Cell lysis and nucleic acid recovery

Abstract

Methods and systems for rapid preparation of nucleic acid samples.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of and priority to U.S. Provisional Patent Application No. 63 / 369,107 filed on July 22, 2022 and U.S. Provisional Patent Application No. 63 / 450,492 filed on March 7, 2023, the entireties of which are incorporated herein by reference. Background Art

[0003] Infectious diseases account for approximately 7% of human mortality in the United States, Canada, and Western Europe, and over 40% in developing regions. Infectious diseases result in a variety of clinical manifestations. Fever, pneumonia, meningitis, diarrhea, and bloody diarrhea are common and obvious manifestations. Although physical manifestations suggest some pathogens and exclude others as etiological agents, a variety of potential causative agents exist, and a clear diagnosis often requires a variety of assays. Traditional microbiological techniques for diagnosing pathogens can take days or weeks, which often delays the appropriate course of treatment.

[0004] In recent years, polymerase chain reaction (PCR) has become the preferred method for rapid diagnosis of infectious agents. PCR can be a fast, sensitive and specific tool for diagnosing infectious diseases. The challenge of using PCR as the primary diagnostic means lies in the diversity of possible pathogenic organisms and the low levels of organisms present in some pathological specimens. It is usually impractical to run a large plate of PCR assays for each possible pathogenic organism, most of which are expected to be negative. The problem is more serious when the pathogen nucleic acid concentration is low and a large volume of sample is required to gather enough reaction templates. In some cases, there are not enough samples to measure all possible pathogenic factors. One solution is to run "multiplex PCR", in which multiple targets of the sample are measured simultaneously 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 of clearly analyzing multiple products. In order to solve these problems, the assay can be divided into multiple secondary PCRs subsequently. Nested secondary reactions within the primary product usually increase robustness. However, this further processing may be expensive and may cause contamination or other problems.

[0005] BioFire Diagnostics, LLC, Salt Lake City, UT is a user-friendly, highly multiplexed PCR system developed for the diagnostic market. The single sample instrument accepts a diagnostic "bag" that integrates 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 multiple organisms simultaneously (e.g., up to 30 or more different organisms and molecular markers). This system is very suitable for pathogen identification, where many different pathogens show similar clinical symptoms. Currently available diagnostic test strips (panels) include respiratory test strips for upper respiratory tract infections, blood culture test strips for bloodstream infections, GI test strips for gastrointestinal infections, meningitis / encephalitis test strips for central nervous system infections, pneumonia test strips for lower respiratory tract infections, and bone and joint test strips for bone and joint infections. Other test strips are under development.

[0006] When PCR first became popular in the late 1980s, the process was slow. The typical protocol was denaturation at 94°C for 1 minute, annealing at 55°C for 2 minutes, and extension at 72°C for 3 minutes. If the transition time between temperatures is included, 8 minute cycles are typical, which results in 4 hours to complete 30 cycles. Over the years, systems have become faster, 30 minute protocols are common, and faster PCR equipment is available. Even with faster PCR protocols, many diagnostic PCR reactions still begin with target cell lysis and extraction / purification of target nucleic acid from the target cells using older techniques.

[0007] As PCR becomes faster, the time spent on sample preparation becomes an increasingly larger part of the processing time. A possible solution is to essentially skip sample preparation, rely on chemical and / or heated lysis to break cells and perform nucleic acid amplification in the original lysate. This does save time, but the adequacy of lysis is not always reliable, and there are many potential inhibitors of nucleic acid amplification in the unpurified lysate. Another possible solution involves mechanical lysis followed by nucleic acid recovery and nucleic acid purification from the lysate. Although this is in line with expectations from the perspective of the cleanliness of the starting template for nucleic acid amplification, it is traditionally quite slow. What is in line with expectations is to use a faster method to prepare samples while maximizing the cleanliness of the starting template for nucleic acid amplification. There is such a demand in the art: keep the cleanliness and robustness of traditional sample preparation (i.e. mechanical lysis, 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

[0008] Methods and systems for cell lysis and nucleic acid recovery are described herein. The method includes combining lysis particles (e.g., zirconium silicate beads) and a first amount of nucleic acid-binding magnetic particles (e.g., silica-coated magnetic beads) with a sample suspected of containing one or more target nucleic acids and lysis buffer in a container (e.g., a tube). The container can then be appropriately placed in a bead beater for a sufficiently long time to produce a lysate. After bead beating is performed to produce a lysate, a second amount of nucleic acid-binding magnetic particles can be appropriately mixed into the lysate. After the second amount of nucleic acid-binding magnetic particles is mixed into the lysate, the first and second amounts of nucleic acid-binding magnetic particles can be recovered from the lysate-with a magnet as an illustration.

[0009] After recovery, magnetic particles can be transferred to another container, released and washed once or many times to remove lysate residues. Then magnetic particles can be recaptured (with magnet as an illustration), washing buffer can be removed, magnetic particles can be released again and mixed with elution buffer to elute captured nucleic acid from magnetic particles. Magnetic particles can be captured again, and elution buffer can be recovered and transferred to a clean tube. Alternatively, magnetic particles can be removed from tubes using magnets and magnetic particles can be disposed of. The material of wash-out can be used for various downstream assays, such as but not limited to the presence of one or more target nucleic acids suspected to be present in the sample by measuring the material of wash-out using one or more of nucleic acid amplification and assay, sequencing, next generation sequencing, etc. In other words, if the nucleic acid in the sample is from a cell (such as a pathogen cell), then assay can be used to identify the cell from which nucleic acid is derived.

[0010] In one example, a method for cell lysis and nucleic acid recovery is disclosed. The method includes the following steps: providing a sample container, placing a certain number of lysis particles in the sample container, adding a sample suspected of containing one or more target nucleic acids and a lysis buffer to the container, adding a first number of nucleic acid-binding magnetic particles to the container, agitating the container with the lysis particles, the sample, the lysis buffer and the first number of nucleic acid-binding magnetic particles for a first period of time to generate a lysate, mixing a second number of nucleic acid-binding magnetic particles into the lysate in the container, and recovering the first and second numbers of nucleic acid-binding magnetic particles from the lysate.

[0011] In another example, a method for cell lysis and nucleic acid recovery is disclosed. The method includes the following steps: providing a sample container comprising a first chamber and a second chamber, combining a certain number of lysis particles, a first number of nucleic acid-binding magnetic particles, a sample suspected of containing one or more target nucleic acids, and a lysis buffer in the first chamber, agitating the first chamber of the sample container having the lysis particles, the sample, the lysis buffer, and the first number of nucleic acid-binding magnetic particles for a period of time sufficient to generate a lysate, mixing a second number of nucleic acid-binding magnetic particles into the lysate, and capturing the first and second numbers of nucleic acid-binding magnetic particles from the lysate in the second chamber using a magnet.

[0012] In yet another example, a method for cell lysis and nucleic acid recovery is disclosed. The method includes the following steps: providing a sample container comprising a plurality of fluidically connected reaction chambers, the reaction chambers including a sample lysis chamber, a nucleic acid recovery chamber, and at least a first nucleic acid amplification chamber, combining a certain number of lysis particles, a first number of nucleic acid binding magnetic particles, a sample suspected of containing one or more target nucleic acids, and a lysis buffer in the sample lysis chamber, agitating the sample lysis chamber of the sample container having the lysis particles, the sample, the lysis buffer, and the first number of nucleic acid binding magnetic particles for a period of time sufficient to generate a lysate, mixing a second number of nucleic acid binding magnetic particles into the lysate, and using a magnet to capture the first and second numbers of nucleic acid binding magnetic particles from the lysate in the nucleic acid recovery chamber. The method further includes: releasing the magnet and washing the first and second numbers of nucleic acid binding magnetic particles with a wash buffer in the nucleic acid recovery chamber, re-capturing the first and second numbers of nucleic acid binding magnetic particles with a magnet and removing the wash buffer, releasing the magnet and eluting nucleic acids from the first and second numbers of nucleic acid binding magnetic particles with an elution buffer in the nucleic acid recovery chamber, and re-capturing the first and second numbers of nucleic acid binding magnetic particles with a magnet and transferring the elution buffer to the first nucleic acid amplification chamber.

[0013] In one aspect of the methods disclosed herein, a first amount of nucleic acid binding magnetic particles is added and then a second amount of nucleic acid binding magnetic particles is added to increase the recovery of RNA and DNA from the sample. Such recovery increase can be represented, for example, by an improvement in Cp (as shown by an earlier Cp) or an improvement in test sensitivity.

[0014] This article describes:

[0015] A1. A method for cell lysis and nucleic acid recovery, comprising:

[0016] Providing a sample container, a certain number of lysis particles, a sample suspected of containing one or more target nucleic acids, a lysis buffer and a first number of nucleic acid-binding magnetic particles,

[0017] placing a sample, a certain number of lysis particles, a lysis buffer and a first number of nucleic acid-binding magnetic particles in a container,

[0018] agitating a container having lysis particles, a sample, a lysis buffer, and a first number of nucleic acid-binding magnetic particles for a first period of time to generate a lysate,

[0019] dispensing a second number of nucleic acid-binding magnetic particles into the lysate in the container, and

[0020] recovering the first and second quantities of nucleic acid-bound magnetic particles from the lysate,

[0021] Wherein adding a first number of nucleic acid binding magnetic particles and then adding a second number of nucleic acid binding magnetic particles is selected to increase the recovery rate of nucleic acid from the sample.

[0022] A2. A method as described in clause A1, wherein the lysis buffer comprises a buffer, a chaotropic salt and a non-ionic surfactant.

[0023] A2.1 A method as described in clause A1 or A2, wherein the lysis buffer is an aqueous buffer comprising a buffer, 50-60% of a chaotropic agent and 10-20% of a non-ionic surfactant.

[0024] A2.2 A method as described in clause A1, A2 or A2.1, wherein the chaotropic agent is a guanidine salt, and the non-ionic surfactant is one of the following: 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.

[0025] A3. A method as described in any of clauses A1-A2.2, wherein the recovery further includes one of the following: releasing the first and second quantities of nucleic acid-bound magnetic particles recovered from the lysate into another sample container, or removing the lysate and lysed particles from the container and releasing the first and second quantities of nucleic acid-bound magnetic particles recovered from the lysate back into the container.

[0026] A4. A method as described in any of clauses A1-A3, further comprising: washing the first and second quantities of nucleic acid-binding magnetic particles with a wash buffer, re-capturing the first and second quantities of nucleic acid-binding magnetic particles with a magnet, and removing the wash buffer.

[0027] A5. A method as described in any of clauses A1-A4, wherein the washing does not include one or more of the following: heating the wash buffer and the magnetic particles before or during washing, vigorously mixing the magnetic particles with the wash buffer, or incubating the magnetic particles with the wash buffer for a period of time greater than 10 seconds.

[0028] A6. A method as described in any of clauses A1-A5, further comprising: releasing the first and second quantities of nucleic acid-binding magnetic particles from the magnet, adding elution buffer to the first and second quantities of nucleic acid-binding magnetic particles, releasing the magnet, and mixing the magnetic particles with the elution buffer, re-capturing the first and second quantities of nucleic acid-binding magnetic particles with the magnet, and transferring the elution buffer to another sample container.

[0029] A7. A method as described in any of clauses A1-A6, further comprising: adding elution buffer to the first and second quantities of nucleic acid-binding magnetic particles and mixing the magnetic particles with the elution buffer, re-capturing the first and second quantities of nucleic acid-binding magnetic particles with a magnet, and transferring the first and second quantities of nucleic acid-binding magnetic particles to another sample container.

[0030] A8. A method as described in any of clauses A1-A7, wherein the elution buffer is configured to elute nucleic acids captured by the nucleic acid-binding magnetic particles into the elution buffer.

[0031] A9. A method as described in any of clauses A1-A8, wherein the amount of the second number of nucleic acid-binding magnetic particles is substantially equal to the amount of the first number of nucleic acid-binding magnetic particles.

[0032] A10. The method of any one of clauses A1-A9, further comprising determining the presence of one or more target nucleic acids suspected to be present in the sample in the elution buffer.

[0033] A11. A method as described in any of clauses A1-A10, wherein the determination includes a nucleic acid amplification step and a step of detecting the amplified nucleic acid.

[0034] A12. The method of any of clauses A1-A11, wherein the agitating step comprises heating the sample and the dispersing step comprises cooling the sample.

[0035] B1. A method for cell lysis and nucleic acid recovery, comprising:

[0036] Provide sample containers,

[0037] combining a number of lysis particles, a first number of nucleic acid-binding magnetic particles, a sample suspected of containing one or more target nucleic acids, and a lysis buffer in a sample container,

[0038] agitating the lysis particles, the sample, the lysis buffer, and the first number of nucleic acid-binding magnetic particles in the sample container for a period of time sufficient to generate a lysate,

[0039] dispersing a second number of nucleic acid-binding magnetic particles into the lysate in the sample container,

[0040] incubating the first and second quantities of nucleic acid-bound magnetic particles in the lysate for a period of time, and

[0041] capturing a first and a second number of nucleic acid-binding magnetic particles from the lysate using a magnet,

[0042] Wherein adding a first number of nucleic acid binding magnetic particles and then adding a second number of nucleic acid binding magnetic particles is selected to increase the recovery rate of nucleic acid from the sample.

[0043] B2. A method as described in clause B1, wherein the time period for incubating the first and second quantities of nucleic acid-bound magnetic particles in the lysate is in the range of 1 second to 1 minute.

[0044] B3. A method as described in clause B1 or B2, wherein the time period is in the range of 20-30 seconds.

[0045] B4. A method as described in any of clauses B1-B3, wherein capturing the first and second quantities of magnetic particles from the lysate further includes: transferring the magnetic particles to a second sample container and releasing the magnetic particles from the magnet into the second sample container, or removing the lysate and lysed particles from the first sample container and releasing the magnetic particles from the magnet back into the first sample container.

[0046] B5. A method as described in any of clauses B1-B4, wherein capturing the first and second quantities of magnetic particles from the lysate preferably does not include capturing lysis particles.

[0047] B6. A method as described in any of clauses B1-B5, further comprising: releasing the first and second quantities of nucleic acid-bound magnetic particles from the magnet, adding a wash buffer to the magnetic particles, washing the magnetic particles with the wash buffer, re-capturing the magnetic particles with a magnet, removing the wash buffer, adding an elution buffer to the magnetic particles, releasing the magnetic particles from the magnet, mixing the magnetic particles with an elution buffer, re-capturing the magnetic particles with a magnet, and transferring the magnetic particles or the elution buffer to another sample container.

[0048] B7. A method as described in any of clauses B1-B6, wherein the washing does not include one or more of the following: heating the wash buffer and the magnetic particles before or during washing, vigorously mixing the magnetic particles with the wash buffer, or incubating the magnetic particles with the wash buffer for a period of time greater than 10 seconds.

[0049] B8. A method as described in any of clauses B1-B7, wherein the steps of the method are completed in <4 minutes.

[0050] B9. A method as described in any of clauses B1-B8, wherein the steps of the method are completed in <3 minutes.

[0051] B10. A method as described in any of clauses B1-B9, wherein the steps of the method are completed in <2 minutes.

[0052] B11. A method as described in any of clauses B1-B10, wherein the steps of the method are completed within 1-3 minutes.

[0053] C1. A method for cell lysis and nucleic acid recovery, comprising:

[0054] providing a sample container comprising a plurality of fluidically connected reaction chambers, the reaction chambers including a sample lysis chamber, a nucleic acid recovery chamber, and at least a first nucleic acid amplification chamber,

[0055] combining a number of lysis particles, a first number of nucleic acid-binding magnetic particles, a sample suspected of containing one or more target nucleic acids, and a lysis buffer in a sample lysis chamber,

[0056] agitating the lysis particles, the sample, the lysis buffer, and the first number of nucleic acid-binding magnetic particles in the lysis chamber for a period of time sufficient to generate a lysate,

[0057] mixing a second quantity of nucleic acid-binding magnetic particles into the lysate,

[0058] transferring at least a portion of the lysate having the first and second quantities of nucleic acid-binding magnetic particles suspended therein to a nucleic acid recovery chamber,

[0059] capturing a first and a second number of nucleic acid-binding magnetic particles from the lysate in the nucleic acid recovery chamber using a magnet,

[0060] removing the lysate from the nucleic acid recovery chamber without removing the first and second quantities of nucleic acid-binding magnetic particles,

[0061] releasing the first and second quantities of nucleic acid-binding magnetic particles from the magnet and washing the first and second quantities of nucleic acid-binding magnetic particles with a wash buffer in a nucleic acid recovery chamber,

[0062] recapturing the first and second quantities of nucleic acid-binding magnetic particles with a magnet and removing the wash buffer,

[0063] releasing the magnet and mixing the first and second quantities of nucleic acid-binding magnetic particles with an elution buffer in a nucleic acid recovery chamber to elute the nucleic acids from the magnetic particles,

[0064] recapturing the first and second quantities of nucleic acid-binding magnetic particles with a magnet and transferring an elution buffer to a first nucleic acid amplification chamber,

[0065] Wherein adding a first number of nucleic acid binding magnetic particles and then adding a second number of nucleic acid binding magnetic particles is selected to increase the recovery rate of nucleic acid from the sample.

[0066] C2. The method of clause C1, wherein capturing nucleic acid-bound magnetic particles comprises deploying a magnet adjacent to a nucleic acid recovery chamber to contain the magnetic particles in the chamber.

[0067] C3. A method as described in clause C1 or C2, further comprising: combining an elution buffer with reagents for a nucleic acid amplification reaction in a first nucleic acid amplification chamber to form an amplification mixture, and subjecting the amplification mixture to amplification conditions to determine the presence of one or more target nucleic acids suspected to be present in the sample.

[0068] C4. A method as described in any of clauses C1-C3, wherein the determination is selected from nucleic acid amplification, sequencing and next-generation sequencing.

[0069] C5. A method as described in any of clauses C1-C4, wherein the second number of nucleic acid binding magnetic particles is substantially equal to the first number of nucleic acid binding magnetic particles.

[0070] C6. A method as described in any of clauses C1-C5, wherein the second number of nucleic acid binding magnetic particles is substantially greater than the first number of nucleic acid binding magnetic particles.

[0071] C7. A method as described in any of clauses C1-C6, wherein the second number of nucleic acid-binding magnetic particles is substantially less than the first number of nucleic acid-binding magnetic particles.

[0072] C8. A method as described in any of clauses C1-C7, wherein the agitating step includes heating the sample and the mixing step includes cooling the sample.

[0073] C9. A method as described in any of clauses C1-C8, wherein the washing does not include one or more of the following: heating the wash buffer and the magnetic particles before or during washing, vigorously mixing the magnetic particles with the wash buffer, or incubating the magnetic particles with the wash buffer for a period of time greater than 10 seconds.

[0074] C10. A method as described in any of clauses C1-C9, wherein the steps of the method are completed in <4 minutes.

[0075] C11. A method as described in any of clauses C1-C10, wherein the steps of the method are completed in <3 minutes.

[0076] C12. A method as described in any of clauses C1-C11, wherein the steps of the method are completed in <2 minutes.

[0077] C13. A method as described in any of clauses C1-C12, wherein the steps of the method are completed within 1-3 minutes.

[0078] This Summary is provided to introduce a selection of concepts in a simplified form that are further described below in the Detailed Description. This Summary is not intended to identify key features 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.

[0079] Additional features and advantages will be described in the following description, and in part will be apparent from the description or may be learned by practice of the invention. The features and advantages may be realized and obtained by 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 learned by practice of the invention as set forth below. BRIEF DESCRIPTION OF THE DRAWINGS

[0080] Figure 1 A flexible bag that can be used for stand-alone PCR is shown.

[0081] Figure 2 is with Figure 1 An exploded perspective view of an instrument used with the bag, including Figure 1 bag.

[0082] Figure 3 show Figure 1 The bag together Figure 2 The capsule component.

[0083] Figure 4 show Figure 2 A motor is used in one illustrative embodiment of an apparatus.

[0084] Figures 5A-5E The steps of the method for cell lysis and nucleic acid recovery are described.

[0085] Fig. 6Ais a bar graph illustrating the detection of various concentrations of DNA organisms during bead beating lysis in the presence of silica coated magnetic particles (BBw / MB) or in the absence of silica coated magnetic particles (BBw / o MB).

[0086] Figure 6B is a bar graph illustrating the detection of various concentrations of RNA organisms during bead beating lysis in the presence of silica coated magnetic particles (BBw / MB) or in the absence of silica coated magnetic particles (BB w / o MB).

[0087] Fig. 7A The crossing point (Cp) was compared between a control assay in which magnetic particles were added after lysis (w / o Split Magbeads) and an assay in which a first amount of magnetic particles was added to lysis and a second amount of magnetic particles was added after lysis (w / Split Magbeads).

[0088] Figure 7B Explain the DNA test and Fig. 7A Similar data.

[0089] Figure 7C Description of RNA determination and Fig. 7A Similar data.

[0090] Figure 8 Description Cp comparisons were performed for amplification of nucleic acids recovered from magnetic particles contained in lysate with no wash, one wash or two washes.

[0091] Fig. 9 Shown are the fragment sizes of human genomic DNA detected with or without bead beating for 120 seconds.

[0092] Fig. 10A Illustrated are the average Cp responses for elution temperatures for DNA and RNA assays.

[0093] 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.

[0094] Fig.11 is a bar graph illustrating specific examples of sample preparation time. DETAILED DESCRIPTION

[0095] 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 understood as being limited to the exemplary embodiments set forth herein. On the contrary, these exemplary embodiments are provided to make the present disclosure comprehensive and complete, and to convey the scope of the present disclosure to those skilled in the art. In the accompanying drawings, the sizes and relative sizes of layers and regions may be exaggerated for clarity. Throughout the description, similar reference numerals refer to similar elements.

[0096] Unless otherwise defined, all terms used herein (including technical and scientific terms) have the same meaning as commonly understood by those of ordinary skill in the art to which the present disclosure belongs. It should also be further understood that terms such as those defined in common dictionaries should be interpreted as having a meaning consistent with their meanings in the context of the present application and the relevant field, and should not be interpreted in an idealized or overly formal sense, unless clearly defined as such herein. The terms used in the description of the invention herein are only used for the purpose of describing a specific embodiment, and are not intended to limit the present invention. Although many methods and materials similar or equivalent to the methods and materials described herein can be used in the practice of the present disclosure, only some exemplary materials and methods are described herein.

[0097] 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.

[0098] Various aspects of the present disclosure, including devices, systems, methods, etc., may be described with reference to one or more exemplary embodiments. As used herein, the terms "exemplary" and "illustrative" mean "serving as an example, instance, or illustration," and should not necessarily be construed as being preferred or advantageous over other embodiments disclosed herein. In addition, reference to an "embodiment" or "embodiment" of the present disclosure or invention includes specific reference to one or more embodiments thereof, and vice versa, and is intended to provide illustrative examples without limiting the scope of the invention, which is indicated by the appended claims rather than the following description.

[0099] It is worth noting 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 tiles. Similarly, references to multiple referents should be understood to include a single referent and / or multiple referents unless the content and / or context clearly dictates otherwise. Thus, reference to a "tile" does not necessarily require a plurality of such tiles. Rather, it should be understood that regardless of inflection; one or more tiles are contemplated herein.

[0100] As used throughout this application, the words "can" and "may" are used in a permissive sense (i.e., meaning having the potential to...) rather than in 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, 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.

[0101] As used herein, directions and / or any terms, such as "top", "bottom", "left", "right", "up", "down", "upper", "lower", "inner", "outer", "inner", "outer", "interior", "exterior", "proximal", "distal", "forward", "reverse", etc., may be used alone to indicate relative directions and / or orientations and may not otherwise be intended to limit the scope of the present disclosure (including this specification, invention and / or claims).

[0102] It should be understood that when an element is referred to as being "coupled", "connected" or "responsive" to another element or "on another element", it can be directly coupled, connected or responsive to another element or on another element, or there may be intervening elements. Conversely, when an element is referred to as being "directly coupled", "directly connected" or "directly responsive" to another element or "directly on another element", there are no intervening elements.

[0103] Exemplary embodiments of the inventive concept are described herein with reference to cross-sectional diagrams, which are schematic diagrams of idealized embodiments (and intermediate structures) of the exemplary embodiments. Therefore, variations in the shapes illustrated due to manufacturing techniques and / or tolerances are to be expected. Therefore, the exemplary embodiments of the inventive concept should not be understood as being limited to the specific shapes of the regions described herein, but should include deviations in shapes such as those caused by manufacturing. Therefore, the regions illustrated in the figures are schematic in nature and their shapes are not intended to illustrate the actual shapes of the regions of the equipment, and are not intended to limit the scope of the exemplary embodiments.

[0104] It should 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 teaching of this embodiment, the "first" element may be referred to as the "second" element.

[0105] It should also be understood that the various embodiments described herein can be combined 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, equipment, devices, systems, methods, processes, compositions and / or kits according to certain embodiments of the present disclosure may include, incorporate or otherwise contain the characteristics, features, parts, components, elements, steps and / or the like (including systems, methods, devices and / or the like) described in other embodiments disclosed herein. Therefore, mentioning a specific feature associated with an embodiment should not be understood as being limited to the application in the embodiment.

[0106] The headings used herein are for organizational purposes only and are not intended to limit the scope of the specification or claims. For ease of understanding, similar reference numerals have been used to designate similar elements shared in the figures, where possible. In addition, similar element numbers have been used in the various figures, where possible. In addition, alternative configurations of specific elements may each include a separate letter attached to the element number.

[0107] The term "about" as used herein means approximately, in the region of ..., roughly or about. When the term "about" is used in conjunction with a numerical range, it modifies the range by extending the upper and lower limits of the numerical values ​​set forth. Generally speaking, the term "about" is used herein to modify a numerical value to a 5% change 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 a specific value forms another embodiment. It should be further understood that the endpoints of each range are important relative to the other endpoints and are independent of the other endpoints.

[0108] As used herein, the word "or" means any one member of a particular list and also includes any combination of members of that list.

[0109] In one aspect, as described in further detail herein, the microorganisms from the sample or growth medium can be separated and interrogated to characterize and / or identify the microorganisms present in the sample. As used herein, the term "separation" is intended to encompass any microbial sample that has been removed, concentrated or otherwise separated from its original state or from a growth or culture matrix. For example, according to the present invention, microorganisms (e.g., as separated samples) can be separated from non-microorganisms or non-microorganism components, which otherwise may interfere with characterization and / or identification. The term can include microorganisms separated from a mixture by centrifugation, filtration or any other separation technology known in the art. Therefore, the separated microbial sample can include a collection of microorganisms and / or its components that are more concentrated than the original sample or otherwise separated from the original sample, and the range can be from a dense microorganism mass of tightly packed microorganisms to a diffusion layer of microorganisms. The non-microorganism components separated from the microorganism can include non-microorganism cells (e.g., blood cells and / or other tissue cells) and / or any of its components. In one aspect, microorganisms are separated from a lysate mixture comprising cracked non-microorganism cells and substantially intact microorganism cells.

[0110] In some embodiments, microbial sample is incomplete from its original state or from the separation of growth or culture matrix. In other words, remove, concentrate or otherwise make microorganism separate from its original state and do not separate microbial sample from other components of sample completely or from growth or culture matrix. In some cases, there is from sample or from the minute amount fragment of growth or culture matrix. For example, the amount of the fragment or growth or culture matrix existing in the sample of separation may not be enough to disturb the identification of microorganism or characterize or the further growth of microorganism. In some embodiments, 99% is pure contamination element in the sample of separation, but it may also be 95% pure, 90% pure, 80% pure, 70% pure, 60% pure, 50% pure or have minimum purity, which still allows to identify the microorganism in the sample of separation via downstream identification technology.

[0111] Although the present invention refers to the testing of microorganisms and viruses, the methods proposed herein can be used for a wide variety of sample types and a wide variety of nucleic acid tests. Therefore, "sample" means an animal; a tissue or organ from an animal; a cell (in a subject, collected directly from a subject, or 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 (such as, 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.

[0112] In another aspect described in further detail herein, the microorganisms from the sample or growth substrate can be precipitated (pellet) and interrogated to characterize and / or identify the microorganisms present in the sample. As used herein, the term "precipitation" is intended to encompass any microorganism sample compressed or deposited into a microorganism block. For example, the microorganisms from the sample can be compressed or deposited into a block at the bottom of the tube by centrifugation or other methods known in the art. The term includes a collection of microorganisms (and / or its components) at the bottom and / or side of the container after centrifugation. According to the present invention, microorganisms can be precipitated out (e.g., as a substantially purified microorganism precipitate) from non-microorganisms or non-microorganism components that may otherwise interfere with characterization and / or identification.

[0113] As used herein, the phrase "nucleic acid" refers to naturally occurring or synthetic oligonucleotides or polynucleotides, whether DNA or RNA or DNA-RNA hybrids, single-stranded or double-stranded, sense or antisense, which can hybridize with complementary nucleic acids by Watson-Crick base pairing. The nucleic acids of the present invention may also include nucleotide analogs (e.g., BrdU) and non-phosphodiester internucleoside bonds (e.g., peptide nucleic acids (PNA) or thiodiester bonds). Specifically, nucleic acids may include, but are not limited to, DNA, RNA, mRNA, rRNA, cDNA, gDNA, ssDNA, dsDNA, or any combination thereof.

[0114] "Probe", "primer" or "oligonucleotide" means a sequence-defined single-stranded nucleic acid molecule that can base pair with a second nucleic acid molecule containing a complementary sequence ("target"). The stability of the resulting hybrid depends on the length, GC content, and 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 stringency of the hybridization conditions. The stringency of hybridization is affected by parameters such as temperature, salt concentration, and the concentration of organic molecules (e.g., formamide), and is determined by methods known to those skilled in the art. Probes, primers, and oligonucleotides can be detectably labeled (radioactively, fluorescently, or non-radioactively) by methods well known to those skilled in the art. dsDNA can be detected using dsDNA binding dyes. It should 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.

[0115] "dsDNA binding dye" means a dye that fluoresces differently when bound to double-stranded DNA than when bound to single-stranded DNA or free in solution, and generally fluoresces more strongly. Although dsDNA binding dyes are mentioned, it should be understood that any suitable dye may be used herein, some non-limiting illustrative dyes of which are described in U.S. Pat. No. 7,387,887, which is incorporated herein by reference. Other signal-generating substances may be used to detect nucleic acid amplification and melting, as exemplified by enzymes, antibodies, and the like known in the art.

[0116] "Specifically hybridizes" 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, while not substantially base pairing with other nucleic acids.

[0117] "High stringency conditions" are typically meant to occur at about the melting temperature (Tm) minus 5°C (ie, 5° below the Tm of the probe). Functionally, high stringency conditions are used to identify nucleic acid sequences that have at least 80% sequence identity.

[0118] "Lysing particles" means 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 fall within the scope of this term, including glass and sand lysing particles. The term "cell lysis component" can include lysing particles, but may also include other components known in the art, such as components for chemical lysis.

[0119] Although PCR is the amplification method used in the examples herein, it should be 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 isothermal amplification of DNA (LAMP); isothermal and chimeric primer-initiated amplification of nucleic acids (ICAN); target-based helicase-dependent amplification (HDA); 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 when measuring in cycles, doubling times, or crossing points (Cp), and additional reaction time can be added when additional PCR cycles are added in the embodiments described herein. It should be understood that the protocol may need to be adjusted accordingly.

[0120] Although various examples herein refer to human targets and human pathogens, these examples are illustrative only. 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 samples, veterinary samples, industrial samples and environmental samples.

[0121] Various embodiments disclosed herein use stand-alone nucleic acid analysis bags to assay samples for the presence of various biological substances, with antigens and nucleic acid sequences as illustrations, as illustrations in a single closed system. Such systems, including bags and instruments used with bags, are disclosed in more detail in U.S. Patent Nos. 8,394,608; and 8,895,295; and U.S. Patent No. 10,464,060, which are incorporated herein by reference. However, it should be understood that such bags are only illustrative, and the nucleic acid preparation and amplification reactions discussed herein can be performed in any of the various open or closed system sample containers known in the art using various nucleic acid purification and amplification systems known in the art, including 96-well plates, plates of other configurations, arrays, rotating trays, etc. Although the terms "sample well", "sample container", "amplification well", "amplification container", etc. are used herein, these terms are intended to cover bubble wells, tubes, and various other reaction containers used in these amplification systems. In one embodiment, the bag is used to assay a variety of pathogens. The bag may include one or more bubbles used as sample wells, as illustrations in a closed system. For illustration, various steps, including nucleic acid preparation, primary large-volume multiplex PCR, dilution of primary amplification products, and secondary PCR can be performed in an optional disposable bag, with optional real-time detection or post-amplification analysis such as melting curve analysis as the end point. In addition, it should 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 multiple reactions for the first stage amplification, it should be understood that this is only illustrative, and in some embodiments, the first stage amplification can be monoplex. In an illustrative example, the first stage monoplex amplification targets a housekeeping gene, and the second stage amplification uses the difference of the housekeeping gene for identification. Therefore, although various embodiments discuss the first stage multiplex amplification, it should be understood that this is only illustrative.

[0122] Figure 1Show can be used for the illustrative bag 510 of 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 similar item numbers are identical.Accessory 590 provides inlet channel 515a to 515l, which is also used as a reagent reservoir or waste reservoir. For illustration, reagent can be freeze-dried in accessory 590 and rehydrated before use.Blisters 522,544,546,548,564 and 566 and their respective passages 514,538,543,552,553,562 and 565 are similar to the blisters of the same number in Figure 15 of U.S. Patent No. 8,895,295. Figure 1 The second stage reaction zone 580 in is similar to the second stage reaction zone in U.S. Patent No. 8,895,295, but the second stage holes 582 of the high density array 581 are arranged in a somewhat different pattern. Figure 1 The more annular pattern of the high density array 581 in the embodiment 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 provides 102 second stage holes 582. The bag 510 is suitable for use in Instrument (BioFire Diagnostics, LLC, Salt Lake City, UT). However, it should be understood that the bag embodiment is illustrative only.

[0123] Although other containers can be used, for illustration, bag 510 can be formed by two layers of flexible plastic film or other flexible materials, such as polyester, polyethylene terephthalate (PET), polycarbonate, polypropylene, polymethyl methacrylate, its mixture, combination and layer, which can be made by any process known in the art, including extrusion, plasma deposition and lamination. For example, each layer can be composed of a single type or multiple types of materials of one or more layers 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 channels are known in the art. If plastic film is used, each layer can be bonded together illustratively by heat sealing. For illustration, the material has low nucleic acid binding and low protein binding capacity.

[0124] For embodiments employing 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 film thicknesses. For plastics with aluminum or other foil laminations, the portion of the bag to be read by the fluorescence detection device may not have 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 do not have 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. For illustration, the bag 510 can be made of a transparent material capable of transmitting approximately 80%-90% of the incident light.

[0125] In illustrative embodiments, the material is moved between the blisters by applying pressure, illustratively pneumatic pressure, to the blisters and channels. Thus, in embodiments employing pressure, illustratively, the bag material is flexible enough to allow the pressure to have the desired effect. The term "flexible" is used herein to describe the physical characteristics of the bag material. The term "flexible" is defined herein as a pressure level that allows for easy deformation without cracking, breaking, splitting, etc., as used herein. For example, a thin plastic sheet such as Saran TM Packaging film and The bag and thin metal foil such as aluminum foil are flexible. However, even in the embodiment using pneumatic pressure, only certain areas of the blister and the channel need to be flexible. In addition, only one side of the blister and the channel needs to be flexible, as long as the blister and the channel are easily deformable. Other areas of the bag 510 can be made of a rigid material or can be reinforced with a rigid material. Therefore, it should be understood that when the term "flexible bag" or "flexible sample container" or the like is used, only a portion of the bag or sample container needs to be flexible.

[0126] As an illustration, plastic film can be used for bag 510. Metal sheets can be milled or otherwise cut to create a mold with a pattern of surface protrusions, and the metal is illustrated with aluminum or other suitable materials. When assembled into a pneumatic press (illustrated with A-5302-PDS, Janesville Tool Inc., Milton WI), as an illustration, the pneumatic press is regulated to be at a working temperature of 195° C. The pneumatic press works similarly to a printing press, and only melts the sealing surface of the plastic film at the place where the mold contacts the film. Similarly, laser cutting and welding equipment can be used to cut and weld the plastic film used for bag 510. As bag 510 is formed, various components such as PCR primers (illustrated with points on the film and drying), antigen binding substrates, magnetic beads and zirconium silicate beads can be sealed in various blisters. Reagents for sample processing can be collectively or individually applied to the film before sealing. In one embodiment, nucleoside triphosphates (NTPs) are applied to the film separately from polymerase and primers, and the activity of the polymerase is basically eliminated until the reaction can be hydrated by an aqueous sample. If the aqueous sample is heated prior to hydration, this will create conditions for true hot-start PCR and reduce or eliminate the need for expensive chemical hot-start components. In another embodiment, the components may be provided in powder or pellet form and placed into blisters prior to final sealing.

[0127] Bag 510 can be used in a manner similar to that described in U.S. Pat. No. 8,895,295. In one illustrative 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 adjacent to 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 channels (not shown) provided in fitting 590 to hydrate up to 11 different reagents, each of which was previously provided in dry form at inlet channels 515b to 515l. Illustrative methods and apparatus for injecting samples and hydrating 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, however it should be understood that these methods and apparatus are merely illustrative and that other ways of introducing samples and hydrating fluids into bag 510 fall within the scope of the present disclosure. These reagents may illustratively include freeze-dried PCR reagents, DNA extraction reagents, washing solutions, immunoassay reagents or other chemical entities. For illustration, 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 1In the embodiment shown, the sample solution in one injection port and the water in the other injection port are all that needs to be injected. 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.

[0128] After injection, the sample can move from injection channel 515a to lysis bubble 522 via channel 514. Lysis bubble 522 provides beads or particles 534, such as ceramic beads or other beating elements, and lysis bubble 522 is configured for use via The impact of the rotating blade or paddle provided in the instrument is vortexed. By bead grinding for shaking, vortexing, ultrasonic treatment and similar treatment of the sample in the presence of cracking particles such as zirconium silicate (ZS) beads 534, it is an effective method to form a lysate. It should be understood that as used herein, terms such as "lyse", "lysing" and "lysate" are not limited to broken cells, but such terms include ruptured 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 lysis in this embodiment and in other embodiments described herein.

[0129] Figure 4 A bead beating motor 819 is shown, which includes a motor that can be mounted on Figure 2Paddle 821 on first side 811 of support member 802 of shown instrument 800. Paddle can extend through slot 804 to contact bag 510. However, it should be understood that motor 819 can be mounted to other structures of instrument 800. In an illustrative embodiment, motor 819 is a Mabuchi RC-280SA-2865DC motor (Chiba, Japan) mounted to support member 802. In an illustrative embodiment, the motor rotates at 5,000rpm to 25,000rpm, more illustratively at 10,000rpm to 20,000rpm, and more illustratively at about 15,000rpm to 18,000rpm. For Mabuchi motor, it was found that 7.2V provides enough rpm for lysis. However, it should be understood that when paddle 821 hits bag 510, the actual speed may be slightly slower. Other voltages and speeds can be used for lysis, depending on the motor and paddle used. Optionally, a controlled small volume of air can be provided in the capsule 822 adjacent to the lysis bubble 522. It is 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 can be used to limit the bag 510 during the lysis, and the another structure is illustratively a rigid or compliant gasket or other retaining structure around the lysis bubble 522. It should also be understood that the motor 819 is only illustrative, and other equipment can be used for grinding, shaking or vortexing samples. In some embodiments, in addition to mechanical lysis or instead of mechanical lysis, chemicals or heat can be used or chemicals or heating can be used.

[0130] Once the sample material is fully lysed, the sample is moved to the nucleic acid extraction area, for example, through channel 538, bubble cap 544 and channel 543 to bubble cap 546, where the sample is mixed with a nucleic acid binding substance, such as a magnetic bead 533 coated with silica. Alternatively, the magnetic bead 533 can be rehydrated, for example, using a fluid provided from one of the inlet channels 515c-515e, and then moved to bubble cap 544 through channel 543, and then moved to bubble cap 522 through channel 538. The mixture is allowed to incubate for an appropriate length of time, for example, about 10 seconds to 10 minutes. A retractable magnet located in the instrument adjacent to bubble cap 546 captures the magnetic bead 533 from the solution, forming a precipitate close to the inner surface of bubble cap 546. If incubation occurs in bubble cap 522, it may be necessary to move multiple portions of the solution to bubble cap 546 for 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 container. One or more wash buffers from one or more 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 beads back and forth from bubble 544 to 546 via channel 543. Once the magnetic beads 533 are washed, the magnetic beads 533 are recaptured in bubble 546 by activation of the magnet, and the wash solution is then 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.

[0131] After washing, the elution buffer stored in the injection channel 515f is moved to the bubble cap 548, and the magnet is retracted. The solution circulates between the bubbles 546 and 548 via the channel 552, breaking up the precipitation of the magnetic beads 533 in the bubble cap 546 and allowing the captured nucleic acid to dissociate from the beads and enter the solution. The magnet is activated again, capturing the magnetic beads 533 in the bubble cap 546, and moving the eluted nucleic acid solution to the bubble cap 548.

[0132] The first stage PCR master mix from injection channel 515g is mixed with the nucleic acid sample in bubble 548. Optionally, the mixture is mixed by forcing the mixture through channel 553 between 548 and 564. After several mixing cycles, the solution is contained in bubble 564, where a precipitate of first stage PCR primers is provided, at least one set of primers for each target, and a first stage multiplex PCR is performed. If an RNA target is 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 lasts for 15-20 cycles for illustration, although other levels of amplification may also be desirable, depending on the requirements of the specific application. The first stage PCR master mix can be any of a variety of master mixes as known in the art. In an illustrative example, the first stage PCR master mix can be any of the chemistries disclosed in U.S. Patent No. 9,932,634 for a PCR protocol that takes 20 seconds or less per cycle, which is incorporated herein by reference.

[0133] After the first stage PCR has progressed to 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 in bubble 564, and the second stage PCR master mix added 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 reserved, illustratively, for sequencing or for other post-PCR analysis, and the second stage PCR master mix from injection channel 515h is then added to some or all of the diluted amplified sample. It should 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 master mix, which contains components for amplification, with polymerase, dNTPs and suitable buffer as an illustration, although other components may also be suitable, especially for non-PCR amplification methods. If necessary, the mixture of this sample and second stage PCR master mix can be preheated in bubble 564 and then moved to second stage well 582 for second stage amplification. Such preheating can avoid the need for hot start components (antibodies, chemicals or other) in the second stage PCR mixture.

[0134] In one embodiment, the illustrative second stage PCR master mix 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 master mix may lack other components (e.g., polymerase, Mg 2+The second stage PCR master mix may lack other reaction components, and these components may be preloaded in the second stage hole 582. Each primer pair may be similar or identical to the first stage PCR primer pair or may be nested in the first stage primer pair. The movement of the sample from the bubble cap 564 to the second stage hole 582 makes the PCR reaction mixture complete. Once the high-density array 581 is filled, the individual second stage reaction is sealed in their respective second stage bubbles by any means as known in the art. The illustrative mode of filling and sealing the high-density array 581 without cross contamination is discussed in U.S. Patent No. 8,895,295, which has been incorporated by reference. For illustration, the various reactions in the hole 582 of the high-density array 581 are illustratively performed with one or more Peltier (Peltier) devices simultaneously or individually for thermal cycling, but other means for thermal cycling are also known in the art.

[0135] 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 should be understood that this dye is illustrative only, and other signals can be used, including other dsDNA binding dyes and probes labeled fluorescently, radioactively, chemiluminescently, enzymatically, etc., as known in the art. Alternatively, wells 582 of array 581 can be provided without a signal, wherein the results are reported by subsequent processing.

[0136] In one embodiment, when pneumatic pressure is used to move the material within bag 510, a "bladder" may be employed. Bladder assembly 810, a portion of which is shown in FIG. Figure 2-3 In, including capsule plate 824, capsule plate 824 accommodates multiple inflatable capsules 822, 844, 846, 848, 864 and 866, each of which can be individually inflatable, inflated by a compressed gas source for illustration. Since capsule assembly 810 may be subjected to compressed gas and used multiple times, capsule assembly 810 can be made of harder or thicker material than the bag. Alternatively, capsules 822, 844, 846, 848, 864 and 866 can be formed by a series of plates fixed 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 bubbles. US2019-0344269 describes in detail the system of mechanical seals and actuators that can be applied to the instrument described herein, and its entire contents have been incorporated by reference.

[0137] The success of the secondary PCR reaction depends on the template generated by the multiple first-stage reactions. Typically, PCR is performed using high-purity DNA. Methods such as phenol extraction or commercial DNA extraction kits provide high-purity DNA. The sample processed by bag 510 may need to be adapted to compensate for the preparation of lower purity. PCR may be inhibited by components of the biological sample, which is a potential obstacle. For illustration, hot start PCR, higher concentrations of Taq polymerase, adjustment of MgCl2 concentration, adjustment of primer concentration, addition of engineered enzymes resistant to inhibitors, and addition of adjuvants (e.g., DMSO, TMSO, or glycerol) can be optionally used to compensate for lower nucleic acid purity. Although purity issues may be more worthy of attention regarding the first-stage amplification, it should be understood that similar adjustments can also be provided in the second-stage amplification.

[0138] When the bag 510 is placed in the instrument 800, the bladder assembly 810 is pressed against one side of the bag 510 so that if a particular bladder is inflated, the pressure forces 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 can also have additional pneumatic actuators corresponding to the various channels of the bag 510, such as bladders or pneumatically driven pistons. Figure 2-3Illustrative multiple pistons or hard seals 838, 843, 852, 853 and 865 corresponding to the channels 538, 543, 553 and 565 of the bag 510 are shown, as well as seals 871, 872, 873, 874 that minimize the backflow into the fitting 590. When activated, the hard seals 838, 843, 852, 853 and 865 form a pinch valve to pinch off and close the corresponding channel. In order to confine the liquid in the specific bubble of the bag 510, the hard seal is activated on the channel leading to the bubble and out of the bubble, so that the actuator performs the pinch valve function to pinch off the channel. For illustration, in order to mix the 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 off more than one channel at a time. Although pneumatic actuators are discussed herein, it should 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 various methods of reversibly or irreversibly closing the channel. These include twisting the bag across the channel, heat sealing, rolling the actuator, and various physical valves such as butterfly valves and ball valves that are sealed into the channel. In addition, a small Peltier device or other temperature regulator can be placed adjacent to the channel and set at a temperature that is sufficient to freeze the fluid, effectively forming a seal. In addition, although Figure 1 The design is suitable for automated instruments featuring actuator elements positioned on each blister and channel, but it is also contemplated that the actuator can remain stationary and the bag 510 can be transferred 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 in which the bag 510 is transferred between stations. Therefore, although pneumatic actuators are used in the presently disclosed embodiments, when the term "pneumatic actuator" is used herein, it should be understood that other actuators and other ways of providing pressure may be used, depending on the configuration of the bag and instrument.

[0139] Back to Figure 2 , each pneumatic actuator is connected to a compressed air source 895 via a valve 899. Figure 2Only a few hoses 878 are shown, but it should be understood that each pneumatic fitting is connected to a compressed gas source 895 via the hoses 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 required. Other compressed gas sources are within the scope of the present invention. Similar pneumatic controls can be provided, such as for controlling the movement of fluids in the bags described herein, or other actuators, servos, etc. can be provided.

[0140] 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, for illustration, deployed and retracted using gas from the compressed gas source 895 via the hose 878, although other methods of moving the magnet 850 are also known in the art. The magnet 850 is located in a groove 851 of the support member 802. It should be understood that the groove 851 can be a passage 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 should be understood that the groove 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 deployed, and the magnet 850 does not significantly affect any magnetic beads 533 present in the blister 546 when the magnet 850 is fully retracted. Although reference is made to retracting the magnet 850, it should be understood that an electromagnet may be used and may be activated and deactivated by controlling the current through the electromagnet. Thus, although the present specification discusses retracting or retracting the magnet, it should be understood that these terms are broad enough to incorporate other ways of retracting the magnetic field. It should be understood that the pneumatic connection may be a pneumatic hose or a pneumatic air manifold, thus reducing the number of hoses or valves required. It should be understood that similar magnets and methods of activating magnets may be used in other embodiments.

[0141] The various pneumatic pistons 868 of the pneumatic piston array 869 are also connected to a compressed gas source 895 via hoses 878. Although only two hoses 878 are shown connecting the pneumatic pistons 868 to the compressed gas source 895, it should be understood that each pneumatic piston 868 is connected to a compressed gas source 895. Twelve pneumatic pistons 868 are shown.

[0142] 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 heats 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, printing element heaters, positive temperature coefficient heaters, and combinations thereof. The temperature control element may include a plurality of heaters, coolers, Peltier devices, etc. In one aspect, 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, wherein a separate resistance heater is applied to the top and / or bottom surface of the Peltier device. Although the term "heater" is used throughout the specification, it should be understood that other temperature control elements may be used to adjust the temperature of the sample.

[0143] As discussed above, the first stage heater 886 can be positioned to heat and cool the contents of the bubble cap 564 for the first stage PCR. Figure 2 As can be seen, 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 second stage PCR. However, it should be understood that these heaters can also be used for other heating purposes and other heaters can be included, depending on the specific application.

[0144] As discussed above, although the Peltier device that performs thermal cycling between two or more temperatures is effective for PCR, in some embodiments, it may be desirable to maintain the heater at a constant temperature. For illustration, this can be used to reduce the run 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 only necessary to perform thermal cycling on smaller samples and sample containers rather than on a much larger (more thermal mass) Peltier device. For example, an instrument can include multiple heaters (i.e., two or more) under a temperature setting for, for example, annealing, extension, denaturation, which are positioned relative to a bag to complete thermal cycling. Two heaters may be sufficient for many applications. In various embodiments, the heater can be moved, the bag can be moved relative to the heater, or the fluid can be moved relative to the heater to complete thermal cycling. For illustration, the heater can be arranged linearly, in a ring, etc. Suitable heater types have been discussed above regarding the first phase PCR.

[0145] When fluorescence detection is required, an optical array 890 may be provided. Figure 2As shown, the optical array 890 includes a light source 898 and a camera 896, wherein the light source 898 is illustrated as a filtered LED light source, filtered white light, or laser illumination. The camera 896 illustratively has a plurality of photodetectors that each correspond to the second stage holes 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 divided into separate areas corresponding to each of the second stage holes 582. Depending on the configuration, the optical array 890 can be stationary, or the optical array 890 can be placed on a mover connected to one or more motors and moved to obtain a signal from each individual second stage hole 582. It should be understood that other arrangements are possible. Some embodiments of the second stage heater are provided at the bag 510 and Figure 2 Heaters on opposite sides shown. Such orientation is illustrative only and may be dictated by space constraints within the instrument. Provided that the second stage reaction zone 580 is provided in an optically transparent material, the photodetectors and heaters may be on either side of the array 581.

[0146] As shown, computer 894 controls valve 899 of compressed air source 895, and thus controls all pneumatic parts of instrument 800. In addition, in other embodiments, many pneumatic systems in the instrument can be replaced by mechanical actuators, pressure application means, etc. Computer 894 also controls heaters 886 and 888 and optical array 890. These components are electrically connected to each other, with electrical connection via cable 891 as an illustration, although other physical or wireless connections are within the scope of the present invention. It should be understood that computer 894 can be housed in instrument 800 or can be outside of 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 laptop computers, to receive and display data from optical arrays. An interface can be provided, with a keyboard interface as an illustration, the interface includes keys for inputting information and variables such as temperature, cycle time, etc. For illustration, a display 892 is also provided. For example, display 892 can be an LED, LCD or other such display.

[0147] 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, containing the 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 embodiments described herein, waste from the cell lysis and waste from all other steps are retained in a sealed bag. Still, it should be understood that the bag contents can be removed for further testing.

[0148] Back to Figure 2 , the instrument 800 includes a support member 802 that can form a housing wall or be mounted within the housing. The instrument 800 can also include a second support member (not shown) that is optionally movable relative to the support member 802 to allow insertion and retrieval of the bag 510. By way of illustration, once the bag 510 is inserted into the instrument 800, the cover can cover the bag 510. In another embodiment, both support members can be fixed, wherein the bag 510 is fixed in place by other mechanical means or by pneumatic pressure.

[0149] In the illustrative example, heaters 886 and 888 are mounted to support member 802. However, it should be understood that this arrangement is only illustrative and other arrangements are possible. Illustrative heaters include Peltier heaters and other block heaters, resistive heaters, electromagnetic heaters and film heaters as known in the art to thermally cycle the contents of bubble cap 864 and second stage reaction zone 580. The capsule plate 810 with capsules 822, 844, 846, 848, 864, 866, hard seals 838, 843, 852, 853 and seals 871, 872, 873, 874 form capsule assembly 808, which can be illustratively mounted to a removable support structure that can be moved toward bag 510 so that the 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 bag assembly 810 and the various seals of the assembly 808, so that activation of the pneumatic actuator can force liquid out of 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 as follows: Figure 3 More details are given in .

[0150] Methods for cell lysis and nucleic acid recovery

[0151] Disclosed herein are methods and systems for preparing nucleic acid samples. The methods and systems described herein are designed for rapid preparation of nucleic acid samples. 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 acids from lysates with media such as silica-coated magnetic particles, washing of recovery media, and elution of nucleic acids from recovery media) to shorten sample preparation as much as possible while still providing high-quality extracted nucleic acids for downstream amplification or other analysis. The methods described herein include focusing on one or more of the following: rapid mechanical lysis, preferably lysis in the presence of silica-coated magnetic particles, rapid recovery of silica-coated magnetic particles from lysates, rapid washing of magnetic particles, and rapid and efficient elution of captured nucleic acids from silica-coated magnetic particles. The methods and systems described herein are also designed for rapid preparation of nucleic acid samples using aqueous, alcohol-free or organic solvent-free buffer compositions. For illustration, 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 shelf-stable liquid, which can be stored under ambient conditions and / or provided as a ready-to-use dry powder composition, which can be hydrated with a rehydration liquid (e.g., water) for use in performing the steps of the method. Successful sample preparation (i.e., lysis, nucleic acid recovery from the lysate, washing, and elution) is important for maximizing the sensitivity of molecular assays while ensuring reliable and consistent results. Specifically, 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 of molecular assays becomes increasingly shorter, reducing the time for sample preparation is important for reducing the overall assay time. The methods and systems described herein greatly reduce the time required for sample preparation using mechanical lysis without sacrificing the integrity, purity, and concentration of the nucleic acid template input to ensure reliable and consistent results.

[0152] An illustrative method for preparing a nucleic acid sample comprises the following steps: providing a sample container comprising a first chamber, providing a sample (e.g., nasal swab, saliva sample, sputum sample, blood, urine, etc.) suspected of containing one or more target nucleic acids (e.g., target nucleic acids in one or more microorganisms) and a sample buffer comprising a buffer, a chaotropic salt, and a nonionic surfactant. The sample and the sample buffer (first mixture) are combined with a certain number of lysis particles (e.g., zirconium silicate) and a first number of magnetic silica particles. The sample, the sample buffer, the lysis particles, and the magnetic particles can be placed in the first chamber, and can be appropriately stirred (e.g., bead beating) in the first chamber for a time period sufficient to produce a lysate. In various embodiments, a mixture comprising a sample, a sample buffer, lysis particles, and magnetic particles can be combined, and then placed in the first chamber, the first chamber can include one or more components (e.g., lysis particles and / or magnetic particles), the sample and buffer can be added to the first chamber comprising the lysis particles and then magnetic particles are added, or any conceivable combination thereof. The sample, buffer, lysis 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 commercially available bead beater instrument that can be used to agitate a vessel containing such a mixture to produce a lysate. Figure 4 The bead beater discussed is another example of a bead beater instrument that may be suitably used for bead beating in the methods described herein.

[0153] After bead beating is performed to produce a lysate, a second quantity of nucleic acid-binding magnetic particles can be mixed into the lysate. After the second quantity of nucleic acid-binding magnetic particles is mixed into the lysate and the magnetic particles are optionally incubated in the lysate for a period of time (e.g., 5 seconds to 2 minutes), the first and second quantities of nucleic acid-binding magnetic particles can be recovered from the lysate - with recovery using a magnet as an illustration. A pickpen is an example of an apparatus that can be used to recover magnetic particles from a solution. Reference Figure 2The magnet 850 discussed is another example of a magnet that can be used to reclaim magnetic particles from a lysate. After recovery, the magnetic particles can be transferred to another container, released and washed once or multiple times to remove the lysate residue. The magnetic particles can then be recaptured (with a magnet recaptured as an illustration) so that washing buffer can be removed, the magnetic particles can be released again and mixed with elution buffer to elute the captured nucleic acid from the magnetic particles. The magnetic particles can be recaptured, and the elution buffer can be recovered and transferred to a clean pipe. Alternatively, a magnet can be used to remove the magnetic particles from the pipe and the magnetic particles can be disposed of. In any case, the nucleic acid recovered in the elution buffer can be used for various downstream assays, such as but not limited to the presence of one or more target nucleic acids suspected to be present in the sample in the elution buffer with one or more assays in nucleic acid amplification and detection, sequencing, next generation sequencing, etc. In other words, if the nucleic acid in the sample is from a cell or virus (such as a pathogen cell), the assay can be used to identify the cell or virus from which the nucleic acid is derived.

[0154] The inventors of the present invention have found that lysis and nucleic acid recovery are more efficient when lysis is performed in the presence of magnetic silica particles. Figures 6A-7C is described in. Fig. 6A and 6B is a bar graph illustrating the analysis of the assay set ( ) based on amplification from DNA during bead beating lysis in the presence or absence of silica coated magnetic particles (BB w / MB) and in the absence of silica coated magnetic particles (BB w / oMB). Fig. 6A ) and a panel of assays based on amplification from RNA ( Figure 6B ) at various concentrations of the organisms detected. Fig. 6A It was shown that the detection percentage of the DNA-based assay was consistently better at 1x LOD, 0.1x LOD, and 0.01x LOD when bead beating was performed in the presence of silica-coated magnetic particles. However, the RNA-based assay appeared to be approximately equivalent when silica-coated magnetic particles were added post-lysis or when bead beating was performed in the presence of silica-coated magnetic particles. Fig. 7AIllustrate the average crossing point (Cp) of a mixed DNA and RNA assay in which a first quantity of magnetic particles is added to the sample prior to forming a lysate and a second quantity of magnetic particles is added after lysis (split magnetic beads) compared to a control assay in which the magnetic particles are added after lysis (no split magnetic beads). An earlier Cp (compared to the control) means that fewer amplification cycles are required in order to detect amplification. In general, an earlier Cp than the control means that the nucleic acid added to the reaction in the case of "split magnetic beads" is more concentrated than the nucleic acid obtained when the magnetic particles are added after lysis. Assuming 100% template amplification efficiency, an improvement in Cp for one cycle represents an increase in the input concentration of template nucleic acid to about 2-fold, an improvement in Cp for two cycles represents an increase to about 4-fold, an improvement in Cp for three cycles represents an increase to about 8-fold, etc. (By general formula, an improvement in Cp for n cycles represents an increase in the input concentration of target cell or template nucleic acid to about 2 n times). Fig. 7A The data in show that the average Cp of the mixed DNA and RNA assays where a first amount of magnetic particles are added to the sample before forming the lysate and a second amount of magnetic particles (split magnetic beads) are added after lysis is better than the control where the magnetic particles are added after forming the lysate. The improvement in Cp shows that the 'split magnetic beads' protocol improves Cp by about 0.42 cycles compared to the control where the magnetic particles are added after lysis is complete, which means that the nucleic acid concentration increases by about 1.34 times on average when magnetic particles are included in the lysis. Figure 7B and 7C Description of DNA-dependent assays ( Figure 7B ) and RNA-dependent assays ( Figure 7C )’s average Cp data. Figure 7B A DNA-dependent assay is illustrated, which shows an improvement of about 1 Cp, representing an overall increase of about 2-fold in DNA recovery from the lysate when a first amount of magnetic particles is added prior to lysate formation and a second amount of magnetic particles is added after lysis. Figure 7C This indicates that the Cp performance of RNA recovered from magnetic particles present during lysis is approximately equivalent to that of the control. In summary, these data demonstrate that nucleic acids (i.e., DNA and RNA) recovered when a first number of magnetic particles are included during lysis formation and a second number of magnetic particles are added after lysis formation are more concentrated and potentially of higher quality than nucleic acids recovered when silica-coated magnetic particles are added after lysis. Furthermore, while a change in Cp may not represent a large change in the number of cycles required to detect a given nucleic acid, the higher concentrations of nucleic acids obtained when magnetic particles are included in lysis may mean that fewer missed detections may occur when the organism is at or near the limit of detection. This is reflected in Fig. 6A and Figure 6B middle.

[0155] These data are surprising and unexpected, because those of ordinary skill in the art traditionally do not approve of the inclusion of magnetic silica particles in mechanical lysis. It has long been believed that the presence of silica-coated magnetic particles during mechanical lysis may damage the magnetic particles by detaching the silica coating from the particles. It is believed that the loss of silica from silica-coated magnetic particles may reduce the nucleic acid binding capacity of the magnetic particles, and in addition, nucleic acids may be lost due to binding to non-recoverable silica pieces that are detached from the magnetic particles. However, when the organism is mechanically lysed (e.g., by bead beating) in the presence of magnetic silica particles, nucleic acid binding and recovery seem to be better. Nucleic acid binding to magnetic silica particles may occur during lysis, and nucleic acid binding may be more efficient and the recovery rate may be higher. In fact, at least for DNA, nucleic acid binding and nucleic acid recovery seem to be significantly better. The inclusion of magnetic particles in lysis does not necessarily contribute to RNA determination, but they will not be harmed. It also seems that when magnetic silica particles are included in lysis, the quality of the recovered nucleic acid is better. For example, it is believed that proteins and other inhibitors co-isolated with nucleic acids may be less, and nucleic acids may be more concentrated. This is evidenced by the fact that less washing of the magnetic silica particles is required when the magnetic silica particles are included in the lysis.

[0156] Figure 8 Explains the difference in washing, Figure 8 Compare the Cp of the amplification of the nucleic acid recovered from the magnetic particles contained in the lysis under the conditions of no washing, one washing and two washings; the samples are the same except the number of washings. Compared with the one-wash condition, the no-wash condition is obviously harmful to both DNA and RNA determinations. This may be due to the presence of PCR inhibitors co-separated with nucleic acids under the no-wash condition. The one-wash condition improves the Cp of both RNA and DNA determinations, which shows that one wash is enough to remove most PCR inhibitors without reducing the amount of nucleic acids that can be recovered from the magnetic particles. The two-wash condition seems to indicate that it may be slightly better for DNA determination and slightly worse for RNA determination, which indicates that some RNA may be washed away. In any case, for RNA determination, the two-wash condition is better than the no-wash condition. When the same determination is performed under the condition of adding magnetic particles after lysis, at least three washes are required to clean nucleic acids and remove PCR inhibitors, and a later Cp (data not shown) is observed compared with one or two washes with magnetic particles in lysis. Without being bound by one theory, it is believed that a faster sample preparation protocol that includes magnetic particles during mechanical lysis may result in fewer inhibitors being captured by the magnetic particles, and thus may require less washing than a slower and more prolonged binding process when magnetic particles are added only after lysis is complete.

[0157] Typically, magnetic silica particles for nucleic acid recovery are added only after bead beating for lysis is completed. It has long been believed that the presence of silica-coated magnetic silica particles during manual lysis can damage the magnetic particles by causing the silica coating to fall off during lysis. If this damage occurs, the nucleic acid will bind to free silica that is no longer associated with the magnetic core, and 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 observed the opposite phenomenon. Compared with nucleic acids obtained from lysed organisms by conventional methods, the recovery of nucleic acids appears to be more efficient when magnetic silica particles are included in the lysis formation and the recovered nucleic acids are co-purified with fewer contaminants and they are more concentrated. It is also believed that bead beating magnetic particles with nucleic acids bound to bead beating magnetic particles may over-shear and damage the nucleic acids. If this damage occurs, the nucleic acid may be damaged and the amount available for amplification is less. Therefore, the method described herein is atypical and contrary to the usual teachings in the art. The data presented herein (see, e.g. Figure 6A-8 ) demonstrated that the addition of magnetic silica particles to lysis could increase the recovery of nucleic acids from samples and that the nucleic acids could be co-isolated with fewer PCR inhibitors. Fig. 9 The data presented in demonstrate 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 may be detected in a shorter period of time (e.g., after fewer amplification cycles), or the limit of detection for a given cell type may be lower (i.e., an assay for a given cell may be more sensitive because the recovery rate of nucleic acids from the cell is increased), or both.

[0158] Without being bound by theory, it is believed that the recovery of samples in which bead beating is performed in the presence of some or all of the magnetic particles is improved, and the improved nucleic acid recovery is attributed to the following: better mixing and binding kinetics between magnetic silica particles, less recovery of proteins and other PCR inhibitors, more recovery of nucleic acids, and the fragmentation of magnetic particle aggregates, which provides additional surface area for nucleic acids to bind. It is also believed that the mechanical action of bead beating may change the silica surface and increase the surface area of ​​individual magnetic silica particles (e.g., by forming microscopic scratches on the surface of the magnetic particles), thereby increasing the possibility of positive binding interactions between magnetic silica particles and nucleic acids. In addition, since the contact time between the lysate and the magnetic particles is shorter when 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 a theory, it is believed that the rapid and rigorous movement during bead beating may change the way 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 when compared to a scheme in which magnetic particles are added only after lysis is complete.

[0159] In addition, the scheme in which some or all of the magnetic particles are present during the mechanical lysis has certain speed advantages compared to the traditional scheme in which the magnetic particles are added after the bead beating. These advantages include, but are not limited to, the aggregation is fragmented, which means that fewer aggregates may settle to the bottom of the container, making it faster and easier to separate from the lysed particles. Similarly, the fragmentation of the aggregate may increase the surface area of ​​the beads that can be used for nucleic acid binding. In addition, since some or all of the magnetic particles are present during the lysis, these schemes can reduce or eliminate the time spent in the rehydration or resuspension of the magnetic particles. In addition, since the magnetic particles are present during the lysis and are constantly mixed, the interaction with the nucleic acid increases, so the nucleic acid binding may be much faster. Lysis in the presence of magnetic particles can also increase the possibility that a given magnetic particle has a nucleic acid to bind to it. On the other hand, since the binding kinetics are faster and the interaction time is correspondingly shorter when magnetic particles are included in the lysis, it is unlikely that the magnetic particles will collect protein and other inhibitors of downstream nucleic acid amplification. In addition, the lysate is simply heated up due to friction during the mechanical bead beating, and this heating can contribute to faster diffusion kinetics and more efficient lysis, but this may potentially harm some binding kinetics, causing loosely bound particles to detach. Since lysis in the presence of magnetic particles results in shorter bead beating times and faster recovery, the sample is less likely to heat excessively and affect recovery.

[0160] Method described herein may suitably include adding a first quantity of magnetic particles before mechanical lysis (e.g., bead beating), and then adding a second quantity of magnetic particles after mechanical lysis. It is believed that some fragments, with double-stranded DNA as an illustration, can be tightly bound to magnetic particles or other silica surfaces, and this combination can even continue in the whole lysis agitation step. It is believed that these fragments are preferentially bound to magnetic particles in the lysis and mixing stages. Other nucleic acids, such as single-stranded RNA, tend to be more loosely combined, and the combination may not survive the agitation step. Adding the second quantity of magnetic particles with fresh nucleic acid binding sites can be combined with unbound nucleic acids quickly and efficiently after mechanical agitation. It is also believed that magnetic particles are not completely homogeneous, and may have local physical or chemical features that are conducive to combining nucleic acids with different configurations. Once the binding site of a particular type is saturated, beads may lose the tendency to make nucleic acids be bound to the remaining binding site. The magnetic particles of fresh quantity also provide a more variety of fresh injections of binding sites for any unbound nucleic acid to be combined.

[0161] Since the lysate may heat up during mechanical bead beating simply due to friction, and this may be harmful, a fresh injection of cooler magnetic particles 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 magnetic particle addition both before and after mechanical lysis advantageously promotes binding of both those molecules that are tightly bound to the surface and those that are more loosely bound.

[0162] The aforementioned 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, where magnetic particles may be extracted from a fluid. The consumed lysate without magnetic particles may then be returned to the lysis container or to another waste container. The method described herein may further include washing the nucleic acid-bound magnetic particles with a wash buffer to remove waste materials from the lysate from the magnetic particles. Preferably, the wash buffer may remove residual lysate, contaminants, etc. from the magnetic particles without eluting a considerable 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 consumed wash buffer from the container or transferring the magnetic particles to another container. For some sample types, washing may not be necessary. Therefore, the method described herein may include zero washes. Typically, however, the method described herein may include 1-3 washes. The washing performed in the methods described herein suitably may not include one or more of: heating the wash buffer and / or the magnetic particles prior to or during washing, vigorously mixing the magnetic particles with the wash buffer, or incubating the magnetic particles with the 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 the magnetic particles with the wash buffer for a period of time of about 2-3 seconds.

[0163] After washing, the methods described herein may suitably include: releasing the magnetic particles from the capture magnet (in the container after removing the wash buffer 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 occur during the lysis and / or washing steps so that the elution buffer is already heated at the time of elution. For example, the elution buffer may be heated to a temperature of 35°C to 105°C (e.g., 50°C to 100°C) prior to mixing it with the magnetic particles, depending on the sample type and other factors. The inventors of the present invention have found that the heated elution temperature and duration can elute RNA / DNA quickly and efficiently and leave proteins and inhibitors on the magnetic particles. For example, this may be done in a vacuum chamber. Fig. 10A In the description, Fig. 10A The average Cp improvement for DNA and RNA assays is shown as the preheat temperature of the elution buffer is increased from about 35°C to about 105°C. Fig. 10A As can be seen in Figure 2, 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 Fig. 10C The same trend is shown for both DNA and RNA assays. The best fit lines for DNA and RNA data show an average Cp improvement of about 2 Cp units for the DNA assay, and an average Cp improvement of about 1.5 Cp units for the RNA assay. This represents an increase of about 4-fold in the amount of DNA eluted from the magnetic particles, and an increase of about 3-fold in the amount of RNA eluted from the magnetic particles.

[0164] Although reference is made in various embodiments discussed herein to moving the magnetic particles to the second chamber, it will be appreciated that an alternative embodiment is to retain the magnetic particles in the first chamber and remove unbound lysate from this chamber.

[0165] Reference now Figures 5A-5E , illustrating exemplary methods 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 5BAs shown, the contents of the container 5000 can be appropriately bead beaten (as schematically depicted by arrow 5009) for a time sufficient to create a lysate 5008. In one aspect, bead beating can be made more efficient by pressing the container against the bead beating device with an appropriate amount of pressure. The time required to prepare the lysate can vary depending on a variety of factors, such as, but not limited to, the sample material to be lysed, the number of samples, the speed or frequency of the bead beating motor, the pressure applied to the bead beater, and the bead lysis instrument. Reducing air from the chamber (illustratively, by evacuating air from the container via vacuum suction on the container or by using pressure applied to the container) and performing the bead beating under pressure helps to impart more energy to the sample and reduce foam generation for more efficient lysis. By way of illustration, 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 shorter bead lysis times. Times of a few seconds to a few minutes are typical. With Figure 4 The bead beating apparatus described in Figure 1 and Figure 2 For instruments and bags in the apparatus, a few seconds of bead beating time 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). It is preferred to perform bead beating with as much force and / or intensity as the system allows, and for no longer than is necessary to lyse the material in the sample. This not only saves time in sample preparation, but too much bead beating time may shear nucleic acids and ultimately reduce sample quality. This is particularly important in Fig.11 To explain, Fig.11 Shown are the fragment sizes of human genomic DNA detected with or without bead beating for 120 seconds. As the bead beating time increases, the length of the largest fragment may decrease and a higher concentration of smaller fragments may be detected.

[0166] The scheme discussed herein does not significantly shear nucleic acids or reduce sample quality. In general, organisms that are more difficult to lyse, such as Cryptosporidium and yeast, prefer high RPM and long bead beating duration, while other organisms, such as Escherichia coli and viral targets, prefer lower RPM combined with shorter bead beating duration. The best combination of RPM and duration is assay specific. Optimizing RPM and duration for organisms that are easier to lyse may leave un-lysed organisms that are difficult to lyse; and optimizing for the most difficult organisms to lyse may result in shearing nucleic acids from organisms that are easy to lyse. In addition to shearing, extended bead beating at elevated speeds may cause the sample to heat up, which may reduce yield. In general, it is best to optimize to a point that balances the lysis requirements of all organisms in the test strip. Bead beating for the shortest possible time (e.g., 20-60 seconds) at high speeds (e.g., 10,000-12,000 RPM) can maximize lysis efficiency and sample quality, while saving considerable sample preparation time and overall result time.

[0167] return Figure 5C , after bead beating, a second quantity of magnetic particles 5010 can be combined with the lysate 5008. The second quantity can be substantially the same as the first quantity, or it can be substantially larger or substantially smaller. For illustration, the amount of magnetic particles in the first and second quantities is substantially the same. In addition, the first quantity and the second quantity can be the same type of silica-coated magnetic particles, or they can vary the thickness of the silica as desired to favor the recovery of RNA, DNA, or both.

[0168] For example, the second number of magnetic particles 5010 can be combined with the lysate 5008 by agitation (as schematically depicted by arrow 5011). Agitation 5011 can be achieved by a bead beater used in the lysis step, although the duration of agitation may be shorter and less intense than the agitation used to produce the lysate. The first and second numbers of magnetic particles 5004 and 5010 can be incubated in the lysate 5008 for a selected period of time (e.g., a few seconds to a few minutes) sufficient to capture nucleic acids from the solution. Figure 5D and 5E As shown, lysis particles 5006, which are typically much larger than magnetic particles 5004 and 5010, can settle much faster than the magnetic particles. Magnet 5015 can be used to recover magnetic particles 5004 and 5010 from lysate 5008, such as Figure 5E In one embodiment, the magnetic particles 5004 and 5010 can be appropriately kept in suspension to facilitate recovery by gently mixing the container. Since the lysed particles are much larger than the magnetic particles, such gentle mixing can promote the sedimentation of the lysed particles while preferably keeping the magnetic particles in suspension and facilitating the separation of the magnetic particles from the lysed particles (not shown).

[0169] Example 1

[0170] The inventors of the present invention have found that lysis and nucleic acid recovery are more efficient when lysis is performed in the presence of magnetic silica particles. Figures 6A-7C is described in. Fig. 6A and Figure 6B is a bar graph illustrating the analysis of the assay set ( BB w / MB) based on amplification from DNA during bead beating lysis in the presence of silica-coated magnetic particles (BB w / MB) or in the absence of silica-coated magnetic particles (BB w / o MB). Fig. 7A ) and a panel of assays based on amplification from RNA ( Figure 7B ) at various concentrations of the organisms detected. Fig. 7A It was shown that the detection percentage of the DNA-based assay was consistently better at 1x LOD, 0.1x LOD, and 0.01x LOD when bead beating was performed in the presence of silica-coated magnetic particles. However, the RNA-based assay appeared to be approximately equivalent when silica-coated magnetic particles were added after lysis or when bead beating was performed in the presence of silica-coated magnetic particles.

[0171] Fig. 7A The data in show that the average Cp for DNA and RNA assays where a first amount of magnetic particles are added to the sample before forming the lysate and a second amount of magnetic particles are added after lysis (split magnetic beads) is better than a control where the magnetic particles are added after forming the lysate. The improvement in Cp shows that the 'split magnetic beads' protocol improves Cp by about 0.42 cycles compared to the control where the magnetic particles are added after lysis is complete, which represents an average increase in nucleic acid concentration of about 1.34 times when magnetic particles are included in the lysis. Figure 7B and Figure 7C Description of DNA-dependent assays ( Figure 7B ) and RNA-dependent assays ( Figure 7C )’s average Cp data. Figure 7B It is illustrated that the DNA-dependent assay showed an improvement of about 1 Cp, which represents an overall increase of about 2-fold in DNA recovery from the lysate when a first amount of magnetic particles was added prior to lysate formation and a second amount of magnetic particles was added after lysis. Figure 7C The Cp performance of RNA recovered from magnetic particles present during lysis was shown to be approximately equivalent to that of the control. These data demonstrate that nucleic acids (i.e., DNA and RNA) recovered when lysis was performed in the presence of magnetic particles are more concentrated and likely of higher quality than nucleic acids recovered when silica-coated magnetic particles are added after lysis.

[0172] This is surprising and unexpected, because those of ordinary skill in the art traditionally do not approve of including magnetic silica particles in mechanical lysis, 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 detach from the particles. It is believed that the loss of silica from silica-coated magnetic particles may reduce the nucleic acid binding ability of the magnetic particles, and in addition, nucleic acids may be lost due to the combination of non-recoverable silica pieces that detach from the magnetic particles. However, when mechanical lysis (e.g., by bead beating) is performed on the organism in the presence of magnetic silica particles, the combination of nucleic acids and magnetic silica particles can occur during lysis, and nucleic acid binding may be more efficient and the recovery rate may 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 nucleic acid recovered also seems to be better.

[0173] Example 2

[0174] Figure 8 The Cp of amplification of DNA (-●-) and RNA (-*-) recovered from magnetic particles included in the lysis under no wash, one wash, and two washes were compared; the samples were identical except for the number of washes. Compared with the one wash condition, the no wash condition was clearly harmful to both DNA and RNA determinations. This may be due to the presence of PCR inhibitors co-separated with nucleic acids under the no wash condition. The one wash condition improved the Cp of both RNA and DNA determinations, 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 conditions seemed to be slightly better for DNA determinations and slightly worse for RNA determinations, indicating that some RNA may be washed away. In any case, for RNA determinations, the two wash conditions were better than the no wash conditions. When the same determination was performed with the addition of magnetic particles after lysis, at least three washes were required to clean the nucleic acids and remove PCR inhibitors, and a later Cp was observed compared to one or two washes with magnetic particles in lysis (data not shown).

[0175] Example 3

[0176] Reference now Fig.11 , Fig.11A bar graph illustrating time savings for multiple specific examples in FilmArray sample preparation is shown in the Figure. Over a number of years, sample preparation time was 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 the ability to reduce sample preparation time from approximately 13 minutes to approximately 2 minutes was surprising and unexpected. Comparing the RP2.1 time (4 minutes 42 seconds) to the R / ST time (1 minute 58 seconds), additional time was saved at each step. The R / ST time represents the time savings that can be achieved using the methods described in this application. Comparing the RP2.1 sample preparation time to the R / ST sample preparation time shows an absolute time difference of 2 minutes 44 seconds and a 58% time reduction. As shown in the Figure. Fig.11 As can be seen in the , each sample preparation step saves time, especially in collecting magnetic particles from lysates and in reducing bead beating time. By combining multiple lysis mechanisms with pressure control and motor feedback mechanisms, nucleic acid release from cells can be accelerated. More stringent control and reduced differences also allow for reduced buffer times to ensure robust performance from the easiest to the most difficult to lyse organism types. By performing multiple steps in parallel and improving the dynamics and efficiency of nucleic acid binding magnetic particles and magnetic particles binding magnets, binding and magnetic bead recovery are reduced from 7 minutes to 30 seconds most drastically. Comparing only the bead beating and magnetic particle collection times between RP2.1 and R / ST, it is shown that it is reduced from 2 minutes and 53 seconds in RP2.1 to 1 minute and 13 seconds in R / ST, with an absolute time saving of 1 minute and 40 seconds and a time saving of 57% for only these two steps. Performing bead beating in the presence of magnetic particles allows faster nucleic acid binding and faster recovery of nucleic acids from lysates. Washing time is reduced from 40 seconds to about 8 seconds, and elution time is reduced from 1 minute and 09 seconds to 22 seconds.

[0177] The present invention may be implemented in other specific forms without departing from its spirit or essential characteristics. The described embodiments should be considered in all respects as illustrative only and not restrictive. Therefore, the scope of the present invention is indicated by the appended claims rather than by the foregoing description. Although certain embodiments and details are included herein and in the attached disclosure of the invention 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 falling within the equivalent meaning and scope of the claims should be included within their scope.

Claims

1. A method for cell lysis and nucleic acid recovery, the method comprising: Providing a sample container, a certain number of lysis particles, a sample suspected of containing one or more target nucleic acids, a lysis buffer and a first number of nucleic acid-binding magnetic particles, placing a sample, a certain number of lysis particles, a lysis buffer and a first number of nucleic acid-binding magnetic particles in a container, agitating a container having lysis particles, a sample, a lysis buffer, and a first number of nucleic acid-binding magnetic particles for a first period of time to generate a lysate, dispensing a second number of nucleic acid-binding magnetic particles into the lysate in the container, and recovering the first and second quantities of nucleic acid-bound magnetic particles from the lysate, Wherein adding a first number of nucleic acid binding magnetic particles and then adding a second number of nucleic acid binding magnetic particles is selected to increase the recovery rate of nucleic acid from the sample.

2. The method of claim 1, wherein the lysis buffer comprises a buffer, a chaotropic salt and a non-ionic surfactant.

3. The method of claim 2, wherein the lysis buffer is an aqueous buffer comprising a buffer, 50-60% of a chaotropic agent and 10-20% of a non-ionic surfactant.

4. The method of claim 3, wherein the chaotropic agent is a guanidine salt and the nonionic surfactant is one of the following: Triton X-100, polydocanol (Thesit), Triton X-114, NP-40, Arlasolve 200, BrijO10, octyl β-D-pyranoglucoside, saponin, monododecyl nonaglycone ether and combinations thereof.

5. The method of claim 1, wherein the recovering further comprises one of: releasing the first and second quantities of nucleic acid-bound magnetic particles recovered from the lysate into another sample container, or removing the lysate and the lysed particles from the container and releasing the first and second quantities of nucleic acid-bound magnetic particles recovered from the lysate back into the container.

6. The method of claim 5, further comprising: The first and second numbers of nucleic acid-binding magnetic particles are washed with a wash buffer, the first and second numbers of nucleic acid-binding magnetic particles are recaptured with a magnet, and the wash buffer is removed.

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

8. The method of claim 6, further comprising: Release the first and second quantities of nucleic acid-binding magnetic particles from the magnet, add an elution buffer to the first and second quantities of nucleic acid-binding magnetic particles, release the magnet, and mix the magnetic particles with the elution buffer, recapture the first and second quantities of nucleic acid-binding magnetic particles with the magnet, and transfer the elution buffer to another sample container.

9. The method of claim 6, further comprising: Adding elution buffer to the first and second quantities of nucleic acid-binding magnetic particles and mixing the magnetic particles with the elution buffer, recapturing the first and second quantities of nucleic acid-binding magnetic particles with a magnet, and transferring the first and second quantities of nucleic acid-binding magnetic particles to another sample container.

10. The method of any one of claims 8 or 9, wherein the elution buffer is configured to elute nucleic acids captured by the nucleic acid binding magnetic particles into the elution buffer.

11. The method of any one of claims 1 to 10, wherein the amount of the second number of nucleic acid binding magnetic particles is substantially equal to the amount of the first number of nucleic acid binding magnetic particles.

12. The method of any one of claims 8-10, further comprising assaying the elution buffer for the presence of one or more target nucleic acids suspected to be present in the sample.

13. The method of claim 12, wherein the assay comprises a nucleic acid amplification step and a step of detecting the amplified nucleic acid produced in the nucleic acid amplification step.

14. The method of any one of claims 1 to 13, wherein the agitating step comprises heating the sample and the dispersing step comprises cooling the sample.

15. A method for cell lysis and nucleic acid recovery, the method comprising: Provide sample containers, combining a number of lysis particles, a first number of nucleic acid-binding magnetic particles, a sample suspected of containing one or more target nucleic acids, and a lysis buffer in a sample container, agitating the lysis particles, the sample, the lysis buffer, and the first number of nucleic acid-binding magnetic particles in the sample container for a period of time sufficient to generate a lysate, dispersing a second number of nucleic acid-binding magnetic particles into the lysate in the sample container, incubating the first and second quantities of nucleic acid-bound magnetic particles in the lysate for a period of time, and capturing a first and a second number of nucleic acid-binding magnetic particles from the lysate using a magnet, Wherein adding a first number of nucleic acid binding magnetic particles and then adding a second number of nucleic acid binding magnetic particles is selected to increase the recovery rate of nucleic acid from the sample.

16. The method of claim 15, wherein the first and second quantities of nucleic acid-bound magnetic particles are incubated in the lysate for a period of time ranging from 1 second to 1 minute.

17. The method of claim 16, wherein the time period is in the range of 20-30 seconds.

18. The method of claim 15, wherein capturing the first and second quantities of magnetic particles from the lysate further comprises: The magnetic particles are transferred to a second sample container and the magnetic particles are released from the magnet into the second sample container, or the lysate and lysed particles are removed from the first sample container and the magnetic particles are released from the magnet back into the first sample container.

19. The method of claim 15 or claim 18, wherein capturing the first and second quantities of magnetic particles from the lysate preferably does not include capturing lysis particles.

20. The method of any one of claims 15, 18 or 19, further comprising: Release the first and second quantities of nucleic acid-binding magnetic particles from the magnet, add a wash buffer to the magnetic particles, wash the magnetic particles with the wash buffer, recapture the magnetic particles with the magnet, remove the wash buffer, add an elution buffer to the magnetic particles, release the magnetic particles from the magnet, mix the magnetic particles with the elution buffer, recapture the magnetic particles with the magnet, and transfer the magnetic particles or the elution buffer to another sample container.

21. The method of claim 20, wherein the washing does not comprise one or more of: heating the wash buffer and magnetic particles prior to or during washing, vigorously mixing the magnetic particles with the wash buffer, or incubating the magnetic particles with the wash buffer for a period of time greater than 10 seconds.

22. The method of any one of claims 15-21, wherein the steps of the method are completed in <4 minutes.

23. The method of any one of claims 15-22, wherein the steps of the method are completed in < 3 minutes.

24. The method of any one of claims 15-23, wherein the steps of the method are completed in <2 minutes.

25. The method of any one of claims 15-24, wherein the steps of the method are completed within 1-3 minutes.

26. A method for cell lysis and nucleic acid recovery, the method comprising: providing a sample container comprising a plurality of fluidically connected reaction chambers, the reaction chambers including a sample lysis chamber, a nucleic acid recovery chamber, and at least a first nucleic acid amplification chamber, combining a number of lysis particles, a first number of nucleic acid-binding magnetic particles, a sample suspected of containing one or more target nucleic acids, and a lysis buffer in a sample lysis chamber, agitating the lysis particles, the sample, the lysis buffer, and the first number of nucleic acid-binding magnetic particles in the lysis chamber for a period of time sufficient to generate a lysate, mixing a second quantity of nucleic acid-binding magnetic particles into the lysate, transferring at least a portion of the lysate having the first and second quantities of nucleic acid-binding magnetic particles suspended therein to a nucleic acid recovery chamber, capturing a first and a second number of nucleic acid-binding magnetic particles from the lysate in the nucleic acid recovery chamber using a magnet, removing the lysate from the nucleic acid recovery chamber without removing the first and second quantities of nucleic acid-binding magnetic particles, releasing the first and second quantities of nucleic acid-binding magnetic particles from the magnet and washing the first and second quantities of nucleic acid-binding magnetic particles with a wash buffer in a nucleic acid recovery chamber, recapturing the first and second quantities of nucleic acid-binding magnetic particles with a magnet and removing the wash buffer, releasing the magnet and mixing the first and second quantities of nucleic acid-binding magnetic particles with an elution buffer in a nucleic acid recovery chamber to elute the nucleic acids from the magnetic particles, recapturing the first and second quantities of nucleic acid-binding magnetic particles with a magnet and transferring an elution buffer to a first nucleic acid amplification chamber, Wherein adding a first number of nucleic acid binding magnetic particles and then adding a second number of nucleic acid binding magnetic particles is selected to increase the recovery rate of nucleic acid from the sample.

27. The method of claim 26, wherein capturing nucleic acid-bound magnetic particles comprises deploying a magnet adjacent to a nucleic acid recovery chamber to contain the magnetic particles in the chamber.

28. The method of claim 26, further comprising combining an elution buffer with reagents for a nucleic acid amplification reaction in a first nucleic acid amplification chamber to form an amplification mixture, and subjecting the amplification mixture to amplification conditions to determine the presence of one or more target nucleic acids suspected of being present in the sample.

29. The method of claim 28, wherein the assay is selected from the group consisting of nucleic acid amplification, sequencing, and next generation sequencing.

30. The method of claim 26, wherein the second number of nucleic acid binding magnetic particles is substantially equal to the first number of nucleic acid binding magnetic particles.

31. The method of claim 26, wherein the second number of nucleic acid binding magnetic particles is substantially greater than the first number of nucleic acid binding magnetic particles.

32. The method of claim 26, wherein the second number of nucleic acid binding magnetic particles is substantially less than the first number of nucleic acid binding magnetic particles.

33. The method of any one of claims 26-32, wherein the agitating step comprises heating the sample and the mixing step comprises cooling the sample.

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

35. The method of claim 26, wherein the steps of the method are completed in < 4 minutes.

36. The method of claim 26, wherein the steps of the method are completed in < 3 minutes.

37. The method of claim 26, wherein the steps of the method are completed in <2 minutes.

38. The method of claim 26, wherein the steps of the method are completed within 1 to 3 minutes.

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