Rapid cell lysis and nucleic acid recovery

Abstract

Methods and systems for rapid preparation of nucleic acid samples.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of and priority to U.S. Provisional Patent Application No. 63 / 369,124 filed on July 22, 2022 and U.S. Provisional Patent Application No. 63 / 450,494 filed on March 7, 2023, the entire contents of which are incorporated herein by reference. Background Art

[0002] Infectious diseases account for approximately 7% of human mortality in the United States, Canada, and Western Europe, and more than 40% in developing regions. Infectious diseases lead to a variety of clinical manifestations. Common overt manifestations are fever, pneumonia, meningitis, diarrhea, and bloody diarrhea. Although physical manifestations suggest some pathogens and eliminate others as causative agents, there are still a variety of potential pathogens, and a clear diagnosis usually requires multiple assays. Traditional microbiological techniques for diagnosing pathogens can take days or weeks, often delaying the appropriate course of treatment.

[0003] In recent years, polymerase chain reaction (PCR) has become the method of choice for rapid diagnosis of infectious agents. PCR can be a rapid, sensitive and specific tool for diagnosing infectious diseases. The challenge of using PCR as the primary diagnostic tool is the diversity of possible pathogenic organisms and the low levels of organisms present in some pathological specimens. It is usually impractical to run large-scale PCR assays, each for a possible pathogenic microorganism (most of which are expected to be negative). This problem is particularly serious when pathogen nucleic acids are at low concentrations and large volumes of samples are required to collect enough reaction templates. In some cases, there are not enough samples to measure all possible pathogenic factors. The solution is to run "multiplex PCR", in which multiple targets of a sample are simultaneously measured in a single reaction. Although multiplex PCR has been proven to be valuable in some systems, there are shortcomings in the robustness of high-level multiple reactions and the difficulty in clearly analyzing multiple products. In order to solve these problems, the assay can then be divided into multiple secondary PCRs. Nesting secondary reactions within primary products usually improves robustness. However, this further processing can be expensive and may cause contamination or other problems.

[0004] (BioFire Diagnostics, LLC, Salt Lake City, UT) is a user-friendly, highly multiplexed PCR system developed for the diagnostic market. The single-sample instrument houses a diagnostic "bag" of integrated sample preparation and nested multiplexed PCR. Integrated sample preparation provides ease of use, while highly multiplexed PCR provides the sensitivity of PCR and the ability to test many organisms (e.g., up to 30 different organisms and molecular markers) simultaneously. The system is well suited for pathogen identification, where many different pathogens exhibit similar clinical symptoms. Currently available diagnostic panels include respiratory panels for upper respiratory tract infections, blood culture panels for bloodstream infections, gastrointestinal panels for GI infections, meningitis / encephalitis panels for central nervous system infections, pneumonia panels for lower respiratory tract infections, and bone and joint panels for bone and joint infections. Other panels are under development.

[0005] When PCR first became popular in the late 1980s, the process was slow. A typical protocol was denaturation at 94°C for one minute, annealing at 55°C for two minutes, and extension at 72°C for three minutes. When transition times between temperatures are included, 8-minute cycles are typical, which results in 30 cycles completed in 4 hours. Over the years, systems have become faster, 30-minute protocols are common, and faster PCR devices have become available. Even with faster PCR protocols, many diagnostic PCR reactions begin with cell lysis and extraction / purification of nucleic acids from the cells using older techniques.

[0006] As PCR becomes faster, the time spent by sample preparation accounts for an increasing proportion in the processing time. A possible solution is to fundamentally skip sample preparation, which relies on chemical and / or heated cracking to destroy cells and perform nucleic acid amplification in the original lysate. This really saves time, but the adequacy of cracking is not always reliable, and there are many potential nucleic acid amplification inhibitors in the unpurified lysate. Another possible solution relates to mechanical cracking, and nucleic acid is subsequently recovered and purified from the lysate. Although this is desirable from the perspective of the cleanliness of the starting template for nucleic acid amplification, the method is traditionally quite slow. It is desirable to use a faster method to prepare samples, while maximizing the cleanliness of the starting template for nucleic acid amplification. This area needs to keep the cleanliness and robustness of traditional sample preparation (i.e. mechanical cracking, nucleic acid recovery, and nucleic acid purification before starting nucleic acid amplification), while also shortening the time required for sample preparation. Summary of the invention

[0007] Disclosed herein are methods and systems for preparing nucleic acid samples. The methods and systems described herein are designed to rapidly prepare nucleic acid samples, for example, less than 5 minutes, less than 4 minutes, less than 3 minutes, less than 2 minutes, or within the range of about 1-3 minutes. The methods and systems described herein can utilize the kinetic efficiency of certain parts of sample preparation (i.e., cracking, recovering nucleic acid from lysate, washing and eluting) to shorten sample preparation as much as possible, while still providing high-quality extracted nucleic acid for downstream amplification or other analysis. The methods described herein include focusing on rapid mechanical cracking (preferably cracking is carried out in the presence of silica-coated magnetic beads), rapidly recovering silica-coated magnetic beads from lysate, rapidly washing magnetic beads, and rapidly and effectively eluting one or more of the captured nucleic acids from silica-coated magnetic beads. The methods and systems described herein are also designed to rapidly prepare nucleic acid samples using aqueous buffer compositions that do not contain alcohol or organic solvents. Exemplarily, one or more buffer compositions used in the method described herein can be suitably provided as a ready-to-use dry powder composition, which can be rehydrated with a rehydration fluid (e.g., with deionized water) to use when carrying out the step of the method. Successful sample preparation (i.e., cracking, recovering nucleic acid from the lysate, washing and elution) is important for maximizing the sensitivity of molecular assays while ensuring reliable, consistent results. As the time to obtain results in molecular assays is constantly shortened, reducing the time of sample preparation is important for reducing the total assay time. Method and system described herein greatly reduce the time required for sample preparation with mechanical cracking, without sacrificing the integrity of the sample to ensure reliable, consistent results.

[0008] An exemplary method for preparing a nucleic acid sample comprises the following steps: providing a sample container comprising a first container, providing a sample suspected of containing one or more target nucleic acids, a sample buffer comprising a buffer, a chaotropic salt and a nonionic surfactant, an amount of lysing particles and an amount of nucleic acid-binding magnetic particles, and placing the sample, the sample buffer, the lysing particles and the nucleic acid-binding magnetic particles in the first container to form a lysate mixture. The method further comprises the following steps: bead milling the lysate mixture in the first container for a period of time sufficient to produce a lysate, wherein the bead milling is performed in the presence of nucleic acid-binding magnetic particles, capturing magnetic particles from the lysate with a magnet and transferring the magnetic particles or unbound lysate to a second container, and releasing the magnetic particles from the magnet and adding an elution buffer to the magnetic particles, mixing the magnetic particles with the elution buffer, re-capturing the magnetic particles with a magnet, and separating the elution buffer from the magnetic particles. The steps of the method can be suitably completed in <5 minutes, <4 minutes, <3 minutes, <2 minutes, or preferably in 1-3 minutes.

[0009] Another exemplary method for preparing a nucleic acid sample comprises the following steps: providing a sample container comprising a first container, providing a sample suspected of containing one or more target nucleic acids, a sample buffer comprising a buffer, a chaotropic salt and a nonionic surfactant, and a certain amount of lysing particles, combining the sample, the sample buffer and the lysing particles to form a lysing mixture, and placing the lysing mixture in the first container. The method further comprises the following steps: bead milling the lysing mixture in the first container for a period of time sufficient to produce a lysate, adding a certain amount of nucleic acid-bound magnetic particles to the lysate, percussively beating the first container to break the bubbles and foam formed during bead milling and keep the magnetic particles suspended while allowing the lysing particles to settle, capturing magnetic particles from the lysate with a magnet and transferring the magnetic particles or unbound lysate to a second container, and releasing the magnetic particles from the magnet and adding an elution buffer to the magnetic particles, mixing the magnetic particles with the elution buffer, re-capturing the magnetic particles with a magnet, and separating the elution buffer from the magnetic particles. The steps of the method can be suitably completed in <5 minutes, <4 minutes, <3 minutes, <2 minutes, or preferably in 1-3 minutes. The nucleic acid binding magnetic particles may suitably be added to the lysate after bead beating, the nucleic acid binding magnetic particles may suitably be added to the sample before bead beating, or some nucleic acid binding magnetic particles may suitably be added before, during and / or after bead beating.

[0010] Another exemplary method for preparing a nucleic acid sample comprises the following steps: providing a sample container comprising a first container, providing a sample suspected of containing one or more target nucleic acids, a sample buffer comprising a buffer, a chaotropic salt and a nonionic surfactant, a certain amount of lysing particles, and a certain amount of nucleic acid-binding magnetic particles, placing the sample, sample buffer, lysing particles and nucleic acid-binding magnetic particles in the first container to form a lysing mixture, and bead milling the lysing mixture in the first container for a period of time sufficient to produce a lysate, wherein the bead milling is performed in the presence of nucleic acid-binding magnetic particles. The method further comprises the following steps: tapping the first container to break bubbles and foam formed during bead milling and keep the magnetic particles suspended while allowing the lysate particles to settle, capturing the magnetic particles from the lysate with a magnet and transferring the magnetic particles or unbound lysate to a second container, and releasing the magnetic particles from the magnet and adding an elution buffer to the magnetic particles, mixing the magnetic particles with the elution buffer, re-capturing the magnetic particles with a magnet, and separating the elution buffer from the magnetic particles. The steps of the method can be suitably completed in <5 minutes, <4 minutes, <3 minutes, <2 minutes, or preferably in 1-3 minutes. The nucleic acid binding magnetic particles may suitably be added to the sample prior to bead beating; however, the amount of nucleic acid binding magnetic particles may suitably be added before, during and / or after bead beating.

[0011] Another exemplary method for preparing nucleic acid samples includes the following steps: providing a sample suspected of containing one or more target nucleic acids, and a sample buffer comprising a buffer, a chaotropic salt and a nonionic surfactant, wherein the sample and the sample buffer are combined to form a first mixture, the first mixture and a certain amount of lysate particles are combined in the first chamber, and the first chamber bead mill is sufficient to produce a lysate for a period of time. The method further includes mixing a certain amount of nucleic acid binding magnetic particles into the lysate, capturing the magnetic particles with a magnet and transferring the magnetic particles or unbound lysate to the second chamber, washing the nucleic acid binding magnetic particles with a wash buffer, recapturing the magnetic particles with a magnet, and removing the wash buffer, releasing the magnetic particles and adding elution buffer to the magnetic particles, mixing the magnetic particles with the elution buffer, recapturing the magnetic particles with a magnet, and separating the elution buffer from the magnetic particles. Sample buffer, wash buffer and elution buffer can be suitably aqueous buffers, and the buffer does not contain organic solvents and alcohols. Preferably, sample buffer, wash buffer and elution buffer can be suitably provided as dry compositions, and each can be suitably rehydrated with aqueous rehydration fluids when in use. The steps of the method may suitably be completed in <5 minutes, <4 minutes, <3 minutes, <2 minutes, or preferably in 1-3 minutes.

[0012] Another exemplary method for preparing a nucleic acid sample comprises the following steps: providing a first chamber and a second chamber, providing a sample suspected of containing one or more target organisms and a sample buffer comprising a buffer, a chaotropic salt, and a nonionic surfactant, wherein the sample and the sample buffer are combined to form a first mixture, the first mixture and a certain amount of lysing particles are combined in the first chamber, and the first chamber is bead milled for a period of time sufficient to produce a lysate. The method further comprises mixing a certain amount of nucleic acid-bound magnetic particles into the lysate, capturing the magnetic particles with a magnet and removing the lysate and the lysate from the first chamber, releasing the magnetic particles from the magnet into the second chamber and washing the nucleic acid-bound magnetic particles with a wash buffer, recapturing the magnetic particles with a magnet, and removing the wash buffer from the second chamber, releasing the magnetic particles from the magnet and adding an elution buffer to the magnetic particles, mixing the magnetic particles with the elution buffer, recapturing the magnetic particles with a magnet, and separating the elution buffer from the magnetic particles. In one embodiment, the one or more target organisms may include a target nucleic acid, which can be used, for example, to identify the target organism. The nucleic acid-bound magnetic particles can be used to recover the target nucleic acid from the lysate. Sample buffer, wash buffer and elution buffer can be suitably aqueous buffer respectively, and this buffer does not contain organic solvent and alcohol.Preferably, sample buffer, wash buffer and elution buffer can be suitably provided as dry composition, and can be suitably rehydrated with aqueous rehydration fluid when using respectively.The step of the method can be suitably completed in <5 minutes, <4 minutes, <3 minutes, <2 minutes or preferably in 1-3 minutes.

[0013] Another exemplary method of preparing a nucleic acid sample includes the following steps: providing a sample container, the sample container including a first chamber and a second chamber connected by a plurality of channel fluids and a plurality of reagent wells, the plurality of dried reagent wells including at least a dried nucleic acid wash buffer and a dried nucleic acid elution buffer; and a device comprising: an opening for receiving the container (the opening including a bead mill positioned to interact with the container), one or more actuators configured to manipulate the container to move fluid in the container, and a magnet for capturing magnetic particles in the container. The method further includes providing a sample suspected of containing one or more target nucleic acids and a sample buffer comprising a buffer, a chaotropic agent, and a non-ionic surfactant, wherein the sample and the sample buffer are combined to form a first mixture, the first mixture is combined with a certain amount of lysed particles in the first chamber of the container, a rehydration fluid is introduced into the container and the dried nucleic acid wash buffer and the dried nucleic acid elution buffer are rehydrated, and the container is placed in the opening of the device, and a first reaction blister is contacted with the bead mill for a period of time sufficient to produce a lysate. The method further includes mixing a certain amount of nucleic acid-binding magnetic particles into the lysate, activating a magnet and an actuator associated with the first chamber to recover the nucleic acid-binding magnetic particles from the lysate and deposit the nucleic acid-binding magnetic particles into a second chamber, transferring a first amount of rehydrated wash buffer to the nucleic acid-binding magnetic particles for washing, wherein washing includes stopping the magnet, agitating the nucleic acid-binding magnetic particles with an actuator associated with the second chamber, reactivating the magnet to recapture the nucleic acid-binding magnetic particles, and plunging the wash buffer into the first chamber with the actuator associated with the second chamber, and eluting nucleic acids from the nucleic acid-binding magnetic particles by transferring a certain amount of rehydrated elution buffer to the nucleic acid in the second and / or third reaction bubbles, wherein elution includes stopping the magnet, agitating the nucleic acid-binding magnetic particles with an actuator associated with the second chamber, reactivating the magnet to recapture the nucleic acid-binding magnetic particles, and plunging the elution buffer with the eluted nucleic acids therein into the third chamber or reagent well. Sample buffer, wash buffer and elution buffer can be suitably aqueous buffer respectively, and this buffer does not contain organic solvent and alcohol.Preferably, sample buffer, wash buffer and elution buffer can be suitably provided as dry composition, and can be suitably rehydrated with aqueous rehydration fluid when using respectively.The step of the method can be suitably completed in <5 minutes, <4 minutes, <3 minutes, <2 minutes or preferably in 1-3 minutes.

[0014] The methods described herein include the step of mixing a certain amount of nucleic acid-binding magnetic particles into the lysate. These nucleic acid-binding magnetic particles (e.g., silica-coated magnetic particles) can be added before bead milling (i.e., so that the lysate is formed in the presence of the magnetic particles), after bead milling, or in a combination of before and after bead milling.

[0015] It describes:

[0016] A1. A method for preparing a nucleic acid sample, comprising: providing a sample container comprising a first container, providing a sample suspected of containing one or more target nucleic acids, a sample buffer comprising a buffer, a chaotropic salt and a non-ionic surfactant, a certain amount of lysing particles, and a certain amount of nucleic acid-binding magnetic particles, placing a sample, a sample buffer, lysis particles, and nucleic acid-binding magnetic particles in a first container to form a lysis mixture, bead-milling the lysis mixture in the first container for a period of time sufficient to produce a lysate, wherein the bead-milling is performed in the presence of the nucleic acid-binding magnetic particles, capturing the magnetic particles from the lysate with a magnet and transferring the magnetic particles or unbound lysate to a second container, and releasing the magnetic particles from the magnet and adding an elution buffer to the magnetic particles, mixing the magnetic particles with the elution buffer, recapturing the magnetic particles with a magnet, and separating the elution buffer from the magnetic particles, Wherein the steps of the method are completed in <5 minutes.

[0017] A2. The method of clause A1, wherein one or more of the sample, sample buffer, lysis particles, and magnetic particles are combined in a first container to form a lysis mixture.

[0018] A3. The method of one of clauses A1 or A2, further comprising releasing the magnetic particles from the magnet and washing the magnetic particles with a wash buffer, recapturing the magnetic particles with a magnet and removing the wash buffer, and releasing the magnetic particles into an elution buffer after washing.

[0019] A3.1. The method of claim A3, wherein washing does not include one or more of: heating the wash buffer and magnetic particles before or during washing, vigorously mixing the magnetic particles and wash buffer, or incubating the magnetic particles and wash buffer for a period of time greater than 1 second, 2 seconds, 3 seconds, 4 seconds, 5 seconds, 6 seconds, 7 seconds, 8 seconds, 9 seconds, 10 seconds, or any time in between.

[0020] A4. The method of any one of clauses A1 to A3, further comprising mixing a second amount of magnetic particles into the lysate after bead beating and before capturing.

[0021] A5. The method of any of clauses A1-A4, wherein the time period for bead beating is in the range of 10 seconds to 1 minute.

[0022] A6. The method of any of clauses A1-A5, wherein the time period for bead beating is 20 seconds.

[0023] A7. The method of any one of clauses A1-A6, wherein the nucleic acid binding magnetic particles are silica-coated magnetic particles.

[0024] A8. The method of any one of clauses A1-A7, wherein the nucleic acid-bound magnetic particles are not substantially degraded by bead beating.

[0025] A9. The method of any of clauses A1-A8, wherein the sample buffer is an aqueous buffer comprising a buffer, 50-60% of a chaotropic agent and 10-20% of a non-ionic surfactant.

[0026] A10. A method according to any of clauses A1-A9, wherein the chaotropic agent is a guanidine salt and the nonionic surfactant is one of TritonX-100, polydocanol (Thesit), Triton X-114, NP-40, Arlasolve 200, Brij O10, octyl β-D-pyranoglucoside, saponin, monododecyl nonaglycone ether and a combination thereof.

[0027] A11. The method of any of clauses A1-A10, wherein the wash buffer and the elution buffer are provided as dried buffer compositions and are rehydrated during the steps of the method.

[0028] A12. The method of any of clauses A1-A11, wherein separating the elution buffer from the magnetic particles comprises recapturing the magnetic particles with a magnet and transferring the elution buffer to a third chamber.

[0029] A13. The method of any of clauses A1-A12, wherein separating the elution buffer from the magnetic particles comprises recapturing the magnetic particles with a magnet and transferring the magnetic particles to another chamber.

[0030] A14. The method of any of clauses A1-A13, wherein the steps of the method are completed in <4 minutes.

[0031] A15. The method of any of clauses A1-A14, wherein the steps of the method are completed in <3 minutes.

[0032] A16. The method of any of clauses A1-A15, wherein the steps of the method are completed in <2 minutes.

[0033] A17. The method of any one of clauses A1-A16, wherein the steps of the method are completed within 1-3 minutes.

[0034] A18. The method of any of clauses A1-A17, wherein the elution buffer is heated before adding the elution buffer to the magnetic particles.

[0035] A19. The method of any of clauses A1-A18, further comprising agitating the lysate to keep the magnetic particles suspended while allowing the lysed particles to settle.

[0036] A20. The method of any one of clauses A1-A19, wherein the sample buffer, wash buffer and elution buffer are each aqueous buffers and are free of organic solvents and alcohols.

[0037] A21. The method of any of clauses A1-A20, wherein the sample buffer, wash buffer, and elution buffer are provided as dried compositions and each buffer is configured to be rehydrated with an aqueous rehydration fluid.

[0038] B1. A method for preparing a nucleic acid sample, comprising: providing a sample container comprising a first container, providing a sample suspected of containing one or more target nucleic acids, a sample buffer comprising a buffer, a chaotropic salt and a non-ionic surfactant, and a quantity of lysing particles, combining the sample, sample buffer, and lysing particles to form a lysis mixture, placing the lysis mixture in a first container, bead milling the lysis mixture in the first container for a period of time sufficient to produce a lysate, Add a certain amount of nucleic acid-binding magnetic particles to the lysate. tapping the first container to break bubbles and foam formed during bead beating and to keep the magnetic particles suspended while allowing the lysed particles to settle, capturing the magnetic particles from the lysate with a magnet and transferring the magnetic particles or unbound lysate to a second container, and releasing the magnetic particles from the magnet and adding an elution buffer to the magnetic particles, mixing the magnetic particles with the elution buffer, recapturing the magnetic particles with a magnet, and separating the elution buffer from the magnetic particles, Wherein the steps of the method are completed in <5 minutes.

[0039] B2. The method of clause B1, wherein tapping comprises slapping or hitting the first container.

[0040] B3. The method of clause B1 or B2, wherein striking comprises inflating the inflatable member against the first container in a time range of 1-250 milliseconds.

[0041] B4. The method of one of clauses B1-B3, wherein one or more of the sample, the sample buffer, and the lysing particles are combined in a first container to form a lysis mixture.

[0042] B5. The method of any one of clauses B1 to B4, further comprising releasing the magnetic particles from the magnet and washing the magnetic particles with a wash buffer, recapturing the magnetic particles with a magnet, and removing the wash buffer, and

[0043] After washing the magnetic particles are released into the elution buffer.

[0044] B5.1. The method of claim B5, wherein washing does not include one or more of: heating the wash buffer and magnetic particles before or during washing, vigorously mixing the magnetic particles and wash buffer, or incubating the magnetic particles and wash buffer for a period of time greater than 1 second, 2 seconds, 3 seconds, 4 seconds, 5 seconds, 6 seconds, 7 seconds, 8 seconds, 9 seconds, 10 seconds, or any time in between.

[0045] B6. The method of any of clauses B1 to B5, further comprising mixing an amount of magnetic particles into the lysis mixture prior to bead beating, and optionally, mixing a second amount of magnetic particles into the lysate after bead beating and prior to capture.

[0046] B7. The method of one of clauses B1-B6, wherein the time period for bead milling is in the range of 1 second to 1 minute.

[0047] B8. The method of any of clauses B1-B7, wherein the time period for bead beating is 20 seconds.

[0048] B9. The method of any of clauses B1-B8, wherein the sample buffer is an aqueous buffer comprising a buffer, 50-60% of a chaotropic agent and 10-20% of a non-ionic surfactant.

[0049] B10. A method according to any of clauses B1-B9, wherein the chaotropic agent is a guanidine salt and the nonionic surfactant is one of Triton X-100, polydocanol (Thesit), Triton X-114, NP-40, Arlasolve 200, Brij O10, octyl β-D-pyranoglucoside, saponin, monododecyl nonaglycone ether and a combination thereof.

[0050] B11. The method of any of clauses B1 to B10, wherein capture of the magnetic particles from the lysate is facilitated by bursting bubbles and foam formed during bead beating and by keeping the magnetic particles suspended while allowing the lysed particles to settle.

[0051] B12. The method of any of clauses B1-B11, wherein the steps of the method are completed in <4 minutes.

[0052] B13. The method of any of clauses B1-B12, wherein the steps of the method are completed in <3 minutes.

[0053] B14. The method of any of clauses B1-B13, wherein the steps of the method are completed in <2 minutes.

[0054] B15. The method of any one of clauses B1-B14, wherein the steps of the method are completed within 1-3 minutes.

[0055] B16. The method of any of clauses B1-B15, wherein the elution buffer is heated before adding the elution buffer to the magnetic particles.

[0056] B17. The method of any one of clauses B1-B16, wherein the sample buffer, wash buffer and elution buffer are each aqueous buffers and are free of organic solvents and alcohols.

[0057] B18. The method of any of clauses B1-B17, wherein the sample buffer, wash buffer, and elution buffer are provided as dried compositions and each buffer is configured to be rehydrated with an aqueous rehydration fluid.

[0058] C1. A method for preparing a nucleic acid sample, comprising: providing a sample container including a first container, providing a sample suspected of containing one or more target nucleic acids, a sample buffer comprising a buffer, a chaotropic salt and a non-ionic surfactant, a certain amount of lysing particles, and a certain amount of nucleic acid-binding magnetic particles, placing a sample, a sample buffer, lysis particles, and nucleic acid-binding magnetic particles in a first container to form a lysis mixture, bead-milling the lysis mixture in the first container for a period of time sufficient to produce a lysate, wherein the bead-milling is performed in the presence of the nucleic acid-binding magnetic particles, tapping the first container to break bubbles and foam formed during bead beating and to keep the magnetic particles suspended while allowing the lysed particles to settle, capturing the magnetic particles from the lysate with a magnet and transferring the magnetic particles or unbound lysate to a second container, and releasing the magnetic particles from the magnet and adding an elution buffer to the magnetic particles, mixing the magnetic particles with the elution buffer, recapturing the magnetic particles with a magnet, and separating the elution buffer from the magnetic particles, Wherein the steps of the method are completed in <5 minutes.

[0059] C2. The method of clause C1, wherein capture of the magnetic particles from the lysate is facilitated by bursting bubbles and foam formed during bead beating and by keeping the magnetic particles suspended while allowing the lysed particles to settle.

[0060] C3. The method of clause C1 or C2, wherein tapping comprises slapping or hitting the first container.

[0061] C4. The method of any of clauses C1 to C3, wherein striking comprises inflating the inflatable member against the first container in a time range of 1-250 milliseconds.

[0062] C5. The method of any one of clauses C1 to C4, further comprising mixing a second amount of magnetic particles into the lysate after bead beating and before capturing.

[0063] C6. The method of any of clauses C1-C5, wherein one or more of the sample, sample buffer, lysis particles, and magnetic particles are combined in a first container to form a lysis mixture.

[0064] C7. The method of any one of clauses C1 to C6, further comprising releasing the magnetic particles from the magnet and washing the magnetic particles with a wash buffer, recapturing the magnetic particles with a magnet, and removing the wash buffer, and

[0065] After washing the magnetic particles are released into the elution buffer.

[0066] C7.1. The method of claim C7, wherein washing does not include one or more of: heating the wash buffer and magnetic particles before or during washing, vigorously mixing the magnetic particles and wash buffer, or incubating the magnetic particles and wash buffer for a period of time greater than 1 second, 2 seconds, 3 seconds, 4 seconds, 5 seconds, 6 seconds, 7 seconds, 8 seconds, 9 seconds, 10 seconds, or any time in between.

[0067] C8. The method of any of clauses C1-C7, wherein the time period for bead milling is in the range of 1 second to 1 minute.

[0068] C9. The method of any of clauses C1-C8, wherein the time period for bead beating is 20 seconds.

[0069] C10. The method of any one of clauses C1 to C9, wherein the nucleic acid binding magnetic particles are silica-coated magnetic particles.

[0070] C11. The method of any one of clauses C1 to C10, wherein the nucleic acid-binding magnetic particles are not sufficiently degraded by bead beating to reduce efficiency.

[0071] C12. The method of any of clauses C1-C11, wherein the sample buffer is an aqueous buffer comprising a buffering agent, 50-60% of a chaotropic agent and 10-20% of a non-ionic surfactant.

[0072] C13. A method according to any of clauses C1-C12, wherein the chaotropic agent is a guanidine salt and the nonionic surfactant is one of Triton X-100, polydocanol (Thesit), Triton X-114, NP-40, Arlasolve 200, Brij O10, octyl β-D-pyranoglucoside, saponin, monododecyl nonaglycone ether and combinations thereof.

[0073] C14. The method of any of clauses C1-C13, wherein the steps of the method are completed in <4 minutes.

[0074] C15. The method of any of clauses C1-C14, wherein the steps of the method are completed in <3 minutes.

[0075] C16. The method of any of clauses C1-C15, wherein the steps of the method are completed in <2 minutes.

[0076] C17. The method of any of clauses C1-C16, wherein the steps of the method are completed within 1-3 minutes.

[0077] C18. The method of any of clauses C1-C17, wherein the elution buffer is heated before adding the elution buffer to the magnetic particles.

[0078] C19. The method of any one of clauses C1-C18, wherein the sample buffer, the wash buffer, and the elution buffer are each aqueous buffers and are free of organic solvents and alcohols.

[0079] C20. The method of any of clauses C1-C19, wherein the sample buffer, wash buffer, and elution buffer are provided as dried compositions and each buffer is configured to be rehydrated with an aqueous rehydration fluid.

[0080] D1. A method for preparing a nucleic acid sample, comprising: providing a sample container comprising a first chamber, providing a sample suspected of containing one or more target nucleic acids and a sample buffer comprising a buffering agent, a chaotropic salt, and a nonionic surfactant, wherein the sample and the sample buffer are combined to form a first mixture, combining a first mixture and an amount of lysing particles in a first chamber, The first chamber is beaded for a period of time sufficient to produce a lysate, A certain amount of nucleic acid-bound magnetic particles is mixed into the lysate, the magnetic particles are captured with a magnet and the magnetic particles or unbound lysate are transferred to a second chamber, releasing the magnetic particles from the magnet and washing the magnetic particles with a wash buffer, recapturing the magnetic particles with a magnet, and removing the wash buffer, releasing the magnetic particles and adding elution buffer to the magnetic particles, mixing the magnetic particles with the elution buffer, recapturing the magnetic particles with a magnet, and separating the elution buffer from the magnetic particles, wherein the steps of the method are completed in < 5 minutes, and The sample buffer, the washing buffer and the elution buffer are each an aqueous buffer, and the buffer does not contain an organic solvent and alcohol.

[0081] D2. The method of clause D1, wherein the sample buffer is an aqueous buffer comprising a buffer, 50-60% of a chaotropic agent and 10-20% of a non-ionic surfactant.

[0082] D3. The method of clause D1 or D2, wherein the chaotropic agent is a guanidine salt and the nonionic surfactant is one of Triton X-100, Thesit, Triton X-114, NP-40, Arlasolve 200, Brij O10, octyl β-D-pyranoglucoside, saponin, monododecyl nonaglycone ether and combinations thereof.

[0083] D4. The method of any one of clauses D1-D3, wherein the wash buffer and the elution buffer are provided as dried buffer compositions and are rehydrated during the steps of the method.

[0084] D5. The method of any one of clauses D1-D4, wherein separating the elution buffer from the magnetic particles comprises recapturing the magnetic particles with a magnet and transferring the elution buffer to a third chamber.

[0085] D6. The method of any one of clauses D1-D5, wherein separating the elution buffer from the magnetic particles comprises recapturing the magnetic particles with a magnet and transferring the magnetic particles to another chamber.

[0086] D7. The method of any of clauses D1-D6, wherein the steps of the method are completed in <4 minutes.

[0087] D8. The method of any of clauses D1-D7, wherein the steps of the method are completed in <3 minutes.

[0088] D9. The method of any of clauses D1-D8, wherein the steps of the method are completed in <2 minutes.

[0089] D10. The method of any one of clauses D1-D9, wherein the steps of the method are completed within 1-3 minutes.

[0090] D11. The method of any of clauses D1-D10, wherein the bead beating time sufficient to produce the lysate is in the range of 1 second to 1 minute.

[0091] D12. The method of any of clauses D1-D11, wherein the elution buffer is heated.

[0092] D13. The method of any one of clauses D1-D12, wherein mixing a certain amount of nucleic acid-binding magnetic particles into the lysate comprises one or more of the following: mixing a certain amount of nucleic acid-binding magnetic particles into the first mixture before bead beating, and mixing a certain amount of nucleic acid-binding magnetic particles into the lysate after bead beating.

[0093] E1. A method for preparing a nucleic acid sample, comprising: providing a first chamber and a second chamber, providing a sample suspected of containing one or more target organisms and a sample buffer comprising a buffering agent, a chaotropic salt, and a nonionic surfactant, wherein the sample and the sample buffer are combined to form a first mixture, combining a first mixture and an amount of lysing particles in a first chamber, The first chamber is beaded for a period of time sufficient to produce a lysate, mixing a certain amount of nucleic acid-binding magnetic particles into the lysate, capturing the magnetic particles with a magnet and removing the lysate particles and the lysate from the first chamber, releasing the magnetic particles from the magnet into a second chamber and washing the nucleic acid-bound magnetic particles with a wash buffer, recapturing the magnetic particles with the magnet, and removing the wash buffer from the second chamber, releasing the magnetic particles from the magnet and adding an elution buffer to the magnetic particles, mixing the magnetic particles with the elution buffer, recapturing the magnetic particles with the magnet, and separating the elution buffer from the magnetic particles, wherein the steps of the method are completed in < 5 minutes, and The sample buffer, the washing buffer and the elution buffer are each an aqueous buffer, and the buffer does not contain an organic solvent and alcohol.

[0094] E2. The method of clause E1, wherein the sample buffer is an aqueous buffer comprising a buffer, 50-60% of a chaotropic agent and 10-20% of a non-ionic surfactant.

[0095] E3. The method of clause E1 or E2, wherein the chaotropic agent is a guanidine salt and the nonionic surfactant is one of Triton X-100, Thesit, Triton X-114, NP-40, Arlasolve 200, Brij O10, octyl β-D-pyranoglucoside, saponin, monododecyl nonaglycone ether and combinations thereof.

[0096] E4. The method of any one of clauses E1-E3, wherein the wash buffer and elution buffer are provided as dried buffer compositions and are rehydrated as part of the steps of the method.

[0097] E5. The method of any of clauses E1-E4, wherein separating the elution buffer from the magnetic particles comprises recapturing the magnetic particles with a magnet and transferring the elution buffer to a third chamber.

[0098] E6. The method of any of clauses E1-E5, wherein separating the elution buffer from the magnetic particles comprises recapturing the magnetic particles with a magnet and transferring the magnetic particles to another chamber.

[0099] E7. The method of any of clauses E1-E6, wherein the steps of the method are completed in <4 minutes.

[0100] E8. The method of any of clauses E1-E7, wherein the steps of the method are completed in <3 minutes.

[0101] E9. The method of any of clauses E1-E8, wherein the steps of the method are completed in <2 minutes.

[0102] E10. The method of any one of clauses E1-E9, wherein the steps of the method are completed within 1-3 minutes.

[0103] E11. The method of any of clauses E1-E10, wherein the bead beating time sufficient to produce the lysate is in the range of about 10 seconds to about 1 minute.

[0104] E12. The method of any one of clauses E1-E11, wherein mixing a certain amount of nucleic acid-binding magnetic particles into the lysate comprises one or more of the following: mixing a certain amount of nucleic acid-binding magnetic particles into the first mixture before bead beating, and mixing a certain amount of nucleic acid-binding magnetic particles into the lysate after bead beating.

[0105] E13. The method of any of clauses E1-E12, wherein the bead milling step further comprises applying pressure on the sample container sufficient to increase frictional heating to aid thermal lysis.

[0106] E14. The method of any of clauses E1-E13, wherein the pressure is regulated using a feedback mechanism.

[0107] E15. The method of any of clauses E1-E14, further comprising, after the bead milling step, providing an impact pressure on the sample container to force the foam to the top of the sample container and collapse the foam bubbles.

[0108] E16. The method of any of clauses E1-E15, wherein the elution buffer is heated.

[0109] F1. A method for preparing a nucleic acid sample, comprising: providing a sample container, the sample container comprising a first chamber and a second chamber connected by a plurality of channel fluids and a plurality of reagent wells, the plurality of dried reagent wells comprising at least a dried nucleic acid wash buffer and a dried nucleic acid elution buffer, a device comprising: an opening for receiving the container (the opening comprising a bead beater positioned to interact with the container), one or more actuators configured to manipulate the container to move fluid in the container, and a magnet for capturing magnetic particles in the container, providing a sample suspected of containing one or more target nucleic acids and a sample buffer comprising a buffering agent, a chaotropic agent and a non-ionic surfactant, wherein the sample and the sample buffer are combined to form a first mixture, combining the first mixture with an amount of lysing particles in a first chamber of the container, introducing a rehydration fluid into the container and rehydrating the dried nucleic acid wash buffer and the dried nucleic acid elution buffer, placing the container in an opening of the device, and contacting the first reaction bubble with the bead beater for a period of time sufficient to produce a lysate, A certain amount of nucleic acid-binding magnetic particles is mixed into the lysate. activating a magnet and an actuator associated with the first chamber to recover the nucleic acid binding magnetic particles from the lysate and deposit the nucleic acid binding magnetic particles into a second chamber, transferring a first amount of rehydrated wash buffer to the nucleic acid binding magnetic particles for washing, wherein washing comprises deactivating the magnet, agitating the nucleic acid binding magnetic particles with an actuator associated with the second chamber, reactivating the magnet to recapture the nucleic acid binding magnetic particles, and pushing the wash buffer into the first chamber with the actuator associated with the second chamber, and eluting nucleic acids from the nucleic acid binding magnetic particles by transferring an amount of rehydrated elution buffer to the nucleic acid binding in the second and / or third reaction blisters, wherein eluting comprises deactivating the magnet, agitating the nucleic acid binding magnetic particles with an actuator associated with the second chamber, reactivating the magnet to recapture the nucleic acid binding magnetic particles, and pushing the elution buffer with the eluted nucleic acids therein into the third chamber or reagent well, wherein the steps of the method are completed in < 5 minutes, and The sample buffer, the washing buffer and the elution buffer are each an aqueous buffer and the buffer does not contain an organic solvent and alcohol.

[0110] F2. The method of clause F1, wherein the sample buffer is an aqueous buffer comprising a buffer, 50-60% of a chaotropic agent and 10-20% of a non-ionic surfactant.

[0111] F3. The method of clause F1 or F2, wherein the chaotropic agent is a guanidine salt and the nonionic surfactant is one of Triton X-100, Thesit, Triton X-114, NP-40, Arlasolve 200, Brij O10, octyl β-D-pyranoglucoside, saponin, monododecyl nonaglycone ether and combinations thereof.

[0112] F4. The method of any of clauses F1-F3, wherein the steps of the method are completed in <4 minutes.

[0113] F5. The method of any of clauses F1-F4, wherein the steps of the method are completed in <3 minutes.

[0114] F6. The method of any of clauses F1-F5, wherein the steps of the method are completed in <2 minutes.

[0115] F7. The method of any one of clauses F1-F6, wherein the steps of the method are completed within 1-3 minutes.

[0116] F8. The method of any of clauses F1-F7, wherein the bead beating time sufficient to produce the lysate is in the range of about 5 seconds to about 1 minute.

[0117] F9. The method of any one of clauses F1-F8, wherein mixing a certain amount of nucleic acid-binding magnetic particles into the lysate comprises one or more of the following: mixing a certain amount of nucleic acid-binding magnetic particles into the first mixture before bead beating, and mixing a certain amount of nucleic acid-binding magnetic particles into the lysate after bead beating.

[0118] F10. The method of any of clauses F1-F9, wherein the elution buffer is heated.

[0119] F11. The method of any one of clauses F1-F10, wherein the wash buffer further comprises a binding buffer.

[0120] This summary is provided to introduce a series of concepts in a simplified form, which will be further described in the detailed description below. This summary is not intended to identify key features or essential features of the claimed subject matter, nor is it intended to be used as an aid in determining the scope of the claimed subject matter.

[0121] Additional features and advantages will be set forth in the following description, and in part will be apparent from the description, or may be understood through the practice of the invention. These features and advantages may be realized and obtained by means of the instruments and combinations particularly pointed out in the appended claims. These and other features will become more fully apparent from the following description and the appended claims, or may be understood through the practice of the invention as described below. BRIEF DESCRIPTION OF THE DRAWINGS

[0122] Figure 1 A flexible bag for self-contained PCR is shown.

[0123] Figure 2 is with Figure 1 An exploded perspective view of an instrument used with the bag, including Figure 1 bag.

[0124] Figure 3 Shows Figure 1 Bags and Figure 2 The capsule component.

[0125] Figure 4 Shown in Figure 2 A motor is used in one illustrative embodiment of an apparatus.

[0126] Figures 5A-5J The steps of the method for cell lysis and nucleic acid recovery are shown.

[0127] Figure 6 is a bar graph showing sample preparation time.

[0128] Fig. 7A is a bar graph showing the detection of various concentrations of DNA organisms in the presence (BB w / MB) or absence (BB w / o MB) of silica-coated magnetic particles during bead beating lysis.

[0129] Figure 7B is a bar graph showing the detection of various concentrations of RNA organisms in the presence (BB w / MB) or absence (BB w / o MB) of silica-coated magnetic particles during bead beating lysis.

[0130] Figure 8 is a graph showing the average ΔCp of DNA and RNA organisms at various concentrations in the presence of silica-coated magnetic particles during bead beating lysis.

[0131] Fig. 9 Shown is a comparison of the Cp for amplification of nucleic acids recovered from magnetic particles contained in lysates that were not washed, washed once, or washed twice.

[0132] Fig. 10A Shown are the average Cp responses for the elution temperatures for DNA and RNA assays.

[0133] Fig. 10B and 10C The Cp response to elution temperature for DNA and RNA assays is shown separately, with the best fit line showing the trend of the data.

[0134] Fig.11Shown are the fragment sizes of human genomic DNA detected with or without bead beating for 120 seconds. DETAILED DESCRIPTION

[0135] Exemplary embodiments are described below with reference to the accompanying drawings. Without departing from the spirit and teachings of the present disclosure, many different forms and embodiments are possible, and therefore the present disclosure should not be construed as being limited to the exemplary embodiments described herein. On the contrary, these exemplary embodiments are provided so that the present disclosure will be thorough and complete, and the scope of the present disclosure will be conveyed to those skilled in the art. In the accompanying drawings, the size and relative size of layers and regions may be exaggerated for clarity. Throughout the specification, the same reference numerals refer to the same elements.

[0136] Unless otherwise defined, all terms used herein (including technical and scientific terms) have the same meanings as those of ordinary skill in the art to which the present disclosure belongs. It should also be understood that terms (such as those defined in common dictionaries) should be interpreted as having the same meanings as those in the context of the present application and the related art, and should not be interpreted in an idealized or overly formal sense, unless explicitly defined in this article. The terms used in the invention description herein are only used to describe the purpose of a specific embodiment, and are not intended to limit the present invention. Although many methods and materials similar or equivalent to those described herein can be used in the practice of the present disclosure, only some exemplary materials and methods are described herein.

[0137] All publications, patent applications, patents, or other references mentioned herein are incorporated by reference in their entirety. In the event of a conflict in terminology, the present specification controls.

[0138] Various aspects of the disclosure, including devices, systems, methods, etc., may be described with reference to one or more exemplary embodiments. The terms "exemplary" and "illustrative" as used herein mean "serving as an example, instance, or illustration," and are not necessarily to be construed as preferred or advantageous over other embodiments disclosed herein. Furthermore, reference to an "embodiment" or "implementation" of the disclosure or invention includes specific reference to one or more embodiments thereof, and vice versa, and are intended to provide illustrative examples without limiting the scope of the invention, which is indicated by the appended claims rather than the following description.

[0139] It is noted that, as used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to "a tile" includes one, two, or more tile elements. Similarly, reference to multiple referents should be interpreted to include a single referent and / or multiple referents unless the content and / or context clearly dictate otherwise. Thus, reference to a "tile element" does not necessarily require a plurality of such tile elements. Rather, it should be understood that regardless of inversion; one or more tile elements are contemplated herein.

[0140] As used throughout this application, the words "can" and "may" are used in an optional sense (i.e., meaning there is a possibility) rather than a mandatory sense (i.e., meaning must). In addition, the terms "including", "having", "involving", "containing", "characterized by", variations thereof (e.g., "includes", "has", "involves", "contains", etc.), and similar terms as used herein (including the claims) shall be inclusive and / or open-ended and shall have the same meaning as the word "comprising" and variations thereof (e.g., "comprise" and "comprises"), and illustratively do not exclude additional, unrecited elements or method steps.

[0141] As used herein, directional and / or any terms, such as "top", "bottom", "left", "right", "up", "down", "upper", "lower", "inside", "outside", "inner", "external", "interior", "exterior", "proximal", "distal", "front", "back", etc. may be used only to indicate relative direction and / or orientation, and are not intended to limit the scope of the present disclosure (including the specification, invention and / or claims) in any other way.

[0142] It is to be understood that when an element is referred to as being "coupled," "connected," or "responsive to" or "on" another element, it can be directly coupled, connected, or responsive to or on another element, or intervening elements may also be present. In contrast, when an element is referred to as being "directly coupled," "directly connected," or "directly responsive to" or "directly on" another element, there are no intervening elements.

[0143] The exemplary embodiments of the present invention are described herein with reference to cross-sectional diagrams, which are schematic diagrams of idealized embodiments (and intermediate structures) of the exemplary embodiments. Therefore, changes in the shapes of the diagrams caused by, for example, manufacturing techniques and / or tolerances are expected. Therefore, the exemplary embodiments of the present invention should not be interpreted as being limited to the specific shapes of the regions shown herein, but include shape deviations caused by, for example, manufacturing. Therefore, the regions shown in the figures are schematic in nature, and their shapes are not intended to illustrate the actual shapes of the device regions, and are not intended to limit the scope of the exemplary embodiments.

[0144] It is to be understood that although the terms "first", "second", etc. may be used herein to describe various elements, these elements should not be limited by these terms. These terms are only used to distinguish one element from another element. Therefore, without departing from the teachings of the present embodiment, the "first" element may be referred to as the "second" element.

[0145] It is also understood that the various embodiments described herein can be used in combination with any other embodiments described or disclosed without departing from the scope of the present disclosure. Therefore, without departing from the scope of the present disclosure, the products, components, elements, devices, equipment, systems, methods, processes, compositions and / or kits according to certain embodiments of the present disclosure may include, incorporate or otherwise contain the properties, features, components, components, elements, steps and / or the like described in other embodiments (including systems, methods, equipment and / or the like) disclosed herein. Thus, the mention of a specific feature associated with an embodiment should not be interpreted as being limited to the application within the embodiment.

[0146] The headings used herein are for organizational purposes only and are not meant to limit the scope of the specification or claims. To facilitate understanding, the same reference numerals are used, where possible, to represent the same elements shared by the figures. In addition, the same element numbers are used, where possible, in the various figures. In addition, alternative configurations of a particular element may each include a separate letter in addition to the element number.

[0147] The term "about" is used herein to mean approximately, within the range of, roughly, or around. When the term "about" is used in conjunction with a numerical range, it modifies the range by extending the boundaries above and below the numerical values. Typically, the term "about" is used herein to modify the numerical values ​​of 5% variance above and below the value. When such a range is expressed, another embodiment includes from a specific value and / or to another specific value. Similarly, when a value is expressed as an approximation by using the antecedent "about", it should be understood that the specific value forms another embodiment. It is further understood that the endpoints of each range are effective relative to the other endpoint and independently of the other endpoint.

[0148] As used herein, the word "or" means any one member of a particular list, and also includes any combination of members of that list.

[0149] On the one hand, as described in further detail herein, can separate and detect the microorganism from sample or growth medium, to characterize and / or identify the microorganism present in sample.Term " separation " as used in this article is intended to contain any microorganism sample that has been removed, concentrated or otherwise separated from its original state or from growth medium or culture medium.For example, according to the present invention, can separate (for example as separated sample) from non-microorganism or non-microorganism component that otherwise may interfere with characterization and / or identification.This term can include the microorganism separated from mixture by centrifugation, filtration or any other separation technique known in the art.Thus, the microorganism sample of separation can include the microorganism and / or its component set that is more concentrated than original sample or otherwise separated from original sample, and scope can be from the dense clot of close packing of microorganism to the diffusion layer of microorganism.The non-microorganism component separated from microorganism can include non-microorganism cell (for example blood cell and / or other tissue cell) and / or any component thereof.On the one hand, separate microorganism from the lysate mixture that comprises non-microorganism cell of cracking and substantially intact microorganism cell.

[0150] In some embodiments, microbial sample is incomplete from its original state or from the separation in growth medium or culture medium.In other words, microorganism is removed, concentrated or otherwise separated from its original state and is not completely separated from other components of microbial sample and sample or from growth medium or culture medium.In some cases, there is from sample or from the trace fragment of growth medium or culture medium.For example, the amount of the fragment or growth medium or culture medium existing in the sample of separation can be not enough to disturb the identification or characterization of microorganism, or the further growth of microorganism.In some embodiments, the sample of separation is 99% pure of contaminating elements, but it can also be 95% pure, 90% pure, 80% pure, 70% pure, 60% pure, 50% pure or still allow to identify the minimum purity of the microorganism in the sample of separation via downstream identification technology.

[0151] Although the testing of microorganisms and viruses is mentioned, the methods provided herein can be used for a variety of sample types and a variety of nucleic acid tests. Thus, "sample" means an animal; a tissue or organ from an animal; a cell (in a subject, taken directly from a subject, or a cell maintained in culture or from a cultured cell line); a cell lysate (or lysate fraction) or a cell extract; a solution containing one or more molecules derived from cells, cellular materials, or viral materials; or other samples containing nucleic acids. The sample can also be any body fluid or excreta (for example, but not limited to, blood, urine, feces, saliva, tears, bile, or cerebrospinal fluid), which may or may not contain host or pathogen cells, cell components, or nucleic acids.

[0152] On the other hand, further described in detail herein, can precipitate and detect the microorganism from sample or growth medium to characterize and / or identify the microorganism present in the sample. As used herein, the term "precipitate (pellet)" is intended to contain any microbial sample that has been compressed or deposited into a microorganism mass. For example, the microorganism from the sample can be compressed or deposited into a mass at the bottom of the container by centrifugal or other known methods in the art. The term includes the collection of microorganisms (and / or its components) at the bottom and / or on the side of the container after centrifugation. According to the present invention, microorganisms can be precipitated out (for example, as a substantially purified microbial precipitate) from non-microorganisms or non-microorganism components that may otherwise interfere with characterization and / or identification.

[0153] As used herein, the phrase "nucleic acid" refers to a naturally occurring or synthetic oligonucleotide or polynucleotide, whether DNA or RNA or DNA-RNA hybrid, single-stranded or double-stranded, sense or antisense, which is capable of hybridizing to a complementary nucleic acid by Watson-Crick base pairing. The nucleic acids of the present invention may also include nucleotide analogs (e.g., BrdU) and non-phosphodiester internucleoside linkages (e.g., peptide nucleic acids (PNA) or thiodiester linkages). In particular, nucleic acids may include, but are not limited to, DNA, RNA, mRNA, rRNA, cDNA, gDNA, ssDNA, dsDNA, or any combination thereof.

[0154] "Probe", "primer" or "oligonucleotide" refers to a single-stranded nucleic acid molecule with a defined sequence that can base pair with a second nucleic acid molecule ("target") containing a complementary sequence. The stability of the resulting hybrid depends on the length, GC content, and the degree of base pairing that occurs. The degree of base pairing is affected 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 affected by parameters such as temperature, salt concentration, and the concentration of organic molecules (such as formamide), and is determined by methods known to those skilled in the art. Probes, primers, and oligonucleotides can be detectably labeled with radioactivity, fluorescence, or non-radioactivity by methods known to those skilled in the art. dsDNA binding dyes can be used to detect dsDNA. It is to be understood that a "primer" is specifically configured to be extended by a polymerase, while a "probe" or "oligonucleotide" may or may not be so configured.

[0155] "dsDNA binding dye" refers to a dye that fluoresces differentially when bound to double-stranded DNA, typically by fluorescing more strongly, than when bound to single-stranded DNA or free in solution. Although reference is made to dsDNA binding dyes, it is to be understood that any suitable dye may be used herein, some non-limiting exemplary dyes being described in U.S. Pat. No. 7,387,887, which is incorporated herein by reference. As is known in the art, other signal-generating substances may be used to detect nucleic acid amplification and melting, exemplified by enzymes, antibodies, and the like.

[0156] "Specifically hybridize" means that the probe, primer or oligonucleotide recognizes and physically interacts (ie, base pairs) with a substantially complementary nucleic acid (eg, a sample nucleic acid) under high stringency conditions, and does not substantially base pair with other nucleic acids.

[0157] "High stringency conditions" are generally those occurring at about the melting temperature (Tm) minus 5°C (ie, 5°C below the Tm of the probe). Functionally, high stringency conditions are used to identify nucleic acid sequences having at least 80% sequence identity.

[0158] "Lysing particles" refers to various particles or beads used to lyse cells, viruses, spores, and other materials that may be present in a sample. Various examples use zirconium silicate ("Zr") or ceramic beads, but other lysing particles are known and are within the scope of the term, including glass and sand lysing particles. As is known in the art, the term "cell lysis component" may include lysing particles, but may also include other components, such as components used for chemical lysis.

[0159] Although PCR is the amplification method used in the examples herein, it is understood that any amplification method using primers may be suitable. Such suitable procedures include polymerase chain reaction (PCR); strand displacement amplification (SDA); nucleic acid sequence-based amplification (NASBA); cascade rolling circle amplification (CRCA), loop-mediated DNA isothermal amplification (LAMP); isothermal and chimeric primer-initiated nucleic acid amplification (ICAN); target-based helicase-dependent amplification (HAD); transcription-mediated amplification (TMA), etc. Therefore, when the term PCR is used, it should be understood to include other alternative amplification methods. For amplification methods without discrete cycles, reaction time can be used, where it is measured in cycles, doubling times, or crossing points (Cp), and when additional PCR cycles are added in the embodiments described herein, additional reaction time can be added. It is understood that the scheme may need to be adjusted accordingly.

[0160] "Cp" or "crossing point" as used herein refers to the number of cycles or fractional cycles required for obtaining a fluorescent signal higher than a predetermined threshold for PCR. For example, a threshold value can be a point at which the measured fluorescence of a detection level or reaction reaches an intensity higher than the background fluorescence level. Cp can be experimentally determined based on a threshold value set manually, although other methods for determining Cp are known in the art. Other points can also be used, such as using a first-order, second-order or n-order derivative, illustratively as taught in U.S. Patent No. 6,303,305 (which is incorporated herein by reference in its entirety). As known in the art, other points can also be used, and any such point can replace Cp in any method discussed herein. The terms "Ct", "crossing threshold" and "Cq" are usually synonymous with crossover point (Cp), and these terms can be used interchangeably.

[0161] Although various examples herein refer to human targets and human pathogens, these examples are merely illustrative.The methods, kits and devices described herein can be used to detect or sequence a variety of nucleic acid sequences from a variety of samples, including human, veterinary, industrial and environmental samples.

[0162] Various embodiments disclosed herein illustratively use a self-contained nucleic acid analysis bag in a single closed system to determine the presence of various biological substances (illustratively antigens and nucleic acid sequences) in a sample. Such systems (including bags and instruments used with bags) are disclosed in more detail in U.S. Patent Nos. 8,394,608, which are incorporated herein by reference; and 8,895,295; and U.S. Patent No. 10,464,060. However, it is to be understood that such bags are merely exemplary, and the nucleic acid preparation and amplification reactions discussed herein can be performed in any of various open or closed system sample containers (including 96-well plates, plates of other configurations, arrays, turntables, etc.) as known in the art using various nucleic acid purification and amplification systems as known in the art. Although the terms "sample well", "sample container", "amplification well", "amplification container", etc. are used herein, these terms are intended to cover blister wells, tubes, and various other reaction containers as used in these amplification systems. In one embodiment, the bag is used to determine a variety of pathogens. The bag may illustratively include one or more blisters used as sample wells in a closed system. Illustratively, various steps can be performed in an optional disposable bag, including nucleic acid preparation, primary large-volume multiplex PCR, dilution of primary amplification products, and secondary PCR, and finally optional real-time detection or post-amplification analysis, such as melting curve analysis. In addition, it is to be understood that, although various steps can be performed in the bag of the present invention, one or more steps can be omitted for certain uses, and the bag configuration can be changed accordingly. Although many embodiments herein use multiplex reactions for the first stage amplification, it is to be understood that this is merely illustrative, and in some embodiments, the first stage amplification can be monoplex. In an illustrative example, the first stage monoplex amplification targets housekeeping genes, and the second stage amplification uses differences in housekeeping genes for identification. Thus, although various embodiments discuss the first stage multiplex amplification, it is to be understood that this is merely illustrative.

[0163] Figure 1 Shown is an exemplary bag 510 that can be used in various embodiments or can be reconfigured for various embodiments. Bag 510 is similar to Figure 15 of U.S. Patent No. 8,895,295, wherein the same item number is the same. Accessory 590 is provided with inlet channel 515a to 515l, which is also used as a reagent reservoir or waste reservoir. Illustratively, reagents can be freeze-dried in accessory 590 and rehydrated before use. Blisters 522, 544, 546, 548, 564 and 566 and their corresponding channels 514, 538, 543, 552, 553, 562 and 565 are similar to the blisters of the same number of Figure 15 of U.S. Patent No. 8,895,295. Figure 1The second stage reaction zone 580 of is similar to the second stage reaction zone of US Pat. No. 8,895,295, but the second stage wells 582 of the high density array 581 are arranged in a slightly different pattern. Figure 1 The more circular pattern of the high density array 581 eliminates holes in the corners and can result in a more uniform filling of the second stage holes 582. As shown, the high density array 581 is provided with 102 second stage holes 582. The bag 510 is suitable for Instrument (BioFire Diagnostics, LLC, Salt Lake City, UT). However, it is to be understood that this bag embodiment is merely exemplary.

[0164] Although other containers can be used, illustratively, bag 510 can be formed by two layers of flexible plastic films or other flexible materials, such as polyester, polyethylene terephthalate (PET), polycarbonate, polypropylene, polymethyl methacrylate, mixtures thereof, combinations and layers that can be manufactured by any method known in the art (including extrusion, plasma deposition and lamination). For example, each layer can be composed of a single type or more than one type of one or more layers of materials laminated together. Metal foil or plastics with aluminum lamination can also be used. Other barrier materials that can be sealed together to form blisters and passages are known in the art. If plastic film is used, these layers can be illustratively combined together by heat sealing. Exemplarily, the material has low nucleic acid binding capacity and low protein binding capacity.

[0165] For embodiments using fluorescence monitoring, plastic films with sufficiently low absorbance and autofluorescence at the operating wavelength are preferred. Such materials can be identified by testing different plastics, different plasticizers and compounding ratios, and different thicknesses of the film. For plastics with aluminum or other foil laminations, the portion of the bag to be read by the fluorescence detection device can be without foil. For example, if fluorescence is monitored in the second stage hole 582 of the second stage reaction zone 580 of the bag 510, one or two layers at the hole 582 will be without foil. In the example of PCR, a film laminate consisting of a polyester (Mylar, DuPont, Wilmington DE) of about 0.0048 inches (0.1219 mm) thick and a polypropylene film of 0.001-0.003 inches (0.025-0.076 mm) thick performs well. Illustratively, the bag 510 can be made of a transparent material capable of transmitting about 80%-90% of the incident light.

[0166] In exemplary embodiments, the material is moved between the blisters by applying pressure (illustratively pneumatic pressure) on the blisters and the channels. Thus, in embodiments that employ pressure, the bag material is illustratively sufficiently flexible to allow the pressure to have the desired effect. The term "flexible" is used herein to describe the physical properties of the bag material. The term "flexible" is defined herein as being easily deformable by the pressure levels used herein without cracking, breaking, crazing, etc. For example, a thin plastic sheet (such as Saran TM membrane and Bags) and thin metal foils (such as aluminum foil) are flexible. However, even in embodiments that employ pneumatic pressure, only certain areas of the blisters and channels need to be flexible. In addition, only one side of the blisters and channels need to be flexible, as long as the blisters and channels can be easily deformed. Other areas of the bag 510 can be made of rigid materials or can be reinforced with rigid materials. Therefore, it is to be understood that when the terms "flexible bag" or "flexible sample container" or the like are used, only portions of the bag or sample container need to be flexible.

[0167] Illustratively, plastic film can be used for bag 510. Sheets of metal (illustratively aluminum) or other suitable materials can be milled or otherwise cut to produce a mold with a raised surface pattern. When assembled to a pneumatic press (illustratively A-5302-PDS, Janesville Tool Inc., Milton WI) regulated illustratively at an operating temperature of 195°C, the pneumatic press works like a printing press, melting the sealing surface of the plastic film only at the place where the mold contacts the film. Similarly, laser cutting and welding devices can be used to cut and weld the plastic film used for bag 510. When forming bag 510, various components, such as PCR primers (illustratively sampled on the film and dried), antigen binding substrates, magnetic beads and zirconium silicate beads can be sealed in various blisters. Reagents for sample processing can be sampled on the film together or individually before sealing. In one embodiment, nucleoside triphosphates (NTPs) are spotted onto the membrane separately from the polymerase and primers, which essentially eliminates the activity of the polymerase until the reaction can be hydrated with an aqueous sample. If the aqueous sample is heated before hydration, this creates conditions for true hot-start PCR and reduces or eliminates the need for expensive chemical hot-start components. In another embodiment, the components can be provided in powder or pill form and placed in blisters before final sealing.

[0168] Bag 510 can be used in a manner similar to that described in U.S. Pat. No. 8,895,295. In an exemplary embodiment, 300 μl of a mixture containing a sample to be tested (100 μl) and a lysis buffer (200 μl) can be injected into an injection port (not shown) in fitting 590 near inlet channel 515a, and the sample mixture can be drawn into inlet channel 515a. Water can also be injected into a second injection port (not shown) in fitting 590 adjacent to inlet channel 515l and dispensed via a channel (not shown) provided in fitting 590, thereby hydrating up to eleven different reagents, each of which was previously provided in dry form at inlet channels 515b to 515l. Exemplary methods and apparatus for injecting samples and hydration fluids (e.g., water or buffer) are disclosed in U.S. Pat. No. 10,464,060, which is incorporated herein by reference in its entirety, but it is to be understood that these methods and apparatus are merely exemplary and that other ways of introducing samples and hydration fluids into bag 510 are within the scope of the present disclosure. Illustratively, these reagents may include freeze-dried PCR reagents, DNA extraction reagents, wash solutions, immunoassay reagents, or other chemical entities. Illustratively, the reagents are used for nucleic acid extraction, first stage multiplex PCR, dilution of multiplex reactions, and preparation of second stage PCR reagents and control reactions. Figure 1 In the embodiment shown in , all that needs to be injected is the sample solution in one injection port and water in the other injection port. After injection, both injection ports can be sealed. For more information on various configurations of bag 510 and accessory 590, see U.S. Patent No. 8,895,295, which has been incorporated by reference.

[0169] After injection, the sample can move from injection channel 515a to lysis bubble 522 via channel 514. Lysis bubble 522 is provided with beads or particles 534, such as ceramic beads or other grinding elements, and is configured for use with a device disposed at The rotating blade or blade in the instrument vortexes via collision. In the presence of cracking particles such as zirconium silicate (ZS) beads 534, bead milling by shaking, vortexing, ultrasonic treatment and similar sample treatment is an effective method for forming lysate. It is to be understood that terms such as "lyse", "lysing" and "lysate" as used herein are not limited to cell rupture, but such terms include the rupture of non-cellular particles (such as viruses). In another embodiment, a paddle beater using a reciprocating or alternating paddle, such as those described in US2019-0344269 (which is incorporated herein by reference in its entirety), can be used for the cleavage in this embodiment and in other embodiments described herein.

[0170] Figure 4 A bead mill motor 819 is shown, which includes a motor that can be mounted on Figure 2 800 is shown in the apparatus 800. The blade 821 on the first side 811 of the support member 802. The blade can extend through the slot 804 to contact the bag 510. However, it is understood that the motor 819 can be mounted on other structures of the apparatus 800. In an exemplary embodiment, the motor 819 is a Mabuchi RC-280SA-2865DC Motor (Chiba, Japan) mounted on the support member 802. In an exemplary embodiment, the motor rotates at 5,000 to 25,000 rpm, more exemplarily 10,000 to 20,000 rpm, and more exemplarily about 15,000 to 18,000 rpm. For the Mabuchi motor, it has been found that 7.2V provides sufficient rpm for lysis. However, it is understood that when the blade 821 hits the bag 510, the actual speed may be slightly slower. Depending on the motor and blade used, other voltages and speeds can be used for lysis. Optionally, a controlled small volume of air can be provided in the capsule 822 adjacent to the lysis bubble 522. It has been found that in some embodiments, partially filling the adjacent capsule with one or more small volumes of air helps to position and support the lysis bubble during the lysis process. Alternatively, another structure, illustratively a rigid or plastic gasket or other retaining structure around the lysis bubble 522, can be used to constrain the bag 510 during lysis. It is also understood that the motor 819 is only illustrative, and other devices can be used to grind, shake or vortex the sample. In some embodiments, chemicals or heat can also be used in addition to mechanical lysis, or chemicals or heat can be used instead of mechanical lysis.

[0171] Once the sample material has been fully cracked, the sample is moved to the nucleic acid extraction area, illustratively by passage 538, blister 544 and passage 543 to arrive blister 546, where the sample is mixed with nucleic acid binding material such as the magnetic beads 533 of silica coating. Alternatively, magnetic beads 533 can illustratively use the fluid rehydration provided by one of inlet channels 515c-515e, and then move to blister 544 by passage 543, and then move to blister 522 by passage 538. Make the mixture incubate the time of appropriate length, illustratively for about 10 seconds to 10 minutes. The retractable magnet in the instrument adjacent to blister 546 captures magnetic beads 533 from the solution, against the inner surface of blister 546 to form a precipitate. If incubated in blister 522, it may be necessary to move multiple solutions to blister 546 to capture. The liquid is then moved out of bubble 546 and back through bubble 544 and into bubble 522, which now serves as a waste receiver. One or more wash buffers from one or more of the injection channels 515c to 515e are provided to bubble 546 via bubble 544 and channel 543. Optionally, the magnet is retracted and the magnetic beads 533 are washed by moving the magnetic beads 533 back and forth from bubbles 544 and 546 via channel 543. Once the magnetic beads 533 are washed, the magnetic beads 533 are recaptured in bubble 546 by activating the magnet and then the wash solution is moved to bubble 522. This process can be repeated as needed to wash the lysis buffer and sample fragments from the nucleic acid binding magnetic beads 533.

[0172] After washing, the elution buffer stored at the injection channel 515f is moved to the bubble 548, and the magnet is retracted. The solution circulates between the bubbles 546 and 548 via the channel 552, destroying the precipitate of the magnetic beads 533 in the bubble 546, and allowing the captured nucleic acid to separate from the beads and enter the solution. The magnet is activated again, the magnetic beads 533 are captured in the bubble 546, and the eluted nucleic acid solution is moved to the bubble 548.

[0173] The first stage PCR premix from injection channel 515g is mixed with the nucleic acid sample in bubble 548. Optionally, the mixture is mixed by pushing the mixture between 548 and 564 through channel 553. After several mixing cycles, the solution is contained in bubble 564, a pellet of first stage PCR primers is provided in bubble 564, at least one primer set for each target, and the first stage multiplex PCR is performed. If RNA targets are present, a reverse transcription (RT) step can be performed before or simultaneously with the first stage multiplex PCR. The first stage multiplex PCR temperature cycle in the instrument illustratively performs 15-20 cycles, although other amplification levels may be desirable depending on the requirements of the specific application. The first stage PCR premix may be any of a variety of premixes as known in the art. In an illustrative example, the first stage PCR premix may be any chemistries disclosed in U.S. Patent No. 9,932,634 (which is incorporated herein by reference) for use with a PCR protocol that takes 20 seconds or less per cycle.

[0174] After the first stage PCR is performed for the desired number of cycles, the sample can be diluted, illustratively by forcing most of the sample back into bubble 548, leaving only a small amount of sample in bubble 564, and adding the second stage PCR premix from injection channel 515i. Alternatively, the dilution buffer from 515i can be moved to bubble 566 and then mixed with the amplified sample in bubble 564 by moving the fluid back and forth between bubbles 564 and 566. If desired, the dilution can be repeated several times using the dilution buffer from injection channels 515j and 515k, or injection channel 515k can be retained, illustratively for sequencing or for other post-PCR analysis, and the second stage PCR premix from injection channel 515h can then be added to some or all of the diluted amplified sample. It is to be understood that the dilution level can be adjusted by changing the number of dilution steps or by changing the percentage of sample discarded before mixing with dilution buffer or second stage PCR premix, which contains components for amplification, illustratively polymerase, dNTPs and suitable buffer, although other components may also be suitable, especially for non-PCR amplification methods. If desired, this mixture of sample and second stage PCR premix can be preheated in blister 564 before moving to second stage well 582 for second stage amplification. Such preheating can avoid the need for hot start components (antibodies, chemicals, etc.) in the second stage PCR mixture.

[0175] In one embodiment, the exemplary second stage PCR premix is ​​incomplete, lacking a primer pair, and each of the 102 second stage wells 582 is preloaded with a specific PCR primer pair. In other embodiments, the premix may lack other components (e.g., polymerase, Mg, 2+The second stage PCR premix can be preloaded with other reaction components, and these components can also be preloaded in the second stage holes 582. Each primer pair can be similar or identical to the first stage PCR primer pair, or can be nested in the first stage primer pair. The movement of the sample from the bubble cap 564 to the second stage holes 582 makes the PCR reaction mixture complete. As known in the art, once the high-density array 581 is filled, each second stage reaction is sealed in their corresponding second stage bubbles by any number of means. The exemplary mode of filling and sealing the high-density array 581 without cross contamination is discussed in the U.S. Patent No. 8,895,295 incorporated by reference. Exemplarily, the various reactions in the holes 582 of the high-density array 581 are exemplarily carried out thermal cycling with one or more Peltier devices simultaneously or individually, although other means for thermal cycling are known in the art.

[0176] In certain embodiments, the second stage PCR master mix contains a dsDNA binding dye Plus (BioFire Diagnostics, LLC) to generate a signal indicating amplification. However, it is to be understood that this dye is merely exemplary and other signals can be used, including other dsDNA binding dyes and fluorescent, radioactive, chemiluminescent, enzymatically labeled probes, etc. as known in the art. Alternatively, the wells 582 of the array 581 can be provided without a signal, wherein the results are reported by subsequent processing.

[0177] When pneumatic pressure is used to move the material within bag 510, in one embodiment, a "bladder" may be used. Bladder assembly 810 (a portion of which is shown in FIG. Figure 2-3 ) includes a bladder plate 824 that accommodates multiple inflatable bladders 822, 844, 846, 848, 864 and 866 (each bladder can be illustratively inflated separately by a compressed gas source). Because the bladder assembly 810 can withstand compressed gas and be used multiple times, the bladder assembly 810 can be made of a tougher or thicker material than the bag. Alternatively, bladders 822, 844, 846, 848, 864 and 866 can be formed by a series of plates fastened together with gaskets, seals, valves and pistons. Other arrangements are within the scope of the present invention. Alternatively, arrays or mechanical actuators and seals can be used to seal channels and guide the movement of fluids between blisters. A system of mechanical seals and actuators that can be suitable for the instrument described herein is described in detail in US2019-0344269, the entire contents of which have been incorporated by reference.

[0178] The success of the secondary PCR reaction depends on the template generated by the multiple first stage reactions. Typically, PCR is performed using highly purified DNA. Methods such as phenol extraction or commercial DNA extraction kits provide highly purified DNA. The sample processed by bag 510 may need to be adjusted to compensate for less pure preparations. PCR may be inhibited by components of biological samples, which is a potential obstacle. Exemplarily, hot start PCR, higher concentrations of Taq polymerase, adjustment of MgCl2 concentration, adjustment of primer concentrations, addition of engineered enzymes resistant to inhibitors, and addition of adjuvants (such as DMSO, TMSO or glycerol) may be optionally used to compensate for lower nucleic acid purity. Although purity issues may be more concerned in the first stage amplification, it is to be understood that similar adjustments may also be provided in the second stage amplification.

[0179] When the bag 510 is placed within the instrument 800, the bladder assembly 810 is pressed against one face of the bag 510 so that if a particular bladder is inflated, the pressure will force liquid out of the corresponding blister in the bag 510. In addition to bladders corresponding to the many blisters of the bag 510, the bladder assembly 810 may have additional pneumatic actuators corresponding to the various channels of the bag 510, such as bladders or air-driven pistons. Figure 2-3 Shown are exemplary multiple pistons or hard seals 838, 843, 852, 853 and 865 corresponding to the channels 538, 543, 553 and 565 of the bag 510, and seals 871, 872, 873, 874 that minimize backflow into the fitting 590. When activated, the hard seals 838, 843, 852, 853 and 865 form a pinch valve to pinch and close the corresponding channel. In order to confine the liquid in a specific bubble of the bag 510, the hard seal is activated on the channel leading to and from the bubble so that the actuator acts as a pinch valve to clamp the channel closed. Exemplarily, in order to mix two volumes of liquid in different bubbles, the pinch valve actuator that seals the connecting channel is activated, and the pneumatic capsule on the bubble is alternately pressurized, forcing the liquid to pass back and forth through the channel connecting the bubble to mix the liquid therein. The pinch valve actuator can have a variety of shapes and sizes and can be configured to pinch more than one channel at a time. Although pneumatic actuators are discussed herein, it is to be understood that other ways of providing pressure to the bag are contemplated, including various electromechanical actuators, such as linear stepper motors, motor-driven cams, rigid paddles driven by pneumatic, hydraulic, or electromagnetic forces, rollers, rocker arms, and in some cases, cocked springs. In addition, in addition to applying pressure perpendicular to the axis of the channel, there are a variety of methods for reversibly or irreversibly closing the channel. These methods include kinking the bag across the channel, heat sealing, rolling actuators, and various physical valves sealed in the channel, such as butterfly valves and ball valves. In addition, a small Peltier device or other temperature regulator can be placed adjacent to the channel and set at a temperature sufficient to freeze the fluid, effectively forming a seal. In addition, although Figure 1 The design is suitable for automated instruments that feature actuator elements positioned on each of the blisters and channels, and it is also contemplated that the actuators can remain stationary and the bag 510 can be translated, so that a small number of actuators can be used for several processing stations, including sample disruption, nucleic acid capture, first and second stage PCR, and processing stations for other applications of the bag 510 (such as immunoassays and immuno-PCR). Rollers acting on the channels and blisters may prove particularly useful in configurations where the bag 510 translates between stations. Thus, while pneumatic actuators are used in the presently disclosed embodiments, when the term "pneumatic actuator" is used herein, it is to be understood that other actuators and other ways of providing pressure may be used, depending on the configuration of the bag and instrument.

[0180] Back to Figure 2 Each pneumatic actuator is connected to a compressed air source 895 via a valve 899. Figure 2 Only a few hoses 878 are shown, it is to be understood that each pneumatic fitting is connected to a compressed gas source 895 via a hose 878. The compressed gas source 895 can be a compressor, or alternatively, the compressed gas source 895 can be a compressed gas cylinder, such as a carbon dioxide cylinder. Compressed gas cylinders are particularly useful if portability is desired. Other compressed gas sources are within the scope of the present invention. Similar pneumatic controls can be provided, for example, for controlling the movement of fluids in the bags described herein, or other actuators, servos, etc. can be provided.

[0181] Several other components of the instrument are also connected to the compressed gas source 895. The magnet 850 mounted on the second side 814 of the support member 802 is illustratively extended and retracted 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 in the support member 802. It is understood that the recess 851 can be a passageway through the support member 802 so that the magnet 850 can contact the blister 546 of the bag 510. However, depending on the material of the support member 802, it is understood that the recess 851 does not need to extend all the way through the support member 802, as long as the magnet 850 is close enough to provide a sufficient magnetic field at the blister 546 when the magnet 850 is extended, and when the magnet 850 is fully retracted, the magnet 850 does not significantly affect any magnetic beads 533 present in the blister 546. Although reference is made to retracting magnet 850, it is to be understood that an electromagnet may be used and that the electromagnet may be activated and deactivated by controlling the current flowing through the electromagnet. Thus, although the present specification discusses withdrawing or retracting the magnet, it is to be understood that these terms are broad enough to encompass other ways of withdrawing the magnetic field. It is to be understood that the pneumatic connection may be a pneumatic hose or a pneumatic air manifold, thereby reducing the number of hoses or valves required. It is to be understood that similar magnets and methods of activating magnets may be used in other embodiments.

[0182] Each pneumatic piston 868 of the pneumatic piston array 869 is also connected to a compressed gas source 895 via a hose 878. Although only two hoses 878 connecting the pneumatic pistons 868 to the compressed gas source 895 are shown, it is understood that each pneumatic piston 868 is connected to a compressed gas source 895. Twelve pneumatic pistons 868 are shown.

[0183] A pair of temperature control elements are mounted on the second side 814 of the support member 802. As used herein, the term "temperature control element" refers to a device that adds heat to a sample or removes heat from a sample. Illustrative examples of temperature control elements include, but are not limited to, heaters, coolers, Peltier devices, resistance heaters, induction heaters, electromagnetic heaters, film heaters, printed element heaters, positive temperature coefficient heaters, and combinations thereof. The temperature control element may include a plurality of heaters, coolers, Peltiers, etc. On the one hand, a given temperature control element may include more than one type of heater or cooler. For example, an illustrative example of a temperature control element may include a Peltier device and a separate resistance heater applied to the top and / or bottom surface of the Peltier. Although the term "heater" is used throughout the specification, it is to be understood that other temperature control elements may be used to adjust the temperature of the sample.

[0184] As described above, the first stage heater 886 may be positioned to heat and cool the contents of the bubble cap 564 for the first stage PCR. Figure 2 As seen in the figure, the second stage heater 888 can be positioned to heat and cool the contents of the second stage blisters of the array 581 of bags 510 for the second stage PCR. However, it is to be understood that these heaters can also be used for other heating purposes and can include other heaters suitable for a particular application.

[0185] As described above, although the Peltier device of thermal cycle between two or more temperatures is effective for PCR, it may be desirable in some embodiments to keep the heater at a constant temperature. Illustratively, this can be used to reduce the running time by eliminating the time required for the conversion heater temperature that exceeds the time required for the conversion sample temperature. In addition, such an arrangement can improve the electrical efficiency of the system, because it is necessary to thermally cycle only smaller samples and sample containers, while thermal cycling of the Peltier device of much larger (larger heat capacity) is not. For example, an instrument may include multiple heaters (i.e., two or more) positioned relative to the bag at a temperature set for, for example, annealing, extension, and denaturation to achieve thermal cycling. For many applications, two heaters may be sufficient. In various embodiments, the heater can be moved, the bag can be moved, or the fluid can be moved relative to the heater to achieve thermal cycling. Illustratively, the heater can be arranged linearly, arranged in a circular arrangement, and the like. The type of suitable heater has been discussed above with reference to the first stage PCR.

[0186] When fluorescence detection is required, an optical array 890 may be provided. Figure 2 As shown, the optical array 890 includes a light source 898 (illustratively a filtered LED light source, filtered white light, or laser illumination) and a camera 896. Illustratively, the camera 896 has a plurality of photodetectors, each photodetector corresponding to a second stage hole 582 in the bag 510. Alternatively, the camera 896 can capture an image containing all of the second stage holes 582, and the image can be separated into separate fields corresponding to each second stage hole 582. Depending on the configuration, the optical array 890 can be stationary, or the optical array 890 can be placed on a mover attached to one or more motors and moved to obtain a signal from each individual second stage hole 582. It is to be understood that other arrangements are possible. Some embodiments of the second stage heater are located at the same level as the bag 510. Figure 2 A heater is provided on the opposite side shown. This orientation is merely illustrative and may be determined by space constraints within the instrument. If the second stage reaction area 580 is disposed in an optically transparent material, the photodetectors and heaters may be on either side of the array 581.

[0187] As shown, computer 894 controls valve 899 of compressed air source 895, and thereby controls all pneumatics of instrument 800. In addition, in other embodiments, many pneumatic systems in the instrument can be replaced with mechanical actuators, pressure applying devices, etc. Computer 894 also controls heaters 886 and 888 and optical array 890. These components are each illustratively electrically connected via cable 891, although other physical or wireless connections are also within the scope of the present invention. It is to be understood that computer 894 can be housed in instrument 800, or can be outside instrument 800. In addition, computer 894 can include built-in circuit boards that control some or all components, and can also include external computers, such as desktop or portable PCs, to receive and display data from optical arrays. An interface can be provided, illustratively a keyboard interface, which includes keys for inputting information and variables such as temperature, cycle time, etc. Illustratively, a display 892 is also provided. Display 892 can be, for example, an LED, LCD or other such display.

[0188] Other instruments known in the art teach PCR in sealed flexible containers. See, for example, U.S. Patent Nos. 6,645,758, 6,780,617, and 9,586,208, which are incorporated herein by reference. However, including cell lysis in a sealed PCR container can improve ease of use and safety, particularly if the sample to be tested may contain biohazards. In the embodiment shown herein, waste from cell lysis and waste from all other steps are retained in a sealed bag. Nevertheless, it is understood that the bag contents can be removed for further testing.

[0189] Back to Figure 2 , the instrument 800 includes a support member 802, which may form a wall of the housing or be mounted within the housing. The instrument 800 may also include a second support member (not shown), which is optionally movable relative to the support member 802 to enable insertion and removal of the bag 510. Illustratively, a cover may cover the bag 510 once the bag 510 is inserted into the instrument 800. In another embodiment, the two support members may be fixed, with the bag 510 being held in place by other mechanical means or by pneumatic pressure.

[0190] In the illustrative example, heaters 886 and 888 are mounted on support member 802. However, it is to be understood that this arrangement is merely illustrative and other arrangements are possible. As known in the art, exemplary heaters include Peltier and other block heaters, resistive heaters, electromagnetic heaters, and film heaters to thermally cycle the contents of bubble cap 864 and second stage reaction zone 580. A capsule plate 810 with capsules 822, 844, 846, 848, 864, 866, hard seals 838, 843, 852, 853, and seals 871, 872, 873, 874 forms capsule assembly 808, which can be illustratively mounted on a movable support structure that can move toward bag 510 so that a pneumatic actuator is placed in contact with bag 510. When the bag 510 is inserted into the instrument 800 and the movable support member is moved toward the support member 802, the various blisters of the bag 510 are positioned adjacent to the various bladders of the bladder assembly 810 and the various seals of the assembly 808, so that activation of the pneumatic actuator can push liquid from one or more blisters of the bag 510, or can form a pinch valve with one or more channels of the bag 510. The relationship between the blisters and channels of the bag 510 and the bladders and seals of the assembly 808 is shown in more detail in FIG. Figure 3 middle. Methods and systems for sample preparation

[0191] Disclosed herein are methods and systems for preparing nucleic acid samples. The methods and systems described herein are designed to rapidly prepare nucleic acid samples, for example, less than 5 minutes, less than 4 minutes, less than 3 minutes, less than 2 minutes, within the range of about 1-3 minutes (e.g., 2 minutes), or any time range within the aforementioned range. As will be explained in more detail below, the methods and systems described herein can utilize the kinetic efficiency of certain parts of sample preparation (i.e., sample lysis, recovery of nucleic acid from lysate with medium such as silica-coated magnetic particles, washing recovery medium, and elution of nucleic acid from recovery medium) to shorten sample preparation as much as possible, while still providing high-quality extracted nucleic acid for downstream amplification or other analysis. The methods described herein include focusing on rapid mechanical lysis (preferably lysis is carried out in the presence of silica-coated magnetic beads), rapid recovery of silica-coated magnetic beads from lysate, rapid washing of magnetic beads, and rapid and effective elution of captured nucleic acid from silica-coated magnetic beads One or more. The methods and systems described herein are also designed to rapidly prepare nucleic acid samples with aqueous and alcohol-free or organic solvent-free buffer compositions. Illustratively, one or more buffer compositions used in the methods described herein can be suitably provided in a sample preparation device as a ready-to-use storage stable liquid that can be stored under ambient conditions and / or as a ready-to-use dry powder composition that can be rehydrated with a rehydration fluid (e.g., water) to be used when carrying out the steps of the method. Successful sample preparation (i.e., cracking, recovering nucleic acid from the lysate, washing and eluting) is important for maximizing the sensitivity of molecular assays while ensuring reliable, consistent results. In particular, PCR fidelity and efficiency depend on the purity and concentration of the nucleic acid template added to the reaction. As the time to obtain results in molecular assays is constantly shortened, reducing the time of sample preparation is important for reducing the total assay time. Methods and systems described herein greatly reduce the time required for sample preparation with mechanical cracking, without sacrificing the integrity, purity and concentration of the input nucleic acid template to ensure reliable, consistent results.

[0192] An exemplary method for preparing a nucleic acid sample comprises the following steps: providing a sample container comprising a first container, providing a sample (e.g., a nasal swab, a saliva sample, a sputum sample, blood, urine, etc.) suspected of containing one or more target nucleic acids (e.g., a target nucleic acid in one or more microorganisms) and a sample buffer comprising a buffer, a chaotropic salt and a non-ionic surfactant, wherein the sample and the sample buffer are combined to form a first mixture; and the first mixture is combined with a certain amount of lysate particles (e.g., zirconium silicate) and a certain amount of magnetic silica particles. The sample, sample buffer, lysate particles and magnetic beads can be placed in the first chamber, and can be suitably stirred (e.g., bead milling) in the first chamber for a period of time sufficient to produce a lysate. In various embodiments, a mixture comprising a sample, a sample buffer, lysate particles and magnetic beads can be combined and then placed in the first chamber, the first chamber can contain one or more components (e.g., lysate particles and / or magnetic beads), the sample and buffer can be added to the first chamber including the lysate particles and magnetic beads can be added thereafter, or any possible combination thereof. The sample, buffer, lysate particles and magnetic particles can be stirred using any method or device known in the art for producing a lysate. The Roche MagnaLyser is an example of a commercial bead milling instrument that can be used to agitate a vessel containing such a mixture to produce a lysate. Figure 4 The bead mill discussed is another example of a bead milling instrument that can be suitably used for bead milling in the methods described herein.

[0193] In the present case, the inventors have found that lysis and nucleic acid recovery are more efficient when lysis is performed in the presence of magnetic silica particles. Figure 7A-8 Shown in. Fig. 7A and 7B Figure 2 shows the presence (BBw / MB) or absence (BB w / o MB) of silica-coated magnetic particles during bead beating lysis in an assay set based on amplification from DNA ( Fig. 7A ) and a set of assays based on amplification from RNA ( Figure 7B ) is a bar graph showing the percentage of detection of organisms at various concentrations in the culture medium. Fig. 7A It was shown that the detection percentage of the DNA-based assay was consistently better at 1×LOD, 0.1×LOD, and 0.01×LOD when bead beating was performed in the presence of silica-coated magnetic particles. However, the RNA-based assay appeared to be roughly equivalent when silica-coated magnetic particles were added after lysis or when bead beating was performed in the presence of silica-coated magnetic particles. Figure 8is a graph showing the change in Cp performance when bead-beating in the presence of magnetic particles compared to a control (bead-beating in the absence of magnetic particles). A negative ΔCp means an earlier Cp (i.e., fewer amplification cycles to detect amplification) compared to the control. Assuming 100% template amplification efficiency, a one-cycle Cp improvement represents an increase in the input concentration of template nucleic acid by about 2-fold, a two-cycle Cp improvement represents an increase by about 4-fold, a three-cycle Cp improvement represents an increase by about 8-fold, and so on (by general formula, an n-cycle Cp improvement represents an increase in the input concentration of target cells or template nucleic acid by about 2-fold). n times). Figure 8 It is shown that at 3 different organism concentrations, the DNA template consistently has better Cp performance than the control. The improvement in Cp indicates that the scheme including magnetic particles in the lysis gives about 2-4 times more input DNA template than the control in which the magnetic particles are added after the lysis is complete. The RNA Cp performance of RNA recovered from the magnetic particles present during the lysis is roughly equivalent to the control. In summary, these data indicate that nucleic acids recovered when lysis is performed in the presence of magnetic particles are more concentrated and may be of higher quality than nucleic acids (i.e., DNA and RNA) recovered when silica-coated magnetic particles are added after lysis.

[0194] This is unexpected and unforeseen, because the inclusion of magnetic silica particles in mechanical lysis is traditionally not favored by those of ordinary skill in the art, because it has long been believed that the presence of silica-coated magnetic particles during mechanical lysis may damage the magnetic particles by causing the silica coating to fall off the particles. It is believed that the loss of silica from silica-coated magnetic particles can reduce the nucleic acid binding capacity of the magnetic particles, and in addition, nucleic acids can be lost by binding to non-recoverable silica fragments that fall off the magnetic particles. However, when the organism is mechanically lysed (e.g., by bead milling) in the presence of magnetic silica particles, the binding of nucleic acids to magnetic silica particles can occur during lysis, and nucleic acid binding can be more effective, and the recovery rate can be higher. In fact, at least for DNA, nucleic acid binding and nucleic acid recovery seem to be significantly better. When magnetic silica particles are included in the lysis, the quality of the recovered nucleic acids seems to be better. For example, it is believed that proteins and other inhibitors co-isolated with nucleic acids can be less, and nucleic acids can be more concentrated. This is demonstrated by the fact that less washing of the magnetic silica particles is required when the magnetic silica particles are included in the lysis.

[0195] This is shown in Fig. 9, which compares the Cp of amplifying nucleic acids recovered from magnetic particles contained in lysis that were not washed, washed once, and washed twice; the samples were identical except for the number of washes. Compared with the one-wash condition, the unwashed condition was clearly unfavorable for both the DNA assay and the RNA assay. This may be due to the presence of PCR inhibitors that co-isolate with the nucleic acids under the unwashed condition. The one-wash condition improved the Cp for both the RNA assay and the DNA assay, indicating that one wash was sufficient to remove most PCR inhibitors without reducing the amount of nucleic acid that could be recovered from the magnetic particles. The two-wash condition showed that it might be slightly better for the DNA assay and slightly worse for the RNA assay, indicating that some RNA might have been washed away. In any case, for the RNA assay, the two-wash condition was superior to the unwashed condition. When the same assay was performed with magnetic particles added after lysis, at least three washes were required to clean the nucleic acids and remove PCR inhibitors, and a later Cp was observed (data not shown), compared with one or two washes performed with magnetic particles in the lysis. Without being bound by theory, it is believed that a faster sample preparation protocol including magnetic particles during mechanical lysis may result in less inhibitor capture by the magnetic particles, thus potentially requiring less washing than a slower and longer binding process when magnetic particles are added only after lysis is complete.

[0196] Typically, magnetic silica particles for nucleic acid recovery are added only after the bead milling for lysis is completed. It has long been believed that the presence of silica-coated magnetic silica particles during manual lysis may damage the magnetic beads by causing the silica coating to fall off during lysis. If this damage occurs, the nucleic acid will then be combined with free silica that is no longer associated with the magnetic core, and the subsequent separation of the magnetic particles will not be able to recover the nucleic acid bound to the free silica. In this case, the inventors have observed the opposite phenomenon. Compared with the nucleic acid obtained from the organism of lysis by conventional methods, when magnetic silica particles are included in the lysis formation, the recovery of nucleic acid seems to be more effective, and the recovered nucleic acid is co-purified with fewer pollutants and they are more concentrated. It is also believed that the magnetic beads to which the bead milling nucleic acid is combined may over-shear and damage the nucleic acid. If this damage occurs, the nucleic acid may be damaged, and a smaller amount can be used for amplification. Thus, the method described herein is atypical and contrary to the conventional teachings of the art. The data presented herein (see, e.g. Figure 7A-9 ) showed that adding magnetic silica particles to lysis could facilitate the recovery of nucleic acids from samples and that the nucleic acids might be co-isolated with fewer PCR inhibitors. Fig.11The data presented in show that bead beating lysis in the presence of magnetic silica particles does not cause nucleic acid shearing. This means that for a given assay, unknown nucleic acids can be detected in a shorter period of time (e.g., after fewer amplification cycles) or the detection limit for a given cell type can be lower (i.e., an assay for a given cell can be more sensitive because nucleic acid recovery from that cell is enhanced), or both.

[0197] Without being bound by theory, it is believed that the improved recovery in samples bead-milled in the presence of some or all of the magnetic beads improves nucleic acid recovery due to better mixing and binding kinetics between the magnetic silica particles, recovery of less protein and other PCR inhibitors, recovery of more nucleic acids, and breaking up of magnetic bead aggregates (which provides additional surface area for nucleic acids to bind). In addition, it is believed that the mechanical action of bead milling can change the silica surface and increase the surface area of ​​individual magnetic silica particles (e.g., by forming microscopic scratches in the surface of the magnetic particles), thereby increasing the likelihood of positive binding interactions between the magnetic silica particles and nucleic acids. In addition, because the contact time between the lysate and the magnetic particles is shorter when the magnetic particles are included in the lysis, the magnetic particles appear to co-isolate fewer PCR inhibitors with the nucleic acids. Again, without being bound by theory, it is believed that the rapid and tight motion during bead milling can change how nucleic acids or proteins and inhibitors bind to the magnetic particles, which may result in the capture of more nucleic acids and fewer proteins and / or inhibitors compared to solutions where the magnetic particles are added only after the lysis is complete.

[0198] In addition, the scheme of some or all magnetic beads during mechanical lysis has certain speed advantages compared with the traditional scheme of adding magnetic beads after bead milling. These advantages include but are not limited to breaking aggregates, which means that fewer aggregates may settle to the bottom of the container, which is conducive to faster and easier separation from the lysis beads. Similarly, breaking aggregates may increase the available surface area for nucleic acid binding on the beads. In addition, since some or all magnetic beads are present during lysis, these schemes can reduce or eliminate the time spent in the rehydration or resuspension of magnetic beads. In addition, because magnetic beads exist and are constantly mixed during lysis, the interaction with nucleic acid increases, so nucleic acid binding can be much faster. In the presence of magnetic particles, lysis can also increase the possibility of given magnetic particles being combined with nucleic acids. On the other hand, because the binding kinetics are faster and the interaction time is correspondingly shorter when magnetic particles are included in the lysis, it is unlikely that magnetic particles will collect protein and other downstream nucleic acid amplification inhibitors. In addition, the lysate solution can be simply heated by friction during mechanical bead milling, and this heating can contribute to faster diffusion kinetics and more effective lysis, but this may be potentially disadvantageous for some binding kinetics, causing loosely bound particles to fall off. Because lysis in the presence of magnetic beads results in shorter bead beating times and faster recovery, the sample is less likely to overheat and affect recovery.

[0199] In some embodiments, the method described herein may be suitably included in the addition of a first amount of magnetic beads before mechanical lysis (e.g., bead grinding) and subsequently adding a second amount of magnetic beads after mechanical lysis. It is believed that some fragments (illustratively double-stranded DNA) can be tightly bound to magnetic beads or other silica surfaces, and this combination can even persist in the entire lysis agitation step. It is believed that these fragments will preferentially bind to magnetic beads during the lysis and mixing stages. Other nucleic acids (such as single-stranded RNA) tend to be more loosely combined, and the combination may not be maintained in the agitation step. Adding a second amount of magnetic beads with fresh nucleic acid binding sites can quickly and effectively bind to unbound nucleic acids after mechanical agitation. It is also believed that magnetic beads are not completely homogeneous, and may have local physical or chemical properties that are conducive to binding nucleic acids in different configurations. Once a particular type of binding site is saturated, beads may lose the tendency to bind nucleic acids to the remaining binding sites. The magnetic beads of fresh amount also provide more diverse fresh injections of binding sites for binding for any unbound nucleic acid.

[0200] Because the lysate solution can heat up simply by friction during mechanical bead beating, and this can be disadvantageous, fresh injection of cooler magnetic beads and subsequent airflow through the instrument to cool the solution can facilitate stronger binding kinetics for loosely bound nucleic acid molecules. Thus, the combination of adding magnetic beads before and after mechanical lysis advantageously promotes binding of both those molecules that will bind tightly to the surface and those that are more loosely bound.

[0201] Another exemplary method of preparing a nucleic acid sample comprises the steps of providing a sample container, providing a sample suspected of containing one or more target nucleic acids and a sample buffer comprising a buffering agent, a chaotropic salt, and a nonionic surfactant, wherein the sample and the sample buffer are combined to form a first mixture, the first mixture and an amount of lysing particles are combined in a first chamber, and the first chamber is bead milled for a period of time sufficient to produce a lysate. The Roche MagnaLyser is an example of a commercial bead milling instrument that can be used to agitate a container containing such a mixture to produce a lysate. Reference above Figure 4 The bead mill discussed is another example of a bead milling apparatus that can be suitably used for bead milling in the methods described herein. The methods described herein using a closed system (illustratively bag 510) can be used to reduce or minimize foaming, thereby reducing or eliminating the need for defoaming agents, and these methods can tolerate higher concentrations of surfactants without excessive foaming.

[0202] Illustrative examples of nonionic surfactants are Triton X-100, Thesit, Triton X-114, NP-40, Arlasolve 200, Brij O10, octyl β-D-pyranoglucoside, saponin, monododecyl nonaglycol ether, and combinations thereof. However, it is to be understood that other suitable detergents are known and can be used.

[0203] The sample buffer can suitably include a sufficient amount of detergent to crack most cell walls and denature most cell proteins, to accelerate the release and protection of nucleic acids from cells when cell lysis, whether it is used alone for chemical lysis, or in collaboration with other lysis techniques such as heat, machinery, sound waves, etc. The detergent dosage that is sufficient to work on a specific sample type or a variety of sample types (whether host or non-host) that may contain high cell counts is selected, and the sample types include but are not limited to blood, sputum, swabs, etc. Although some methods will be limited to the excessive foaming of detergents from high concentration levels, the exemplary methods herein can tolerate higher than usual detergent levels before foaming becomes a problem, illustratively>1% by weight,>5% by weight,>10% by weight, or>15% by weight of surfactant. In a specific example, the lysis buffer may include 10-20% by weight of surfactant, and when combined with a sample to be lysed, the buffer-sample combination may include 5-15% by weight of surfactant. This is generally considered to be a very high surfactant amount for the lysis buffer. The advantage of such high surfactant amounts is that there is a sufficient amount of detergent to lyse most cell walls and denature most cell proteins to accelerate the release and protection of nucleic acids from cells upon cell lysis. However, such high surfactant amounts can cause significant foaming. The methods disclosed herein include steps and procedures to address such foaming.

[0204] The method further includes knocking the sample container at the end of the lysis to help reduce foaming. Knocking (which is usually less powerful and gentler than bead milling) can force the foam to reach the top of the sample container and cause the foam bubbles to burst. It is usually desirable to burst the foam bubbles in the lysate because the bubbles (in the case of aggregation) can introduce air into the downstream process, which may be undesirable. Knocking can be too strong to keep the lysis beads suspended, and can actually help the lysis beads to settle faster, thereby reducing the time spent in sample preparation. In the application of magnetic silica particles (which can be added before lysis, added after lysis, or a combination thereof) for nucleic acid collection in the lysate, knocking can keep the magnetic silica particles suspended while accelerating the sedimentation of the lysis particles. Foam can also interfere with the effective collection of magnetic silica particles. Particles may be trapped in the foam, and collecting particles without collecting air can be challenging and time-consuming. Breaking the foam formed during the lysis can promote the rapid and effective collection of magnetic silica particles.

[0205] In one aspect, the tapping can be suitably performed with a bead milling device that is turned down, the bead milling device is turned up to a slower speed (e.g., 10-100 rpm or reciprocating motion relative to 1000 RPM or reciprocating motion for bead milling) and, if applicable, optionally a reduced striking force. Suitable examples of bead milling devices that can be turned down for tapping include, but are not limited to, Roche MagNA Lyser, Figure 4 The bead mill apparatus shown in , or the paddle bead mill apparatus shown in U.S. Pat. No. 11,090,652 (the entire contents of which are incorporated herein by reference). The action of the turned-down bead mill can suitably force the foam to the top of the sample container and cause the foam bubbles to collapse while keeping the magnetic particles suspended and allowing the lysis beads to settle more quickly. The paddle bead mill apparatus shown in U.S. Pat. No. 11,090,652 can be particularly desirable because the paddles can be extended together to "slap" the sample container.

[0206] When cracking in a closed sample container such as bubble cap 522, the powerful opening and closing pressure from bubble cap 822 can help reduce foaming by forcing any foam to arrive at the top of the bubble cap and causing bubble burst. This impact pressure can also help the cracking beads to settle faster, thus reducing the time spent by sample preparation. For example, capsule 822 can be inflated against bubble cap 522 in about 1-500 millisecond (for example, about 1-250 millisecond, about 1-200 millisecond, about 1-150 millisecond, about 1-100 millisecond, etc.). The feature of this fast filling can be similar to the slapping or slapping of capsule 822 against bubble cap 522. This impact slapping or slapping can make the foam bubble burst, and simultaneously counter-intuitively makes larger cracking beads settle faster and separate from the liquid fraction. It is usually good that the cracking beads are effectively separated from the lysate, because for example the cracking particles can interfere with downstream processing. The impact slapping or slapping can also stretch bubble cap 522, for the cracking beads sedimentation produces a better geometry. For example, the lysis beads may be able to settle to the bottom of the blister 522 without being caught along the edge of the blister 522 .

[0207] Another exemplary method for preparing a nucleic acid sample comprises the following steps: providing a sample container, providing a sample suspected of containing one or more target nucleic acids and a sample buffer comprising a buffer, a chaotropic salt and a nonionic surfactant. The sample and the sample buffer are combined to form a first mixture, the first mixture is combined with a certain amount of magnetic silica particles and a certain amount of lysate particles in a first chamber, and the first chamber is bead milled for a period of time sufficient to produce a lysate. As discussed above, including magnetic silica in the lysate formation has many unexpected advantages, including but not limited to: more effective binding, faster nucleic acid recovery, and higher nucleic acid recovery. Nucleic acid binding magnetic particles can be added before lysis, or a certain amount of magnetic particles can be added before, during and after lysis. The method may further include capturing magnetic particles with a magnet and removing magnetic particles from the lysate. The foaming may make it more difficult and time-consuming to recover magnetic silica particles from the lysate. The method described herein can solve this foaming so that the magnetic silica particles can be quickly recovered. Impact slapping or slapping can burst foam bubbles and keep the magnetic particles suspended, while counterintuitively making larger lysate beads settle faster. Combining the step of adding magnetic silica particles to the lysate with a post-lysis step of tapping the sample container to remove foam, settle the lysed particles, and keep the magnetic silica particles suspended can accelerate the separation of the lysed particles from the magnetic particles.

[0208] The above method described above may further include capturing magnetic particles with a magnet and transferring the magnetic particles to another container using a magnet. Alternatively, a lysate containing suspended magnetic particles may be flowed into another container, in which the magnetic particles may be extracted from the fluid. The waste lysate without magnetic particles may then flow back into the lysis container or another waste container. The method described herein may further include washing the nucleic acid-bound magnetic particles with a wash buffer to remove waste from the lysate from the magnetic particles. Preferably, the wash buffer may remove residual lysate, contaminants, etc. from the magnetic particles without eluting a significant amount of nucleic acid from the magnetic particles. Exemplary washing may include adding a wash buffer to the magnetic particles, gently stirring the magnetic particles for a period of time (e.g., a few seconds), re-capturing the magnetic particles with a magnet, and removing the waste wash buffer from the container or transferring the magnetic particles to another container. For some sample types, it may be necessary not to wash. Therefore, the method described herein may include zero washes. However, typically, the method described herein may include 1-3 washes. The washing carried out in the methods described herein may suitably not include one or more of the following: heating wash buffer and / or magnetic particles before or during washing, vigorously mixing magnetic particles and wash buffer, or incubating magnetic particles and wash buffer for a period of time greater than 1 second, 2 seconds, 3 seconds, 4 seconds, 5 seconds, 6 seconds, 7 seconds, 8 seconds, 9 seconds, 10 seconds or any time therebetween. Preferably, the wash cycle may include incubating magnetic particles and wash buffer for a period of time of about 2-3 seconds. The specific examples of the washing schemes are found in the table of Example 4.

[0209] After washing, the methods described herein may suitably include releasing the magnetic particles from the capture magnet (in the container after the wash buffer has been removed or in a new container) and adding an elution buffer to the magnetic particles, mixing the magnetic particles with the elution buffer, re-capturing the magnetic particles with a magnet, and separating the elution buffer from the magnetic particles. Optionally, the elution buffer may be heated before adding it to the magnetic particles. This preheating may be performed during the lysis and / or washing steps so that the elution buffer is already heated during elution. Illustratively, the elution buffer may be heated to a temperature between 35°C and 105°C (e.g., 50°C to 100°C) prior to mixing it with the magnetic particles, depending on factors such as the sample type. In the present case, the inventors have found that the heated elution temperature and duration can quickly and effectively elute RNA / DNA and leave proteins and inhibitors on the magnetic particles. This is shown, for example, in Fig. 10A , which shows that the average Cp of DNA and RNA assays improves as the preheat temperature of the elution buffer increases from about 35°C to about 105°C. Fig. 10AIt can be seen that the average Cp improves by up to about 1.5 to 2.5 Cp units as the temperature of the elution buffer increases from about 60° C. to about 100° C. This represents an increase of about 3-5 times in the amount of nucleic acid eluted from the magnetic particles. Fig. 10B and 10C The same trend was shown for both DNA and RNA assays. The best fit lines for the DNA and RNA data showed an average Cp improvement of about 2 Cp units for the DNA assay and about 1.5 Cp units for the RNA assay. This represents an improvement of about 4-fold in the amount of DNA eluted from the magnetic particles and about 3-fold in the amount of RNA.

[0210] While reference is made in various embodiments discussed herein to moving the magnetic particles to the second chamber, it is to be understood that an alternative embodiment is to retain the magnetic particles in the first chamber and remove unbound lysate from that chamber.

[0211] Another exemplary method for preparing a nucleic acid sample includes the following steps: providing a sample container, the sample container comprising a first chamber and a second chamber connected by a plurality of channel fluids and a plurality of reagent wells, the plurality of dried reagent wells comprising at least a dried nucleic acid wash buffer and a dried nucleic acid elution buffer, and a device comprising: an opening for receiving the container (the opening comprising a bead mill positioned to interact with the container), one or more actuators configured to manipulate the container to move fluid in the container, and a magnet for capturing magnetic particles in the container. The method further includes providing a sample suspected of containing one or more target nucleic acids and a sample buffer comprising a buffer, a chaotropic agent, and a non-ionic surfactant, wherein the sample and the sample buffer are combined to form a first mixture, the first mixture is combined with a certain amount of lysed particles in the first chamber of the container, a rehydration fluid is introduced into the container and the dried nucleic acid wash buffer and the dried nucleic acid elution buffer are rehydrated, and the container is placed in the opening of the device and the first reaction bubble is contacted with the bead mill for a period of time sufficient to produce a lysate. It should be noted that the energy from the bead mill can be regulated by adjusting the pressure in a capsule (such as capsule 882), thereby pressing the container with a variable amount of pressure to accelerate the dynamics of mechanical lysis, and thereby inducing more frictional heating to help thermal lysis. After the bead mill, the impact action as discussed above can be used to reduce foam and help settle the lysis particles. The method further includes mixing a certain amount of nucleic acid-bound magnetic particles into the lysate. As discussed in the above method, magnetic particles can be suitably added before lysis, after lysis, or a certain amount of magnetic particles can be added before, during, and after lysis, depending on factors such as sample type. The method may further include activating a magnet and an actuator associated with the first chamber to recover the nucleic acid-binding magnetic particles from the lysate and deposit the nucleic acid-binding magnetic particles into a second chamber, transferring a first amount of rehydrated wash buffer to the nucleic acid-binding magnetic particles for washing, wherein washing optionally includes deactivating the magnet, gently agitating the nucleic acid-binding magnetic particles with an actuator associated with the second chamber to exchange the lysate solution and release loosely bound proteins and inhibitors but not release more strongly bound nucleic acids, reactivating the magnet to recapture the nucleic acid-binding magnetic particles, and pushing the wash buffer into the first chamber with the actuator associated with the second chamber, and eluting nucleic acids from the nucleic acid-binding magnetic particles by transferring a certain amount of rehydrated elution buffer to the nucleic acid binding in the second and / or third reaction bubbles, wherein elution includes deactivating the magnet, vigorously agitating the nucleic acid-binding magnetic particles with an actuator associated with the second chamber to release tightly bound nucleic acids, reactivating the magnet to recapture the nucleic acid-binding magnetic particles, and pushing the elution buffer with the eluted nucleic acids therein into the third chamber or reagent well. Optionally, the elution buffer is heated prior to adding it to the magnetic particles.This preheating can be performed during the lysis and / or washing steps so that the elution buffer is already heated at the time of elution. The heat generated during bead beating can be used to heat the elution buffer.

[0212] Reference now Figures 5A-5J , showing an exemplary method of cell lysis and nucleic acid recovery. Figure 5A As shown, sample plus lysis buffer 5002 is combined with lysis particles 5006 and magnetic particles 5004 in container 5000. Figure 5B As shown, the contents of container 5000 can be suitably bead milled with a bead mill (as schematically depicted by arrow 5009) for a sufficient time to produce a lysate. On the one hand, bead milling can be performed more efficiently by pressing the container against the bead mill with an appropriate amount of pressure. The time required to prepare the lysate can vary depending on factors such as, but not limited to, the sample material to be lysed, the sample amount, the speed or frequency of the bead mill motor, the pressure applied to the bead mill, and the bead lysis instrument. Reducing the air from the chamber (illustratively by evacuating the container or by using pressure applied to the container to exhaust air from the container) and bead milling under pressure helps to provide more energy to the sample and reduce foam generation for more efficient lysis. Illustratively, a pressure-regulated feedback control mechanism and an electronically controlled motor can be used to deliver more power and adjust the lysis energy of the system to achieve a shorter bead lysis time. Typically a few seconds to a few minutes. Using Figure 4 The bead milling apparatus shown in Figure 1 and Figure 2 For instruments and bags of this type, a bead milling time of a few seconds may be sufficient (e.g., 1 second, 5 seconds, 10 seconds, 20 seconds, 25 seconds, 30 seconds, 40 seconds, 45 seconds, 50 seconds, 55 seconds to 1 minute). Preferably, the bead milling is performed with as much force and / or intensity as the system allows and for no longer than is required to lyse the material in the sample. This not only saves time in sample preparation, but excessive bead milling time may shear nucleic acids and ultimately reduce sample quality. This is shown in Fig.11 In, it shows the fragment size of human genomic DNA detected with or without bead beating for 120 seconds. In the case of bead beating, the length of the largest fragment is reduced, and a higher concentration of smaller fragments is detected.

[0213] The scheme discussed herein does not significantly shear nucleic acids or reduce sample quality. Generally, organisms that are difficult to lyse, such as Cryptosporidium and yeast, prefer high RPM and long bead milling duration, while other organisms such as E. coli and viral targets prefer lower RPMs combined with shorter bead milling durations. The best combination of RPM and duration is specific to the assay. Optimizing RPM and duration for organisms that are more easily lysed may prevent organisms that are difficult to lyse from being lysed; and optimizing for the most difficult organisms to lyse may result in the shearing of nucleic acids from organisms that are easy to lyse. In addition to shearing, extended bead milling at an increased speed may result in heating of the sample, which may reduce yield. Generally, the best is to optimize to the point of lysing requirements of all organisms in the balance panel. The shortest duration (e.g., 20-60 seconds) possible with high-speed (e.g., 10,000-12,000RPM) bead milling can maximize lysis efficiency and sample quality, while saving considerable sample preparation time and the total time to obtain results.

[0214] Back to Figure 5C , after bead beating, a second amount of magnetic particles 5010 can be combined with the lysate 5008. Figure 5A and 5C Shown is the magnetic particle that adds the first and second amount before and after cracking, but this is only a kind of selection.Can add all magnetic particles before cracking, after cracking, or can add a certain amount of magnetic particles before, during and after cracking, this depends on sample type and application.The magnetic particle 5010 of the second amount can for example be combined with lysate 5008 (as schematically depicted by arrow 5011) by stirring.Stirring 5011 can be realized by the bead mill that uses in the cracking step, although the duration of stirring can be shorter and intensity is lower than the stirring that is used to produce lysate.The magnetic particles 5004 and 5010 of the first and second amount can be incubated in lysate 5008 to be enough to capture the selected time period (for example, several seconds to several minutes) of nucleic acid from solution.Magnetic particles can be incubated in lysate to be enough to capture the selected time period (for example, several seconds to several minutes) of nucleic acid from solution.

[0215] like Figure 5DAs shown, a tap (schematically shown by arrow 5012) can be applied to the sample container 5000 after bead milling to break any foam that may have formed during the lysis process (e.g., bead milling) and promote sedimentation of the lysis beads while keeping the magnetic particles 5004 (and optionally 5010) suspended, as depicted at 5014. The lysis particles 5006, which are typically much larger than the magnetic particles, can settle much faster than the magnetic particles. The tapping 5012 of the container 5000 can promote sedimentation of the lysis beads 5006 and promote separation of the magnetic particles 5004 from the lysis beads 5006. This ensures that the magnetic particles can be quickly and efficiently recovered from the lysate without inadvertently collecting the lysis beads and without losing the magnetic particles in the lysis beads. Breaking the foam can also enable faster recovery of the magnetic particles and without collecting trapped air. As Figure 5E As shown, a magnet (schematically shown at 5015) can be used to recover magnetic particles 5016 from lysate 5008. Figure 5D Magnetic particles 5004 may be kept suspended to facilitate recovery, illustratively by adjustable gentle or vigorous mixing (e.g., beating) of the container, which promotes faster magnetic bead recovery kinetics.

[0216] In the exemplary example performed in bag 510, a longer magnet actuation time for collecting magnetic particles not only from the immediately adjacent blister but also from the adjacent blister unexpectedly resulted in faster removal of magnetic particles from the liquid moved to and fro by blister 522. In this exemplary example, the capture time was reduced from 3 seconds to 0.5 seconds.

[0217] Optionally, a first amount of magnetic particles can be combined with the sample and lysing particles prior to bead beating ( Figure 5A ), while a second amount of magnetic particles can be combined with the lysate after bead beating ( Figure 5C ). The second amount can be substantially the same as the first amount, or it can be substantially larger or smaller. Illustratively, the amount of magnetic particles in the first and second amounts is substantially the same. In addition, the first amount and the second amount can be the same type of silica-coated magnetic particles, or they can vary as needed in terms of the thickness of the silica to favor the recovery of RNA, DNA, or both, and to favor the recovery of double-stranded or single-stranded nucleic acids or other configurations of nucleic acids. Although it is shown that the magnetic particles can be added before cleavage and then the second amount can be added after cleavage, without departing from the spirit and intent of the methods described herein, the magnetic particles can also be added all before cleavage or all after cleavage.

[0218] Reference now Fig. 5F ,exist Figure 5EThe recovered magnetic particles 5016 can be released into a new container 5020 and combined with a wash buffer as shown at 5022. The wash buffer can remove residues from the lysate and improve the overall quality and yield of nucleic acids from the magnetic particles. Fig. 5F Only one wash is shown, but in some cases multiple washes (e.g., 2-5 washes) may be performed. In some cases omitting the wash step may also be an option (i.e., zero washes may be performed). Figure 5G As shown, the magnetic particles can be recaptured (e.g., with an external magnet, not shown), and the spent wash buffer 5025 can be removed. Figure 5H As shown, the magnetic particles can be released from the external magnet and combined with an elution buffer 5026 to elute the material (e.g., captured nucleic acid) from the magnetic particles. After elution, the magnetic particles 5028 can be captured again ( Fig.5I ), and the elution buffer with the eluted nucleic acid 5029 can be transferred to a clean container 5030 ( Figure 5J ). Alternatively, after elution, the magnetic particles may be captured and removed from the container (not shown). Example 1

[0219] In the present case, the inventors have found that lysis and nucleic acid recovery is more efficient when lysis is performed in the presence of magnetic silica particles. This is shown, for example, in Figure 7A-8 middle. Fig. 7A and 7B Figure 2 shows the presence (BBw / MB) or absence (BB w / o MB) of silica-coated magnetic particles during bead beating lysis in an assay set based on amplification from DNA ( Fig. 7A ) and a set of assays based on amplification from RNA ( Figure 7B ) is a bar graph showing the percentage of detection of organisms at various concentrations in the culture medium. Fig. 7A It was shown that the detection percentage of the DNA-based assay was consistently better at 1×LOD, 0.1×LOD, and 0.01×LOD when bead beating was performed in the presence of silica-coated magnetic particles. However, the RNA-based assay appeared to be roughly equivalent when silica-coated magnetic particles were added after lysis or when bead beating was performed in the presence of silica-coated magnetic particles.

[0220] Figure 8is a graph showing the change in Cp performance when bead-beating in the presence of magnetic particles compared to a control (bead-beating in the absence of magnetic particles). A negative ΔCp means an earlier Cp (i.e., fewer amplification cycles to detect amplification) compared to the control. Assuming 100% template amplification efficiency, a one-cycle Cp improvement represents an increase in the input concentration of template nucleic acid by about 2-fold, a two-cycle Cp improvement represents an increase by about 4-fold, a three-cycle Cp improvement represents an increase by about 8-fold, and so on (by general formula, an n-cycle Cp improvement represents an increase in the input concentration of target cells or template nucleic acid by about 2-fold). n times). Figure 8 It is shown that at 3 different organism concentrations, the DNA template consistently had better Cp performance than the control. The improvement in Cp indicates that the protocol including magnetic particles in the lysis gave about 2-4 times more input DNA template than the control in which the magnetic particles were added after the lysis was complete. The RNA Cp performance of RNA recovered from the magnetic particles present during the lysis was roughly equivalent to the control. These data indicate that the nucleic acids recovered when the lysis was performed in the presence of magnetic particles are more concentrated and may be of higher quality than the nucleic acids (i.e., DNA and RNA) recovered when the silica-coated magnetic particles were added after the lysis.

[0221] This is unexpected and unforeseen, because including magnetic silica particles in mechanical lysis is traditionally not favored by those of ordinary skill in the art, because it has long been believed that the presence of silica-coated magnetic particles during mechanical lysis may damage the magnetic particles by causing the silica coating to fall off the particles. It is believed that the loss of silica from silica-coated magnetic particles can reduce the nucleic acid binding capacity of the magnetic particles, and in addition, nucleic acids can be lost by binding to non-recoverable silica fragments that fall off the magnetic particles. However, when an organism is mechanically lysed (e.g., by bead milling) in the presence of magnetic silica particles, binding of nucleic acids to magnetic silica particles can occur during lysis, and nucleic acid binding can be more efficient, and the recovery rate can be higher. In fact, at least for DNA, nucleic acid binding and nucleic acid recovery appear to be significantly better. When magnetic silica particles are included in the lysis, the quality of the recovered nucleic acids appears to be better. Example 2

[0222] Fig. 9The Cp of DNA (-●-) and RNA (-*-) recovered from magnetic particles included in lysis without washing, washing once, and washing twice were compared; the samples were identical except for the number of washes. Compared with the one-wash condition, the unwashed condition was clearly unfavorable for both the DNA assay and the RNA assay. This may be due to the presence of PCR inhibitors co-separated with nucleic acids under the unwashed condition. The one-wash condition improved the Cp for both the RNA assay and the DNA assay, indicating that one wash was sufficient to remove most PCR inhibitors without reducing the amount of nucleic acid that can be recovered from the magnetic particles. The two-wash condition showed that it may be slightly better for the DNA assay and slightly worse for the RNA assay, indicating that some RNA may have been washed away. In any case, for the RNA assay, the two-wash condition was better than the unwashed condition. When the same assay was performed with magnetic particles added after lysis, at least three washes were required to clean the nucleic acids and remove PCR inhibitors compared to one or two washes performed with magnetic particles in the lysis, and a later Cp was observed (data not shown). Example 3

[0223] Fig. 10A It was shown that the average Cp of the DNA assay and the RNA assay improved as the preheat temperature of the elution buffer was increased from about 35°C to about 105°C. Fig. 10A It can be seen that the average Cp improves by up to about 1.5 to 2.5 Cp units as the temperature of the elution buffer increases from about 60° C. to about 100° C. This represents an increase of about 3-5 times in the amount of nucleic acid eluted from the magnetic particles. Fig. 10B and 10C The same trend was shown for both DNA and RNA assays. The best fit lines for the DNA and RNA data showed an average Cp improvement of about 2 Cp units for the DNA assay and about 1.5 Cp units for the RNA assay. This represents an improvement of about 4-fold in the amount of DNA eluted from the magnetic particles and about 3-fold in the amount of RNA. Example 4

[0224] Reference now Figure 6, shows a bar graph illustrating time time savings for many specific examples in FilmArray sample preparation. Over a number of years, sample preparation times were reduced from approximately 13 minutes to approximately 2 minutes. That is a reduction of 11 minutes in sample preparation time. FilmArray is a state-of-the-art system, and being able to reduce sample preparation times from approximately 13 minutes to approximately 2 minutes was unexpected and unanticipated. Comparing the RP2.1 times (4:42) to the R / ST times (1:58), time savings are again seen at each step. The R / ST times represent the time savings that can be achieved using the methods described in this application. Comparing the RP2.1 sample preparation times to the R / ST sample preparation times shows an absolute time difference of 2:44 and a 58% time reduction. As can be seen from Figure 6 As can be seen in the figure, time is saved in each sample preparation step, especially in collecting magnetic particles from lysate and reducing bead grinding time. The release of nucleic acid from cells can be accelerated by combining multiple lysis mechanisms with pressure control and motor feedback mechanisms. More stringent control and reduced changes also make it possible to reduce buffer time to ensure robust performance in the case of the easiest to most difficult organism types to be cracked. By carrying out multiple steps in parallel and improving the kinetics and efficiency of nucleic acid binding to magnetic particles and magnetic particles binding to magnets, binding and magnetic bead recovery are greatly reduced from 7 minutes to 30 seconds. Only comparing the bead grinding and magnetic particle collection time between RP2.1 and R / ST shows a reduction from 2:53 in RP2.1 to 1:13 in R / ST, an absolute time saving of 1:40, and only for these two steps time is reduced by 57%. Washing time is reduced from 40 seconds to about 8 seconds, and elution time is reduced from 1:09 to 22 seconds.

[0225] The table included herein entitled "Sample Purification Specifications" describes the steps of a method for cell lysis and nucleic acid recovery using FilmArray bags and FilmArray instruments. Figure 6 In addition to the time savings mentioned above, considerable time is saved by performing some sample preparation steps simultaneously. See, for example, steps 5.1.04-5.1.07 and 5.1.15 and 5.1.16.

[0226] An exemplary protocol for use on an instrument such as instrument 800 is as follows. However, it is to be understood that the protocol may be modified depending on the sample type, the type of nucleic acid to be tested (RNA, DNA, etc.), and other assay-specific factors. It is also to be understood that the protocol may be adapted for use on other instruments. Extraction and binding of nucleic acids Nucleic acid collection washing Elution

[0227] The present invention may be implemented in other specific forms without departing from the spirit or essential features of the present invention. The described embodiments are considered to be merely illustrative and not restrictive in all respects. Therefore, the scope of the present invention is indicated by the appended claims rather than by the foregoing description. Although certain embodiments and details have been included in this article and the attached invention disclosure for the purpose of illustrating the present invention, it is obvious to those skilled in the art that various changes may be made to the methods and apparatus disclosed herein without departing from the scope of the present invention defined in the appended claims. All changes within the meaning and scope of the equivalents of the claims are included within their scope.

Claims

1. A method for preparing a nucleic acid sample, comprising: providing a sample container comprising a first container, providing a sample suspected of containing one or more target nucleic acids, a sample buffer comprising a buffer, a chaotropic salt and a non-ionic surfactant, and a quantity of lysing particles, combining the sample, sample buffer, and lysing particles to form a lysis mixture, placing the lysis mixture in the first container, bead milling the lysis mixture in the first container for a period of time sufficient to produce a lysate, Adding a certain amount of nucleic acid-binding magnetic particles to the lysate, tapping the first container to break bubbles and foam formed during bead beating and to keep the magnetic particles suspended while allowing the lysed particles to settle, capturing the magnetic particles from the lysate with a magnet and transferring the magnetic particles or unbound lysate to a second container, and releasing the magnetic particles from the magnet and adding an elution buffer to the magnetic particles, mixing the magnetic particles with the elution buffer, recapturing the magnetic particles with a magnet, and separating the elution buffer from the magnetic particles, The steps of the method are completed in <5 minutes.

2. The method of claim 1, wherein tapping comprises slapping or hitting the first container.

3. The method of claim 1 or 2, wherein striking comprises inflating the inflatable member against the first container within a time range of 1-250 milliseconds.

4. The method of claim 1, wherein one or more of the sample, sample buffer, and lysing particles are combined in the first container to form a lysis mixture.

5. The method of any one of claims 1 to 4, further comprising releasing the magnetic particles from the magnet and washing the magnetic particles with a wash buffer, recapturing the magnetic particles with a magnet and removing the wash buffer, and releasing the magnetic particles into an elution buffer after washing.

6. The method of claim 5, wherein the washing does not comprise one or more of: heating the wash buffer and magnetic particles before or during washing, vigorously mixing the magnetic particles and wash buffer, or incubating the magnetic particles and wash buffer for a period of time greater than 10 seconds.

7. The method of any one of claims 1 to 5, further comprising mixing an amount of magnetic particles into the lysis mixture prior to bead beating, and optionally, mixing a second amount of magnetic particles into the lysate after bead beating and prior to capture.

8. The method of claim 1, wherein the time period for bead milling is in the range of 1 second to 1 minute.

9. The method of claim 8, wherein the time period for bead beating is 20 seconds.

10. The method of claim 1, wherein the sample buffer is an aqueous buffer comprising a buffer, 50-60% of a chaotropic agent, and 10-20% of a nonionic surfactant.

11. The method of claim 10, wherein the chaotropic agent is a guanidine salt and the nonionic surfactant is one of TritonX-100, polydocanol (Thesit), Triton X-114, NP-40, Arlasolve 200, Brij O10, octyl β-D-pyranoglucoside, saponin, monododecyl nonaglycone ether and a combination thereof.

12. The method of any one of claims 1 to 11, wherein capturing the magnetic particles from the lysate is facilitated by bursting bubbles and foam formed during bead beating and by keeping the magnetic particles suspended while allowing the lysed particles to settle.

13. The method of any one of claims 1-12, wherein the steps of the method are completed in <4 minutes.

14. The method of any one of claims 1-13, wherein the steps of the method are completed in < 3 minutes.

15. The method of any one of claims 1-14, wherein the steps of the method are completed in <2 minutes.

16. The method of any one of claims 1-14, wherein the steps of the method are completed within 1-3 minutes.

17. The method of any one of claims 1 to 16, wherein the elution buffer is heated prior to adding the elution buffer to the magnetic particles.

18. The method of any one of claims 1 to 17, wherein the sample buffer, wash buffer and elution buffer are each an aqueous buffer and are free of organic solvents and alcohols.

19. The method of claim 18, wherein the sample buffer, wash buffer, and elution buffer are provided as a dried composition and each buffer is configured to be rehydrated with an aqueous rehydration fluid.

20. A method for preparing a nucleic acid sample, comprising: providing a sample container comprising a first container, Providing a sample suspected of containing one or more target nucleic acids, a sample buffer comprising a buffer, a chaotropic salt and a non-ionic surfactant, a certain amount of lysing particles, and a certain amount of nucleic acid-binding magnetic particles, placing the sample, sample buffer, lysis particles and nucleic acid binding magnetic particles in the first container to form a lysis mixture, bead beating the lysis mixture in the first container for a period of time sufficient to produce a lysate, wherein the bead beating is performed in the presence of nucleic acid-binding magnetic particles, tapping the first container to break bubbles and foam formed during bead beating and to keep the magnetic particles suspended while allowing the lysed particles to settle, capturing the magnetic particles from the lysate with a magnet and transferring the magnetic particles or unbound lysate to a second container, and releasing the magnetic particles from the magnet and adding an elution buffer to the magnetic particles, mixing the magnetic particles with the elution buffer, recapturing the magnetic particles with a magnet, and separating the elution buffer from the magnetic particles, The steps of the method are completed in <5 minutes.

21. The method of claim 20, wherein capturing the magnetic particles from the lysate is facilitated by collapsing bubbles and foam formed during bead beating and by maintaining the magnetic particles in suspension while allowing the lysed particles to settle.

22. The method of claim 20 or 21, wherein tapping comprises slapping or hitting the first container.

23. The method of any one of claims 20 to 22, wherein striking comprises inflating the inflatable member against the first container within a time range of 1-250 milliseconds.

24. The method of any one of claims 20 to 23, further comprising mixing a second amount of magnetic particles into the lysate after bead beating and before capturing.

25. The method of claim 20, wherein one or more of the sample, sample buffer, lysis particles, and magnetic particles are combined in the first container to form a lysis mixture.

26. The method of any one of claims 20 to 25, further comprising releasing the magnetic particles from the magnet and washing the magnetic particles with a wash buffer, recapturing the magnetic particles with a magnet and removing the wash buffer, and releasing the magnetic particles into an elution buffer after washing.

27. The method of claim 26, wherein the washing does not comprise one or more of: heating the wash buffer and magnetic particles before or during washing, vigorously mixing the magnetic particles and wash buffer, or incubating the magnetic particles and wash buffer for a period of time greater than 10 seconds.

28. The method of any one of claims 20-27, wherein the time period for bead beating is in the range of 1 second to 1 minute.

29. The method of claim 28, wherein the time period for bead beating is 20 seconds.

30. The method of any one of claims 20 to 29, wherein the nucleic acid binding magnetic particles are silica coated magnetic particles.

31. The method of any one of claims 20 to 30, wherein the nucleic acid binding magnetic particles are not significantly degraded by bead beating.

32. The method of claim 20, wherein the sample buffer is an aqueous buffer comprising a buffering agent, 50-60% of a chaotropic agent, and 10-20% of a nonionic surfactant.

33. The method of claim 32, wherein the chaotropic agent is a guanidine salt and the nonionic surfactant is one of TritonX-100, polydocanol (Thesit), Triton X-114, NP-40, Arlasolve 200, Brij O10, octyl β-D-pyranoglucoside, saponin, monododecyl nonaethylene glycol ether and combinations thereof.

34. The method of any one of claims 20-33, wherein the steps of the method are completed in < 4 minutes.

35. The method of any one of claims 20-34, wherein the steps of the method are completed in < 3 minutes.

36. The method of any one of claims 20-35, wherein the steps of the method are completed in <2 minutes.

37. The method of any one of claims 20-35, wherein the steps of the method are completed within 1-3 minutes.

38. The method of any one of claims 20-37, wherein the elution buffer is heated prior to adding the elution buffer to the magnetic particles.

39. The method of any one of claims 20-38, wherein the sample buffer, wash buffer and elution buffer are each an aqueous buffer and are free of organic solvents and alcohols.

40. The method of claim 39, wherein the sample buffer, wash buffer, and elution buffer are provided as dried compositions and each buffer is configured to be rehydrated with an aqueous rehydration fluid.

41. A method for preparing a nucleic acid sample, comprising: providing a sample container comprising a first container, Providing a sample suspected of containing one or more target nucleic acids, a sample buffer comprising a buffer, a chaotropic salt and a non-ionic surfactant, a certain amount of lysing particles, and a certain amount of nucleic acid-binding magnetic particles, placing the sample, sample buffer, lysis particles and nucleic acid binding magnetic particles in the first container to form a lysis mixture, bead beating the lysis mixture in the first container for a period of time sufficient to produce a lysate, wherein the bead beating is performed in the presence of nucleic acid-binding magnetic particles, capturing the magnetic particles from the lysate with a magnet and transferring the magnetic particles or unbound lysate to a second container, and releasing the magnetic particles from the magnet and adding an elution buffer to the magnetic particles, mixing the magnetic particles with the elution buffer, recapturing the magnetic particles with a magnet, and separating the elution buffer from the magnetic particles, The steps of the method are completed in <5 minutes.

42. The method of claim 41, wherein one or more of the sample, sample buffer, lysis particles, and magnetic particles are combined in the first container to form a lysis mixture.

43. The method of any one of claims 41 or 42, further comprising releasing the magnetic particles from the magnet and washing the magnetic particles with a wash buffer, recapturing the magnetic particles with a magnet and removing the wash buffer, and releasing the magnetic particles into an elution buffer after washing.

44. The method of claim 43, wherein the washing does not comprise one or more of: heating the wash buffer and / or magnetic particles before or during washing, vigorously mixing the magnetic particles and wash buffer, or incubating the magnetic particles and wash buffer for a period of time greater than 10 seconds.

45. The method of any one of claims 41 to 44, further comprising mixing a second amount of magnetic particles into the lysate after bead beating and before capture by the magnetic particles.

46. ​​The method of claim 41, wherein the time period for bead beating is in the range of 1 second to 1 minute.

47. The method of claim 46, wherein the time period for bead beating is 20 seconds.

48. The method of any one of claims 41-47, wherein the nucleic acid binding magnetic particles are silica-coated magnetic particles.

49. The method of any one of claims 41-48, wherein the nucleic acid-binding magnetic particles are not significantly degraded by bead beating.

50. The method of claim 41, wherein the sample buffer is an aqueous buffer comprising a buffering agent, 50-60% of a chaotropic agent, and 10-20% of a non-ionic surfactant.

51. The method of claim 50, wherein the chaotropic agent is a guanidine salt and the nonionic surfactant is one of TritonX-100, polydocanol (Thesit), Triton X-114, NP-40, Arlasolve 200, Brij O10, octyl β-D-pyranoglucoside, saponin, monododecyl nonaglycone ether and combinations thereof.

52. The method of claim 43, wherein the wash buffer and elution buffer are provided as dried buffer compositions and are rehydrated during the steps of the method.

53. The method of claim 41, wherein separating the elution buffer from the magnetic particles comprises recapturing the magnetic particles with a magnet and transferring the elution buffer to a third chamber.

54. The method of claim 41, wherein separating the elution buffer from the magnetic particles comprises recapturing the magnetic particles with a magnet and transferring the magnetic particles to another chamber.

55. The method of any one of claims 41-54, wherein the steps of the method are completed in <4 minutes.

56. The method of any one of claims 41-55, wherein the steps of the method are completed in < 3 minutes.

57. The method of any one of claims 41-56, wherein the steps of the method are completed in <2 minutes.

58. The method of any one of claims 41-56, wherein the steps of the method are completed within 1-3 minutes.

59. The method of any one of claims 41-58, wherein the elution buffer is heated prior to adding the elution buffer to the magnetic particles.

60. The method of any one of claims 41 to 59, further comprising agitating the lysate to keep the magnetic particles suspended while allowing the lysed particles to settle, preferably agitating the lysate by tapping.

61. The method of any one of claims 41-60, wherein the sample buffer, wash buffer, and elution buffer are each aqueous buffers and are free of organic solvents and alcohols.

62. The method of claim 61, wherein the sample buffer, wash buffer, and elution buffer are provided as dried compositions and each buffer is configured to be rehydrated with an aqueous rehydration fluid.

63. A method for preparing a nucleic acid sample, comprising: providing a sample container comprising a first chamber, providing a sample suspected of containing one or more target nucleic acids and a sample buffer comprising a buffering agent, a chaotropic salt and a nonionic surfactant, wherein the sample and the sample buffer are combined to form a first mixture, combining the first mixture and an amount of lysed particles in the first chamber, bead milling the first chamber for a period of time sufficient to produce a lysate, A certain amount of nucleic acid-binding magnetic particles is mixed into the lysate, capturing the magnetic particles with a magnet and transferring the magnetic particles or unbound lysate to a second chamber, releasing the magnetic particles from the magnet and washing the magnetic particles with a wash buffer, recapturing the magnetic particles with a magnet, and removing the wash buffer, releasing the magnetic particles and adding an elution buffer to the magnetic particles, mixing the magnetic particles with the elution buffer, recapturing the magnetic particles with a magnet, and separating the elution buffer from the magnetic particles, wherein the steps of the method are completed in < 5 minutes, and The sample buffer, the washing buffer and the elution buffer are each an aqueous buffer, and the buffer does not contain an organic solvent and alcohol.

64. The method of claim 63, wherein the sample buffer is an aqueous buffer comprising a buffering agent, 50-60% of a chaotropic agent, and 10-20% of a non-ionic surfactant.

65. The method of claim 64, wherein the chaotropic agent is a guanidine salt and the nonionic surfactant is one of TritonX-100, polydocanol (Thesit), Triton X-114, NP-40, Arlasolve 200, Brij O10, octyl β-D-pyranoglucoside, saponin, monododecyl nonaglycone ether and combinations thereof.

66. The method of claim 63, wherein the wash buffer and elution buffer are provided as dried buffer compositions and are rehydrated during the steps of the method.

67. The method of claim 63, wherein separating the elution buffer from the magnetic particles comprises recapturing the magnetic particles with a magnet and transferring the elution buffer to a third chamber.

68. The method of claim 63, wherein separating the elution buffer from the magnetic particles comprises recapturing the magnetic particles with a magnet and transferring the magnetic particles to another chamber.

69. The method of any one of claims 63-68, wherein the steps of the method are completed in <4 minutes.

70. The method of any one of claims 63-69, wherein the steps of the method are completed in < 3 minutes.

71. The method of any one of claims 63-70, wherein the steps of the method are completed in <2 minutes.

72. The method of any one of claims 63-70, wherein the steps of the method are completed within 1-3 minutes.

73. The method of claim 63, wherein the bead beating time sufficient to produce the lysate is in the range of 1 second to 1 minute.

74. The method of any one of claims 63-73, wherein the elution buffer is heated.

75. The method of claim 63, wherein mixing a certain amount of nucleic acid-binding magnetic particles into the lysate comprises one or more of the following: mixing a certain amount of nucleic acid-binding magnetic particles into the first mixture before bead beating, and mixing a certain amount of nucleic acid-binding magnetic particles into the lysate after bead beating.

76. A method for preparing a nucleic acid sample, comprising: Provide a first room and a second room, providing a sample suspected of containing one or more target organisms and a sample buffer comprising a buffering agent, a chaotropic salt, and a nonionic surfactant, wherein the sample and the sample buffer are combined to form a first mixture, combining the first mixture and an amount of lysed particles in the first chamber, bead milling the first chamber for a period of time sufficient to produce a lysate, A certain amount of nucleic acid-binding magnetic particles is mixed into the lysate, capturing the magnetic particles with a magnet and removing the lysate and the lysate from the first chamber, releasing the magnetic particles from the magnet into the second chamber and washing the nucleic acid-binding magnetic particles with a wash buffer, recapturing the magnetic particles with a magnet, and removing the wash buffer from the second chamber, releasing the magnetic particles from the magnet and adding an elution buffer to the magnetic particles, mixing the magnetic particles with the elution buffer, recapturing the magnetic particles with a magnet, and separating the elution buffer from the magnetic particles, wherein the steps of the method are completed in < 5 minutes, and The sample buffer, the washing buffer and the elution buffer are each an aqueous buffer, and the buffer does not contain an organic solvent and alcohol.

77. The method of claim 76, wherein the sample buffer is an aqueous buffer comprising a buffering agent, 50-60% of a chaotropic agent, and 10-20% of a non-ionic surfactant.

78. The method of claim 77, wherein the chaotropic agent is a guanidine salt and the nonionic surfactant is one of TritonX-100, polydocanol (Thesit), Triton X-114, NP-40, Arlasolve 200, Brij O10, octyl β-D-pyranoglucoside, saponin, monododecyl nonaglycone ether and combinations thereof.

79. The method of claim 76, wherein the wash buffer and elution buffer are provided as dried buffer compositions and are rehydrated as part of the steps of the method.

80. The method of claim 76, wherein separating the elution buffer from the magnetic particles comprises recapturing the magnetic particles with a magnet and transferring the elution buffer to a third chamber.

81. The method of claim 76, wherein separating the elution buffer from the magnetic particles comprises recapturing the magnetic particles with a magnet and transferring the magnetic particles to another chamber.

82. The method of any one of claims 76-81, wherein the steps of the method are completed in <4 minutes.

83. The method of any one of claims 76-82, wherein the steps of the method are completed in < 3 minutes.

84. The method of any one of claims 76-84, wherein the steps of the method are completed in <2 minutes.

85. The method of any one of claims 76-84, wherein the steps of the method are completed within 1-3 minutes.

86. The method of claim 76, wherein the bead beating time sufficient to produce the lysate is in the range of about 1 second to about 1 minute.

87. The method of claim 76, wherein mixing a certain amount of nucleic acid-binding magnetic particles into the lysate comprises one or more of the following: mixing a certain amount of nucleic acid-binding magnetic particles into the first mixture before bead beating, and mixing a certain amount of nucleic acid-binding magnetic particles into the lysate after bead beating.

88. The method of any one of claims 76-87, wherein the bead milling step further comprises applying pressure on the sample container sufficient to increase frictional heating to aid thermal lysis.

89. The method of claim 88, wherein the pressure is regulated using a feedback mechanism.

90. The method of any one of claims 76-89, further comprising, after the bead milling step, providing impact pressure on the sample container to force the foam to the top of the sample container and collapse the foam bubbles.

91. The method of any one of claims 76-90, wherein the elution buffer is heated.

92. A method for preparing a nucleic acid sample, comprising: A sample container is provided, wherein the sample container comprises a first chamber and a second chamber connected by a plurality of channel fluids and a plurality of reagent wells, wherein the plurality of dry reagent wells comprises at least a dry nucleic acid washing buffer and a dry nucleic acid elution buffer, providing an apparatus comprising: an opening for receiving the container, the opening comprising a bead beater positioned to interact with the container, one or more actuators configured to manipulate the container to move fluid in the container, and a magnet for capturing magnetic particles in the container, providing a sample suspected of containing one or more target nucleic acids and a sample buffer comprising a buffering agent, a chaotropic agent and a nonionic surfactant, wherein the sample and the sample buffer are combined to form a first mixture, combining the first mixture with an amount of lysing particles in a first chamber of the container, introducing a rehydration fluid into the container and rehydrating the dried nucleic acid wash buffer and the dried nucleic acid elution buffer, placing the container in the opening of the apparatus and contacting the first reaction bubble with the bead mill for a period of time sufficient to produce a lysate, A certain amount of nucleic acid-binding magnetic particles is mixed into the lysate, activating a magnet and an actuator associated with the first chamber to recover the nucleic acid-binding magnetic particles from the lysate and deposit the nucleic acid-binding magnetic particles into the second chamber, transferring a first amount of rehydrated wash buffer to the nucleic acid binding magnetic particles for washing, wherein the washing comprises deactivating the magnet, agitating the nucleic acid binding magnetic particles with an actuator associated with the second chamber, reactivating the magnet to recapture the nucleic acid binding magnetic particles, and pushing the wash buffer into the first chamber with the actuator associated with the second chamber, and binding to the nucleic acid in the second and / or third reaction blisters to elute the nucleic acid from the nucleic acid binding magnetic particles, wherein the elution comprises deactivating the magnet, agitating the nucleic acid binding magnetic particles with an actuator associated with the second chamber, reactivating the magnet to recapture the nucleic acid binding magnetic particles, and pushing the elution buffer having the eluted nucleic acid therein to the third chamber or reagent well, wherein the steps of the method are completed in < 5 minutes, and The sample buffer, the washing buffer and the elution buffer are each an aqueous buffer and the buffer does not contain an organic solvent and alcohol.

93. The method of claim 92, wherein the sample buffer is an aqueous buffer comprising a buffer, 50-60% of a chaotropic agent, and 10-20% of a non-ionic surfactant.

94. The method of claim 93, wherein the chaotropic agent is a guanidine salt and the nonionic surfactant is one of TritonX-100, polydocanol (Thesit), Triton X-114, NP-40, Arlasolve 200, Brij O10, octyl β-D-pyranoglucoside, saponin, monododecyl nonaglycone ether and combinations thereof.

95. The method of any one of claims 92-94, wherein the steps of the method are completed in <4 minutes.

96. The method of any one of claims 92-95, wherein the steps of the method are completed in <3 minutes.

97. The method of any one of claims 92-96, wherein the steps of the method are completed in <2 minutes.

98. The method of any one of claims 92-96, wherein the steps of the method are completed within 1-3 minutes.

99. The method of claim 92, wherein the bead beating time sufficient to produce the lysate is in the range of about 5 seconds to about 1 minute.

100. The method of claim 92, wherein mixing a certain amount of nucleic acid-binding magnetic particles into the lysate comprises one or more of the following: mixing a certain amount of nucleic acid-binding magnetic particles into the first mixture before bead beating, and mixing a certain amount of nucleic acid-binding magnetic particles into the lysate after bead beating.

101. The method of any one of claims 92-100, wherein the elution buffer is heated.

102. The method of any one of claims 92-101, wherein the wash buffer further comprises a binding buffer.

Images