example, as a result of manufacturing techniques and / or tolerances, are to be expected. Therefore, examples of conceptual embodiments of the present invention should not be construed as being limited to specific shapes of the areas illustrated herein, but will include, for example, deviations in shape due to manufacturing. Accordingly, the areas illustrated in the drawings are essentially schematic, and their shapes are not intended to illustrate the actual shapes of any area of ​​the apparatus, nor are they intended to limit the scope of the examples of embodiments.

[0138] While terms such as "first" and "second" may be used herein to describe various elements, it will be understood that these elements should not be limited by these terms. These terms are simply used to distinguish one element from another. Therefore, a "first" element can be referred to as a "second" element without departing from the teachings of this embodiment.

[0139] Furthermore, it is understood that the various practices described herein can be used in combination with any other practices described or disclosed herein without departing from the scope of this disclosure. Accordingly, products, members, elements, devices, apparatus, systems, methods, processes, compositions, and / or kits that conform to a particular practice of this disclosure may include, encompass, or otherwise comprise the characteristics, features, components, members, elements, steps, etc., described herein without departing from the scope of this disclosure. Accordingly, specific features relating to one practice should not be construed to be limited to their application in that practice only.

[0140] The headings used herein are for structuring purposes only and are not intended to limit the scope of the description or claims. To facilitate understanding, similar elements common to the drawings are designated, where possible, using similar reference numbers. Furthermore, where possible, similar element numbering is used across different drawings. Additionally, alternative designs for particular elements may include separate letters assigned to their element numbers.

[0141] The term “about” is used herein to mean roughly or approximately within a range of about. When the term “about” is used in conjunction with a numerical range, it modifies the range by extending to the upper and lower boundaries of the numerical value being described. Generally, the term “about” is used herein to modify the numerical values ​​above and below a 5% variation of a given value. When such a range is expressed, an alternative embodiment includes one specific value and / or the other specific value. Similarly, it will be understood that a specific value forms an alternative embodiment when it is expressed as an approximation by the use of “about” preceded by the value. Furthermore, it will be understood that each endpoint of such range is significant in relation to the other endpoint, and independently of the other endpoint.

[0142] When used herein, the term "or" means any one member of a given list, and further includes any combination of members of that list.

[0143] As used herein, the term “microorganism” is intended to encompass, but is not limited to, unicellular organisms that can be cultivated and handled in a laboratory, including Gram-positive or Gram-negative bacteria, yeasts, fungi, and parasites. Non-limiting examples of Gram-negative bacteria in this invention include bacteria of the following genera: Pseudomonas, Escherichia, Salmonella, Shigella, Enterobacter, Klebsiella, Serratia, Proteus, Campylobacter, Haemophilus, Morganella, Vibrio, Yersinia, Acinetobacter, Stenotrophomonas, and Brevundimonas. Examples include evundimonas, Ralstonia, Achromobacter, Fusobacterium, Prevotella, Branhamella, Neisseria, Burkholderia, Citrobacter, Hafnia, Edwardsiella, Aeromonas, Moraxella, Brucella, Pasteurella, Providencia, and Legionella.Non-limiting examples of Gram-positive bacteria in the present invention include bacteria of the following genera: Enterococcus, Streptococcus, Staphylococcus, Bacillus, Paenibacillus, Lactobacillus, Listeria, Peptostreptococcus, Propionibacterium, Clostridium, Bacteroides, Gardnerella, Kocuria, Lactococcus, Leuconostoc, Micrococcus, Mycobacteria, and Corynebacteria. Non-limiting examples of yeasts and fungi of the present invention include yeasts and fungi of the following genera: Candida, Cryptococcus, Nocardia, Penicillium, Alternaria, Rhodotorula, Aspergillus, Fusarium, Saccharomyces, and Trichosporon. Non-limiting examples of parasites of the present invention include parasites of the following genera: Trypanosoma, Babesia, Leishmania, Plasmodium, Wucheria, Brugia, Onchocerca, and Naegleria.

[0144] In one embodiment, as described in further detail herein, microorganisms from a sample or growth medium can be isolated and examined to characterize and / or identify the microorganisms present in the sample. As used herein, the term “isolate” is intended to encompass any sample of microorganisms that has been removed, concentrated, or otherwise isolated from its original state or from the growth medium or culture medium. For example, according to the present invention, microorganisms may be isolated (e.g., as separate samples) from non-microorganisms or non-microbial components that would normally interfere with characterization and / or identification. The term may also include microorganisms isolated from a mixture by centrifugation, filtration, or any other separation technique known in the art. As such, an isolated microbial sample may include a collection of microorganisms and / or components thereof that are concentrated from the original sample, or otherwise isolated from the original sample, and may range from densely packed, high-density aggregates of microorganisms to a diffusion layer of microorganisms. Non-microbial components isolated from microorganisms may include non-microbial cells (e.g., blood cells and / or other tissue cells) and / or components thereof. In one embodiment, microorganisms are isolated from a lysate mixture containing lysed non-microbial cells and substantially intact microbial cells.

[0145] In some embodiments, the isolation of a microbial sample from its original state or from its growth or culture medium is incomplete. In other words, due to removal, concentration, or otherwise modification that deviates from the microbial sample from its original state, the microbial sample is not completely isolated from other components of the sample or from its growth or culture medium. In some cases, a minimal amount of debris from the sample or from its growth or culture medium is present. For example, the amount of debris or growth or culture medium present in the isolated sample may be insufficient to identify or characterize the microbial or interfere with its further growth. In some embodiments, the isolated sample is 99% pure from contaminating components, but it may also be 95% pure, 90% pure, 80% pure, 70% pure, 60% pure, 50% pure, or a minimum purity that still allows for identification of the microbial in the isolated sample by downstream identification techniques.

[0146] In further different embodiments described in more detail herein, microorganisms from a sample or growth medium can be pelletized and examined to characterize and / or identify the microorganisms present in the sample. As used herein, the term “pellet” is intended to encompass any sample of microorganisms compressed or accumulated in a mass of microorganisms. For example, microorganisms from a sample can be compressed or accumulated in a mass at the bottom of a tube by centrifugation or by other known methods in the art. The term includes the collection of microorganisms (and / or their components) on the bottom and / or sides of the container after centrifugation. In accordance with the present invention, microorganisms can be pelletized (e.g., as substantially purified microbial pellets) from non-microorganisms or non-microorganismal components that would normally interfere with characterization and / or identification.

[0147] When used herein, the term "nucleic acid" refers to naturally occurring or synthetic oligonucleotides or polynucleotides of single-stranded or double-stranded sense or antisense DNA, RNA, or DNA-RNA hybrids that are capable of hybridization with complementary nucleic acids by Watson-Crick base pairing. The nucleic acids of the present invention may also include nucleotide analogs (e.g., BrdU) and non-phosphated diester nucleoside bonds (e.g., peptide nucleic acids (PNA) or thiodiester bonds). In particular, nucleic acids may include, but are not limited to, DNA, RNA, mRNA, rRNA, cDNA, gDNA, ssDNA, dsDNA, or any combination thereof.

[0148] "Probe," "primer," or "oligonucleotide" means a single-stranded nucleic acid molecule with a defined sequence that can base-pair with a second nucleic acid molecule containing a complementary sequence ("target"). The stability of the resulting hybrid depends on the length, GC content, and range of base pairings. The range of base pairings is influenced by parameters such as the degree of complementarity between the probe and the target molecule and the degree of stringency of the hybridization conditions. The degree of hybridization stringency is influenced by parameters such as temperature, salt concentration, and concentration of organic molecules such as formamide, and is determined by methods known to those skilled in the art. Probes, primers, and oligonucleotides may be labeled detectably by radioactive, fluorescent, or non-radioactive means by methods known to those skilled in the art. dsDNA may be detected using dsDNA-binding dyes. It is understood that while "primers" are specifically designed to be extended by polymerase, "probes" or "oligonucleotides" may or may not be designed in this way.

[0149] "dsDNA-binding dye" means a dye that fluoresces, usually more strongly, when bound to double-stranded DNA than when bound to single-stranded DNA or when free in solution. When referring to a dsDNA-binding dye, any suitable dye may be used herein, and it is understood that several non-limiting exemplary dyes are described in U.S. Patent No. 7,387,887, which is incorporated herein by reference. As is known in the art, other signal-generating substances, such as enzymes and antibodies, may be used to detect nucleic acid amplification and fusion.

[0150] "Specifically hybridizing" means that, under high stringency conditions, the probe, primer, or oligonucleotide recognizes a substantially complementary nucleic acid (e.g., sample nucleic acid), physically interacts with it (i.e., base pairs with it), and substantially does not form base pairs with other nucleic acids.

[0151] "High stringency conditions" typically mean that the analysis is performed at approximately 5°C below the melting temperature (Tm) (i.e., 5° lower than the probe's Tm). Functionally, using high stringency conditions allows for the identification of nucleic acid sequences with at least 80% sequence identity.

[0152] "Dissolving particles" refers to various particles or beads for dissolving cells, viruses, spores, and other materials that may be present in a sample. In various examples, zirconium ("Zr") silicate or ceramic beads are used, but other dissolving particles, including glass and sand dissolving particles, are known and fall within the scope of this term. The term "cell lysing component" may include dissolving particles as is known in the art, but may further include other components such as components for chemical dissolution.

[0153] While PCR is the amplification method used in the examples herein, it is understood that any amplification method using primers may be preferred. Such preferred methods include polymerase chain reaction (PCR); strand displacement amplification (SDA); nucleic acid sequence-based amplification (NASBA); cascade rolling circle amplification (CRCA); loop-mediated isothermal amplification of DNA (LAMP); isothermal and chimeric primer-initiated amplification of nucleic acids (ICAN); target-based helicase-dependent amplification (HDA); and transcription-mediated amplification (TMA). Therefore, when the term PCR is used, it should be understood that it includes other alternative amplification methods. In amplification methods without separation cycles, reaction time may be used if measurements are taken at cycles, doubling times, or crossing points (Cp), and additional reaction time may be added if additional PCR cycles are added in the embodiments described herein. Therefore, it is understood that the protocol may need to be modified.

[0154] While various examples in this specification refer to human targets and human pathogens, these examples are merely illustrative. The methods, kits, and apparatus described herein can be used to detect or sequence a wide variety of nucleic acid sequences from a wide variety of samples, including human, animal, industrial, and environmental samples.

[0155] In various embodiments disclosed herein, a self-contained nucleic acid analysis pouch is used, exemplary, for assaying a sample for the presence of various biological substances, exemplary, antigens and nucleic acid sequences, within a single, closed system. Such systems, including pouches and apparatus for use with pouches, are disclosed in detail in U.S. Patent No. 8,394,608; U.S. Patent No. 8,895,295; and U.S. Patent No. 10,464,060, which are incorporated herein by reference. However, it is understood that such pouches are merely exemplary, and the nucleic acid preparation and amplification reactions considered herein may be carried out in any of the various open or closed sample containers known in the art, including 96-well plates, plates of other shapes, arrays, carousels, etc., using various nucleic acid purification and amplification systems known in the art. While terms such as “sample well,” “amplification well,” and “amplification container” are used herein, these terms mean to encompass wells, tubes, and various other reaction vessels, as used in these amplification systems. In one embodiment, a pouch is used to assay for multiple pathogens. The pouch may, exemplary, contain one or more blisters used as sample wells within a closed system. Various steps, exemplary, including nucleic acid preparation, primary high-volume multiplex PCR, dilution of the primary amplification product, and secondary PCR, and ending with optional real-time detection or post-amplification analysis such as melting curve analysis, may be optionally performed within the disposable pouch. Furthermore, while various steps may be performed within the pouch of the present invention, it is understood that one or more steps may be omitted in specific uses, and accordingly, the pouch configuration may be modified. While a multiplex reaction is used in the first-stage amplification in many embodiments herein, this is merely illustrative, and it is understood that in some embodiments the first-stage amplification may be singleplex. In one exemplary example, the first-stage singleplex amplification targets a housekeeping gene, and the second-stage amplification uses differences in the housekeeping gene for identification.Therefore, while first-stage multiplex amplification is considered in various embodiments, it should be understood that this is merely illustrative.

[0156] Figure 1 shows an exemplary pouch 510, which may be used in various embodiments or redesigned in various embodiments. Pouch 510 is similar to Figure 15 of U.S. Patent No. 8,895,295, with similar items numbered similarly. Fitting 590 is provided with entry channels 515a-515l, which also serve as reagent reservoirs or waste reservoirs. Exemplarily, reagents may be lyophilized within fitting 590 and rehydrated before use. Blisters 522, 544, 546, 548, 564, and 566, along with their respective channels 514, 538, 543, 552, 553, 562, and 565, are similar to the numbered blisters in Figure 15 of U.S. Patent No. 8,895,295. The second-stage reaction zone 580 in Figure 1 is similar to that in U.S. Patent No. 8,895,295, except that the second-stage wells 582 of the high-density array 581 are arranged in a slightly different pattern. The more circular pattern of the high-density array 581 in Figure 1 may result in a more uniform filling of the second-stage wells 582 by removing the wells at the edges. As shown, the high-density array 581 provides 102 second-stage wells 582. The pouch 510 is suitable for use in a FilmArray® device (BioFire Diagnostics, LLC, Salt Lake City, UT). However, it should be understood that the embodiment of the pouch is merely illustrative.

[0157] While other containers may be used, the pouch 510 may, exemplary, be formed of two layers of a flexible plastic film or other flexible material, e.g., polyester, polyethylene terephthalate (PET), polycarbonate, polypropylene, polymethyl methacrylate, mixtures thereof, combinations thereof, and layers, which can be manufactured by any process known in the art, including extrusion molding, plasma deposition, and lamination. For example, each layer may consist of one or more layers of a single type or two or more types of material that are laminated together. Alternatively, metal foil or plastic with an aluminum lamination may be used. Other barrier materials are known in the art and can be sealed together to form blisters and channels. When a plastic film is used, the layers may, exemplary, be bonded together by heat sealing. Exemplarily, the material has low nucleic acid binding and low protein binding capacity.

[0158] In embodiments using fluorescence monitoring, plastic films with sufficiently low absorbance and autofluorescence at the effective wavelength are preferred. Such materials can be identified by testing different plastics, different plasticizers, and composite ratios, as well as different film thicknesses. In the case of plastics with aluminum or other foil laminations, the portion of the pouch to be read by the fluorescence detector can be made foil-free. For example, if fluorescence is monitored in the second-stage well 582 of the second-stage reaction zone 580 of pouch 510, one or both layers in well 582 will be made foil-free. In the PCR example, film laminations made of polyester with a thickness of approximately 0.0048 inches (0.1219 mm) (Mylar, DuPont, Wilmington DE) and polypropylene films with a thickness of 0.001 to 0.003 inches (0.025 to 0.076 mm) are effective. Exemplarily, pouch 510 may be made from a transparent material capable of transmitting approximately 80% to 90% of incident light.

[0159] In exemplary embodiments, the material is moved between the blisters by the application of pressure, exemplary pneumatic pressure, to the blisters and channels. Thus, in embodiments using pressure, the pouch material is exemplary sufficiently flexible to allow the pressure to have the desired effect. The term “flexibility” is used herein to describe the physical characteristics of the pouch material. The term “flexibility” is defined herein as being easily deformable by the level of pressure used herein, without cracking, breaking, or fissureing. For example, thin plastic sheets such as Saran Wrap and Ziploc Bags, and thin metal foils such as aluminum foil, are flexible. However, flexibility is required only in specific areas of the blisters and channels, even in embodiments using pneumatic pressure. Furthermore, flexibility is required only on one side of the blisters and channels, insofar as the blisters and channels are easily deformable. Other areas of the pouch 510 may be made of a rigid material or may be reinforced with a rigid material. Thus, when terms such as “flexible pouch” or “flexible sample container” are used, it is understood that flexibility is required only in certain parts of the pouch or sample container.

[0160] Exemplary, a plastic film may be used for the pouch 510. A sheet of metal, exemplary aluminum, or other suitable material may be rolled or otherwise cut to produce a mold having a raised surface pattern. When fitted to an air press (exemplary, A-5302-PDS, Janesville Tool Inc., Milton WI) and exemplary regulated to an effective temperature of 195°C, the air press operates like a printing press, dissolving the sealing surface of the plastic film only where the mold contacts the film. Similarly, the plastic films used for the pouch 510 may be cut and welded together using laser cutting and welding equipment. Various components such as PCR primers, antigen-binding substrates, magnetic beads, and zirconium silicate beads (exemplary, spotted on the film and dried) may be sealed inside various blisters when the pouch 510 is formed. Reagents for sample processing can be spotted on the film together or separately before sealing. In one embodiment, nucleotide triphosphates (NTPs) are spotted separately on a film along with the polymerase and primers, and the polymerase activity is essentially removed until the reactants can be hydrated by the aqueous sample. If the aqueous sample is heated before hydration, this provides conditions for true hot-start PCR, reducing or eliminating the need for expensive chemical hot-start components. In another embodiment, the components may be supplied in powder or pill form and placed in a blister pack before final sealing.

[0161] Pouch 510 may be used in a manner similar to that described in U.S. Patent No. 8,895,295. In one exemplary embodiment, a 300 μl mixture containing the sample under test (100 μl) and lysis buffer (200 μl) may be injected into an injection port (not shown) in equipment 590 near entry channel 515a, and the sample mixture may be directed into entry channel 515a. Alternatively, water may be injected into a second injection port (not shown) of entry channel 515l adjacent to equipment 590, and the water is distributed through channels (not shown) provided within equipment 590, thereby hydrating up to 11 different reagents, each of which is provided in a dry form in entry channels 515b-515l. Exemplary methods and apparatus for injecting samples and hydrates (e.g., water or buffer) are disclosed in U.S. Patent Application Publication No. 2014-0283945 (which is incorporated herein by reference in its entirety), but it should be understood that these methods and apparatus are merely illustrative, and other methods for introducing samples and hydrates into pouch 510 are within the scope of this disclosure. These reagents may, exemplarily, include lyophilized PCR reagents, DNA extraction reagents, washing solutions, immunoassay reagents, or other chemical entities. Exemplarily, the reagents are for nucleic acid extraction, first-stage multiplex PCR, dilution of multiplex reactions, and preparation of second-stage PCR reagents, as well as control reactions. In the embodiment shown in Figure 1, all that needs to be injected is the sample solution in one injection port and water in the other injection port. After injection, the two injection ports may be sealed. For further information regarding various stereochemistry of pouch 510 and fitting 590, see U.S. Patent No. 8,895,295, which is already incorporated by reference.

[0162] After injection, the sample may be transferred from injection channel 515a through channel 514 to dissolution blister 522. Dissolution blister 522 is provided with beads or particles 534, such as ceramic beads or other abrasive elements, and is designed to be vortexed through embedding using a rotating blade or paddle provided within the FilmArray® apparatus. Bead milling by shaking, vortexing, sonication, and similar treatment of the sample in the presence of dissolution particles such as zirconium silicate (ZS) beads 534 is an effective method for forming a lysate. When used herein, terms such as “dissolve,” “dissolve,” and “lysate” are not limited to the destruction of cells, but such terms are understood to include the destruction of non-cellular particles such as viruses. In another embodiment, for example, a paddle beater using a reciprocating or alternating paddle, as described in U.S. Patent Application Publication No. 2019-0344269 (which is incorporated herein by reference in its entirety), may be used for dissolution in this embodiment and for dissolution in other embodiments described herein.

[0163] Figure 4 shows a bead-beating motor 819, including a blade 821 that can be mounted on the first side surface 811 of the support member 802 of the apparatus 800 shown in Figure 2. The blade may extend through a slot 804 and contact the pouch 510. However, it is understood that the motor 819 may be mounted on other structures of the apparatus 800. In one exemplary embodiment, the motor 819 is a Mabuchi Motor RC-280SA-2865 DC motor (Chiba, Japan) mounted on the support member 802. In one exemplary embodiment, the motor rotates at 5,000 to 25,000 rpm, more exemplary, 10,000 to 20,000 rpm, and even more exemplary, about 15,000 to 18,000 rpm. For the Mabuchi Motor motor, 7.2V has been found to provide sufficient rpm for melting. However, it is understood that the actual speed may be somewhat slower when the blade 821 is affecting the pouch 510. Depending on the motor and paddle used, other potentials and speeds may be used for dissolution. Optionally, a controlled small amount of air may be supplied to the dissolution blister 522 adjacent to the bladder 822. In some embodiments, it has been found that partially filling the adjacent bladder with one or more small amounts of air assists in positioning and supporting the dissolution blister during the dissolution process. Alternatively, the pouch 510 can be confined during dissolution using another structure, exemplary, a robust or compliant gasket or other retaining structure around the dissolution blister 522. It is also understood that the motor 819 is merely illustrative, and other devices may be used for milling, shaking, or vortexing the sample. In some embodiments, chemicals or heat may be used in addition to, or instead of, mechanical dissolution.

[0164] Once the sample material is sufficiently dissolved, the sample is transferred to the nucleic acid extraction zone, exemplary, through channel 538, blister 544, and channel 543 to blister 546, where the sample is mixed with a nucleic acid binding material such as silica-coated magnetic beads 533. Alternatively, the magnetic beads 533 may be exemplary rehydrated with a liquid provided from one of the entry channels 515c-515e, then transferred through channel 543 to blister 544, and then through channel 538 to blister 522. The mixture may be incubated for an appropriate time, exemplary, about 10 seconds to about 10 minutes. A retractable magnet located inside blister 546 adjacent to the apparatus captures the magnetic beads 533 from the solution, forming a pellet against the inner surface of blister 546. If incubation is performed in blister 522, multiple portions of the solution may need to be transferred to blister 546 for capture. Next, the liquid is transferred from blister 546 to the outside and returned to blister 522 through blister 544, where it is used as a waste container. One or more wash buffers from one or more injection channels 515c-515e are supplied to blister 546 through blister 544 and channel 543. Optionally, a magnet is placed inside, and the magnetic beads 533 are washed by moving the beads back and forth through channel 543 from blisters 544 and 546. Once the magnetic beads 533 are washed, they are recaptured in blister 546 by magnet activation, and the wash solution is then transferred to blister 522. This process may be repeated as needed to wash the lysis buffer and sample residue from the nucleic acid-bound magnetic beads 533.

[0165] After washing, the elution buffer stored in the injection channel 515f is transferred to the blister 548, and the magnet is stored inside. The solution is circulated between blisters 546 and 548 via channel 552, which breaks up the pellet of magnetic beads 533 in blister 546, dissociating the captured nucleic acids from the beads and into a solution. When the magnet is reactivated, the magnetic beads 533 are captured in blister 546, and the eluted nucleic acid solution is transferred to blister 548.

[0166] The first-stage PCR master mix from injection channel 515g is mixed with the nucleic acid sample in blister 548. Optionally, the mixture is mixed by forcing it between 548 and 564 via channel 553. After several mixing cycles, the solution is contained within blister 564, where a pellet of first-stage PCR primers is provided, with at least one primer set provided for each target, and first-stage multiplex PCR is performed. If an RNA target is present, a reverse transcription (RT) step may be performed before or concurrently with the first-stage multiplex PCR. Temperature cycling of the first-stage multiplex PCR in a FilmArray® instrument is typically performed between 15 and 20 cycles, although other levels of amplification may be desirable depending on the requirements of the specific application. The first-stage PCR master mix may be any of the various master mixes known in the art. In one example, the first-stage PCR master mix may be any of the chemicals disclosed in U.S. Patent No. 9,932,634, which are incorporated herein by reference, when used in a PCR protocol taking 20 seconds or less per cycle.

[0167] After the first-stage PCR has progressed for the desired number of cycles, the sample can be diluted, exemplary, by forcing the majority of the sample back into blister 548, leaving only a small amount in blister 564, and adding the second-stage PCR master mix through injection channel 515i. Alternatively, the dilution buffer from 515i can be transferred to blister 566, and then mixed with the amplified sample in blister 564 by moving the liquid back and forth between blisters 564 and 566. If necessary, the dilution may be repeated several times using the dilution buffers from injection channels 515j and 515k, or, exemplary, injection channel 515k may be prepared for sequencing or other post-PCR analysis, and then the second-stage PCR master mix may be added to some or all of the diluted amplified sample through injection channel 515h. It is understood that the level of dilution may be adjusted by changing the number of dilution steps, or by changing the percentage of sample discarded before mixing with the dilution buffer or the components for amplification, exemplary, particularly polymerase, dNTPs, and a suitable buffer (however, in non-PCR amplification methods, other components may be suitable). If necessary, this mixture of sample and second-stage PCR master mix may be preheated in blister 564 before being transferred to second-stage well 582 for second-stage amplification. Such preheating can eliminate the need for a hot-start component (antibody, chemical, or otherwise) in the second-stage PCR mixture.

[0168] In one embodiment, the exemplary second-stage PCR master mix is ​​incomplete and lacks primer pairs, and each of the 102 second-stage wells 582 is pre-filled with a specific PCR primer pair. In another embodiment, the master mix contains other components (e.g., polymerase, Mg 2+The second-stage PCR master mix may lack components such as , and the missing components may be pre-filled in the array. If necessary, the second-stage PCR master mix may lack other reaction components, and similarly, these components may be pre-filled in the second-stage wells 582. Each primer pair may be similar to or identical to the first-stage PCR primer pair, or may be nested within the first-stage primer pair. The PCR reaction mixture is completed by transferring the sample from blister 564 to the second-stage wells 582. Once the high-density array 581 is filled, the individual second-stage reactants are sealed in their respective second-stage blisters by several means, as known in the art. Exemplary methods for filling and sealing the high-density array 581 without cross-contamination are discussed in U.S. Patent No. 8,895,295, which has already been incorporated by reference. Exemplary, various reactions within the wells 582 of the high-density array 581 are thermally circulated, either simultaneously or individually, using one or more Peltier devices, although other means for thermal circulation are known in the art.

[0169] In certain embodiments, the second-stage PCR master mix includes the dsDNA-binding dye LCGreen® Plus (BioFire Diagnostics, LLC) to generate a signal signifying amplification. However, it is understood that this dye is merely illustrative, and other signals may be used, including other dsDNA-binding dyes and probes labeled with fluorescence, radioactivity, chemiluminescence, enzymes, etc., as is known in the art. Alternatively, well 582 of array 581 may be provided under conditions without a signal, and the results may be reported through subsequent processing.

[0170] In one embodiment, when pneumatic pressure is used to move material within the pouch 510, a “bladder” may be utilized. A bladder assembly 810, partly shown in Figures 2-3, exemplifies a bladder plate 824 that houses a plurality of expandable bladders 822, 844, 846, 848, 864, and 866, each of which may be individually expandable, via a compressed gas source. Since the bladder assembly 810 receives compressed gas and may be used multiple times, the bladder assembly 810 may be made from a material that is rigider or thicker than the pouch. Alternatively, the bladders 822, 844, 846, 848, 864, and 866 may be formed from a series of plates that are firmly fixed together with gaskets, seals, valves, and pistons. Other arrangements are within the scope of the present invention. Alternatively, an array or mechanical actuator and seal can be used to seal the channel and guide the movement of liquid between the blisters. A system of mechanical seals and actuators suitable for the apparatus described herein is described in detail in U.S. Patent Application Publication No. 2019-0344269, which is incorporated in its entirety by reference.

[0171] The success of a secondary PCR reaction depends on the template generated by the multiplex first-step reaction. Typically, PCR is performed using high-purity DNA. Methods such as phenol extraction or commercially available DNA extraction kits provide high-purity DNA. Samples processed through pouch 510 may require adjustments to compensate for lower purity preparations. PCR can be inhibited by components of the biological sample, which are a potential barrier. Exceptionally, lower nucleic acid purity can be compensated for using hot-start PCR, higher concentrations of Taq polymerase enzyme, adjustment of MgCl2 concentration, adjustment of primer concentration, addition of modified enzymes resistant to inhibitors, and optional addition of adjuvants (such as DMSO, TMSO, or glycerol). While purity challenges are likely to be a greater concern in first-step amplification, it is understood that similar adjustments can be provided in second-step amplification.

[0172] When the pouch 510 is placed within the device 800, the bladder assembly 810 is pressed against one side of the pouch 510, so that when a particular bladder is inflated, the liquid from the corresponding blister within the pouch 510 is pressurized. In addition to the bladders corresponding to many of the blisters in the pouch 510, the bladder assembly 810 may have additional pneumatic actuators, such as bladders or pneumatically driven pistons, corresponding to various channels in the pouch 510. Figures 2-3 show exemplary pistons or hard seals 838, 843, 852, 853, and 865 corresponding to channels 538, 543, 553, and 565 of the pouch 510, as well as seals 871, 872, 873, and 874 that minimize backflow into the equipment 590. When activated, the hard seals 838, 843, 852, 853, and 865 form pinch valves for pinching off and closing the corresponding channels. To contain the liquid within a specific blister of the pouch 510, the hard seal is activated more than the channels leading to and from the blister, so that the actuator functions as a pinch valve to pinch and close the channels. Exemplaryly, to mix two volumes of liquid in different blisters, a pinch valve actuator sealing the connecting channels is activated, and a pneumatic bladder above the blister is alternately compressed, pressurizing the liquid back and forth through the channels connecting the blisters and mixing the liquids within. The pinch valve actuator may be of various shapes and sizes and may be designed to pinch off two or more channels simultaneously. While pneumatic actuators are considered herein, it is understood that other methods of pressurizing the pouch are contemplated, including various electromechanical actuators such as linear stepping motors, motor-driven cams, rigid paddles, rollers, rocker arms driven by air pressure, hydraulic or electromagnetic forces, and in some cases, cock springs. Furthermore, in addition to applying normal pressure to the channel axes, there are various methods for reversibly or irreversibly closing the channels.These include twisting the bag through the channel, heat sealing, rotating the actuator, and sealing the channel with various physical valves such as butterfly valves and ball valves. In addition, a seal can be effectively formed by placing a small Peltier device or other temperature control device adjacent to the channel and setting it to a temperature sufficient to freeze the liquid. It is also intended that while the design of Figure 1 is adapted to an automated apparatus characterized in that the actuator elements are positioned on the blister and the channel respectively, the actuator can maintain steady operation and the pouch 510 can be modified so that a small number of actuators can be used in several processing stations, including processing stations for sample disruption, nucleic acid capture, first and second-stage PCR, and other applications of the pouch 510 such as immunoassays and immunoPCR. Rollers acting on the channel and blister can be proven to be particularly useful in designs in which the pouch 510 is translated between stations. Thus, while pneumatic actuators are used in embodiments of this disclosure, it is understood that other actuators and other methods of providing pressure may be used depending on the design of the pouch and apparatus when the term “pneumatic actuator” is used herein.

[0173] Returning to Figure 2, each pneumatic actuator is connected to a compressed gas source 895 via a valve 899. While only some hoses 878 are shown in Figure 2, it is understood that each pneumatic fitting is connected to the compressed gas source 895 via a hose 878. The compressed gas source 895 may be a compressor, or alternatively, a compressed gas cylinder such as a carbon dioxide cylinder. Compressed gas cylinders are particularly useful when portability is desired. Other compressed gas sources are within the scope of the present invention. For example, similar pneumatic controls may be provided for controlling liquid movement in the pouch described herein, or other actuators, servos, etc.

[0174] In addition, several other components of the apparatus are connected to a compressed gas source 895. The magnet 850 mounted on the second side 814 of the support member 802 is deployed and retracted, exemplary, using gas from the compressed gas source 895 via a hose 878, although other methods of moving the magnet 850 are known in the art. The magnet 850 is located in a recess 851 within the support member 802. It is understood that the recess 851 may be a passage through the support member 802 so that the magnet 850 can contact the blister 546 of the pouch 510. However, depending on the material of the support member 802, it is understood that the recess 851 does not need to extend through the support member 802, provided that when the magnet 850 is deployed, it is close enough to provide a sufficient magnetic field in the blister 546, and when the magnet 850 is fully retracted, it does not need to significantly affect any magnetic beads 533 present in the blister 546. While the storage of magnet 850 is mentioned, it is understood that electromagnets may be used, and the operation and deactivation of the electromagnets may be controlled by controlling the flow of electricity through them. Therefore, while the removal or storage of magnets is considered herein, it is understood that these terms are broad enough to encompass other methods of removing magnetic fields. It is understood that pneumatic connections may be pneumatic hoses or pneumatic air manifolds, and thus the number of hoses or valves required may be reduced. It is understood that similar magnets and methods for operating them may be used in other embodiments.

[0175] Furthermore, the various pneumatic pistons 868 of the pneumatic piston array 869 are connected to a compressed gas source 895 via hoses 878. It is shown that only two hoses 878 connect the pneumatic pistons 868 to the compressed gas source 895, while it is understood that each of the pneumatic pistons 868 is connected to the compressed gas source 895. Twelve pneumatic pistons 868 are shown.

[0176] A pair of temperature control elements is mounted on the second side surface 814 of the support member 802. As used herein, the term “temperature control element” refers to a device that adds heat to or removes heat from a sample. Examples of temperature control elements include, but are not limited to, heaters, coolers, Peltier devices, resistance heaters, induction heaters, electromagnetic heaters, thin-film heaters, printed element heaters, positive temperature coefficient heaters, and combinations thereof. A temperature control element may include multiple heaters, coolers, Peltiers, etc. In one embodiment, a given temperature control element may include two or more types of heaters or coolers. For example, an example of a temperature control element may be a Peltier device in which separate resistance heaters are applied to the upper and / or lower surfaces of the Peltier. While the term “heater” is used throughout this specification, it is understood that other temperature control elements may be used to regulate the temperature of a sample.

[0177] As discussed above, the first-stage heater 886 may be positioned to heat and cool the contents of the blister 564 in the first-stage PCR. As is clear from Figure 2, the second-stage heater 888 may be positioned to heat and cool the contents of the second-stage blister of the array 581 of the pouch 510 in the second-stage PCR. However, it is understood that these heaters can also be used for other heating purposes, and other heaters may be included as they are suitable for specific applications.

[0178] As discussed above, while Peltier devices that circulate heat between two or more temperatures are effective for PCR, in some embodiments it may be desirable to maintain the heater at a constant temperature. For example, this can be used to reduce run time by eliminating the time required to change the heater temperature that exceeds the time required to change the sample temperature. Furthermore, since it is necessary to circulate heat only smaller samples and sample containers, rather than much larger (greater thermal mass) Peltier devices, such preparation can improve the electrical efficiency of the system. For example, the apparatus may include multiple heaters (i.e., two or more) positioned relative to a pouch for achieving heat circulation at temperatures set for, for example, annealing, extension, or denaturation. Two heaters may be sufficient for many applications. In various embodiments, the heaters, pouches, or liquids may be movable relative to the heaters for achieving heat circulation. For example, the heaters may be arranged linearly, in annular arrangement, or similarly. Preferred heater types are discussed above with reference to first-step PCR.

[0179] When fluorescence detection is desired, an optical array 890 may be provided. As shown in Figure 2, the optical array 890 includes a light source 898, exemplary, a filtered LED light source, filtered white light, or laser illumination, and a camera 896. The camera 896 exemplary has multiple photodetectors, each corresponding to a second-stage well 582 in the pouch 510. Alternatively, the camera 896 can acquire an image encompassing all of the second-stage wells 582, and the image may be divided into separate regions corresponding to each of the second-stage wells 582. Depending on the design, the optical array 890 may be fixed, or it may be mounted on one or more motors and placed on a mover that moves to obtain signals from each individual second-stage well 582. It is understood that other arrangements are possible. In some embodiments of the second-stage heater, the heater is provided on the opposite side of the pouch 510 from the side shown in Figure 2. Such arrangements are merely exemplary and may be determined by spatial constraints within the apparatus. Provided that the second-stage reaction zone 580 is provided with an optically transparent material, the photodetector and heater may be located on either side of the array 581.

[0180] As shown in the figure, computer 894 controls valve 899 of compressed gas source 895 and thus controls all of the pneumatic systems of apparatus 800. Furthermore, in other embodiments, many of the pneumatic systems in the apparatus may be replaced with mechanical actuators, pressure applying means, etc. Computer 894 also controls heaters 886 and 888, and optical array 890. Each of these components is electrically connected, exemplary, via cable 891, but other physical or wireless connections are within the scope of the invention. It is understood that computer 894 may be housed inside apparatus 800 or provided outside apparatus 800. Furthermore, computer 894 may include an integrated circuit board that controls some or all of the components, and may also include an external computer, such as a desktop or laptop PC, to receive and display data from the optical array. An interface, exemplary, a keyboard interface including keys for inputting information and variables such as temperature and cycle time, may be provided. Also exemplary, a display 892 is provided. The display 892 may be, for example, an LED, an LCD, or another such display.

[0181] Other apparatus known in the art teach about PCR in sealed flexible containers. See, for example, U.S. Patent No. 6,645,758, U.S. Patent No. 6,780,617, and U.S. Patent No. 9,586,208, which are incorporated herein by reference. However, cell lysis in a sealed PCR container can improve ease of use and safety, for example, especially when the sample under test may contain biohazards. In the embodiments illustrated herein, waste from cell lysis, as well as waste from all other steps, remains in the sealed pouch. Furthermore, it is understood that the contents of the pouch can be removed in preparation for further testing.

[0182] Returning to Figure 2, the device 800 includes a support member 802 which may form the wall of the casing or may be mounted within the casing. The device 800 may also optionally include a second support member (not shown) which is movable relative to the support member 802 to allow insertion and removal of the pouch 510. Exemplaryly, once the pouch 510 is inserted into the device 800, a lid may cover the pouch 510. In another embodiment, both support members may be fixed, and the pouch 510 may be fixed in place by other mechanical means or pneumatically.

[0183] In the example, heaters 886 and 888 are mounted on a support member 802. However, it is understood that this arrangement is merely illustrative and other arrangements are possible. Exemplary heaters include Peltier and other block heaters, resistance heaters, electromagnetic heaters, and thin-film heaters, as are known in the art, for circulating heat within the blister 864 and the second-stage reaction zone 580. The bladder plate 810, together with bladders 822, 844, 846, 848, 864, 866, hard seals 838, 843, 852, 853, and seals 871, 872, 873, 874, forms a bladder assembly 808, which may exemplary be mounted on a movable support structure that can move toward the pouch 510, where a pneumatic actuator is positioned in contact with the pouch 510. When the pouch 510 is inserted into the device 800 and the possible support member is moved toward the support member 802, the various blisters of the pouch 510 are positioned adjacent to the various bladders of the bladder assembly 810 and the various seals of the assembly 808, so that by the operation of a pneumatic actuator, liquid can be pressed from one or more of the blisters of the pouch 510, or together with one or more channels of the pouch 510, a pinch valve can be formed. The relationship between the blisters and channels of the pouch 510 and the bladder and seals of the assembly 808 is illustrated in detail in Figure 3.

[0184] Isolation, concentration, characterization, and / or identification of microorganisms in a sample. The present invention provides methods, systems, and apparatus for the isolation, concentration, characterization, and / or identification of microorganisms in a sample. In one embodiment, the microorganism is a bacterium. In another embodiment, the microorganism is a fungus (e.g., yeast or mold). In a further embodiment, the microorganism is a parasite. In yet another embodiment, the microorganism may be a combination of microorganisms selected from the group consisting of bacteria, yeast, mold, and parasites. The methods, systems, and apparatus may be particularly useful for the isolation, characterization, and / or identification of microorganisms from composite samples such as blood, urine, or cerebrospinal fluid. In a preferred embodiment, the methods, systems, and apparatus of the present invention may be used, for example, to isolate, characterize, and / or identify microorganisms obtained directly from blood in order to rapidly determine whether a patient is septic or pre-septic.

[0185] As used herein, “obtained directly from blood” or “obtained directly from whole blood” means determining the presence of microorganisms by concentrating and / or isolating microorganisms from whole blood and then identifying them, in relation to determining the presence of microorganisms present in a blood sample. “Whole blood” is blood (e.g., human blood) that, when found in a circulating system, has none of its components separated or removed. Blood with added anticoagulants is also generally referred to as whole blood. Microorganisms may preferably be concentrated and / or isolated from whole blood without using a blood culture step of pre-concentration and / or pre-isolation to increase the number of microorganisms in the sample. Microorganisms may preferably be concentrated and / or isolated from blood after a short culture step (e.g., <5 hours, <4 hours, <3 hours, <2 hours, or <1 hour) to increase the number of microorganisms in the sample. After concentrating and / or isolating the microorganisms, they may preferably be identified by several techniques, including, but not limited to, one or more of the following: molecular testing (i.e., nucleic acid-based testing), phenotypic testing, proteomics testing, optical testing, or culture-based testing. After concentrating and / or isolating the microorganisms, they may preferably be cultured for a short period (<5 hours, <4 hours, <3 hours, <2 hours, or <1 hour (e.g., 3 hours)) to increase the number of concentrated / isolated fractions. However, culture may preferably not be necessary in the methods and systems described herein. For example, the methods described herein may preferably work for all bacteria and fungi of interest, including, but not limited to, preferential organisms that do not typically grow sufficiently or rapidly in blood cultures, aerobic and anaerobic organisms that may require different culture conditions, and organisms that may require different culture medium formulations for growth and detection.

[0186] Characterization and / or identification of microorganisms in concentrated microbial samples (e.g., centrifugal pellets) preferably does not involve precise species identification. Characterization encompasses broad categorization or classification of biological particles, as well as the actual identification of a single species. As used herein, “identification” means determining the family, genus, species, and / or lineage to which a microorganism belongs. For example, this includes identifying microorganisms isolated from biological samples (e.g., blood, urine, or cerebrospinal fluid) down to the family, genus, species, and / or lineage level.

[0187] The methods, systems, and apparatus described herein enable the characterization and / or identification of microorganisms more rapidly than the prior art, resulting in a faster diagnosis (for example, in subjects with or suspected of having sepsis). The steps involved in the methods of the present invention can be carried out within a very short timeframe, from obtaining a sample for characterization / identification of a microorganism to obtaining clinically relevant usable information. In certain embodiments, the methods of the present invention can be carried out in less than about 120 minutes, for example, less than about 110 minutes, about 100 minutes, about 95 minutes, about 90 minutes, about 85 minutes, about 80 minutes, about 75 minutes, about 70 minutes, about 65 minutes, about 60 minutes, about 55 minutes, about 50 minutes, about 45 minutes, about 40 minutes, about 35 minutes, about 30 minutes, about 25 minutes, about 20 minutes, about 15 minutes, about 10 minutes, about 5 minutes, about 4 minutes, about 3 minutes, about 2 minutes, about 1 minute, or within any range of the above time points, or between them, or encompassing them. In preferred embodiments, the method of the present invention can be carried out within less than about 90 minutes (e.g., about 75 minutes). For example, the time elapsed from the collection of a whole blood sample from a patient suspected of having sepsis to the completion of analysis and positive identification of infectious pathogens (if present) may be less than about 90 minutes in many scenarios. When faster molecular analysis systems are available, the sampling response time can be significantly reduced. Similar times may be achievable for other composite sample types where low-titer organisms are concentrated from large volumes of blood or other sample types, while sepsis and whole blood are used in the preceding examples. The remarkable speed of the method of the present invention represents an improvement over previous methods. Using this method, any microorganism as described herein can be characterized and / or identified.

[0188] The exemplary workflows relating to the methods, systems, and apparatus described herein are simple and minimize the handling of samples, lysates, and microorganisms. For example, a sample can be mixed with a differential lysis buffer in a single tube for lysing and isolation of microorganisms. In one embodiment, the microorganisms can be recovered from the single tube in a manner that isolates the microbial pellet from the lysate, thereby reducing the risk of handling potentially infectious material and / or contaminating the sample. In addition, the methods of the present invention can be fully automated, thereby further reducing the risk of handling infectious material and / or contaminating the sample.

[0189] Figure 5 is a schematic diagram of one embodiment of components useful for the method, system, and apparatus described herein. The exemplary method can provide a simplified workflow and shorter time to results by comprising a limited number of components and steps and being completed within approximately 20 to 75 minutes from contact of the sample and differential lysis buffer. The exemplary method in Figure 5 includes the steps of obtaining a sample 5000 (e.g., a whole blood sample, a urine sample, a cerebrospinal fluid sample, or an environmental sample), preparing a lysate, recovering microbial cells from the lysate, and characterizing and / or identifying microorganisms in the sample. In one embodiment, sample 5000, which may be a blood sample, may be provided in a standard blood collection tube (e.g., a vacutainer) with or without an anticoagulant. In one embodiment, sample 5000 and the differential lysis buffer may be combined for the lysis of substantially all (e.g., >90%) of the non-microbial cells in sample 5000. In the exemplary embodiment, the lysate may be prepared in a specially designed centrifugal concentrator 5010. In one embodiment, the differential lysis buffer may be provided in a centrifugal concentrator 5010, and the lysis of non-microbial cells in the sample may be initiated simply by adding the sample 5000 to the centrifugal concentrator 5010, thereby combining the sample and the differential lysis buffer in the centrifugal concentrator 5010. In another embodiment, the sample 5000 is mixed with the differential lysis buffer and then placed in the centrifugal concentrator 5010, for example, by pipetting the mixture into the centrifugal concentrator. After combination, the differential lysis buffer and the sample are combined over a period of time (e.g., 1 to 5 minutes) to obtain a lysate. In one embodiment, microorganisms may be recovered from the lysate by centrifugation, filtration, etc. In the case of centrifugation, the microbial cells may be pelleted by centrifugating the centrifugal concentrator 5010 at a range of about 1,000 × g to about 20,000 × g for a period of time ranging from about 4 to 10 minutes.In exemplary embodiments, the recovered microbial cells may be added from the centrifugal concentrator 5010 to an analyzer 5020 designed to characterize and / or identify the microorganisms in the sample at a clinically relevant level. Characterization and / or identification of microorganisms in the illustrated analyzer 5020 can be performed rapidly (e.g., in about 15–60 minutes). However, the illustrated analyzer is merely illustrative. For example, in some embodiments, the microorganisms may be characterized and / or identified by sequencing (e.g., next-generation sequencing).

[0190] sample Samples that may be tested by the methods and systems described herein may include both clinical and non-clinical samples in which the presence and / or growth of microorganisms is suspected or possible, as well as samples of materials routinely or incidentally tested for the presence of microorganisms. The amount of sample used may vary considerably due to the versatility and / or sensitivity of the methods. One advantage of the methods and systems described herein is that complex sample types, such as blood, body fluids, and / or other opaque substances, can be tested directly using systems that require little or no extensive pretreatment.

[0191] "Sample" means animals; tissues or organs from animals, including but not limited to human animals; cells (either cells within a subject directly taken from a subject (e.g., human or non-human animal), or cells maintained in a culture or from a cultured cell line); cell lysates (or lysate fractions) or cell extracts; solutions containing one or more molecules (e.g., polypeptides or nucleic acids) derived from cells, cellular material, or viral material; or solutions containing nucleic acids that do not exist in nature, which are assayed as described herein. Samples that may be tested by the methods and systems described herein may include both clinical and non-clinical samples in which the presence and / or growth of microorganisms is suspected or possible, as well as samples of materials that are routinely or optionally tested for the presence of microorganisms. Clinical samples that may be tested include, but are not limited to, any type of sample typically tested in a clinical or research laboratory, including blood, serum, plasma, blood fractions, synovial fluid, urine, semen, saliva, feces, cerebrospinal fluid, gastric contents, vaginal secretions, tissue homogenates, bone marrow aspirates, bone homogenates, sputum, aspirates, swabs and swab rinsates, other body fluids, blood products (e.g., platelets, serum, plasma, leukocyte fractions, etc.), donor organs or tissue samples, etc. Some specimen samples that may be cultured and subsequently tested may include blood, serum, plasma, platelets, red blood cells, white blood cells, blood fractions, synovial fluid, urine, nasal samples, semen, saliva, feces, cerebrospinal fluid, gastric contents, vaginal secretions, tissue homogenates, bone marrow aspirates, bone homogenates, sputum, aspirates, swabs and swab rinsates, other body fluids, etc. For example, in some embodiments, it may be optional to subject a sample, such as blood from a subject, to a limited culture step (e.g., within the range of 1 minute to 4 hours) before testing to increase the level of detectable microorganisms in the sample. Alternatively, the sample, such as blood, may be cultured before the selective lysis and recovery of microorganisms, the microorganisms may be cultured during the lysis and recovery, or the microorganisms may be cultured from a pellet recovered (e.g., by centrifugation) from a sample that has been selectively lysed (e.g., by growing the organisms in a liquid medium or on a solid plate).The culture of microorganisms (particularly bacteria and fungi) may be more rapid when the cell concentration is higher. Culture from recovered or concentrated microorganisms (e.g., from pellets obtained from a centrifugation step) may be more rapid than blood culture. Furthermore, culture from recovered or concentrated microorganisms may be able to remove antibiotics and defensins that may be present in the blood and promote more rapid growth.

[0192] The present invention finds applications in research, as well as in veterinary and medical applications. Preferred subjects from which clinical samples can be obtained are generally mammals, but can be any animal. The term “mammal” as used herein includes, but is not limited to, humans, non-human primates, cattle, sheep, goats, pigs, horses, cats, dogs, rabbits, rodents (e.g., rats or mice), etc. Human subjects include neonates, infants, young people, adults, and elderly subjects. Subjects from which samples can be obtained include, but is not limited to, mammals, birds, reptiles, amphibians, and fish.

[0193] Non-clinical samples that can be tested also include, but are not limited to, substances such as food, beverages, pharmaceuticals, cosmetics, water (e.g., drinking water, non-drinking water, and wastewater), seawater ballast, air, soil, sewage, plant materials (e.g., seeds, leaves, stems, roots, flowers, fruits), and biological weapons samples. Samples may also include, but are not limited to, environmental samples such as soil, air monitoring system samples (e.g., materials captured in air filter media), surface swabs, and vectors (e.g., mosquitoes, ticks, fleas, etc.). Furthermore, the method is particularly well suited for real-time testing to monitor contamination levels, process control, quality control, etc., in industrial settings. In a preferred embodiment of the present invention, the sample is obtained from a subject (e.g., a patient) that has or is suspected of having a microbial infection. In one embodiment, the subject has or is suspected of having sepsis, e.g., bacteremia or fungemia. Preferably, the sample may be a blood sample that is collected from the subject and then tested directly. That is, the sample is a whole blood sample that has not been added to a blood medium and has not been processed, cultured, or diluted before testing. In another embodiment, the sample may be obtained from a blood culture grown from a sample of a patient's blood, for example, a BacT / ALERT® blood culture. The blood culture sample may be obtained from a positive blood culture, for example, a blood culture showing the presence of microorganisms. In certain embodiments, the sample may be taken from a positive blood culture within a short time after it has turned positive, for example, within about 6 hours, for example, within about 5 hours, about 4 hours, about 3 hours, or about 2 hours, or within about 60 minutes, for example, about 55 minutes, about 50 minutes, about 45 minutes, about 40 minutes, about 35 minutes, about 30 minutes, about 25 minutes, about 20 minutes, about 15 minutes, about 10 minutes, about 5 minutes, about 4 minutes, about 3 minutes, about 2 minutes, or about 1 minute. In one embodiment, the sample may be taken from a culture in which the microorganisms are in logarithmic growth. In another embodiment, the sample may be taken from a culture in which the microorganisms are in a stationary phase. In some embodiments, the whole blood sample may be provided as part of the method within one hour of the whole blood sample being taken from the patient.In yet another embodiment, the sample may be blood that has been cultured for a shorter time than typically required to obtain a positive blood culture result (e.g., between 1 minute and 4 hours), or may contain such blood. In some embodiments, the sample is provided at room temperature for use in the method, while in other embodiments, the sample is cooled after being obtained from the patient and before being provided for use in the method. For example, the sample may be refrigerated after being obtained from the patient until the method can be carried out.

[0194] The present invention provides high sensitivity for the detection and identification of microorganisms. Exemplarily, it enables the detection and identification of microorganisms without the need to first go through the steps of liquid culture, followed by the steps of isolating the microorganisms and growing them on a solid or semi-solid medium, and sampling the growing colonies. Thus, in one embodiment of the present invention, the sample is not obtained from a liquid culture or from a microbial (e.g., bacterial, yeast, or mold) colony grown on a solid or semi-solid surface. To facilitate the identification of promising BSIs, in some embodiments, the method includes the step of lysing the sample without culturing it after the sample is obtained from the patient. In some embodiments, the method described herein can be used even in patients who have been treated with antibiotics before the collection of blood samples. Patients hospitalized with symptoms consistent with sepsis are often immediately treated with antibiotics, after which sepsis may be definitively included or ruled out. While such treatment protocols are consistent with standard care, antibiotics may interfere with blood cultures used in classical sepsis diagnosis. Surprisingly, the methods described herein can also be used to diagnose sepsis in patients undergoing antibiotic treatment when intact microbial cells are still present in the blood.

[0195] The sample volume must be large enough to produce an analyzable pellet of microorganisms after the separation step of the method of the present invention has been carried out. The appropriate volume will depend on the source of the sample, the expected level of microorganisms in the sample, and the analytical method used for characterizing and identifying the microorganisms. For example, whole blood from a patient with BSI typically has a microbial load of about 1 to 100 cfu / ml (e.g., <1 to 10 cfu / ml). Generally, the sample size may be about 50 ml, about 40 ml, about 30 ml, about 20 ml, about 15 ml, about 10 ml, about 5 ml, about 4 ml, about 3 ml, or about 2 ml (e.g., about 10 ml). In certain embodiments, the sample size may be about 1 ml, e.g., about 0.75 ml, about 0.5 ml, or about 0.25 ml. In certain embodiments where separation is performed on a microscale, the sample size may be less than about 200 μl, e.g., about 150 μl, 100 μl, 50 μl, 25 μl, 20 μl, 15 μl, 10 μl, or less than about 5 μl. In some embodiments (e.g., when the sample is expected to contain a small number of microorganisms), the sample size may be about 100 ml or more, e.g., about 250 ml, 500 ml, 750 ml, or 1000 ml or more. Positive blood cultures will contain higher levels of microorganisms per ml, and therefore, a smaller volume of blood medium may be used compared to whole blood.

[0196] While much of the discussion herein relates specifically to whole blood, the methods, systems, and apparatus described herein may be used for other sample types as described in the definition of “sample” above. Two specific examples of further sample types are urine and cerebrospinal fluid (CSF). Urine and CSF often contain leukocytes (WBCs) during infection, which can harbor intracellular pathogens. These WBCs may be produced in the fight against infection, or, in the case of CSF, numerous pathogens may approach the brain / spinal column by hiding inside WBCs or other blood cells, and then cross the blood-brain barrier. Pathogenic blood cells (non-pathogenic cells) can be selectively lysed with the differential lysis buffer disclosed herein to release intracellular pathogens and concentrate them into a pellet that may substantially contain no contaminating eukaryotic host DNA (e.g., contaminating host DNA may be reduced by >95%). In addition, bladder epithelial cells can slough off during infection, shedding pathogen-carrying cells and preventing the spread of infection as a precautionary measure. Similar to leukocytes, these epithelial cells can be lysed with the differential lysis buffer disclosed herein, and intact pathogen cells can be concentrated into a pellet without contamination from bladder cells.

[0197] As will be discussed in more detail elsewhere in this specification, the recovered pathogen cells may be lysed, and nucleic acids from the pathogen cells may be recovered for analysis. Since the pathogen cells are isolated without significant host cell DNA contamination, the recovered pathogen nucleic acids are suitable for downstream molecular assays to characterize and / or identify the pathogen. In some embodiments, the pathogen cells may be used for downstream characterization and / or identification of the pathogen by molecular methods (e.g., PCR amplification of pathogen DNA or RNA and identification of the amplification product), gene sequencing (e.g., by next-generation sequencing technology), or mass spectrometry. The apparatus and methods described herein can remove many or all of the host cell components so that the pathogen signal can be identified by any of these methods.

[0198] Dissolution step The next step in the exemplary method of the present invention, after providing or obtaining a sample, is to lyse any non-microbial cells that may be present in the sample, such as blood cells and / or tissue cells or other eukaryotic host cells. In some embodiments, the method includes selectively lysing cells to allow for the separation of microorganisms from other components of the sample. Separation of microorganisms from other components reduces interference between subsequent investigation steps. If the presence of non-microbial cells in the sample is not expected or is not expected to interfere with the investigation steps, the lysation step may be omitted. In one embodiment, the cells to be lysed are non-microbial cells present in the sample, and any microbial cells that may be present in the sample are little to no lysed. However, in some embodiments, selective lysing of a particular class of microorganisms may be desirable and can therefore be carried out according to the method described herein and as is well known in the art. For example, an undesirable class of microorganisms may be selectively lysed, for example, yeast may be lysed while bacteria are not, or vice versa. In another embodiment, a desired microorganism is lysed to isolate a particular intracellular component of the microorganism, such as the cell membrane or organelles. In one embodiment, all non-microbial cells are lysed. In other embodiments, a portion of the non-microbial cells, for example, enough cells to prevent interference with the investigation step, are lysed. Cell lysis may be carried out by any method known in the art, including, but not limited to, the addition of a differential lysis buffer, sonication, and / or osmotic shock, so as to be effective in selectively lysing cells in or out of the presence of lysing microorganisms.

[0199] A differential lysis buffer can selectively lyse one class of cells, e.g., non-microbial cells and / or some microbial cells (e.g., by solubilizing the eukaryotic cell membrane), but cannot lyse another class of cells, e.g., microorganisms or types of microorganisms. In one embodiment, the differential lysis buffer may comprise an aqueous medium, one or more detergents, a buffer, one or more salts, and may further comprise additional agents. In one embodiment, the differential lysis buffer may further comprise one or more enzymes (e.g., proteases). In one embodiment, the detergent may be an unmodified soluble detergent such as Triton® X-100, Triton® X-100-R, Triton® X-114, NP-40, Igepal® CA630, Arlasolve® 200, Brij O10 (also known as Oleth-10, Brij 96V, Brij 97, Volpo 10NF, Volpo N10) (the name Brij is a registered trademark of Croda International Plc), CHAPS, octyl β-D-glucopyranoside, saponin, and nonaethylene glycol monododecyl ether (also known as C12E9, polidocenol, Brij 35). In one embodiment, the detergent is a nonionic surfactant. Examples of suitable nonionic surfactants include, but are not limited to, Triton X-114, NP-40, Arlasolve200, BrijO10, octyl β-D-glucopyranoside, saponins, nonaethylene glycol monododecyl ethers, and combinations thereof. In preferred embodiments, the nonionic surfactant is a polyoxyethien ether (POE ether). POE ethers are a class of nonionic surfactants that may be used for cell membrane disruption. A POE ether consists of an alkyl chain, a hydrophilic moiety consisting of an "n" oxyethylene unit, and a terminal -OH group. Examples of suitable POE ethers include, but are not limited to, Arlasolve200 (poly(oxy-1,2-ethanediyl)), BrijO10 (and other Brij detergents), and nonaethylene glycol monododecyl ether (Brij35).Optionally, modified solubilating detergents such as sodium dodecyl sulfate, N-lauryl sarcosine, sodium deoxycholate, bile salts, hexadecyltrimethylammonium bromide, SB3-10, SB3-12, amidosulfobetaine-14, and C7BzO may be included. Optionally, solubilizing agents such as Brij98, Brij58, Brij35, Tween® 80, Tween® 20, Pluronic® L64, Pluronic® P84, non-detergent sulfobetaine (NDSB201), Amphipol (PMAL-C8), and methyl-β-cyclodextrin may also be included. Typically, non-modified detergents and solubilizers are used at concentrations higher than their critical micelle concentration (CMC), while modified detergents may be added at concentrations lower than their CMC. For example, the non-denatured soluble detergent can be used at concentrations of approximately 0.010% to 10%, for example, approximately 0.015% to 1.0%, for example, approximately 0.05% to 0.5%, for example, approximately 0.10% to 0.30% (final concentration after sample dilution). The enzymes usable in the differential lysis buffer are not limited to but include enzymes that digest nucleic acids and other membrane fouling materials (e.g., proteinase XXIII, DNase, neuraminidase, polysaccharide, Glucanex®, and Pectinex®). In specific embodiments, the differential lysis buffer does not contain DNase and is not used in combination with DNase. Other usable additives are not limited to but include reducing agents such as 2-mercaptoethanol (2-Me) or dithiothreitol (DTT), and stabilizers such as magnesium, pyruvate, and water-retaining agents.

[0200] Differential lysis buffers can be buffered at any pH suitable for lysing the desired cells, and will depend on several factors, including the type of sample, the cells to be lysed, and the detergent used. In some embodiments, the pH may be in the range of about 2 to about 13, e.g., about 6 to about 10, e.g., about 7 to about 9, e.g., about 7 to about 8. A suitable pH buffer may include any buffer that has the ability to maintain the pH within the desired range. In some embodiments, the buffer may be used outside of its pH buffering range. Suitable examples of buffering materials, but not limited to, may include about 0.005 M to about 1.0 M of CAPS, CAPSO, CHES, CABS, and combinations thereof. In a specific example, the differential lysis buffer has the composition shown in Table 1 below.

[0201] [Table 1]

[0202] In the specific example illustrated in Table 1, the sample is approximately 10 ml of whole blood combined with approximately 30 ml of differential lysis buffer.

[0203] CAPS is a buffering agent; CAPS has a pKa of approximately 10.4 at 25°C and a typical buffering range of approximately 9.7–11.1. Before combining the differential lysis buffer with the blood sample, the CAPS buffer is within its buffering range. However, after combining the differential lysis buffer with the blood sample, the CAPS buffer in this example is well outside its buffering range (e.g., at a pH of approximately 7–8). Surprisingly, it has been found that using CAPS (and chemically similar buffers—e.g., CAPSO, CHES, and CABS) in the differential lysis buffer, which is outside its buffering range, can have a synergistic effect that improves solubility. Unless constrained by one theory, it is possible that CAPS acts like a second detergent to help permeate and dissolve non-microbial cells. For example, at a pH of approximately 7–8 (e.g., 7.6–8), the CAPS buffer becomes almost completely protonated and positively charged. According to the Henderson-Hasselbach formula, for example, in the pH range of approximately 7.0–8.0, the ratio of protonated to aprotonated CAPS species is approximately 250:1 or greater. For CAPS buffers at pH approximately 7.0–8.0, a protonation-to-aprotonation ratio of approximately 250:1 or greater is an example of how it means it is "substantially positively charged." Since cell membranes generally have a net negative charge, it is theorized that the positive charge of CAPS buffers can attract CAPS molecules to the cell surface. CAPS has a phenyl ring that can be inserted into the hydrophobic membrane of non-microbial cells to aid in cell permeabilization. CAPSO, CHES, and CABS have a similar structure to CAPS, and it is expected that CAPSO, CHES, and CABS, as well as combinations of CAPS, CAPSO, CHES, and CABS, and similar buffers may provide similar results. CAPSO has a pKa of approximately 9.6 and a typical buffer range of approximately 8.9–10.3 at 25°C, CHES has a pKa of approximately 9.3 and a typical buffer range of approximately 8.6–10 at 25°C, and CABS has a pKa of approximately 10.7 and a typical buffer range of approximately 10–11.4 at 25°C.For CAPSO, CHES, and CABS, the Henderson-Hasselbach formula provides that, for example in the pH range of approximately 7.0–8.0, the ratio of protonated buffer species to aprotonated buffer species is within the ranges of approximately 500:1 or higher (i.e., CABS at approximately 7.0–8.0 pH), approximately 40:1 or higher (i.e., CAPSO at approximately 7.0–8.0 pH), and 20:1 or higher (i.e., CHES at approximately 7.0–8.0 pH). Therefore, for CAPSO, CHES, and CABS at a pH of approximately 7.0–8.0, a ratio of approximately 40:1 or higher for protonated CAPSO to unprotonated CAPSO, a ratio of approximately 20:1 or higher for protonated CHES to unprotonated CHES, and a ratio of approximately 500:1 or higher for protonated CABS to unprotonated CABS are further examples of how they mean "substantially positively charged." Furthermore, those skilled in the art will understand that the buffer used in a differential lysis buffer may preferably include a combination of CAPS, CAPSO, CHES, and CABS. In the case of such a combination, a ratio of approximately 20:1 or higher for any of the protonated to unprotonated species is a further example of how it means "substantially positively charged."

[0204] In one embodiment, the sample and differential lysis buffer are combined over a period of time sufficient for lysis to occur, for example, about 1 second, 2 seconds, 3 seconds, 4 seconds, 5 seconds, 10 seconds, 15 seconds, 20 seconds, 25 seconds, 30 seconds, 40 seconds, 50 seconds, or 60 seconds, or about 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 15 minutes, or 20 minutes or more, for example, about 1 second to 20 minutes, about 1 second to 5 minutes, or about 1 second to 2 minutes. In one embodiment, up to 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, and 99% of non-microbial cells in the sample can be lysed within 2 to 5 minutes of combining the sample with the differential lysis buffer. In some embodiments, the sample and differential lysis buffer are combined for a sufficient amount of time for cell membrane solubilization to occur. Solubilization of the cell membrane of blood cells (i.e., non-microbial cells) is illustrated in Figure 21. To demonstrate the effectiveness of blood cell lysis at room temperature, the OD500 absorbance (wavelength 500 nm) of whole blood combined with several differential lysis buffer formulations was measured at various time points. The decrease in absorbance as a function of time illustrates the progression of lysis. Buffer / blood combinations containing 0.125% BrijO10 and 50 mM CAPS(○) (i.e., detergent and buffer concentrations after combining 10 ml of whole blood with 30 ml of differential lysis buffer) were ineffective for lysis, likely due to insufficient detergent concentration. Each of the other three buffers tested (0.15% BrijO10 and 100 mM CAPS (△), 0.25% BrijO10 and 10 mM CAPS (□), and 0.25% BrijO10 and 50 mM CAPS (◇)) was effective in lysing blood cells. The decrease in absorbance in these buffer / blood combinations illustrates that lysis was complete within 2 minutes for the 100 mM CAPS buffer and 50 mM CAPS buffer, and within 3 minutes for the 10 mM CAPS buffer. The buffers containing 0.25% BrijO10 and 10 mM CAPS are the buffers exemplified in Table 1 above. As illustrated in Figure 21, the lysis time will depend on the strength of the differential lysis buffer, e.g., the concentration of the detergent and / or the pH of the solution.Generally, weaker lysis buffers are expected to require more time and higher dilutions of the sample to completely or partially solubilize non-microbial cells. The strength of the differential lysis buffer may be selected based on the microorganisms known or suspected to be present in the sample. For microorganisms that are more susceptible to lysis, a weaker differential lysis buffer can be used. Dissolution can occur at temperatures ranging from approximately 2°C to approximately 45°C, for example, approximately 15°C to approximately 40°C, or for example, approximately 20°C to approximately 40°C.

[0205] In one embodiment, the differential lysis buffer can be filled into a syringe so that combination occurs within the syringe, and then the sample can be drawn into the syringe. In one embodiment, the sample and differential lysis buffer can be provided in separate tubes, and they can be combined by pouring one into the other. In one embodiment, the differential lysis buffer can be provided in a centrifugal concentrator so that combination and microbial recovery are performed in the centrifugal concentrator, and the sample can be drawn into the centrifugal concentrator. In some embodiments, mixing is performed by combining the sample and differential lysis buffer in solution. In further embodiments, mixing includes stirring the combined sample and differential lysis buffer. For example, the sample and differential lysis buffer can be combined in a centrifugal concentrator and mixed by tilting or gently shaking the centrifugal concentrator. In another example, a bead beater or sonicator can be used to stir the combined sample and differential lysis buffer.

[0206] In some embodiments, the lysis conditions (e.g., combination and / or combination time), as well as the isolation and / or investigation steps, may be sufficient to kill some or all of the microorganisms in the sample. The methods of the present invention are highly versatile and do not require that the microorganisms be viable when isolation and identification are performed. In certain embodiments, some or all of the microorganisms may be dead, and death may occur before, during, and / or after the steps of the method are performed. In other embodiments, some or all of the microorganisms may survive as a result of the isolation step, thereby allowing for further cultivation of the microorganisms at a culture temperature (e.g., about 37°C for bacteria and about 32°C for a number of fungal species) and in a suitable medium (e.g., bacterial or fungal medium). For example, the microorganisms may survive after the isolation step and then be included in an isolation technique to determine whether the microorganisms are susceptible or resistant to one or more antibiotics, and preferably, the growth of the microorganisms may not be affected by the use of differential lysis buffer.

[0207] Separation step After the sample is lysed, a separation step can be performed to isolate the microorganisms from other components of the sample, and the microorganisms can be concentrated into a pellet that can be investigated for identification and characterization purposes. The isolation does not need to be perfect; i.e., 100% isolation is not required. Exemplarily, the isolation of microorganisms from other components of the sample is sufficient if the investigation of the microorganisms can be carried out without substantial interference from the other components. For example, the isolation can result in a microbial pellet that is at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% or more pure. One contaminant that potentially confounds direct microbial identification from whole blood is human genomic DNA. In one embodiment, the inventors in this case have found that treatment of whole blood with the differential lysis buffer described herein has the ability to remove 98% or more of human genomic DNA when microorganisms subsequently pelletize the blood lysate without DNase treatment.

[0208] In one embodiment, separation is carried out by a centrifugation step in which the sample (e.g., lysed sample) is placed in a centrifugal concentrator and the centrifugal concentrator vessel is centrifuged under conditions that the microbial pellet at the bottom and / or sides of the vessel and other components of the sample in the sample medium (e.g., lysed cellular components) remain in the supernatant. This separation isolates the microorganism from the material in the sample, such as the culture medium, cell fragments, human genomic DNA, and / or other components that may interfere with the investigation of the microorganism (e.g., by amplification and detection of microbial-specific nucleic acids). This separation isolates the microorganism from the bulk volume of the sample, reduces the volume of the microbial portion, and concentrates the microorganism to a small volume (e.g., about 200 μl). In one embodiment, a differential lysis buffer is provided in the centrifugal concentrator, and lysis is initiated by combining the sample and the differential lysis buffer for a sufficient amount of time to dissolve, followed by the recovery of the microorganism by centrifugation. In one embodiment, the centrifugal concentrator does not include a high-density cushion, a physical separator, or any similar medium known in the art. Surprisingly, it has been found that high-density cushioning is not necessary to provide adequate isolation and separation of microorganisms from contaminated debris when used in conjunction with molecular techniques for identification or characterization.

[0209] In one embodiment of the present invention, a centrifugal concentrator is centrifuged with a swinging bucket rotor so that microorganisms form pellets directly at the bottom of the tube. The container is centrifuged for a sufficient amount of time at an acceleration sufficient to pelletize the microorganisms and / or separate them from other components of the sample. The acceleration of the centrifugal separation may be, for example, about 1,000 × g to about 20,000 × g, e.g., about 2,500 × g to about 15,000 × g, e.g., about 7,500 × g to about 12,500 × g. The centrifugal separation time may be, for example, about 30 seconds to about 30 minutes, e.g., about 1 minute to about 15 minutes, e.g., about 1 minute to about 10 minutes. The centrifugal separation may be carried out at a temperature of, for example, about 2°C to about 45°C, e.g., about 15°C to about 40°C, e.g., about 20°C to about 30°C. In one embodiment, the centrifugal concentrator includes a closure, which is applied to the container, and after a seal is formed, it is centrifuged. The presence of a closed section reduces the risks associated with handling microorganisms that are or may be infectious and / or harmful, as well as the risk of contaminating the sample. One advantage of the method of the present invention is the ability to perform one or more of the steps of the method (e.g., dissolution, separation, investigation, and / or identification) using microorganisms in a sealed container (e.g., a tightly sealed container). The method may also include the use of an automated system, thereby avoiding the health and safety risks associated with handling highly toxic microorganisms, such as those that arise with the recovery of microorganisms from samples in direct testing.

[0210] The centrifugal concentrator may be any container having sufficient volume to hold the differential lysis buffer and the sample. In one embodiment, the container is fitted to or can be fitted to a centrifugal rotor. Exemplarily, the volume of the container may be about 0.1 ml to about 100 ml, for example, about 50 ml. If the separation is performed on a microscale, the volume of the container may be about 2 μl to about 100 μl, for example, about 5 μl to about 50 μl. In one embodiment, the container has a wider internal diameter in the upper part for holding the sample and a narrower internal diameter in the lower part where the microbial pellet is collected. A tapered internal diameter section can connect the upper and lower parts. Exemplarily, the tapered section may have an angle of about 20° to about 70°, for example, about 30° to about 60°. In one embodiment, the narrow lower section is less than half the total height of the container, for example, less than 40%, about 30%, about 20%, or about 10% of the total height of the container. The container may have an attached closure device, or may be fitted to receive a closure device (e.g., a cap), allowing the container to be sealed before centrifugation. In certain embodiments, the container is designed so that the microbial pellet can be easily recovered from the container after separation, either manually or in an automated manner (so that the technician is not exposed to the contents of the container). For example, the container may include a removable or peelable portion that contains the pellet and can be separated from the rest of the container. In another embodiment, the container includes one or more structures that allow access to the pellet after separation, such as one or more ports or permeable surfaces for inserting a syringe or other sampling device, or for removing the pellet. In one embodiment, the container is a standalone container, i.e., a device for separating a single sample. In other embodiments, the container is part of a device that includes two or more centrifugal concentrators so that multiple samples can be separated simultaneously. In one embodiment, the apparatus includes 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, 25, 30, 36, 42, 48, 60, 72, 84, 96, or more centrifugal concentrators.

[0211] In another embodiment, separation can be carried out by a filtration step in which the sample (e.g., a lysed sample) is placed in an apparatus equipped with a selective filter or filter set having a pore size that retains microorganisms. Other examples of filtration, but not limited to, include tangential flow filtration and / or buffer exchange, in which microorganisms are separated from the sample, the volume of the sample is reduced, and the microorganisms are concentrated. Preferred examples of filtration techniques that may be used in the methods herein are illustrated in Figures 15-20. The retained microorganisms may be washed by slowly passing a suitable buffer through the filter. The washed microorganisms may then be examined directly on the filter and / or recovered for examination by directly sampling the surface of the filter or by backflowing the filter with a suitable aqueous buffer.

[0212] In one embodiment, the container may be a tube, such as a centrifuge tube. In another embodiment, the container may be a chip or a card. In one embodiment, the inventors have developed a centrifugal concentrator, and related apparatus, systems, and methods that can enable the lysis of non-microbial cells and the recovery of microbial cells in a single tube. Furthermore, a microbial pellet can be generated from the centrifugal concentrator such that the supernatant is isolated and contained within the upper part of the centrifugal concentrator. Specifically, the centrifugal concentrator, and related apparatus, systems, and methods described herein allow the user to separate microorganisms from a sample with fewer operations and in only a single centrifugation step. Also, the centrifugal concentrator, and related apparatus, systems, and methods described herein allow the user to separate and test a sample without handling microorganisms, and thus avoid the health and safety risks associated with handling highly toxic microorganisms.

[0213] Referring to Figures 6A-6F, embodiments of the centrifugal concentrator 5010 and its elements are illustrated. In one embodiment, the centrifugal concentrator is a centrifuge tube designed for the concentration of microorganisms from a sample by centrifugation. The centrifugal concentrator 5010 includes a tubular body 6002 and a closing cap 6006 at the proximal end 6001 of the tubular body 6002. In one embodiment, a protective cap 6004 designed to protect the tube during centrifugation may be located at the distal end 6005 of the tubular body 6002. In one embodiment, differential lysis buffer and a sample (e.g., a whole blood sample) may be added to the tubular body 6002 after the cap 6006 has been removed; the contents may be sealed inside by replacing the cap 6006. Exemplary, the distal 6005 and proximal 6001 ends of the centrifugal concentrator 5010 are sealed during operation to prevent the release of presumed biohazardous material, but the distal end 6005 of the tubular body 6002 may be selectively open to allow the discharge of pelletized microorganisms from the concentrator, as will be described in more detail below.

[0214] In exemplary embodiments, the centrifugal concentrator 5010 includes a plunger 6008. The plunger 6008 may be designed to perform several functions, including, but not limited to, collecting concentrated (e.g., pelletized) microorganisms at or near the distal end of the centrifugal concentrator 5010, opening the distal end 6005 of the centrifugal concentrator 5010, and discharging the pelletized microorganisms from the distal end 6005 of the centrifugal concentrator 5010. The opening of the distal end 6005 of the concentrator and the discharge of the microbial pellets will be discussed in more detail below in relation to Figures 6D and 6E. In one embodiment, the plunger 6008 has a proximal end 6024 including an expanding portion 6009 designed for manual operation of the plunger 6008. For example, the expanding portion 6009 may be operated by the thumb, fingers, or another part of the user's hand, or by a mechanical device, to actuate the plunger 6008 and discharge the pellets. For example, the plunger 6008 may be pushed in by the user's thumb to actuate the plunger and eject the pellet. In another embodiment, the plunger 6008 is actuated in a different manner, for example, by rotating along a threaded screw portion to lower the plunger 6008 and pass it through the centrifugal concentrator. In an exemplary embodiment, the plunger 6008 includes a pair of retaining members 6026 designed to hold the plunger in a fixed "upper" position in a first direction and in a "plunge" position in a second direction. In one embodiment, the retaining members 6026 hold the plunger in a fixed "upper" position by interacting with a corresponding pair of stoppers 6030 on the cap 6006. In an exemplary embodiment, to plunge, the retaining members 6026 are aligned with the passage 6032 by gripping and using the enlarged portion 6009 and twisting the plunger against the cap. In one embodiment, the passage 6032 is designed to push the plunger 6008, allowing the microbial pellet to be discharged from the distal end 6005 of the centrifugal concentrator.

[0215] In one embodiment, the centrifugal concentrator 5010 may have any volume sufficient to hold the differential lysis buffer and sample. In one embodiment, the centrifugal concentrator 5010 is or can be fitted to a centrifugal rotor. Exemplarily, the volume of the centrifugal concentrator 5010 may be about 0.1 ml to about 100 ml. In a specific embodiment, the centrifugal concentrator 5010 has a shape and internal volume similar to a standard 50 ml conical centrifuge tube and is fitted to a centrifugal rotor (fixed angle and swing bucket) designed to fit a 50 ml conical centrifuge tube. In one embodiment, the differential lysis buffer 6003 is provided in the centrifugal concentrator 5010. For example, optionally, about 20 to 40 ml (e.g., about 30 ml) of the differential lysis buffer described herein may be provided in the centrifugal concentrator 5010. While the centrifugal concentrator 5010 may contain differential lysis buffer 6003, exemplary, the centrifugal concentrator may not be provided with a high-density cushion, regardless of whether the differential lysis buffer is provided within the centrifugal concentrator 5010. In one embodiment, the cap 6006 of the centrifugal concentrator 5010 may optionally include a septum 6007 (e.g., a rubber septum) or similar structure that allows a sample (e.g., a whole blood sample) to be added to the centrifugal concentrator 5010 without the need to remove the closing cap 6006. This may be particularly useful if we assume that many of the samples intended to be used with the differential lysis buffer and centrifugal concentrator may be biohazardous and / or infectious. In some embodiments, the sample is mixed with the differential lysis buffer and aseptically packed into the centrifugal concentrator through the septum 6007. This reduces the potential contamination of the sample before analysis.

[0216] Figure 6B shows another drawing of the centrifugal concentrator 5010. Considering Figure 6B, the external protective cap 6004 is removed from the distal end of the tubular body 6002 to show the internal support cap 6010 that caps the pellet collection reservoir (pellet collection reservoir 6014 in Figure 6D). In addition to the external protective cap 6004, the internal support cap 6010 protects the distal end 6005 of the tubular body 6002 and can prevent leakage from the tube, for example, during storage or use, particularly during centrifugation. In some embodiments, the support cap 6010 may be removed. Also, the removal of the distal protective cap 6004 shows support ribs 6012 that may be included to reinforce and protect the distal end 6005 of the tubular body 6002, particularly during centrifugation. The support ribs 6012 may be removed in some embodiments. For example, a specially designed centrifuge bucket insert may be designed to support the distal portion of the tubular body 6002, perhaps by removing the support rib 6012.

[0217] Figure 6C shows a drawing of a centrifugal concentrator 5010 similar to Figure 6B, except that the closing cap 6006 is removed, to illustrate how the closing cap 6006 is attached to the tubular body 6002. In an exemplary embodiment, the proximal end 6001 of the tubular body 6002 includes a thread 6011 that allows the closing cap 6006 to be screwed onto the tubular body 6002. However, the thread 6011 is merely illustrative. The thread 6011 can be replaced with any structure known in the art that performs the same or similar function. For example, the closing cap 6006 can be sealed to the tubular body 6002 by a bayonet mount, friction arrangement, O-ring assembly, etc., on or on the tubular body 6002.

[0218] Referring here to Figures 6D–6F, details of the distal end 6005 of the tubular body 6002 and how the plunger 6008 can discharge the microbial pellet are illustrated. In an exemplary embodiment, the distal end 6005 of the tubular body 6002 includes a pellet collection reservoir 6014. For reference, it is shown that the pellet collection reservoir 6014 is covered by a support cap 6010 in Figures 6B and 6C. As will be discussed in more detail elsewhere in this specification, in some embodiments, the centrifugal concentrator 5010 is designed to be centrifuged in a swing bucket centrifuge such that the microorganisms are pelletized at the bottom of the tube (unlike on the sidewall, which is typical in the case of a fixed-angle centrifuge rotor). Thus, substantially all of the insoluble microorganisms in the sample need to be pelletized in the pellet collection reservoir 6014. In one embodiment, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% of the microorganisms in the sample are pelletizable in the pellet collection reservoir 6014. In one embodiment, the pellet collection reservoir 6014 may be sized and designed to contain substantially all of the microbial pellets (e.g., about 500 μl, about 400 μl, about 300 μl, about 200 μl, 20–200 μl, 40–150 μl, or about 50–100 μl or less). In some embodiments, the tubular body 6002 may include inclined inner sidewalls 6015 designed for pouring microorganisms into the pellet collection reservoir 6014 (see Figures 6E and 6F).

[0219] In some embodiments, the pellet collection reservoir 6014 may include a separation end 6016 designed to allow a portion of the plunger 6008 to be pushed through the pellet collection reservoir 6014 to discharge microbial pellets. Preferred examples of the separation end include, but are not limited to, a thinner molded portion, a thinner molded portion with a fragile area, a foil cap, and the like.

[0220] Referring specifically to Figures 6E and 6F, details are shown of how the plunger 6008 penetrates the pellet reservoir 6014 and discharges the microbial pellet from the distal end 6005 of the tubular body 6002. The plunger 6008 and pellet reservoir 6014 are designed so that the microbial pellet can be discharged from the pellet reservoir while isolating the spent lysate from the tubular body. The plunger 6008 includes a distal portion 6030 having a tip 6022 designed for collecting the microbial pellet and penetrating the end 6016 of the pellet reservoir 6014 (e.g., a fixed foil cap or a fragile separation section). In exemplary embodiments, the tip 6022 is a shovel-shaped tip with a sharp edge 6023 that can penetrate the end 6016 of the pellet reservoir 6014. While the tip 6022 is shown as a shovel shape in exemplary embodiments, other preferred shapes, but not limited to, include blade shapes, smooth ends, and spike ends. When the end of the pellet reservoir 6014 is pierced and the microbial pellet is discharged, it is desirable that spent isolates remain in the tubing to prevent the spent lysate from leaking out and diluting the pellet. In some embodiments, spent lysate may contain potentially infectious or biohazardous material, and for this reason, the presence of spent lysate is an important safety feature. Near the distal end 6030 of the plunger 6008, in one embodiment, there is a portion 6018 that is sized and designed to connect to the internal portion 6020 of the pellet reservoir 6014. In this embodiment, the interface between the portion 6018 of the plunger 6008 and the internal portion 6020 of the pellet reservoir 6014 creates a seal that isolates the spent lysate in the tubing 6002. The interface can also ensure that the microbial pellet is efficiently collected and discharged. While the interface between 6018 and 6020 is shown as a friction fit, part 6018 or part 6020 may include, for example, an O-ring or similar structure for creating a seal to isolate used lysate in the tubular body 6002.In some embodiments, by operating the plunger 6008, pellets are discharged from the distal end of the centrifugal concentrator under pressure, opening the separation end 6016. In this embodiment, as the plunger is pushed in, the interface between part 6018 and the internal part 6020 of the pellet reservoir 6014 causes an increase in pressure within the cavity until the separation end 6016 is penetrated, resulting in the discharge of microbial pellets from the pellet reservoir 6014. This can lead to a larger recovery of microorganisms, as the pressure reduces the opportunity for microorganisms to be retained on the sides of the pellet reservoir.

[0221] Referring here to Figure 6G, the plunger 6008 is shown alone. The plunger 6008 includes a proximal end 6024, a distal end 6030, a retaining member 6026, a portion 6018, and a shovel tip 6022, which are discussed elsewhere in this specification. In one embodiment, the plunger 6008 includes a plunger shaft 6028 sized to be long enough (e.g., substantially the same length as the tubular body 6002) to penetrate the end of the tubular body 6002 and discharge the microbial pellet when the plunger is pushed in. Furthermore, the shaft may optionally include an O-ring 6027 or a similar structure, which may be connected to the cap 6006 and designed to seal the interface between the cap and the plunger. Naturally, the cap may also include a sealing member instead of, or in addition to, the O-ring 6027. It is understood that the centrifugal concentrator 5010 is merely illustrative. The centrifugal concentrator 5010 and its variations, as well as other containers, discussed above, may be used in conjunction with the various methods disclosed herein.

[0222] Investigation Steps Once the microorganisms have been pelletized, the pellets can be examined, and the microorganisms within them can be identified and / or characterized. In one embodiment, the examination may be performed in a non-invasive manner, i.e., while the pellets are examined, they remain in the centrifugal concentrator. Optionally, the ability to identify microorganisms in a non-invasive manner, in conjunction with maintaining container airtightness throughout the entire separation and identification process and automating some or all of the procedures, avoids constant handling of contaminated and / or infectious samples and significantly increases the safety of the entire process.

[0223] In another embodiment, the pellet can be investigated using molecular techniques (e.g., PCR) for amplifying microbial RNA or DNA sequences, which can be used to individually identify each of the types of microorganisms that may be present in the sample. For example, nucleic acid sequences characteristic of each of the individual types that may be present in the sample, such as bacteria and fungi, may be selected, forward and reverse primers may be designed for amplification of those sequences, the microorganisms in the pellet may be lysed, and the lysate (or nucleic acid purified from the lysate) may be combined with primers and other PCR reagents (buffers, polymerase, etc.), thereby amplifying the selected characteristic nucleic acid sequences according to procedures well known in the art. The amplified nucleic acids can be detected and used to identify the presence of one or more microorganisms in the sample according to procedures well known in the art, such as real-time detection or post-amplification analysis such as melting curve analysis, fluorescent labeling, radioactive labeling, chemiluminescence labeling, enzymatic labeling, and other dsDNA-binding dye techniques and probes, as well as, but not limited to, procedures well known in the art. In one embodiment, the pellet can be investigated using a FilmArray system, which is detailed elsewhere in this specification. In one embodiment, the pellet can be investigated using a specially adapted blood culture identification (BCID) panel pouch and protocol. The BCID panel and protocol are described in U.S. Patent No. 10,053,726 (which is incorporated herein by reference in its entirety). However, BCID is merely an example of an assay apparatus. Those skilled in the art will understand that the pellet can preferably be investigated using a specially designed assay having sensitivity and detection limits suitably adapted to, for example, the concentration of organisms in a sample obtained directly from blood.

[0224] In another embodiment, the pellet can be investigated by sequencing the nucleic acids present in the pellet. Microorganisms in the pellet can be identified using characteristic microbial sequences or sequencing of the whole microbial genome. Such sequencing may be carried out according to one or more of the many sequencing techniques known in the art. In one embodiment, the sequencing is Sanger sequencing. In another preferred embodiment, the sequencing includes large-scale parallel or “next-generation” sequencing (NGS) techniques. Large-scale parallel / NGS techniques process hundreds of thousands to millions of DNA fragments in parallel, resulting in a low cost per base of the generated sequence and throughput on a gigabase (Gb) to terabase (Tb) scale in a single sequencing run. As a result, large-scale parallel / NGS techniques can be used to characterize the whole genome at low cost and with high throughput. [Examples]

[0225] Example 1 - Direct detection of microorganisms from whole blood The differential lysis buffers and centrifugal concentrators described herein can be used for the direct detection of microorganisms from whole blood. For example, they can be used to rapidly identify sepsis-causing microorganisms without the need for pre-culturing the blood sample and amplifying the disease-causing organisms before detection. However, as discussed elsewhere in this specification, such direct detection and diagnosis from whole blood has proven difficult for several reasons. Firstly, the number of infectious organisms found in whole blood in BSI is usually low (approximately 1–100 colony-forming units (cfu / ml) per mL of blood, and approximately 1–10 cfu / ml is typical in most individuals with sepsis confirmed by culture), and secondly, blood contains several inhibitors of polymerase chain reaction (PCR) (e.g., hemoglobin and leukocyte-derived genomic DNA, which can be co-purified with microorganisms and interfere with both nucleic acid recovery from target microorganisms and downstream PCR).

[0226] Due to the very small number of organisms and PCR inhibitors present in whole blood, concentration from larger volumes of whole blood (e.g., 1–20 mL) is desired to obtain the quality and quantity of DNA templates required to achieve clinically relevant sensitivity at the microbiological level. In one embodiment, the differential lysis buffer and centrifugal concentrator described herein allow a technician to dissolve non-microbial cells in about 1–20 mL (e.g., about 10 mL) of whole blood and concentrate the microorganisms therein by centrifugation within about 5–20 minutes (e.g., about 15 minutes). Exemplarily, sample lysis does not involve a DNase step, and no high-density cushion is used in the centrifugal concentrator. This makes sample preparation faster, easier, and more reproducible.

[0227] The microbial pellets obtained from the centrifugal concentrator described herein can be directly discharged into a sample vial that can be used to inject the sample into a molecular assay instrument. In a specific example, the microbial pellets obtained from the centrifugal concentrator can be directly discharged into a FilmArray injection vial (FAIV) (as described in U.S. Patent No. 10,464,060, which is incorporated herein by reference in its entirety) and then into a FilmArray pouch. In one embodiment, using an FAIV, the microorganisms obtained from the centrifugal concentrator can be directly injected into a blood culture identification (BCID) panel pouch for the identification of microorganisms in a sample. Currently, the BCID panel assay takes approximately 60 minutes to perform. The BCID panel assay and protocol are described in U.S. Patent No. 10,053,726 (which is incorporated herein by reference in its entirety as described above). In this case, the inventors have used a differential lysis buffer and a centrifugal concentrator in conjunction with the BCID panel assay and a specially modified instrument protocol to achieve a detection limit of approximately 1–10 cfu / ml and an overall sample preparation and analysis time of approximately 75 minutes. However, further improvements in chemical reactions and equipment performance could significantly reduce analysis time.

[0228] Referring now to Figure 7, an example of the sample preparation workflow is illustrated. In the first step 700, a blood sample and a centrifugal concentrator are provided. In one example, a blood sample, which may have a volume of approximately 10 ml, is provided in a standard vacuum container. In another example, the centrifugal concentrator may provide a certain volume (e.g., approximately 30 ml) of differential lysis buffer within it.

[0229] In the second step 702, the blood sample and differential lysis buffer shown in Table 1 were combined for a sufficient amount of time to dissolve substantially all non-microbial cells in the sample (i.e., all blood cells) and obtain a lysate. For example, the blood sample and differential lysis buffer may be combined for about 1 to 5 minutes, but longer or shorter times may be used. Exemplarily, dissolution can be carried out at temperatures of about 2°C to about 45°C, for example, about 15°C to about 40°C, for example, about 30°C to about 40°C. In one embodiment, dissolution may be carried out at room temperature.

[0230] After combining the blood sample with the differential lysis buffer for a sufficient amount of time to obtain a lysate, in step 704, microorganisms may be recovered from the lysate by centrifugation, if present. In one embodiment of the present invention, the centrifugal concentrator may centrifuge in a swinging bucket rotor so that the microorganisms form pellets directly on the sides of the tube. The container is centrifuged at a sufficient acceleration and for a sufficient amount of time so that the microorganisms are pelletized and / or separated from other components of the sample. In one embodiment, the centrifugation time may be about 30 seconds to about 30 minutes, for example, about 10 to 15 minutes. Exemplarily, the centrifugal acceleration may be about 1,000 × g to about 20,000 × g, for example, about 3,000 to 10,000 × g. Exemplarily, the centrifugation may be carried out at a temperature of about 2°C to about 45°C, for example, about 4 to 8°C.

[0231] If the centrifugal concentrator includes an optional distal support cap, the support cap may be removed before plunging, as illustrated in step 706. Steps 708–712 illustrate an example process for discharging the microbial pellet from the centrifugal concentrator. In step 708, at the start of the plunge, the distal end of the plunger moves into the pellet reservoir, allowing the pellet to be isolated from the supernatant. This has already been discussed herein and is illustrated in Figure 6E. As the plunger is further pushed into the pellet reservoir, the isolation end of the pellet reservoir may undergo puncture, separation, crushing, etc., and the pellet may be discharged, as illustrated in step 710. Step 712 in the workflow indicates that the pellet has been completely discharged from the centrifugal concentrator. The discharged pellet can be used in several assays known in the art to characterize and identify the microorganisms in the pellet. As has already been discussed herein, PCR analysis and sequencing are two non-limiting examples of assays that can be used to characterize and identify the microorganisms in the pellet.

[0232] As illustrated in 714, in one embodiment, the distal end of the centrifugal concentrator may be sized and designed to fit directly into a container. In one embodiment, the container is a FilmArray injection vial (FAIV). Thus, the pellet can be directly discharged into the FAIV for characterization and identification of microorganisms in the pellet; the sample can then be injected into the FilmArray assay pouch using the FAIV. In one embodiment, the pellet may be directly discharged into the FAIV, and the microorganisms may be injected into the FilmArray pouch using the FAIV without removing the FAIV from the centrifugal concentrator. After filling the FilmArray pouch with the sample using the FAIV, the entire assembly may be disposed of in a biohazard waste container. This reduces the handling of potentially infectious organisms and potentially biohazardous waste, and limits the risk of contamination.

[0233] In one embodiment, a fixed amount (e.g., about 1 ml) of the original sample may be added to the pellet discharged in step 712, to the vial in step 714 together with the pellet, or directly to the analyzer together with the pellet. The dissolution and centrifugation methods (and related methods) described herein are well suitable for the concentration and detection of microorganisms such as bacteria and yeast, but are not particularly suitable for the detection of viruses. Viruses can also be isolated and detected by adding a fixed amount of the original sample to the pellet and to the analyzer.

[0234] In one aspect of the present invention, some or all of the method steps can be automated. Automating the method steps allows for more efficient testing of a larger number of samples and reduces the risk of human error in handling samples that may contain harmful and / or infectious microorganisms. More importantly, however, automation allows for obtaining definitive results at any time, day or night, without delay. Several studies have shown that faster identification of sepsis-causing organisms correlates with improved patient care, shorter hospital stays, and lower overall costs.

[0235] Referring here to Figure 8, the sample preparation time in the method described herein, using differential lysis buffer and centrifugation, is compared with other methods. As is evident from Figure 8, the method of combining the sample with differential lysis buffer and subsequent centrifugation involves only two steps and a total processing time of approximately 15 minutes. This is significantly faster and easier than other methods tested by the inventors in this case. The MolYsis method discussed in the introductory section of this document involves several complex steps, including a DNase step, and takes approximately 45-50 minutes for eukaryotic cell lysis, DNase treatment, and microbial cell recovery alone. Washing and lysis of microbial cells requires further steps and buffers. Successful use of the MolYsis system requires skilled professionals. Dependence on operator skill creates a risk of interoperator variability in yield and quality of results. Many buffers and manual pipetting steps increase the risk of sample cross-contamination. The step of combining the sample with differential lysis buffer and subsequent centrifugation does not require many of those steps, including the DNase step, nor many of the washing steps. In the method described herein, the microbial cells recovered after centrifugation are suitable for post-centrifugation molecular analysis (e.g., PCR assay, DNA sequencing, or mass spectrometry) without further processing.

[0236] Figure 8 also compares the sample preparation time of the differential lysis and centrifugation method with two other protocols. The Y2 protocol is a procedure that uses saponin-based lysis combined with protease and DNase digestion steps. The Y2 protocol required several complex steps and approximately 90 minutes for sample preparation. The Lycoll+DNase protocol is a procedure that uses saponin-based lysis combined with a Ficoll density gradient in DNase digestion and centrifugation. The Lycoll procedure yielded good biological yields and effectively removed genomic DNA, but involved complex lysate stratification in the Ficoll density gradient and a processing time of approximately 2 hours. Compared to the method claimed herein, which uses differential lysis buffer and subsequent centrifugation, both the Y2 protocol and the Lycoll+DNase procedure involve complex steps and excessive time. The same applies to the comparison between the differential lysis and centrifugation method and the MolYsis method.

[0237] Figure 9 illustrates microbial recovery in a single experimental set, which can be achieved by treating the sample with differential lysis buffer and subsequent centrifugation. The control is the buffer added, and the test sample is the whole blood added. Both the control and test samples were added with the same number of organisms. The control tests the ability to recover the added organisms from the buffer by centrifugation, while the test samples demonstrate the effectiveness of the differential lysis buffer in lysing eukaryotic cells (i.e., RBCs, leukocytes, platelets, etc.) and recovering the added microbial cells from the lysate by centrifugation. Compared to the control, in this experiment, approximately 86% of the microorganisms can be recovered from the whole blood sample by a method including treating the added blood sample with differential lysis buffer and subsequent centrifugation. Recovery rates can exceed 90% in other experiments, as illustrated in Table 2.

[0238] [Table 2]

[0239] In a preferred embodiment, the recovery rate of microorganisms by differential lysis and subsequent centrifugation can be at least 85%, at least 90%, at least 95%, at least 99% or 100%.

[0240] Also, by treating a blood sample with a differential lysis buffer and subsequent centrifugation, genomic DNA is efficiently removed, without any DNase step or other complex or time-consuming processing steps. Table 3 below compares the degree of genomic DNA removal from whole blood achieved with the differential lysis buffer and centrifugation, against the Lycoll and Lycoll+DNase methods. The whole blood control represents the amount of genomic DNA recovered from the lysed whole blood sample. The DNA was purified with the MagnaPure system and quantified using the ThermoFisher Quantifiler Human DNA Quantification Kit.

[0241]

Table 3

[0242] As is clear from Table 3, the amount of genomic DNA recovered by the differential lysis buffer and centrifugation treatment is significantly better than the Lycoll method and equivalent to the Lycoll+DNase method. The differential lysis buffer and centrifugation treatment is significantly faster, easier and more reproducible than the Lycoll or Lycoll+DNase methods, and in the differential lysis buffer and centrifugation treatment, a spectacular reduction of genomic DNA is achieved without the time-consuming DNase step.

[0243] This is demonstrated in a slightly different form in Figure 10, which compares the crossing point (Cp) values in the amplification of various amounts of yeast control in the presence of whole blood material that can be pelleted by centrifugation after treatment of blood with the differential lysis buffer. In this case, the differential lysis buffer (● alkaline) is compared with the Lycoll method ( *The results were compared with those of Lycoll. For the differential lysis buffer experiment, 1, 10, or 100 CFU / mL of yeast control was added to 10 mL of whole blood, and then processed using differential lysis buffer and subsequent centrifugation as described herein. The resulting pellet was transferred to an FAIV, and the sample was run in a FilmArray BCID pouch. The Lycoll experiment was performed similarly, except that 10 mL of added whole blood was processed using the Lycoll method. The CFU control (■) represents the Cp in the absence of any whole blood material in various amounts of yeast DNA. Different yeast CFU amounts were diluted in PBS and pipetted into an FAIV at a level equivalent to 100% concentration of the organism from the differential lysis buffer and 10 mL of added whole blood used in the Lycoll procedure (1 CFU / mL in whole blood = 10 CFU control in FAIV). The WB control (□) is unconcentrated whole blood. In the case of the WB control, 200 μL of added whole blood was pipetteed into FAIV at a level equivalent to 100% of the organism's concentration from 10 mL of added whole blood used in the differential lysis buffer and Lycoll procedure, and after indicating the initial LoD / Cp value of the organism, the enrichment protocol was performed. As is clear from Figure 10, the amplification of yeast DNA in the presence of the Lycoll pellet was delayed by approximately 3 Cp units compared to the CFU and WB controls. In contrast, no detectable inhibition was observed in the pellet obtained using the differential lysis buffer and subsequent centrifugation. That is, the amplification of yeast DNA in the presence of the pellet obtained from the differential lysis buffer was substantially indistinguishable from that of the CFU and WB controls. The increase in Cp in the Lycoll pellet appears to be due to the high levels of hgDNA enriched in the pellet along with the added yeast organism. hgDNA is a known competitive inhibitor with magnetic silica beads and a nonspecific inhibitor of PCR in DNA recovery. Based on the data shown in Table 3, and based on this data, itisconcludedthat pellets obtained from differential lysis buffer do not contain such a large amount of hgDNA in the pellet, and as a result have a more similar yeast DNA Cp, even in the case of whole blood controls or CFU controls that do not contain any matrix.

[0244] Referring now to FIG. 11, data are presented for different differential lysis buffer formulations (LB18-LB21) having various amounts of CAPS and BrijO10. In the case of the lysis buffer test, Escherichia coli (E. Coli), enterobacteria, or yeast was added to 10 ml of whole blood samples, and the samples were processed using the designated differential lysis buffer and centrifugation according to the method described herein. As described in FIG. 10, the resulting pellet was transferred to FAIV and the samples were run on a FilmArray BCID panel. The WB control was the same as that described in FIG. 10. The data presented in FIG. 11 show that the differential lysis buffer can efficiently lyse host cells (i.e., RBCs, white blood cells, platelets, etc.) and the nuclei of host cells, while leaving microbial cells intact and pelletable by centrifugation.

[0245] Using the differential lysis buffer disclosed herein, sample processing involves only two simple steps and the processing time can be reduced to about 15 minutes. The DNase step, which is common in other methods, may be omitted due to the effective disruption of the nuclear membrane. The volume of the pellet obtained by differential lysis buffer and subsequent centrifugation may preferably be <200 μL. BrijO10 and CAPS lysed blood cells completely within seconds or minutes. As shown in Example 1, this buffer is easy to handle, and therefore, the results are required to be more reproducible (FIG. 8). Microbial cells can be rapidly concentrated from whole blood (FIG. 8), a high percentage of microbial cells in the sample can be recovered (FIGS. 9 and Table 2), human genomic DNA can be reduced from the microbial pellet (FIGS. 10 and Table 3), and while effectively lysing host cells and the nuclei of host cells, microbial cells can be recovered intact and pelletable by centrifugation (FIG. 11).

[0246] Example 2 - Microbial Recovery by Species at Low Inoculum Levels (<1 CFU / mL) In previous examples, it was shown that the differential lysis buffer and centrifugal concentrator described herein can be used for the lysis of all blood factors, the recovery of microbial cells, and subsequently the detection of microorganisms. This example builds upon Example 1 and demonstrates the ability to recover and identify microorganisms at low levels (i.e., <1 CFU / mL) from added whole blood. In most cases of bloodstream infection (i.e., sepsis), clinically relevant microbial levels in whole blood range from approximately <1 CFU / mL to up to approximately 10 CFU / mL. This example also demonstrates the ability to recover and identify microorganisms at clinically relevant levels, on a species-by-species basis.

[0247] The blood processing method described herein directly yields a simple workflow comprising the steps of dissolving the pellet, centrifuging, and discharging to recover organisms from the lysate. In this example, a whole blood sample was mixed with differential lysis buffer, lysis was allowed to proceed for approximately 5 minutes, and the lysate was centrifuged at approximately 3000 × g for approximately 30 minutes to recover the microorganisms. The lysis buffer used in this example is shown in Table 4.

[0248] [Table 4]

[0249] The comprehensive test panel included 120 biological strains from 12 species of bacteria and yeasts, most commonly isolated from bloodstream infections. The collected organisms not only maintained viability but also possessed reduced levels of blood debris and contaminated host DNA, facilitating their potential use as inputs to various downstream applications of both growth-based and molecular analyses.

[0250] Figure 12 illustrates the workflow used in this experiment. In steps (a) and (b), a biological stock of an appropriate concentration (e.g., approximately 100 CFU / mL) can be obtained by sequentially diluting the biological stock until the desired concentration is achieved and then seeding. The concentration can be verified by seeding the stock solution onto an agar plate and growing individual colonies on the plate. For example, when seeding 50 μL of 100 CFU / mL stock, 5 colonies / plate should be obtained. The desired concentration of the stock biological solution can be obtained by diluting and seeding several times. In step (c), the stock biological solution (e.g., approximately 100 CFU / mL) was added to whole blood. In the example shown in Figure 12, 150 μL of biological stock was added to 30 mL of whole blood. It was desired to add the organism at a concentration of <1 CFU / mL. In the example shown in Figure 12, the target addition concentration was 0.5 CFU / mL. Addition varied among Gram-negative organisms, Gram-positive organisms, and yeast organisms, as will be described in more detail below. The added whole blood was divided into three 10 mL fractions, combined with 20 mL of LB100 buffer in a centrifugal concentrator, and allowed to dissolve at room temperature for 5 minutes. Steps (d) to (f). In the specific example shown in Figure 12, the blood and lysis buffer were inverted 10 times in the centrifuge concentrator tube, incubated at RT for 5 minutes, vortexed for about 5 seconds, and then centrifuged at 3000 × g for 30 minutes using a swing bucket centrifuge rotor.

[0251] After centrifugation, the pellet from the centrifugal concentrator was transferred to 500 μL of TSB (tryptosoy broth) (step (g)), and 100 μL was plated onto each of five agar plates (step (h)). The plates were incubated at 37°C for 24 hours (step (i)), and CFU from the five plates was added to obtain the total recovery rate (step (j)). In this embodiment, while plating and culture were used for the detection of organisms, various different types of workflows could be used for detection. For example, but not limited to, PCR (e.g., by the FilmArray system as discussed in detail herein), whole-genome sequencing, or molecular detection techniques such as molecular AST could be used. Phenotypic analysis (e.g., Vitek2 AST), proteomics (e.g., maldi-TOF, Vitek MS, etc.), and microscopy techniques may be used to investigate the pellet obtained from the centrifugal concentrator.

[0252] The results of this research study are summarized below.

[0253] Table 5 below shows the percentage recovery rates for all organisms, including Gram-negative, Gram-positive, and yeast. The average overall recovery rate in this study was 80%, which exceeded the target of >70%.

[0254] [Table 5]

[0255] Figure 13 illustrates that although some variability was observed in the recovery rates by organisms, all organisms were recovered and cultured. As is clear in Figure 13, the recovery rate of Streptococcus pneumoniae strains was low in this test. The recovery rate of Gram-positive species increased to 95% when Streptococcus pneumoniae strains were excluded, and the overall recovery rate increased to 84%. Further optimization of methods, such as pH, contact time, and supplementation, may improve the recovery rate of this species. It is also conceivable that their seemingly low relative recovery rates increased when Streptococcus pneumoniae strains had lower viability after recovery from the lysis buffer and detection techniques that do not rely on viable organisms (e.g., molecular techniques) were used for detection.

[0256] The percent recovery by CFU for all organisms, Gram-negative, Gram-positive, and yeast, is shown in Table 6 below.

[0257]

Table 6

[0258] The measured input inoculation levels for all organisms were slightly higher than the target of 5 CFU / 10 mL. Nevertheless, all goals of achieving recovery and detection of <1 CFU / mL from whole blood were met. Figure 14 is an expansion of the data in Table 6 based on each organism.

[0259] Table 7 (below) shows the reduction of contaminating host DNA.

[0260]

Table 7

[0261] An average reduction of 99.7% of host DNA was calculated from the input DNA concentration of 10 mL of blood in the lysate relative to the host DNA in the effluent pellets. The range shown reflects the variation over 15 different blood donors and test days.

[0262] For a comprehensive biological panel, this test demonstrated recovery rates at low addition levels from whole blood (i.e., <1 CFU / mL). Recovery and detection sensitivity in this test were comparable to that of conventional blood cultures (4–8 CFU / 10mL). CAPS-Brij lysis buffer lysates and solubilizes human blood cell membranes, RBCs, and WBCs. CAPS-Brij lysis buffer also reduced blood cell fragments and DNA in the efflux pellet. This processing method concentrates and collects viable organisms, significantly reducing levels of blood debris and host DNA, providing a sample potentially suitable for multiple rapid diagnostic pathways.

[0263] Example 3 - Culture of microbial cells after lysis and collection of whole blood In this embodiment, different differential lysis buffer compositions were compared for detection in a FilmArray BCID assay pouch. Only three organisms were used in this comparison: C. albicans, E. coli, and S. agalactiae. Blood treated with ACD (dextrose citrate anticoagulant) was used in this test. This test demonstrates that (1) cells can be concentrated and all buffer compositions and organisms can be tested by the differential lysis buffer / centrifugation procedure described herein, and (2) cells of organisms isolated from blood and lysis buffers can be concentrated by culturing after centrifugation, allowing for the testing of all selective lysis buffer compositions. All selective lysis buffers tested can lyse blood cells while leaving microbial cells intact and viable.

[0264] The buffer solutions to be tested are listed in Table 8 below.

[0265] [Table 8]

[0266] LB20 is the buffer listed in Table 1, and is the buffer used in the test described in Example 2.

[0267] The data in Table 9 show the improvement in the crossing point (Cp) of the alkaline dissolution / centrifugation method compared to unconcentrated added whole blood. The improvement in Cp is likely due to the removal of substances that interfere with PCR (e.g., hemoglobin) and the concentration of cells in the sample.

[0268] [Table 9]

[0269] For buffers LB16, LB19, and LB20, the apparent enrichment was approximately 8 times, while the enrichment for LB100 was approximately 2.5 times. An improvement in Cp after one cycle represents an increase of approximately 2 times in the input concentration of target cells or template DNA, an improvement after two cycles represents an increase of approximately 4 times, an improvement after three cycles represents an increase of approximately 8 times, and so on (based on the general formula that an improvement in Cp after n cycles represents an increase of approximately 2n times in the input concentration of target cells or template DNA).

[0270] The data in Table 10 show the improvement in Cp resulting from a 3-hour culture of pellets collected from a centrifugal concentrator in culture medium. 150 μL of BHI culture medium was mixed with the pellets from the centrifugal concentrator and incubated at 37°C for 0 or 3 hours. The improvement in Cp shown in Table 10 represents the mean decrease in Cp (i.e., reduction in time to detection) observed in 3-hour cultures compared to samples cultured for 0 hours.

[0271] [Table 10]

[0272] After 3 hours of cell culture, cells from LB16 were concentrated approximately 2-fold, 12-fold, and 8-fold respectively for LB19 and LB20, and for LB100.

[0273] Figure 22 illustrates another experiment comparing lysis and centrifugation culture of organisms recovered from blood treated with ACD anticoagulant and blood treated with SPS anticoagulant. This test was performed on C. albicans, Escherichia coli, K. pneumoniae, S. agalactiae, and Staphylococcus aureus. After lysis in LB20 and cell recovery by centrifugation, 150 μL of BHI culture medium was mixed with the pellet from the centrifugal concentrator and incubated at 37°C for 0 or 3 hours. The improvement in Cp shown in Figure 22 represents the mean decrease in Cp (i.e., reduction in time to detection) observed in 3-hour cultures compared to samples cultured for 0 hours.

[0274] In this study, cells from ACD-treated blood showed an improvement of approximately 5.5 Cp after 3 hours or at 37°C, while cells recovered from SPS blood showed an improvement of approximately 3 Cp after 3 hours or at 37°C. The improvements were most dramatic in Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus). C. albicans, which grows more slowly than bacteria, showed only an improvement of approximately 1 Cp in ACD-treated blood, and in fact performed somewhat worse in SPS blood. No ACD performance data was obtained for Klebsiella pneumoniae in this study. This study exemplifies that certain anticoagulants may affect the growth and culturability of some organisms. For all organisms in this study, ACD appeared to be less detrimental to growth and culturability compared to SPS.

[0275] Example 3 - Flow-through lysis, culture, and volume reduction system In addition to, or in combination with, other devices discussed herein, the flow-through system can be used for cell lysis, culture, and volume reduction. A schematic diagram of an example of such a system 1500 is shown in Figure 15. The flow-through system 1500 comprises three adjacent buffer chambers 1502, 1506, and 1510, each containing a first buffer 1504, a second buffer 1508, and a third buffer 1512. System 1500 further includes a tube or open trough of a buffer exchange membrane in contact with buffers 1504, 1508, and 1512 in the buffer chambers 1502, 1506, and 1510, respectively, via channel 1514. In one embodiment, the first buffer 1504, the second buffer 1508, and the third buffer 1512 may consist of a selective lysis buffer, medium, or nutrient broth for culturing microbial cells (e.g., a nutrient broth for culturing bacterial organisms, fungal organisms, or a culture medium suitable for culturing both bacteria and fungal organisms), and a hypertonic solution / medium for reducing sample volume. In one embodiment, System 1500 also includes, for example, selective lysis, microbial culture, To enhance the volume reduction, a temperature control system (not shown) may be included that can adjust and control the temperatures of the first buffer 1504, the second buffer 1508, and the third buffer 1512 (individually or as a group). For example, selective lysis may be performed at room temperature, microbial culture may be performed at 32-37°C, and volume reduction may be performed at 4°C. A sample (e.g., a whole blood sample) placed in channel 1514 can be selectively exposed to each of the buffers 1504, 1508, and 1512 in any given order, or to two or more buffer chambers simultaneously, to achieve, for example, hemolysis, microbial cell culture, and sample volume reduction / concentration.

[0276] In one embodiment, a whole blood sample containing microbial cells (e.g., a whole blood sample from a subject suspected of having sepsis) may be added to channel 1514, thereby selectively exposing the blood sample to buffers 1504, 1508, and 1512, respectively. Buffer exchange membranes are widely known in the art. A suitable buffer exchange membrane may be selected so that microbial cells are retained while blood cell fragments, hemoglobin, and other products of hemolysis diffuse through the membrane. For example, the buffer exchange membrane may be a dialysis membrane. Dialysis membranes are fabricated and characterized to have different molecular weight cutoffs (MWCOs) in the range of, for example, 1 kilodalton (kDa) to about 1 MDa (i.e., 1 megadalton, or about 1,000,000 Da). The determination of the MWCO is a result of the number and average size of pores created during the fabrication of the dialysis membrane. Typically, the MWCO refers to the minimum average molecular weight of a standard molecule that does not effectively diffuse through the membrane during dilated dialysis. However, it is important to acknowledge that the MWCO of a membrane is not a clearly defined value. Molecules with a mass close to the membrane's MWCO will diffuse through the membrane more slowly than molecules significantly smaller than the MWCO. For molecules to diffuse rapidly through the membrane, it typically needs to be at least 20–50 times smaller than the membrane's MWCO rating. Dialysis tubing in laboratory use is typically made from regenerated cellulose or cellulose ester films. However, dialysis membranes made from polysulfone, polyethersulfone (PES), etched polycarbonate, or collagen are also widely used in certain medical, food, or water treatment applications.

[0277] Because microbial cells are relatively large and cell fragments are relatively small, channel 1514 may also be fabricated from a filtration membrane material. Membrane materials designed to filter bacteria and larger cells from solution are well known in the art. For example, a filtration membrane with a nominal pore size of 0.25–1 μm (e.g., 0.5 μm) can be used to retain microbial cells within channel 1514 while allowing rapid exchange of the sample within channel 1514 with buffers 1504, 1508, and 1512.

[0278] Referring to Figure 15, a sample placed in channel 1514 (e.g., a whole blood sample) may first be exposed to buffer 1504 in chamber 1502, as shown at 1516 in Figure 15A. Next, the sample may be moved to be exposed to buffer 1508 in chamber 1506, as shown at 1518 in Figure 15B, and then to be exposed to buffer 1512 in chamber 1510, as shown at 1520 in Figure 15C. While Figures 15A-15C show the sample being moved from one buffer chamber to the other over time, exposing the sample to each buffer sequentially, it will be understood that the sample can be moved back and forth to be exposed to two or more buffers simultaneously, for example, to be exposed to a buffer two or more times. Similarly, buffers 1504, 1508, and 1512 may be prepared in any given order. Table 11 illustrates some of these options.

[0279] [Table 11]

[0280] In this embodiment, the culture of microbial cells and the reduction of sample volume are discussed using selective lysis buffer, culture medium, and hypertonic solution for lysis, respectively. However, those skilled in the art will understand that these buffers are merely illustrative and that other buffers may be used in System 1500. Similarly, while System 1500 includes three buffer tanks, this is merely illustrative. Alternative versions of System 1500 may include more or less buffer. Furthermore, while Channel 1514 is shown as a linear channel, this is merely illustrative. Channel 1514 may include, for example, detour channels or other modifications known in the art to maximize the surface area of ​​the sample exposed to the buffer.

[0281] In one embodiment, a hypertonic medium may be sufficient to concentrate the microorganisms in the sample and enable identification (e.g., by PCR, whole-genome sequencing, or molecular AST, phenotyping, proteomics, and microscopy). In other embodiments, filtration techniques may be used for concentration / volume reduction. Filtration may be performed before or after one or more exposures of the sample to selective lysis buffer, medium, and hypertonic solution. In yet another embodiment, centrifugation techniques may be used to concentrate the microorganisms in the sample. Centrifugation may be performed before or after one or more exposures of the sample to selective lysis buffer, medium, and hypertonic solution.

[0282] Example 4 - Filtration Technology In some embodiments described herein, the separation of microbial cells from their environment (e.g., separation of bacterial and / or fungal cells from whole blood samples) can be carried out by filtration. Filtration techniques may be designed to retain or allow through selected cells or cell sizes. For example, blood cells (e.g., red blood cells, white blood cells, platelets, etc.) may be captured while microbial cells are allowed to pass through, or microbial cells may be captured, or a combination of filtration media may be used to selectively capture large and small cells at different stages of the filtration apparatus. Differential filtration techniques may also be used to separate larger and smaller cells into different fractions. For example, filtration membranes with different nominal pore sizes may be stacked (or used in a series of separate containers) to allow and / or capture cells having a selected size range. Flow cytometry is also a well-known technique that can sort cells by size. Cells may also be captured or concentrated by activated filtration techniques. For example, most cell types have certain surface factors (e.g., proteins) that can be used in affinity purification by techniques known in the art.

[0283] An example of a differential filtration system is shown in Figure 16. Whole blood samples from subjects suspected of having sepsis may be concentrated for microbial cells by first passing the whole blood sample through a large filter with a pore size of 8–15 μm (e.g., 10 μm) to filter out large cells such as leukocytes (WBCs) and some erythrocytes. Microbial cells would then flow through the first filter. In one embodiment, using a semi-soluble level of Brij detergent (e.g., <0.1%) can ensure that any microbial cells attached to the outside of WBCs are released, reducing the capture of microbial cells on WBCs by the filter. Other detergents may preferably have other semi-soluble concentration levels, i.e., generally 0.1%–1%. A second filter with a smaller pore size (e.g., 5 μm) can be used in tandem to remove more human cells while concentrating microbial cells in the filtrate. Using a final filter with a pore size of less than 1 μm (e.g., 0.45 μm), all microbial cells can be captured, and the sample volume can be significantly reduced. The microbial cells concentrated by the filter can be used directly for identification and diagnosis (e.g., molecular identification by FilmArray, imaging, optical fluorescence, etc., such as metabolic processes or metabolic consumption, conductivity, pH, etc.), the sample can be cultured (e.g., 1-3 hours) to increase the number of microbial cells in the sample, or they can be subjected to alkaline dissolution, centrifugation, and molecular identification to further remove animal cells (e.g., human cells) as described herein.

[0284] In another embodiment, cells can be recovered after selective lysis (i.e., alkaline lysis) with the alkaline / Brij buffer described herein using filtration. However, it was found that proteinase K treatment was necessary to reduce the viscosity of the sample before filtration. In connection with this, alkaline / Brij selective lysis buffer was added to whole blood and incubated for 5 minutes. After 5 minutes, 1 mL of 30 units / mL of proteinase K was added and incubated at RT for approximately 5 minutes. The lysate could then be filtered through a 0.45 μm filter. As in the preceding examples, for example, the filtered microbial cells could be used directly for identification and diagnosis, and they could be cultured (e.g., for 1-3 hours) to increase the number of microbial cells in the sample.

[0285] In addition to the conventional filtration techniques described above, filtration techniques using various types of structures may be employed to selectively concentrate specific cell types in a sample. Such filtration techniques may be used instead of conventional filtration, or in combination with conventional filtration, to concentrate or isolate target microbial cells from blood cells and reduce their volume and inhibitors. Such concentrated or isolated microbial cells may be subjected to culture calls (similar to conventional blood cultures, but likely to be faster due to the concentration of microbial cells in the sample), film array identification, or other investigation techniques. A further desire is to ensure that the bacterial cells are likely to contribute to making the process more economical for the customer (e.g., imaging of metabolic processes, or metabolic consumption, conductivity, or pH, or less expensive tests such as optical fluorescence).

[0286] Various filtration techniques that can be used to concentrate specific cells are illustrated in Figures 17–20. Figure 17A illustrates a weir filter, Figure 17B illustrates a micropillar filter, and Figure 17C illustrates a cross-flow filter. By using the differential flow of larger and smaller cells around these structures, smaller cells can be separated from larger cells. Figure 18 schematically illustrates different types of pillar filters: (18A) polygonal, (18B) U-shaped, and (18C) butterfly-shaped micropillar shapes. Larger cells are immobilized by the trap structure, while smaller cells pass through. Figure 18B also schematically illustrates the concept that micropillars can be formed by structural features (shape, pockets, etc.) to selectively delay the passage of specific cells through the micropillar structure.

[0287] While Figures 17 and 18 show only one set of each of these structures, such structures (and flow directions) can be used sequentially and in combination to achieve a high level of separation. Figures 19 and 20 illustrate this principle. Figure 19 illustrates the separation of large and small cells in a structure with a series of micropillars and crossflow of buffer and cell suspension. The structure in Figure 19 separates large and small cells by decisive lateral displacement. Large cells move streamlined away from small cells due to the modified size and spacing of microposts in the fluid channel. Figure 20 schematically illustrates the concentration of large and small cells by movement along an oval filter unit. The filter unit achieves simultaneous separation of large cells larger than the gap and small cells smaller than the gap. The rotation along the pillars at a relatively low speed, induced by the shear layer of the filtrate, helps prevent clogging by large particles. The systems in Figures 19 and 20 are examples of systems that may be used for lysate removal, buffer exchange, and growth medium addition within a single system. That is, lysate may be introduced to initiate separation, buffer may be added to flush out the lysate, and medium may be added. In addition to, or instead of, the centrifugal concentrators described herein, filtration concentrators, microfluidic concentrators, dielectrophoretic concentrators, FACS (fluorescent cell sorting), or other similar devices may be used, preferably. The benefit of selective lysis may preferably be that it can simplify the filtration concentrator mechanism or allow them to process larger quantities before fouling.

[0288] A workflow that may include one or more of the following: chemistry, filtration, centrifugation, and identification (e.g., molecular identification such as imaging of metabolic processes or metabolic consumption, conductivity, or pH, optical fluorescence, or other techniques, but not limited to these). Route 1: Selective lysis with alkaline / Brij buffer, transfer of lysate to a microfluidic chip for concentration and culture, and detection and identification using a FilmArray. 1. Selectively lyse human cells with an alkaline / Brij selective lysis buffer. 2. Concentrate microbial cells using one or more of the following methods: centrifugal separation, filtration, or microfluidic chip design. a. Sorting techniques (active (e.g., flow cytometry) or passive (Weir filtration, micropillar filtration, or a combination thereof)) b. Capture techniques (active or passive) c. Filtration technology (usually passive, but potentially active) 3. Rinse with growth medium. a. In some embodiments, centrifugation, filtration, microfluidic separation, or a combination thereof may be used for lysate removal, buffer exchange, and growth medium addition within a single system. b. In some embodiments, further sensing techniques may be added for the positive detection of microbial cells. i. Imaging ii. Photofluorescence of metabolic processes or metabolic consumption iii. Conductivity iv. pH v. Microresonator vi. Dielectrophoresis vii. Capacitive Sensing viii.SPR ix.FLIR 4. Release cells from the microfluidic device for FilmArray analysis, culture, or other confirmatory processes. Route 2: Use microfluidic chips / selective filtration for both sorting / concentration and culturing, FilmArray, or other confirmatory processes for detection. 1. Selectively separate microbial cells from human blood cells. a. Active selection i. Flow cytometry (fluorescence activation or optical detection) ii. Dielectrophoresis (DEP) sorting iii. Pneumatic separation b. Passive selection i. Size selection ii. Inertial sorting iii. Dielectric traps iv. Selective protein adhesion process v. Acoustic traps vi. Viscoelastic (or cell hardness) sorting under shear gradient 2. Rinse with growth medium. a. In some embodiments, centrifugation, filtration, microfluidic separation, or a combination thereof may be used for lysate removal, buffer exchange, and growth medium addition within a single system. b. In some embodiments, further sensing techniques may be added for the positive detection of microbial cells. i. Imaging ii. Photofluorescence of metabolic processes or metabolic consumption iii. Conductivity iv. pH v. Microresonator vi. Dielectrophoresis vii. Capacitive Sensing viii.SPR ix.FLIR 4. Release cells from the microfluidic device for FilmArray analysis, culture, or other confirmatory processes.

[0289] The present invention may be illustrated in other specific forms without departing from its spirit or essential features. The embodiments described herein should be interpreted in all respects as merely illustrative and not limiting. Accordingly, the scope of the invention is indicated by the appended claims rather than by the foregoing description. While certain embodiments and details are included herein and in the appended disclosures for the purpose of illustrating the invention, it will be apparent to those skilled in the art that various modifications in the methods and apparatus disclosed herein may be made without departing from the scope of the invention as defined in the appended claims. All modifications made in a sense and scope equivalent to the claims should be included within that scope.

Claims

1. A method for isolating and identifying microorganisms, (a) To provide a blood sample of a certain volume suspected to contain the microorganisms; (b) Mixing the blood sample with differential lysis buffer to obtain a lysate containing lysed blood cells and non-lysed microorganisms; (c) Concentrating the microorganisms from the lysate; (d) Adding the microorganism to an apparatus containing one or more reagents necessary for identifying the microorganism; (e) Identifying the microorganisms present in the blood sample, Includes, If the microorganisms are present, they are concentrated to a degree of 25 to 100 times relative to the volume of the provided blood sample, and If the microorganism is present, it will have a concentration in the provided blood sample in the range of < approximately 1 CFU / ml to approximately 20 CFU / ml, and A method characterized in that the differential lysis buffer comprises a buffering agent, a nonionic surfactant, a salt, and a pH range of about 10 to 11 before the blood sample is mixed with the differential lysis buffer, and the lysate has a pH of about 7.0 to 8.15, and the method does not include a culture step before mixing the blood sample with the differential lysis buffer, or a DNase step for digesting genomic DNA in the lysate.

2. The method according to claim 1, wherein steps (a) to (e) can be completed in less than about 120 minutes, preferably less than about 90 minutes.

3. The method according to claim 1, wherein steps (a) to (c) can be completed within a time range of approximately 10 to 20 minutes.

4. The method according to claim 1, wherein steps (d) and (e) can be completed within a time range of less than 4 hours, preferably less than 3 hours, preferably less than 2 hours, or more preferably less than 1 hour.

5. The method according to claim 1, wherein the microorganism is a bacterium or fungus associated with bloodborne infections.

6. The method according to claim 1, wherein the identification includes one or more molecular tests, phenotypic tests, proteomics tests, optical tests, or culture-based tests.

7. The method according to claim 1, wherein the identification includes the steps of isolating one or more nucleic acids having the characteristics of the microorganism from the microorganism, and analyzing the one or more nucleic acids to identify the microorganism present in the blood sample.

8. The method according to claim 7, further comprising amplifying one or more nucleic acids and then detecting the one or more amplified nucleic acids.

9. The method according to claim 8, wherein the detection of the one or more amplified nucleic acids comprises the use of one or more dsDNA-binding dyes, real-time PCR, a post-amplification nucleic acid lysis step, a nucleic acid sequencing step, a labeled DNA-binding probe, or an unlabeled probe.

10. The method according to any one of claims 7 to 9, wherein the step of identification can be completed within a time range of approximately 5 to 75 minutes.

11. The method according to any one of claims 7 to 9, further comprising performing a culture step for the concentrated microorganism in a culture medium to increase the concentration of the microorganism, and then performing the identification step, wherein the culture step is performed over a period of 4 hours or less, 3 hours or less, or 2 hours or less, preferably 3 hours or less.

12. The method according to claim 1, wherein the differential lysis buffer comprises a buffer, a nonionic surfactant, a salt, and a pH range of about 10 to 11 before mixing the blood sample with the differential lysis buffer.

13. The method according to claim 12, wherein the differential lysis buffer has a pH of approximately 7.6 to 8.0 after mixing the blood sample and the differential lysis buffer.

14. The method according to claim 12, wherein the buffering material is selected from the group consisting of CABS, CAPS, CAPSO, CHES, and combinations thereof, and the buffering material is preferably CAPS.

15. The method according to claim 12, wherein the pH of the differential lysis buffer mixed with the blood sample is approximately 1.5 to 2.5 pH units below the pH buffering range of the buffering material.

16. The method according to claim 12, wherein the nonionic surfactant is one or more of polyoxyethylene (POE) ethers, preferably Arlasolve 200 (also known as poly(oxy-1,2-ethanediyl)), BrijO10, and nonaethylene glycol monododecyl ether (also known as Brij35).

17. The method according to claim 12, wherein the nonionic surfactant is selected from the group consisting of Triton X-114, NP-40, Arlasolve 200, BrijO10 (also known as Brij96 / 97), octyl β-D-glucopyranoside, saponin, nonaethylene glycol monododecyl ether (also known as Brij35), and combinations thereof.

18. The method according to claim 1, wherein the concentration of the microorganisms from the lysate comprises centrifugation, and the concentration further comprises recovering a pellet fraction containing the microorganisms from a supernatant fraction containing a dissolved blood fraction.

19. The blood sample mixed with the differential lysis buffer is placed in a centrifugal concentrator, The centrifugal concentrator is used to concentrate the microorganisms from the blood sample placed in the chamber, The concentrated microorganisms are generated from a second open portion at the second end of the chamber, preferably by aseptically generating a pellet from the second end of the centrifugal concentrator into a vial or assay device. The further includes, where the centrifugal concentrator is A chamber having an open portion at a first end and a sealing portion at a second end, wherein the sealing portion is designed to seal the second open portion at the second end of the chamber; and A plunger, at least partially movable, positioned inside the chamber, and designed to be actuated to release the sealing portion. The method according to claim 1, including the method described in claim 1.

20. The method according to claim 19, wherein the centrifugal concentrator does not include a high-density cushion or physical separator for separating the microorganisms from the lysate.

21. The method according to claim 19, wherein the blood sample and the differential lysis buffer are mixed in a first container, the lysate is then transferred to a centrifugal concentrator containing components other than the blood sample and the differential lysis buffer, the centrifugal concentrator is opened after centrifugation and the supernatant fraction is decanted, one or more of the following steps are not included: a culture step before mixing the blood sample with the differential lysis buffer, or a DNase step for digesting genomic DNA in the lysate.

22. The method according to claim 1, wherein the microorganisms are concentrated from the lysate by filtration technology.

23. The method according to claim 22, further comprising adding a filter having concentrated microorganisms on top to one or more culture or assay devices designed to identify the microorganisms present in the blood sample.

24. The method according to claim 1, wherein the steps of mixing the blood sample with the differential lysis buffer to obtain the lysate, and separating the microorganisms from the lysate are performed in a single tube.

25. The method according to claim 24, wherein the differential lysis buffer is a single buffer provided in the single tube.

26. The method according to claim 24, wherein the differential lysis buffer does not contain DNase or protease, and the method does not include the step of adding exogenous DNase or protease to the single tube.

27. The method according to claim 1, wherein the differential lysis buffer is compatible with an anticoagulant selected from the group consisting of EDTA, citrate, dextrose citrate (ACD), sodium polyanethole sulfate (SPS), heparan, sodium fluoride / oxalate, and combinations thereof.

28. A method for concentrating and identifying microorganisms from blood, (a) To provide a blood sample that is known to contain or may contain the microorganisms mentioned above; (b) Mixing the blood sample with a differential lysis buffer comprising a buffer, a nonionic surfactant, and a salt, thereby obtaining a lysate comprising lysed blood cells and non-lysed microorganisms, wherein the blood sample mixed with the differential lysis buffer has a pH of approximately 7.0 to 8.15, and the buffer has a useful pH buffering range of approximately 8.6 to 11.4; (c) Concentrating the microorganisms from the lysate such that the microorganisms are concentrated to a degree of 25 to 100 times relative to the initial volume of the provided blood sample; (d) Identifying the microorganisms present in the blood sample, wherein the identification is achieved in 4 hours or less, 3 hours or less, 2 hours or less, or preferably 1 hour or less. A method comprising, wherein the method does not include a culture step prior to mixing the blood sample with the differential lysis buffer, or a DNase step for digesting genomic DNA in the lysate.

29. The method according to claim 28, wherein the identification includes one or more molecular tests, phenotypic tests, proteomics tests, optical tests, or culture-based tests.

30. The method according to claim 28, wherein the identification includes the steps of isolating one or more nucleic acids having the characteristics of the microorganism from the microorganism, and analyzing the one or more nucleic acids to identify the microorganism present in the blood sample.

31. The method according to claim 28, wherein the nonionic surfactant is one or more of polyoxyethylene (POE) ethers, preferably Arlasolve 200 (also known as poly(oxy-1,2-ethanediyl)), BrijO10, and nonaethylene glycol monododecyl ether (also known as Brij35).

32. The method according to claim 28, wherein the nonionic surfactant is selected from the group consisting of Triton X-114, NP-40, Arlasolve 200, BrijO10 (also known as Brij96 / 97), octyl β-D-glucopyranoside, saponin, nonaethylene glycol monododecyl ether (also known as Brij35), and combinations thereof.

33. The method according to claim 28, wherein the buffering material is selected from the group consisting of CABS, CAPS, CAPSO, CHES, and combinations thereof.

34. The method according to claim 33, wherein the buffering substance is CAPS, and the CAPS has a pH buffering range of about 9.7 to 11.1 and a pKa of about 10.4 at 25°C.

35. The method according to claim 28, wherein the salt is sodium chloride.

36. The method according to claim 28, wherein it does not include the blood culture step prior to the concentration and the DNase step for digesting the genomic DNA in the lysate.

37. The method according to claim 28, wherein steps (a) to (c) are completed within a time range of approximately 10 to 20 minutes.

38. The method according to claim 30, wherein the isolation and analysis steps can be completed within a time range of approximately 5 to 75 minutes.

39. The method according to claim 28, wherein the time required to obtain the lysate is in the range of about 2 to 10 minutes, preferably about 5 minutes.

40. The method according to claim 28, wherein obtaining the lysate does not involve any additional steps.

41. Blood samples known to contain or potentially containing microorganisms; and A differential lysis buffer to be combined with the blood sample, comprising an aqueous medium, a buffering agent, a nonionic surfactant, and a salt. A composition comprising a buffering substance having a useful pH buffering range of about 8.6 to 11.4 and a pKa in the range of about 9.5 to about 10.7 at 25°C, and having a pH of about 7.0 to 8.

15.

42. The composition according to claim 41, wherein the nonionic surfactant is one or more of polyoxyethylene (POE) ethers, preferably Arlasolve 200 (also known as poly(oxy-1,2-ethanediyl)), BrijO10, and nonaethylene glycol monododecyl ether (also known as Brij35).

43. The composition according to claim 41, wherein the nonionic surfactant is selected from the group consisting of Triton X-114, NP-40, Arlasolve 200, Brij O10 (also known as Brij 96 / 97), octyl β-D-glucopyranoside, saponin, nonaethylene glycol monododecyl ether (C12E9, polidocenol), and combinations thereof.

44. The composition according to claim 41, wherein the buffering material is selected from the group consisting of CABS, CAPS, CAPSO, CHES, and combinations thereof.

45. The composition according to claim 44, wherein the buffering substance is a CAPS having a pH buffering range of about 9.7 to 11.1 and a pKa of about 10.4 at 25°C.

46. The composition according to claim 41, wherein the buffering material is substantially positively charged at a pH of approximately 7.0 to 8.

15.

47. The composition according to claim 41, which does not contain DNase.

48. The blood sample known to contain or potentially containing microorganisms; The differential lysis buffer containing a buffering agent, and The aforementioned nonionic surfactant The composition according to claim 41, wherein the composition has a pH of about 7.0 to 8.15, together with the buffering substance which is a CAPS having the useful pH buffering range of about 9.7 to 11.1 and the pKa of about 10.4 at 25°C.

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