Cell lysis and nucleic acid recovery

Abstract

Methods and systems for rapid preparation of nucleic acid samples.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of and priority to U.S. Provisional Patent Application Nos. 63 / 369,107, filed July 22, 2022, and 63 / 450,492, filed March 7, 2023, the entire contents of which are incorporated herein by reference. [Background technology]

[0002]

[0002] In the United States, Canada, and Western Europe, infectious diseases account for approximately 7% of human mortality, but in developing regions, infectious diseases account for over 40% of human mortality. Infectious diseases cause a variety of clinical manifestations. Common telltale signs include fever, pneumonia, meningitis, diarrhea, and diarrhea containing blood. While physical signs suggest some pathogens and exclude others as etiologies, the potential causative agents are diverse, and a definitive diagnosis often requires the administration of multiple tests. Traditional microbiological methods for diagnosing pathogens can take days or weeks, often delaying appropriate treatment.

[0003] In recent years, polymerase chain reaction (PCR) has become the method of choice for the rapid diagnosis of infectious agents. PCR is a rapid, sensitive, and specific tool for diagnosing infectious diseases. Challenges in using PCR as a primary diagnostic tool include the high diversity of potential pathogenic bacteria and the low levels of bacteria present in some pathology specimens. Performing large PCR assay panels, one for each potential pathogenic bacteria, is often impractical. The problem is exacerbated when pathogen nucleic acids are present in low concentrations, requiring large sample volumes to collect sufficient reaction templates. In some cases, there may not be enough sample to assay all potential pathogenic agents. A solution is to perform "multiplex PCR," in which a sample is simultaneously assayed for multiple targets in a single reaction. While multiplex PCR has proven useful in some systems, drawbacks remain, such as the lack of robustness of high-level multiplex reactions and the difficulty of unambiguously analyzing multiple products. To address these issues, assays can be split into multiple secondary PCRs. Nesting secondary reactions within the primary product often enhances robustness. However, this additional handling is costly and can lead to contamination or other problems.

[0004]

[0004] The FilmArray® (BioFire Diagnostics, LLC, Salt Lake City, UT) is a user-friendly, highly multiplexed PCR system developed for the diagnostic market. This single-sample instrument accepts a diagnostic "pouch" that integrates sample preparation and nested multiplex PCR. Integrated sample preparation provides ease of use, while highly multiplexed PCR offers both the sensitivity of PCR and the ability to simultaneously test for many organisms (e.g., up to 30 or more different organisms and molecular markers). This system is suitable for identifying many different pathogens, all of which have similar clinical manifestations. Currently available diagnostic panels include a respiratory panel for upper respiratory tract infections, a blood culture panel for bloodstream infections, a gastrointestinal panel for gastrointestinal infections, a meningitis / encephalitis panel for central nervous system infections, a pneumonia panel for lower respiratory tract infections, and a bone and joint panel for bone and joint infections. Other panels are in development.

[0005] When PCR first became popular in the late 1980s, the process was slow. A typical protocol involved 1 minute of denaturation at 94°C, 2 minutes of annealing at 55°C, and 3 minutes of extension at 72°C. Including transition times between temperatures, 8-minute cycles were common, with 30 cycles completed in 4 hours. Over the years, systems have sped up, with 30-minute protocols becoming commonplace and faster PCR machines becoming available. Even with faster PCR protocols, many diagnostic PCR reactions begin with lysis of target cells and extraction / purification of target nucleic acids from the target cells using traditional techniques.

[0006]

[0006] As PCR speeds increase, the time spent on sample preparation takes up an increasingly large proportion of processing time. One possible solution would be to essentially eliminate sample preparation and rely on chemical and / or heat lysis to disrupt cells and perform nucleic acid amplification on the raw lysate. While this saves time, the sufficiency of lysis is not always reliable, and the crude lysate is often filled with substances that can inhibit nucleic acid amplification. Another possible solution is to perform mechanical lysis followed by nucleic acid recovery and nucleic acid purification from the lysate. While this is desirable from the perspective of purity of the starting template for nucleic acid amplification, it has traditionally been quite time-consuming. It would be desirable to perform sample preparation in a more rapid manner while maximizing the purity of the starting template for nucleic acid amplification. There is a need in the art to reduce the time required for sample preparation while retaining the purity and robustness of traditional sample preparation (i.e., mechanical lysis, nucleic acid recovery, and nucleic acid purification before initiating nucleic acid amplification). Summary of the Invention

[0007]

[0007] Described herein are methods and systems for cell lysis and nucleic acid recovery. 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 a lysis buffer in a container (e.g., a tube). The container can then be placed in a bead beater for a period of time sufficient to produce a lysis solution. After beating the beads to produce a lysis solution, a second amount of nucleic acid-binding magnetic particles can be mixed with the lysis solution. After mixing the second amount of nucleic acid-binding magnetic particles with the lysis solution, the first amount of nucleic acid-binding magnetic particles and the second amount of nucleic acid-binding magnetic particles can be recovered from the lysis solution, illustratively using a magnet.

[0008] After recovery, the magnetic particles may be transferred to another container, released, and washed one or more times to remove residual lysate. The magnetic particles may then be recaptured (e.g., with a magnet), the wash buffer removed, the magnetic particles released again, and mixed with an elution buffer to elute the captured nucleic acids from the magnetic particles. The magnetic particles may be recaptured, and the elution buffer may be collected and transferred to a clean tube. Alternatively, a magnet may be used to remove the magnetic particles from the tube, and the magnetic particles may be discarded. The eluted material may be used in a variety of downstream assays, including, but not limited to, assaying the eluted material for the presence of one or more target nucleic acids suspected to be present in the sample using one or more of nucleic acid amplification and detection, sequencing, next-generation sequencing, etc. That is, if the nucleic acid in the sample originates from a cell (e.g., a pathogen cell), the assay may be used to identify the cell from which the nucleic acid originated.

[0009]

[0009] As an example, a method for cell lysis and nucleic acid recovery is disclosed, which includes the steps of providing a sample vessel, disposing a quantity of lysis particles in the sample vessel, adding a sample suspected of containing one or more target nucleic acids and a lysis buffer to the vessel, adding a first quantity of nucleic acid-binding magnetic particles to the vessel, agitating the vessel containing the lysis particles, sample, lysis buffer, and first quantity of nucleic acid-binding magnetic particles for a first period of time to produce a lysis solution, mixing a second quantity of nucleic acid-binding magnetic particles into the lysis solution in the vessel, and recovering the first quantity of nucleic acid-binding magnetic particles and the second quantity of nucleic acid-binding magnetic particles from the lysis solution.

[0010] In another example, a method for cell lysis and nucleic acid recovery is disclosed, comprising the steps of providing a sample vessel including a first chamber and a second chamber, combining in the first chamber a quantity of lysis particles, a first quantity of nucleic acid-binding magnetic particles, a sample suspected of containing one or more target nucleic acids, and a lysis buffer, agitating the first chamber of the sample vessel containing the lysis particles, the sample, the lysis buffer, and the first quantity of nucleic acid-binding magnetic particles for a period sufficient to produce a lysis solution, mixing a second quantity of nucleic acid-binding magnetic particles into the lysis solution, and capturing the first quantity of nucleic acid-binding magnetic particles and the second quantity of nucleic acid-binding magnetic particles from the lysis solution in the second chamber using a magnet.

[0011] In yet another example, a method for cell lysis and nucleic acid recovery is disclosed, comprising the steps of providing a sample vessel including multiple fluidly connected reaction chambers, including a sample lysis chamber, a nucleic acid recovery chamber, and at least a first nucleic acid amplification chamber, combining in the sample lysis chamber a quantity of lysis particles, a first quantity of nucleic acid-binding magnetic particles, a sample suspected of containing one or more target nucleic acids, and a lysis buffer, agitating the sample lysis chamber of the sample vessel containing the lysis particles, the sample, the lysis buffer, and the first quantity of nucleic acid-binding magnetic particles for a period sufficient to produce a lysis solution, mixing a second quantity of nucleic acid-binding magnetic particles into the lysis solution, and using a magnet to capture the first quantity of nucleic acid-binding magnetic particles and the second quantity of nucleic acid-binding magnetic particles from the lysis solution in the nucleic acid recovery chamber. The method further includes the steps of releasing the magnet and washing the first amount of nucleic acid-binding magnetic particles and the second amount of nucleic acid-binding magnetic particles with a wash buffer in the nucleic acid recovery chamber, recapturing the first amount of nucleic acid-binding magnetic particles and the second amount of nucleic acid-binding magnetic particles using the magnet and removing the wash buffer, releasing the magnet and eluting the nucleic acids from the first amount of nucleic acid-binding magnetic particles and the second amount of nucleic acid-binding magnetic particles with an elution buffer in the nucleic acid recovery chamber, recapturing the first amount of nucleic acid-binding magnetic particles and the second amount of nucleic acid-binding magnetic particles using the magnet and transferring the elution buffer to the first nucleic acid amplification chamber.

[0012] In one aspect of the methods disclosed herein, adding a first amount of nucleic acid-binding magnetic particles followed by a second amount of nucleic acid-binding magnetic particles is selected to enhance recovery of RNA and DNA from the sample, such enhanced recovery can be expressed, for example, in terms of improved Cp (as indicated by the previous Cp) or improved test sensitivity.

[0013]

[0013] The following is described in this specification:

[0014] A1. A method for cell lysis and nucleic acid recovery, comprising: providing a sample vessel with a quantity of lysis particles, a sample suspected of containing one or more target nucleic acids, a lysis buffer, and a first quantity of nucleic acid-binding magnetic particles; placing a sample, a quantity of lysis particles, a lysis buffer, and a first quantity of nucleic acid-binding magnetic particles in a container; agitating a vessel containing lysis particles, a sample, a lysis buffer, and a first amount of nucleic acid-binding magnetic particles for a first period of time to produce a lysis solution; dispersing a second amount of nucleic acid-binding magnetic particles in a lysis solution in the container; recovering a first amount of nucleic acid-binding magnetic particles and a second amount of nucleic acid-binding magnetic particles from the lysate; wherein adding a first amount of nucleic acid-binding magnetic particles followed by a second amount of nucleic acid-binding magnetic particles is selected to enhance recovery of nucleic acids from the sample.

[0014]

[0015] A2. The method of clause A1, wherein the lysis buffer comprises a buffering agent, a chaotropic salt, and a non-ionic detergent.

[0015]

[0016] A2.1. The method according to clause A1 or A2, wherein the lysis buffer is an aqueous buffer comprising a buffering agent, 50-60% chaotropic agent, and 10-20% non-ionic surfactant.

[0016]

[0017] A2.2. The method of clause A1, A2, or A2.1, wherein the chaotropic agent is a guanidinium salt and the non-ionic detergent is one of Triton X-100, polidocanol (Thesit), Triton X-114, NP-40, Arlasolve 200, Brij O10, octyl β-D-glucopyranoside, saponin, nonaethylene glycol monododecyl ether, and combinations thereof.

[0017]

[0018] A3. A method according to any one of A1 to A2.2, wherein the recovering further comprises one of releasing the first amount of nucleic acid-binding magnetic particles and the second amount of nucleic acid-binding magnetic particles recovered from the lysis solution into another sample container, or removing the lysis solution and the lysis particles from the container and returning the first amount of nucleic acid-binding magnetic particles and the second amount of nucleic acid-binding magnetic particles recovered from the lysis solution to the container.

[0018]

[0019] A4. The method of any one of clauses A1 to A3, further comprising washing the first amount of nucleic acid-binding magnetic particles and the second amount of nucleic acid-binding magnetic particles with a wash buffer, recapturing the first amount of nucleic acid-binding magnetic particles and the second amount of nucleic acid-binding magnetic particles using a magnet, and removing the wash buffer.

[0019]

[0020] A5. The method of any one of clauses A1-A4, wherein washing does not include one or more of heating the wash buffer and magnetic particles before or during washing, vigorously mixing the magnetic particles and wash buffer, or incubating the magnetic particles and wash buffer for a period of more than 10 seconds.

[0020]

[0021] A6. The method of any one of clauses A1 to A5, further comprising releasing the first quantity and the second quantity of nucleic acid-binding magnetic particles from the magnet, adding an elution buffer to the first quantity and the second quantity of nucleic acid-binding magnetic particles, releasing the magnet and mixing the magnetic particles with the elution buffer, recapturing the first quantity and the second quantity of nucleic acid-binding magnetic particles using the magnet, and transferring the elution buffer to another sample container.

[0021]

[0022] A7. The method of any one of clauses A1 to A6, further comprising adding an elution buffer to the first amount of nucleic acid-binding magnetic particles and the second amount of nucleic acid-binding magnetic particles, mixing the magnetic particles with the elution buffer, recapturing the first amount of nucleic acid-binding magnetic particles and the second amount of nucleic acid-binding magnetic particles using a magnet, and transferring the first amount of nucleic acid-binding magnetic particles and the second amount of nucleic acid-binding magnetic particles to another sample container.

[0022]

[0023] A8. The method of any one of clauses A1-A7, wherein the elution buffer is configured to elute the nucleic acids captured by the nucleic acid-binding magnetic particles into the elution buffer.

[0023]

[0024] A9. The method of any one of clauses A1-A8, wherein the amount of the second amount of nucleic acid-binding magnetic particles is substantially equal to the amount of the first amount of nucleic acid-binding magnetic particles.

[0024]

[0025] A10. The method of any one of clauses A1-A9, further comprising assaying the elution buffer for the presence of one or more target nucleic acids suspected of being present in the sample.

[0025]

[0026] A11. The method according to any one of A1 to A10, wherein the assaying comprises a nucleic acid amplification step and a detection step of the amplified nucleic acid.

[0026]

[0027] A12. The method according to any one of A1 to A11, wherein the stirring step comprises heating the sample and the dispersion step comprises cooling the sample.

[0027]

[0028] B1. A method for cell lysis and nucleic acid recovery, comprising: providing a sample container; combining in a sample vessel a quantity of lysing particles, a first quantity of nucleic acid-binding magnetic particles, a sample suspected of containing one or more target nucleic acids, and a lysis buffer; agitating the lysis particles, the sample, the lysis buffer, and the first amount of nucleic acid-binding magnetic particles in the sample container for a period of time sufficient to generate a lysis solution; Dispersing a second amount of nucleic acid-binding magnetic particles in a lysis solution in the sample vessel; Incubating a first amount of nucleic acid-binding magnetic particles and a second amount of nucleic acid-binding magnetic particles in a lysis solution for a period of time; capturing a first amount of nucleic acid-binding magnetic particles and a second amount of nucleic acid-binding magnetic particles from the lysate using a magnet; wherein adding a first amount of nucleic acid-binding magnetic particles and then a second amount of nucleic acid-binding magnetic particles is selected to enhance recovery of RNA and DNA from the sample.

[0028]

[0029] B2. The method of clause B1, wherein the period of incubation of the first amount of nucleic acid-binding magnetic particles and the second amount of nucleic acid-binding magnetic particles in the lysis solution ranges from 1 second to 1 minute.

[0029]

[0030] B3. The method of clause B1 or B2, wherein the period is in the range of 20 to 30 seconds.

[0030]

[0031] B4. A method according to any one of B1 to B3, wherein capturing the first amount of nucleic acid-bound magnetic particles and the second amount of magnetic particles from the lysate further comprises transferring the magnetic particles to a second sample vessel and releasing the magnetic particles from the magnet into the second sample vessel, or removing the lysate and lysed particles from the first sample vessel and returning the magnetic particles from the magnet to the first sample vessel.

[0031]

[0032] B5. The method of any one of B1 to B4, wherein capturing the first quantity of magnetic particles and the second quantity of magnetic particles from the lysate preferably does not include capturing lysed particles.

[0032]

[0033] B6. The method of any one of B1 to B5, further comprising releasing the first amount of nucleic acid-bound magnetic particles and the second amount of nucleic acid-bound magnetic particles from a magnet, adding a wash buffer to the magnetic particles, washing the magnetic particles with the wash buffer, recapturing the magnetic particles using 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 the elution buffer, recapturing the magnetic particles using a magnet, and transferring either the magnetic particles or the elution buffer to another sample container.

[0033]

[0034] B7. The method of any one of clauses B1-B6, wherein washing does not include one or more of heating the wash buffer and magnetic particles before or during washing, vigorously mixing the magnetic particles and wash buffer, or incubating the magnetic particles and wash buffer for a period of more than 10 seconds.

[0034]

[0035] B8. The method of any one of B1-B7, wherein the steps of the method are completed in <4 minutes.

[0035]

[0036] B9. The method of any one of B1-B8, wherein the steps of the method are completed in <3 minutes.

[0036]

[0037] B10. The method of any one of B1-B9, wherein the steps of the method are completed in <2 minutes.

[0037]

[0038] B11. The method of any one of B1-B10, wherein the steps of the method are completed in 1-3 minutes.

[0038]

[0039] C1. A method for cell lysis and nucleic acid recovery, comprising: providing a sample vessel containing a plurality of fluidly connected reaction chambers, including a sample lysis chamber, a nucleic acid recovery chamber, and at least a first nucleic acid amplification chamber; combining in a sample lysis chamber a quantity of lysis particles, a first quantity of nucleic acid-binding magnetic particles, a sample suspected of containing one or more target nucleic acids, and a lysis buffer; agitating the lysis particles, the sample, the lysis buffer, and the first amount of nucleic acid-binding magnetic particles in the lysis chamber for a period of time sufficient to generate a lysis solution; mixing a second amount of nucleic acid-binding magnetic particles into the lysis solution; transferring at least a portion of the lysate in which the first amount of nucleic acid-binding magnetic particles and the second amount of nucleic acid-binding magnetic particles are suspended to a nucleic acid recovery chamber; capturing a first amount of nucleic acid-binding magnetic particles and a second amount 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, but not removing the first amount of nucleic acid-binding magnetic particles and the second amount of nucleic acid-binding magnetic particles; Releasing the first amount of nucleic acid-binding magnetic particles and the second amount of nucleic acid-binding magnetic particles from the magnet, and washing the first amount of nucleic acid-binding magnetic particles and the second amount of nucleic acid-binding magnetic particles with a wash buffer in the nucleic acid recovery chamber; recapturing the first amount of nucleic acid-binding magnetic particles and the second amount of nucleic acid-binding magnetic particles using a magnet and removing the wash buffer; releasing the magnet and mixing the first amount of nucleic acid-binding magnetic particles and the second amount of nucleic acid-binding magnetic particles with an elution buffer in the nucleic acid recovery chamber to elute the nucleic acids from the magnetic particles; recapturing the first amount of nucleic acid-binding magnetic particles and the second amount of nucleic acid-binding magnetic particles using a magnet and transferring the elution buffer to the first nucleic acid amplification chamber; wherein adding the first amount of nucleic acid-binding magnetic particles followed by the second amount of nucleic acid-binding magnetic particles is selected to enhance recovery of RNA and DNA from the sample.

[0039]

[0040] C2. The method of clause C1, wherein capturing the nucleic acid-bound magnetic particles comprises disposing a magnet adjacent to the nucleic acid recovery chamber and containing the magnetic particles within the chamber.

[0040]

[0041] C3. The method of clause C1 or C2, further comprising combining the elution buffer with reagents for a nucleic acid amplification reaction to form an amplification mix in a first nucleic acid amplification chamber and subjecting the amplification mix to amplification conditions to assay for the presence of one or more target nucleic acids suspected of being present in the sample.

[0041]

[0042] C4. The method of any one of C1-C3, wherein the assay is selected from the group consisting of nucleic acid amplification, sequencing, and next-generation sequencing.

[0042]

[0043] C5. The method of any one of clauses C1-C4, wherein the second amount of nucleic acid-binding magnetic particles is substantially equal to the first amount of nucleic acid-binding magnetic particles.

[0043]

[0044] C6. The method of any one of clauses C1-C5, wherein the second quantity of nucleic acid-binding magnetic particles is substantially larger than the first quantity of nucleic acid-binding magnetic particles.

[0044]

[0045] C7. The method of any one of clauses C1-C6, wherein the second quantity of nucleic acid-binding magnetic particles is substantially smaller than the first quantity of nucleic acid-binding magnetic particles.

[0045]

[0046] C8. The method of any one of C1-C7, wherein the stirring step comprises heating the sample and the mixing step comprises cooling the sample.

[0046]

[0047] C9. The method of any one of clauses C1-C8, wherein washing does not include one or more of heating the wash buffer and magnetic particles before or during washing, vigorously mixing the magnetic particles and wash buffer, or incubating the magnetic particles and wash buffer for a period of more than 10 seconds.

[0047]

[0048] C10. The method of any one of C1-C9, wherein the steps of the method are completed in <4 minutes.

[0048]

[0049] C11. The method of any one of C1-C10, wherein the steps of the method are completed in <3 minutes.

[0049]

[0050] C12. The method of any one of C1-C11, wherein the steps of the method are completed in <2 minutes.

[0050]

[0051] C13. The method of any one of C1 to C12, wherein the steps of the method are completed in 1 to 3 minutes.

[0051]

[0052] This Summary is provided to introduce in a simplified form some concepts 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 for use as an aid in determining the scope of the claimed subject matter.

[0052]

[0053] Additional features and advantages will be set forth in the description which follows, and in part will be obvious from the description, or may be learned by the practice of the invention. The features and advantages may be realized and obtained by means of the instruments and combinations particularly pointed out in the appended claims. These and other features will become more fully apparent from the following description and appended claims, or may be learned by the practice of the invention as set forth hereinafter. [Brief explanation of the drawings]

[0053] [Figure 1]

[0054] 1 shows a flexible pouch useful for self-contained PCR. [Figure 2]

[0055] 2 is an exploded perspective view of a device including the pouch of FIG. 1 and for use with the pouch of FIG. 1. [Figure 3]

[0056] The pouch of FIG. 1 is shown with the bladder component of FIG. [Figure 4]

[0057] 3 illustrates a motor used in an exemplary embodiment of the device of FIG. 2. [Figures 5A-5E]

[0058] 1 shows the steps of the method for cell lysis and nucleic acid recovery. [Figure 6A]

[0059] 10 is a bar graph showing detection of DNA organisms at various concentrations in the presence (BB, with MB) or absence (BB, without MB) of silica-coated magnetic particles during bead-beating lysis. [Figure 6B]

[0060] 10 is a bar graph showing detection of RNA organisms at various concentrations in the presence (BB, with MB) or absence (BB, without MB) of silica-coated magnetic particles during bead-beating lysis. [Figure 7A]

[0061] Compare the crossing points (Cps) for a control assay (without divided mag beads) in which magnetic particles were added after lysis, and an assay (with divided mag beads) in which a first amount of magnetic particles was added to lysis and a second amount of magnetic particles was added after lysis. [Figure 7B-7C]

[0062] 7B shows the same data as in FIG. 7A for the DNA assay, and 7C shows the same data as in FIG. 7A for the RNA assay. [Figure 8]

[0064] Comparison of Cps of amplification of nucleic acids recovered from magnetic particles contained in lysis with no wash, one wash, or two washes. [Figure 9]

[0065] Fragment sizes of human genomic DNA detected with and without bead beating for 120 seconds are shown. [Figure 10A]

[0066] The average Cp response of elution temperature in DNA and RNA assays is shown. [Figures 10B-10C] 10B and 10C show the Cp response of elution temperature for DNA and RNA assays, respectively, with lines of best fit showing the trend of the data. [Figure 11]

[0068] A bar graph showing an example of sample preparation time. DETAILED DESCRIPTION OF THE INVENTION

[0054]

[0069] Exemplary embodiments are described below with reference to the accompanying drawings. Because many different configurations and embodiments are possible without departing from the spirit and teachings of the present disclosure, the present disclosure should not be construed as limited to the exemplary embodiments set forth herein. Rather, these exemplary embodiments are provided so that this disclosure will be thorough and complete, and will convey the scope of the disclosure to those skilled in the art. In the drawings, the sizes and relative dimensions of layers and regions may be exaggerated for clarity. Like reference numerals refer to like elements throughout the description.

[0055]

[0070] Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. Furthermore, terms as defined in commonly used dictionaries should be interpreted as having a meaning consistent with the meaning in the context of this application and the related art, and should not be interpreted in an idealized or overly formal sense unless expressly so defined herein. The terms used to describe the present invention are intended only to describe particular embodiments and are not intended to limit the invention. Although many methods and materials similar or equivalent to those described herein can be used in the practice of the present disclosure, only certain exemplary materials and methods are described herein.

[0056]

[0071] 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.

[0057]

[0072] 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 "exemplary" mean "serving as an example, instance, or illustration," and should not necessarily be construed as preferred or advantageous over other embodiments disclosed herein. In addition, references to "implementations" or "embodiments" of the disclosure or invention include specific references to one or more embodiments thereof, and vice versa, and are intended to provide illustrative examples without limiting the scope of the invention, which is indicated by the appended claims rather than by the following description.

[0058]

[0073] Please note that as used in this specification and the appended claims, the singular forms "a," "an," and "the" include the plural forms unless the content clearly contradicts otherwise. Thus, for example, the reference to "tiles" includes one, two, or more tiles. Similarly, references to multiple sources should be construed as consisting of a single source and / or multiple sources unless the content and / or context clearly contradicts otherwise. Thus, a reference to "tiles" does not necessarily require a plurality of such tiles. Instead, it will be understood that one or more tiles are contemplated herein, regardless of the inflection.

[0059]

[0074] As used throughout this application, the words "can" and "may" are used in a permissive sense (i.e., meaning that they could) rather than a mandatory sense (i.e., meaning that they must). Furthermore, as used in this specification, including the claims, the terms "including," "having," "involving," "containing," "characterized by," variations thereof (e.g., "includes," "has," "involves," "contains," etc.), and similar terms are intended to be inclusive and / or open-ended and to have the same meaning as the word "comprising" and variations thereof (e.g., "comprises" and "comprises") and, illustratively, do not exclude additional, unrecited elements or method steps.

[0060]

[0075] As used herein, directional terms such as "top," "bottom," "left," "right," "up," "down," "superior," "inner," "outer," "internal," "external," "medial," "external," "proximal," "distal," "anterior," "posterior," and / or any other terminology may be used to indicate relative directions and / or orientations only and are not otherwise intended to limit the scope of this disclosure, including the specification, invention, and / or claims.

[0061]

[0076] When an element is referred to as being "coupled," "connected," or "responsive" to or "on" another element, it will be understood that it may be directly coupled, connected, or responsive to, or on the other element, or that intervening elements may also be present. In contrast, when an element is referred to as being "directly coupled," "directly connected," or "directly responsive to," or "directly on," another element, there are no intervening elements present.

[0062]

[0077] Exemplary embodiments of the inventive concepts are described herein with reference to cross-section illustrations that are schematic illustrations of idealized embodiments (and intermediate structures) of the exemplary embodiments. As such, variations from the shapes of the illustrations are to be expected as a result, for example, of manufacturing techniques and / or tolerances. Thus, the exemplary embodiments of the inventive concepts should not be construed as limited to the particular shapes of regions illustrated herein and are to include, for example, deviations in shapes that result from manufacturing. Thus, the regions illustrated in the figures are schematic in nature and the shapes are not intended to illustrate the actual shape of a region of a device, nor are they intended to limit the scope of the exemplary embodiments.

[0063]

[0078] In this specification, terms such as "first," "second," etc. may be used to describe various elements, but it should be understood that these elements are not limited by these terms. These terms are used only to distinguish one element from another. Thus, a "first" element could be termed a "second" element without departing from the teachings of the present embodiments.

[0064]

[0079] It is also understood that the various implementations described herein can be utilized in combination with any other implementations described or disclosed without departing from the scope of the present disclosure. Thus, products, components, elements, devices, apparatus, systems, methods, processes, compositions, and / or kits according to a particular implementation of the present disclosure can include, incorporate, or otherwise be configured with properties, features, components, components, elements, steps, and / or the like described in other implementations (including systems, methods, apparatus, and / or the like) disclosed herein without departing from the scope of the present disclosure. Thus, reference to a particular feature with respect to an implementation should not be construed as being limited to application only within that implementation.

[0065]

[0080] Headings used herein are for organizational purposes only and are not intended to limit the scope of the specification or the claims. To facilitate understanding, like reference numerals have been used whenever possible to indicate like elements common to the figures. Furthermore, whenever possible, elements have been similarly numbered in the various figures. Furthermore, alternative configurations of a particular element may each include a different letter appended to the element number.

[0066]

[0081] The term "about" is used herein to mean approximately, in the region of, roughly, or around. When the term "about" is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values. In general, the term "about" is used herein to modify a numerical value above and below the stated value by a variance of 5%. When such a range is expressed, another embodiment includes from the one particular value and / or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent "about," it is understood that the particular value forms a separate embodiment. It will be further understood that each endpoint of a range is significant both in relation to the other endpoint, and independently of the other endpoint.

[0067]

[0082] As used herein, the word "or" means any one member of a particular list and also includes any combination of members of that list.

[0068]

[0083] In one aspect, as described in further detail herein, microorganisms from a sample or growth medium can be separated and tested to characterize and / or identify the microorganisms present in the sample. As used herein, the term "separate" is intended to encompass a sample of microorganisms that has been removed, concentrated, or otherwise separated from its original state or from a growth or culture medium. For example, in accordance with the present invention, microorganisms can be separated (e.g., as a separate sample) from non-microorganisms or non-microorganism components that may interfere with characterization and / or identification. This term may include microorganisms separated from a mixture by centrifugation, filtration, or other separation techniques known in the art. Thus, a separated microbial sample may contain a collection of microorganisms and / or components thereof that is more concentrated than the original sample or that is otherwise separated from the original sample, ranging from a dense mass of closely packed microorganisms to a diffuse layer of microorganisms. Non-microbial components separated from microorganisms may include non-microbial cells (e.g., blood cells and / or other tissue cells) and / or any components thereof. In one aspect, the microorganisms are separated from a lysate mixture comprising lysed non-microbial cells and substantially intact microbial cells.

[0069]

[0084] In some embodiments, the separation of the microorganisms from the original state of the sample or from the growth or culture medium is incomplete. In other words, removing, concentrating, or otherwise separating the microorganisms from their original state does not completely separate the microorganism sample from other components of the sample or from the growth or culture medium. In some cases, only small amounts of residue are present from the sample or from the growth or culture medium. For example, the amount of residue or growth or culture medium present in the separated sample may be insufficient to identify or characterize the microorganism or to interfere with further growth of the microorganism. In some embodiments, the separated sample is 99% pure of contaminating elements, but may be 95% pure, 90% pure, 80% pure, 70% pure, 60% pure, 50% pure, or the minimum purity that allows identification of the microorganism in the separated sample by downstream identification techniques.

[0070]

[0085] Although reference is made to testing for microorganisms and viruses, the methods presented herein can be used for a wide variety of sample types and a wide variety of nucleic acid tests. Thus, "sample" refers to an animal; an animal tissue or organ; a cell (either within a subject, directly from a subject, or maintained in culture or from a cultured cell line); a cell lysate (or lysate fraction) or cell extract; a solution containing one or more molecules derived from cells, cellular material, or viral material; or other sample containing nucleic acid. A sample can also be a bodily fluid or excreta (e.g., but not limited to, blood, urine, stool, saliva, tears, bile, or cerebrospinal fluid) that may or may not contain host or pathogen cells, cellular components, or nucleic acids.

[0071]

[0086] In yet another aspect described in more detail herein, microorganisms from a sample or growth medium can be pelleted and examined to characterize and / or identify the microorganisms present in the sample. As used herein, the term "pellet" is intended to encompass a sample of microorganisms that has been compacted or sedimented into a mass of microorganisms. For example, microorganisms from a sample can be compacted or sedimented as a mass to the bottom of a tube by centrifugation or other methods known in the art. This includes the collection of microorganisms (and / or their components) on the bottom and / or sides of a container after centrifugation. In accordance with the present invention, microorganisms can be pelleted to remove them from non-microorganisms or non-microbial components that may interfere with characterization and / or identification (e.g., as a substantially purified microbial pellet).

[0072]

[0087] As used herein, the term "nucleic acid" refers to a naturally occurring or synthetic oligonucleotide or polynucleotide capable of hybridizing to a complementary nucleic acid by Watson-Crick base pairing, whether DNA or RNA or a DNA-RNA hybrid, single-stranded or double-stranded, sense or antisense. Nucleic acids of the present invention may also contain nucleotide analogs (e.g., BrdU) and non-phosphodiester internucleoside linkages (e.g., peptide nucleic acid (PNA) or thiodiester linkages). In particular, nucleic acids may include, but are not limited to, DNA, RNA, mRNA, rRNA, cDNA, gDNA, ssDNA, dsDNA, or any combination thereof.

[0073]

[0088] "Probe," "primer," or "oligonucleotide" refers to a single-stranded nucleic acid molecule of defined sequence that can base-pair with a second nucleic acid molecule ("target") containing a complementary sequence. The stability of the resulting hybrid depends on the length, GC content, and 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 target molecule and the degree of stringency of the hybridization conditions. The degree of hybridization stringency is affected by parameters such as temperature, salt concentration, and the concentration of organic molecules such as formamide, and is determined by methods known to those skilled in the art. Probes, primers, and oligonucleotides can be detectably labeled, either radioactively, fluorescently, or non-radioactively, by methods well known to those skilled in the art. dsDNA-binding dyes can be used to detect dsDNA. It is understood that while a "primer" is specifically configured to be extended by a polymerase, a "probe" or "oligonucleotide" may or may not be so configured.

[0074]

[0089] "dsDNA binding dye" refers to a dye that, when bound to double-stranded DNA, emits a different fluorescence than when bound to single-stranded DNA or when free in solution, typically exhibiting a stronger fluorescence intensity when bound to double-stranded DNA. Although dsDNA binding dyes are mentioned herein, any suitable dye may be used, and it is understood that some non-limiting exemplary dyes are described in U.S. Patent No. 7,387,887, which are incorporated herein by reference. Other signal-producing substances, such as enzymes, antibodies, and the like, may be used to detect nucleic acid amplification and melting, as known in the art.

[0075]

[0090] By "specifically hybridize" is meant that a probe, primer, or oligonucleotide recognizes and physically interacts (i.e., base-pairs) with a substantially complementary nucleic acid (e.g., a sample nucleic acid) under high stringency conditions, and does not substantially base-pair with other nucleic acids.

[0076]

[0091] "High stringency conditions" typically refer to conditions occurring at the melting temperature (Tm) minus 5°C (i.e., 5°C below the Tm of the probe). Functionally, high stringency conditions are used to identify nucleic acid sequences with at least 80% sequence identity.

[0077]

[0092] "Lysing particles" refers to various particles or beads for lysing cells, viruses, spores, and other materials that may be present in a sample. Various examples use zirconium ("Zr") silicate or ceramic beads, although other lysing particles, including glass and sand lysing particles, are known and are within the scope of the term. The term "cell lysing component" can include lysing particles, but can also include other components as known in the art, such as components for chemical lysis.

[0078]

[0093] PCR is the amplification method used in the examples herein, but it is understood that any amplification method using primers may be suitable. Such suitable procedures include polymerase chain reaction (PCR); strand displacement amplification (SDA); nucleic acid sequence-based amplification (NASBA); cascade rolling circle amplification (CRCA); loop-mediated 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. In amplification methods without discrete cycles, reaction time may be used when measurements are made at cycles, doubling times, or crossover times (Cp), and additional reaction time may be added when additional PCR cycles are added in the embodiments described herein. It is understood that protocols need to be adjusted accordingly.

[0079]

[0094] 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 wide variety of nucleic acid sequences from a wide variety of samples, including human, veterinary, industrial, and environmental.

[0080]

[0095] In various embodiments disclosed herein, self-contained nucleic acid analysis pouches are used to assay samples for the presence of various biological substances, such as antigens and nucleic acid sequences, within a single, closed system. Such systems, including pouches and instruments for use with the pouches, are disclosed in detail in U.S. Pat. Nos. 8,394,608; 8,895,295; and 10,464,060, which are incorporated herein by reference. However, it is understood that such pouches are merely exemplary, and that the nucleic acid preparation and amplification reactions discussed herein can be performed in any of a variety of open or closed sample vessels, including 96-well plates, plates of other configurations, arrays, carousels, and the like, using a variety of nucleic acid purification and amplification systems, as known in the art. Terms such as "sample well," "sample vessel," "amplification well," and "amplification vessel" are used herein, and are meant to encompass blister wells, tubes, and various other reaction vessels, such as those used in these amplification systems. In one embodiment, the pouch is used for assaying multiple pathogens. The pouch may include one or more blisters used as sample wells and is illustratively used in a closed system. Illustratively, various steps can be performed within the optional disposable pouch, including nucleic acid preparation, primary high-volume multiplex PCR, dilution of the primary amplification product, and secondary PCR, optionally concluding with post-amplification analysis such as real-time detection or melting curve analysis. Furthermore, while various steps can be performed in the pouch of the present invention, it is understood that one or more steps may be omitted for specific applications, and the pouch configuration may be modified accordingly. While many embodiments herein use a multiplex reaction for first-stage amplification, it is understood that this is merely exemplary, and that in some embodiments, the first-stage amplification may be singleplex. In one illustrative example, the first-stage singleplex amplification targets housekeeping genes, and the second-stage amplification utilizes differences in the housekeeping genes for identification.Thus, while various embodiments discuss first-stage multiplex amplification, it should be understood that this is by way of example only.

[0081]

[0096] FIG. 1 shows an exemplary pouch 510 that can be used in or reconfigured for various embodiments. Pouch 510 is similar to FIG. 15 of U.S. Patent No. 8,895,295, and like elements are numbered the same. Receptacle 590 is provided with inlet channels 515a-515l, which also function as reagent or waste reservoirs. Illustratively, reagents are lyophilized in receptacle 590 and rehydrated prior to use. Blisters 522, 544, 546, 548, 564, and 566, and their respective channels 514, 538, 543, 552, 553, 562, and 565, are similar to the like-numbered blisters in FIG. 15 of U.S. Patent No. 8,895,295. The second-stage reaction zone 580 in FIG. 1 is similar to that of U.S. Patent No. 8,895,295, although the second-stage wells 582 of the high-density array 581 are arranged in a slightly different pattern. The more circular pattern of the high-density array 581 in FIG. 1 eliminates corner wells and may result in more uniform filling of the second-stage wells 582. As shown, the high-density array 581 is provided with 102 second-stage wells 582. The pouch 510 is suitable for use with the FilmArray® instrument (BioFire Diagnostics, LLC, Salt Lake City, UT). However, it is understood that the pouch embodiment is exemplary only.

[0082]

[0097] Although other containers can be used, illustratively, the pouch 510 can be formed of two layers of flexible plastic film or other flexible material, such as polyester, polyethylene terephthalate (PET), polycarbonate, polypropylene, polymethyl methacrylate, or mixtures, combinations, and layers thereof, 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 one or more layers of a single type or types of material laminated together. Aluminum-laminated metal foil or plastic can also be used. Other barrier materials that can be sealed together to form blisters and channels are also known in the art. If a plastic film is used, the layers can be adhered, illustratively by heat sealing. Illustratively, this material has low nucleic acid and protein binding capacity.

[0083]

[0098] In 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 composite ratios, as well as different film thicknesses. In the case of aluminum or other foil-laminated plastics, the portion of the pouch to be read with the fluorescence detection device can be left without foil. For example, if fluorescence is monitored in the second-stage well 582 of the second-stage reaction zone 580 of the pouch 510, one or both layers of the well 582 are left without foil. In a PCR example, a film laminate composed of approximately 0.0048 inch (0.1219 mm) thick polyester (Mylar, DuPont, Wilmington DE) and 0.001-0.003 inch (0.025-0.076 mm) thick polypropylene film works well. Illustratively, the pouch 510 can be made of a transparent material that can transmit approximately 80%-90% of incident light.

[0084]

[0099] In exemplary embodiments, material is moved between blisters by applying pressure, illustratively air pressure, to the blisters and channels. Thus, in embodiments using pressure, the pouch material illustratively has sufficient flexibility for the pressure to have the desired effect. The term "flexible" is used herein to describe the physical properties of the pouch material. The term "flexible" is defined herein as being easily deformable by the levels of pressure used herein without cracking, breaking, cracking, etc. For example, Saran Wrap TM Thin plastic sheets, such as Ziploc® bags, and thin metal foils, such as aluminum foil, are flexible. However, even in embodiments that use pneumatic pressure, only certain regions of the blisters and channels need to be flexible. Furthermore, as long as the blisters and channels are easily deformable, only one side of the blisters and channels need not be flexible. Other regions of the pouch 510 may be made of a rigid material or may be reinforced with a rigid material. Thus, when terms such as "flexible pouch" or "flexible sample container" are used, it is understood that only a portion of the pouch or sample container need be flexible.

[0085]

[0100] Illustratively, a plastic film can be used for pouch 510. A metal sheet, illustratively aluminum, or other suitable material can be milled or otherwise cut to create a die with a pattern of raised surfaces. When loaded into a pneumatic press (illustratively, A-5302-PDS, Janesville Tool Inc., Milton, WI), illustratively adjusted to an operating temperature of 195°C, the pneumatic press operates like a printing press, melting the sealing surface of the plastic film only where the die contacts the film. Similarly, the plastic film used for pouch 510 can be cut and welded using a laser cutting and welding device. Various components, such as PCR primers (illustratively spotted and dried on the film), antigen-binding substrates, magnetic beads, and zirconium silicate beads, can be sealed inside the various blisters as pouch 510 is formed. Reagents for sample processing can be spotted onto the film, collectively or separately, before sealing. In one embodiment, nucleotide triphosphates (NTPs) are spotted onto the film separately from the polymerase and primers, essentially eliminating polymerase activity until the reaction is hydrated with an aqueous sample. If the aqueous sample is heated prior to hydration, this creates the conditions for true hot-start PCR, reducing or eliminating the need for expensive chemical hot-start components. In another embodiment, the components are provided in powder or pill form and placed into blisters before final sealing.

[0086]

[0101] Pouch 510 may be used similarly as described in U.S. Patent No. 8,895,295. In one exemplary embodiment, a 300 μl mixture containing the sample to be tested (100 μl) and lysis buffer (200 μl) may be injected into an injection port (not shown) in fitting 590 near inlet channel 515 a, and the sample mixture may be drawn into inlet channel 515 a. Water may also be injected into a second injection port (not shown) in fitting 590 adjacent to inlet channel 515 l and distributed through channels (not shown) in fitting 590, thereby hydrating up to 11 different reagents previously provided in dry form in inlet channels 515 b through 515 l, respectively. Exemplary methods and devices for injecting sample and hydration fluid (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 is understood that these methods and devices are merely exemplary and that other ways of introducing sample and hydration fluid into pouch 510 are within the scope of this disclosure. These reagents may illustratively include lyophilized PCR reagents, DNA extraction reagents, wash solutions, immunoassay reagents, or other chemicals. Illustratively, the reagents are for nucleic acid extraction, first-stage multiplex PCR, dilution of multiplex reactions, and preparation of second-stage PCR reagents and control reactions. In the embodiment shown in FIG. 1 , all that needs to be injected is sample solution into one injection port and water into the other. After injection, the two injection ports are sealed. For details regarding various configurations of pouch 510 and fitting 590, see U.S. Pat. No. 8,895,295, previously incorporated by reference.

[0087]

[0102] After injection, the sample can be transferred from injection channel 515a through channel 514 to lysis blister 522. Lysis blister 522 contains beads or particles 534, such as ceramic beads or other abrasive elements, and is configured for vortexing by impaction using rotating blades or paddles provided within the FilmArray® instrument. Bead milling by shaking, vortexing, sonication, and similar processes of the sample in the presence of lysing particles, such as zirconium silicate (ZS) beads 534, is an effective method for forming a lysate. As used herein, terms such as "lyse," "lysing," and "lysate" are understood to be not limited to rupturing cells but also include disruption of non-cellular particles, such as viruses. In another embodiment, a paddle beater using reciprocating or alternating paddles, such as those described in U.S. Patent Publication No. 2019-0344269, incorporated herein by reference in its entirety, can be used for lysis in this and other embodiments described herein.

[0088]

[0103] FIG. 4 illustrates a bead-beating motor 819 including a blade 821 that may be attached to the first surface 811 of the support member 802 of the instrument 800 shown in FIG. 2 . The blade extends through the slot 804 and contacts the pouch 510. However, it is understood that the motor 819 may be attached to other structures on the instrument 800. In one exemplary embodiment, the motor 819 is a Mabuchi RC-280SA-2865 DC motor (Chiba Prefecture, Japan) and is attached to the support member 802. In one exemplary embodiment, the motor rotates at 5,000 to 25,000 rpm, more illustratively 10,000 to 20,000 rpm, and even more illustratively about 15,000 to 18,000 rpm. It has been found that a voltage of 7.2 V provides sufficient rotational speed for lysis with the Mabuchi motor. However, it is understood that the actual speed may be somewhat slower when the blade 821 is impacting the pouch 510. Other voltages and speeds may be used for lysis, depending on the motor and paddles used. Optionally, a small, controlled volume of air may be provided to the bladder 822 adjacent to the lysis blister 522. In some embodiments, partially filling one or more adjacent bladders with a small volume of air has been found to aid in positioning and supporting the lysis blister during the lysis process. Alternatively, another structure, illustratively a rigid or flexible gasket or other retaining structure around the lysis blister 522, may be used to restrain the pouch 510 during lysis. It is also understood that the motor 819 is illustrative only, and other devices may be used to crush, shake, or vortex the sample. In some embodiments, chemicals or heat may be used in addition to or instead of mechanical lysis.

[0089]

[0104] Once the sample material is sufficiently lysed, the sample is moved to a nucleic acid extraction zone, illustratively through channel 538, blister 544, and channel 543, to blister 546, where the sample is mixed with a nucleic acid-binding substance, such as silica-coated magnetic beads 533. Alternatively, the magnetic beads 533 can be rehydrated, illustratively using fluid supplied from one of inlet channels 515c-515e, and then moved through channel 543 to blister 544 and then through channel 538 to blister 522. The mixture is incubated for a suitable time, illustratively approximately 10 seconds to 10 minutes. A retractable magnet located within the device adjacent to blister 546 captures the magnetic beads 533 from the solution and forms a pellet against the inner surface of blister 546. If incubation is performed in blister 522, multiple portions of the solution may need to be transferred to blister 546 for capture. The liquid is then drained from blister 546 and returned through blister 544 to blister 522, which serves as a waste receptacle. One or more wash buffers from one or more of injection channels 515c-515e are provided to blister 546 via blister 544 and channel 543. Optionally, the magnet is retracted and the magnetic beads 533 are washed by transferring the beads back and forth from blisters 544 and 546 via channel 543. Once the magnetic beads 533 have been washed, activation of the magnet recaptures the magnetic beads 533 in blister 546 and transfers the wash solution to blister 522. This process may be repeated as necessary to wash any lysis buffer and sample residue from the nucleic acid-bound magnetic beads 533.

[0090]

[0105] After washing, the elution buffer stored in injection channel 515f is transferred to blister 548 and the magnet is retracted. Solution is circulated between blisters 546 and 548 via channel 552, disrupting the pellet of magnetic beads 533 in blister 546 and allowing the captured nucleic acids to dissociate from the beads and enter solution. The magnet is activated again, capturing the magnetic beads 533 in blister 546 and transferring the eluted nucleic acid solution to blister 548.

[0091]

[0106] First-stage PCR master mix from injection channel 515g is mixed with the nucleic acid sample in blister 548. Optionally, the mixture is force-mixed between 548 and 564 via channel 553. After several cycles of mixing, the solution is contained in blister 564, a pellet of first-stage PCR primers is provided, at least one set of primers is provided for each target, and first-stage multiplex PCR is performed. If an RNA target is present, a reverse transcription (RT) step can be performed prior to or simultaneously with the first-stage multiplex PCR. First-stage multiplex PCR temperature cycling in the FilmArray® instrument is illustratively performed for 15-20 cycles, although other levels of amplification may be desired depending on the requirements of a particular application. The first-stage PCR master mix can be any of a variety of master mixes as known in the art. In one illustrative example, the first-stage PCR master mix can be any of the chemistries disclosed in U.S. Patent No. 9,932,634, incorporated herein by reference, for use in PCR protocols of 20 seconds or less per cycle.

[0092]

[0107] After the first-stage PCR has proceeded for the desired number of cycles, the sample may be diluted, illustratively by forcing most of the sample back into blister 548, leaving only a small amount in blister 564, and adding second-stage PCR master mix from injection channel 515i. Alternatively, dilution buffer from 515i may be moved to blister 566 and then mixed with the amplified sample in blister 564 by pumping fluid back and forth between blisters 564 and 566. If necessary, the dilution may be repeated several times using dilution buffer from injection channels 515j and 515k, or injection channel 515k may be reserved, illustratively for sequencing or other post-PCR analysis, after which second-stage PCR master mix from injection channel 515h may be added to some or all of the diluted amplified sample. The level of dilution can be adjusted by varying the number of dilution steps or by varying the proportion of sample discarded before mixing with a dilution buffer or a second-stage PCR master mix containing components for amplification, illustratively polymerase, dNTPs, and an appropriate buffer, although it is understood that other components may be suitable, particularly for non-PCR amplification methods. If desired, this mixture of sample and second-stage PCR master mix may be preheated in blister 564 before being transferred to second-stage well 582 for second-stage amplification. Such preheating may eliminate the need for hot start components (antibodies, chemicals, etc.) in the second-stage PCR mixture.

[0093]

[0108] In one embodiment, the exemplary second-stage PCR master mix is incomplete, lacking primer pairs, with each of the 102 second-stage wells 582 preloaded with a specific PCR primer pair. In other embodiments, the master mix contains other components (e.g., polymerase, Mg 2+The second-stage PCR master mix may lack other reaction components, such as a first-stage primer pair (e.g., a first-stage primer pair), and the missing components may be pre-loaded onto the array. If desired, the second-stage PCR master mix may lack other reaction components, which may also be pre-loaded into the second-stage wells 582. Each primer pair may be similar or identical to a first-stage PCR primer pair, or may be nested within a first-stage primer pair. Transfer of the sample from blister 564 to second-stage wells 582 completes the PCR reaction mixture. Once the high-density array 581 is filled, the individual second-stage reactions are sealed into their respective second-stage blisters by any number of means, as known in the art. Exemplary ways of filling and sealing the high-density array 581 without cross-contamination are discussed in U.S. Pat. No. 8,895,295, previously incorporated by reference. Illustratively, the various reactions in the wells 582 of the high-density array 581 are simultaneously or individually thermocycled, illustratively using one or more Peltier devices, although other means for thermocycling are known in the art.

[0094]

[0109] In certain embodiments, the second-stage PCR master mix contains the dsDNA binding dye LCGreen® Plus (BioFire Diagnostics, LLC) to generate a signal indicative of amplification. However, it is understood that this dye is exemplary only, and other signals may be used, including other dsDNA binding dyes and probes as known in the art, such as fluorescent, radioactive, chemiluminescent, and enzyme labels. Alternatively, wells 582 of array 581 may be provided without a signal, and subsequent processing may report the results.

[0095]

[0110] When air pressure is used to move material within pouch 510, in one embodiment, a "bladder" may be employed. Bladder assembly 810, a portion of which is shown in FIGS. 2-3 , includes a bladder plate 824 housing multiple inflatable bladders 822, 844, 846, 848, 864, and 866, each of which may be individually inflatable, illustratively by a compressed gas source. Because bladder assembly 810 is exposed to compressed gas and may be used multiple times, bladder assembly 810 may be made of a stronger or thicker material than the pouch. Alternatively, bladders 822, 844, 846, 848, 864, and 866 may be formed from a series of plates secured together with gaskets, seals, valves, and pistons. Other arrangements are within the scope of the present invention. Alternatively, an array of mechanical actuators and seals may be used to seal channels and direct fluid movement between blisters. Mechanical seal and actuator systems that may be adapted for the devices described herein are described in detail in U.S. Patent Publication No. 2019-0344269, previously incorporated by reference in its entirety.

[0096]

[0111] The success of the secondary PCR reaction depends on the template generated by the first-stage reaction of the multiplex. PCR is typically performed using highly pure DNA. Methods such as phenol extraction or commercially available DNA extraction kits provide highly pure DNA. Samples processed through pouch 510 may require adjustments to compensate for less pure preparations. PCR can be inhibited by components of the biological sample, which poses a potential obstacle. For example, to compensate for reduced nucleic acid purity, hot-start PCR, higher concentrations of Taq polymerase enzyme, adjustments to MgCl2 concentration, adjustments to primer concentration, addition of inhibitor-resistant artificial enzymes, and adjuvants (such as DMSO, TMSO, or glycerol) can optionally be used. While purity issues are likely to be more of a concern in the first-stage amplification, it is understood that similar adjustments may be made in the second-stage amplification.

[0097]

[0112] When pouch 510 is placed within device 800, bladder assembly 810 presses against one side of pouch 510, and as a particular bladder expands, the pressure forces liquid out of the corresponding blister within pouch 510. In addition to bladders corresponding to the many blisters in pouch 510, bladder assembly 810 may have additional pneumatic actuators, such as bladders or pneumatically driven pistons, corresponding to various channels in pouch 510. Figures 2-3 show exemplary pistons or hard seals 838, 843, 852, 853, and 865 corresponding to channels 538, 543, 553, and 565 in pouch 510, as well as seals 871, 872, 873, and 874 that minimize backflow into mounting portion 590. When actuated, hard seals 838, 843, 852, 853, and 865 form pinch valves that pinch off and close off the corresponding channels. To confine liquid within a particular blister in the pouch 510, hard seals are actuated on the channels leading to and from the blister, such that the actuators function as pinch valves, closing the channels. Illustratively, to mix two volumes of liquid in different blisters, a pinch valve actuator sealing the connecting channel is actuated, and a pneumatic bladder above the blister is alternately pressurized, forcing the liquid back and forth through the channel connecting the blisters and mixing the liquid therein. Pinch valve actuators come in a variety of shapes and sizes and may be configured to pinch off multiple channels at once. While pneumatic actuators are described herein, it is understood that other ways of providing pressure to the pouch are contemplated, including various electromechanical actuators such as linear stepper motors, motor-driven cams, rigid paddles driven by air, hydraulic, or electromagnetic forces, rollers, rocker arms, and even cocked springs. Furthermore, there are various ways to reversibly or irreversibly close a channel beyond applying pressure perpendicular to the axis of the channel. These include bending a bag across the channel, heat sealing, rotating actuators, and various physical valves sealed into the channel, such as butterfly valves and ball valves.Additionally, a small Peltier device or other temperature regulator may be placed adjacent to the channel and set at a temperature sufficient to freeze the fluid, effectively forming a seal. Also, while the design of FIG. 1 is compatible with automated equipment featuring actuator elements positioned on each of the blisters and channels, it is also contemplated that the actuators may remain stationary and that the pouch 510 may be moved such that a small number of actuators may be used for several processing stations, including processing stations for sample disruption, nucleic acid capture, first- and second-stage PCR, and other uses of the pouch 510, such as immunoassays and immunoPCR. Rollers acting on the channel and blister may prove particularly useful in configurations in which the pouch 510 moves between stations. Thus, while pneumatic actuators are used in the presently disclosed embodiment, when the term "pneumatic actuator" is used herein, it is understood that other actuators and other ways of providing pressure may be used, depending on the pouch and instrument configuration.

[0098]

[0113] Returning to FIG. 2 , each pneumatic actuator is connected to a compressed air source 895 via a valve 899. While only a few hoses 878 are shown in FIG. 2 , it is understood that each pneumatic fitting is connected to a compressed gas source 895 via a hose 878. The compressed gas source 895 may be a compressor, or alternatively, the compressed gas source 895 may be a compressed gas cylinder, such as a carbon dioxide cylinder. Compressed gas cylinders are particularly useful when portability is desired. Other compressed gas sources are within the scope of the present invention. Similar pneumatic controls may be provided, for example, for control of fluid movement within the pouches described herein, and other actuators, servos, and the like may be provided.

[0099]

[0114] Several other components of the device are also connected to a compressed gas source 895. A magnet 850 attached to a second side 814 of the support member 802 is illustratively deployed and retracted using gas from the compressed gas source 895 via a hose 878, although other methods of moving the magnet 850 are known in the art. The magnet 850 fits into a recess 851 in the support member 802. It is understood that the recess 851 can be a passageway through the support member 802 so that the magnet 850 can contact the blister 546 of the pouch 510. However, it is understood that, depending on the material of the support member 802, the recess 851 need not extend all the way through the support member 802, as long as the magnet 850 is close enough to provide a sufficient magnetic field to the blister 546 when deployed and does not significantly affect the magnetic beads 533 present in the blister 546 when the magnet 850 is fully retracted. Although reference is made to a retractable magnet 850, it is understood that an electromagnet may be used and that the electromagnet may be activated and deactivated by controlling the flow of electricity through the electromagnet. Thus, while reference is made herein to withdrawing or retracting the magnet, it is understood that these terms are broad enough to incorporate other ways of withdrawing the magnetic field. It is understood that the pneumatic connections may be pneumatic hoses or pneumatic air manifolds, thus reducing the number of hoses or valves required. It is understood that similar magnets and methods for activating the magnets may be used in other embodiments.

[0100]

[0115] The various pneumatic pistons 868 of pneumatic piston array 869 are also connected to compressed gas source 895 via hoses 878. Although only two hoses 878 are shown connecting pneumatic pistons 868 to compressed gas source 895, it is understood that each of pneumatic pistons 868 is connected to compressed gas source 895. Twelve pneumatic pistons 868 are shown.

[0101]

[0116] A pair of temperature control elements are attached to the second side 814 of the support member 802. As used herein, the term "temperature control element" refers to a device that adds heat to or removes heat from a sample. Examples of temperature control elements include, but are not limited to, heaters, coolers, Peltier devices, resistive heaters, inductive heaters, electromagnetic heaters, thin film heaters, printed element heaters, positive temperature coefficient heaters, and combinations thereof. A temperature control element may include multiple heaters, coolers, Peltiers, etc. In one aspect, a given temperature control element may include multiple types of heaters or coolers. For example, an example temperature control element is a Peltier device with separate resistive heaters applied to the top and / or bottom surfaces of the Peltier. While the term "heater" is used throughout this specification, it is understood that other temperature control elements may be used to regulate the temperature of the ampoule.

[0102]

[0117] As described above, first-stage heater 886 may be positioned to heat and cool the contents of blister 564 for first-stage PCR. As seen in Figure 2, second-stage heater 888 may be positioned to heat and cool the contents of second-stage blisters in array 581 of pouches 510 for second-stage PCR. However, it is understood that these heaters may be used for other heating purposes and may include other heaters suitable for particular applications.

[0103]

[0118] As noted above, while Peltier devices that thermocycle between two or more temperatures are useful for PCR, in some embodiments it may be desirable to maintain a heater at a constant temperature. Illustratively, this can be used to shorten run times by eliminating the time required to transition heater temperatures beyond that required to transition sample temperatures. Such an arrangement can also improve the electrical efficiency of the system, since only small samples and sample containers need to be thermocycled, eliminating the need to thermocycle the much larger (higher thermal mass) Peltier device. For example, an instrument may include multiple heaters (i.e., two or more) with set temperatures for, e.g., annealing, extension, and denaturation, positioned relative to the pouch to achieve thermal cycling. For many applications, two heaters may be sufficient. In various embodiments, the heaters may be moved, the pouch may be moved, or fluids may be moved relative to the heaters to achieve thermal cycling. Illustratively, heaters may be arranged in a linear fashion, an arc, or the like. Suitable heater types are described above with reference to first-stage PCR.

[0104]

[0119] When fluorescence detection is desired, an optical array 890 can be provided. As shown in FIG. 2 , the optical array 890 includes a light source 898, illustratively a filtered LED light source, filtered white light, or laser illumination, and a camera 896. The camera 896 illustratively has multiple photodetectors, each corresponding to a second-stage well 582 in the pouch 510. Alternatively, the camera 896 can capture an image containing all of the second-stage wells 582 and split the image into separate fields corresponding to each of the second-stage wells 582. Depending on the configuration, the optical array 890 can be stationary, or the optical array 890 can be placed on a mover attached to one or more motors and moved to obtain signals from individual second-stage wells 582. It will be understood that other arrangements are possible. In some embodiments of the second-stage heater, the heater is provided on the opposite side of the pouch 510 from that shown in FIG. 2 . Such an orientation is exemplary only and may be dictated by spatial constraints within the instrument. The photodetectors and heaters may be on either side of the array 581, provided that the second-stage reaction zone 580 is provided with an optically transparent material.

[0105]

[0120] As shown, computer 894 controls valve 899 of compressed air source 895, and thus all pneumatics of instrument 800. Furthermore, many of the instrument's pneumatic systems may be replaced in other embodiments with mechanical actuators, pressure application means, and the like. Computer 894 also controls heaters 886 and 888 and the optical array. Each of these components is electrically connected, illustratively via cable 891, although other physical or wireless connections are within the scope of the present invention. It will be understood that computer 894 may be housed within instrument 800 or may be external to instrument 800. Furthermore, computer 894 may include an internal circuit board that controls some or all of the components, or may include an external computer, such as a desktop or laptop PC, for receiving and displaying data from the optical array. An interface, illustratively a keyboard interface, including keys for inputting information and variables such as temperature, cycle time, and the like, may be provided. Illustratively, display 892 is also provided. Display 892 may be, for example, an LED, LCD, or other such display.

[0106]

[0121] Other instruments known in the art teach PCR in a sealed flexible container. See, for example, U.S. Patent Nos. 6,645,758, 6,780,617, and 9,586,208, which are incorporated herein by reference. However, including cell lysis within the sealed PCR container can improve ease of use and safety, especially when the sample being tested may contain a biohazard. In the embodiment illustrated herein, waste from cell lysis, as well as waste from all other processes, remains within the sealed pouch. It is understood, however, that the contents of the pouch may be removed for further testing.

[0107]

[0122] 2, device 800 includes a support member 802, which may form a wall of the casing or may be mounted within the casing. Device 800 may also include a second support member (not shown), which is optionally movable relative to support member 802 to allow for insertion and withdrawal of pouch 510. Illustratively, a lid may cover pouch 510 when pouch 510 is inserted into device 800. In another embodiment, both support members may be fixed and pouch 510 may be held in place by other mechanical means or by air pressure.

[0108]

[0123] In the illustrated example, heaters 886 and 888 are mounted to support member 802. However, it is understood that this arrangement is illustrative only and that other arrangements are possible. Exemplary heaters include Peltier and other block heaters, resistive heaters, electromagnetic heaters, and thin film heaters, as known in the art, for thermally cycling the contents of blister 864 and second-stage reaction zone 580. Bladder plate 810, including bladders 822, 844, 846, 848, 864, 866, hard seals 838, 843, 852, 853, and seals 871, 872, 873, 874, forms bladder assembly 808, which illustratively may be mounted to a movable support structure that may be moved toward pouch 510 so that a pneumatic actuator is placed in contact with pouch 510. When pouch 510 is inserted into instrument 800 and movable support member 802 is moved toward support member 802, the various blisters of pouch 510 are positioned adjacent to the various bladders of bladder assembly 810 and the various seals of assembly 808, and actuation of the pneumatic actuators may force liquid out of one or more of the blisters of pouch 510 or form pinch valves in one or more channels of pouch 510. The relationship of the blisters and channels of pouch 510 to the bladders and seals of assembly 808 is shown in more detail in FIG.

[0109] Methods for cell lysis and nucleic acid recovery

[0124] 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 described in further detail herein below, the methods and systems described herein take advantage of the kinetic efficiency of certain parts of sample preparation (i.e., sample lysis, recovery of nucleic acids from the lysate using a medium such as silica-coated magnetic particles, washing of the recovery medium, and elution of nucleic acids from the recovery medium) to minimize sample preparation while providing high-quality extracted nucleic acids for downstream amplification or other analysis. The methods described herein include focusing on one or more of: rapid mechanical lysis (preferably lysis performed in the presence of silica-coated magnetic particles), rapid recovery of silica-coated magnetic particles from the lysate, rapid washing of the magnetic particles, and fast, efficient elution of captured nucleic acids from the silica-coated magnetic particles. The methods and systems described herein are also designed for rapid preparation of nucleic acid samples using aqueous, alcohol- and organic solvent-free buffer compositions. Illustratively, one or more of the buffer compositions used in the methods described herein may be provided in the sample preparation device as a ready-to-use, shelf-stable liquid that can be stored under ambient conditions, and / or as a ready-to-use dry powder composition that can be rehydrated with a rehydration fluid (e.g., water) for use in performing the method steps. Successful sample preparation (i.e., lysis, nucleic acid recovery from the lysate, washing, and elution) is critical to maximizing the sensitivity of molecular assays and ensuring reliable and consistent results. The fidelity and efficiency of PCR, in particular, depend on the purity and concentration of the nucleic acid template added to the reaction. With increasingly shorter time-to-result in molecular assays, reducing sample preparation time is critical to shortening the overall assay time. The methods and systems described herein significantly reduce the time required for sample preparation by mechanical lysis, ensuring reliable and consistent results, without sacrificing the integrity, purity, and concentration of the input nucleic acid template.

[0110]

[0125] One exemplary method for preparing a nucleic acid sample includes providing a sample container including a first chamber, a sample (e.g., a 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 including a buffering agent, a chaotropic salt, and a non-ionic surfactant. The sample and sample buffer (first mixture) are combined with a quantity of lysing particles (e.g., zirconium silicate) and a first quantity of magnetic silica particles. The sample, sample buffer, lysing particles, and magnetic particles may be placed in the first chamber and preferably agitated (e.g., bead-beating) in the first chamber for a period of time sufficient to produce a lysing solution. In various embodiments, a mixture containing a sample, a sample buffer, lysing particles, and magnetic particles may be combined and then placed in a first chamber, which may contain one or more components (e.g., lysing particles and / or magnetic particles), and the sample and buffer may be added to the first chamber containing the lysing particles, the magnetic particles may be added subsequently, or any conceivable combination thereof. The sample, buffer, lysing particles, and magnetic particles may be agitated using any method or device known in the art for generating 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 generate a lysate. The bead beater described herein above with reference to FIG. 4 is another example of a bead beater instrument that may be suitable for use in bead beating in the methods described herein.

[0111]

[0126] After stirring the beads to generate a lysate, a second amount of nucleic acid-binding magnetic particles can be mixed with the lysate. After mixing the second amount of nucleic acid-binding magnetic particles with the lysate and optionally incubating the magnetic particles in the lysate for a period of time (e.g., 5 seconds to 2 minutes), the first amount of nucleic acid-binding magnetic particles and the second amount of nucleic acid-binding magnetic particles can be recovered from the lysate, illustratively using a magnet. A Pickpen is an example of a device that can be used to recover magnetic particles from a solution. The magnet 850 described with reference to FIG. 2 is another example of a magnet that can be used to recover magnetic particles from a lysate. After recovery, the magnetic particles can be transferred to another container, released, and washed one or more times to remove residual lysate. The magnetic particles can then be recaptured (e.g., with a magnet), the wash buffer removed, and the magnetic particles released again and mixed with an elution buffer to elute the captured nucleic acids from the magnetic particles. The magnetic particles can be recaptured, and the elution buffer can be recovered and transferred to a clean tube. Alternatively, a magnet can be used to remove the magnetic particles from the tube, and the magnetic particles can be discarded. In either case, the recovered nucleic acid in the elution buffer can be used in a variety of downstream assays, including, but not limited to, assaying the elution buffer for the presence of one or more target nucleic acids suspected to be present in the sample using one or more of nucleic acid amplification and detection, sequencing, next-generation sequencing, etc. That is, if the nucleic acid in the sample is derived from a cell or virus (e.g., a pathogen cell), the assay can be used to identify the cell or virus from which the nucleic acid originated.

[0112]

[0127] We have found that lysis and nucleic acid recovery are more efficient when lysis is performed in the presence of magnetic silica particles. This is shown, for example, in Figures 6A-7C. Figures 6A and 6B are bar graphs showing the detection rates of organisms at various concentrations in the presence (with BB and MB) or absence (without BB and MB) of silica-coated magnetic particles during bead-beating lysis for a set of assays based on amplification from DNA (Figure 6A) and a set of assays based on amplification from RNA (Figure 6B). Figure 6A shows that when bead-beating is performed in the presence of silica-coated magnetic particles, the detection rates of the DNA-based assays are consistently improved at 1x LOD, 0.1x LOD, and 0.01x LOD. However, the RNA-based assays appear to be nearly equivalent when silica-coated magnetic particles are added after lysis or when bead-beating is performed in the presence of silica-coated magnetic particles. Figure 7A shows the average crossing point (Cp) for a mixed DNA and RNA assay (with divided Mag beads) in which a first amount of magnetic particles was added to the sample before forming the lysate and a second amount of magnetic particles was added after lysis, compared to a control assay (without divided Mag beads) in which the magnetic particles were added after lysis. A faster Cp (relative to the control) indicates that fewer amplification cycles were required to detect amplification. Generally, a faster Cp than the control indicates that the nucleic acid added to the "with divided Mag beads" reaction was more concentrated than that obtained when the magnetic particles were added after lysis. Assuming 100% template amplification efficiency, a 1-cycle Cp improvement represents approximately a 2-fold increase in the input concentration of template nucleic acid; a 2-cycle Cp improvement represents approximately a 4-fold increase; a 3-cycle Cp improvement represents approximately an 8-fold increase, etc. (By the general formula, an n-cycle Cp improvement represents approximately 2-fold increase in the input concentration of target cells or template nucleic acid). n(This represents a fold increase). The data in Figure 7A show that the average Cp of the combined DNA and RNA assay (with divided Mag beads) in which a first amount of magnetic particles was added to the sample before forming the lysate and a second amount of magnetic particles was added after lysis was better than the control in which the magnetic particles were added after lysate formation. The Cp improvement demonstrates that the "with divided Mag beads" protocol improved Cp by approximately 0.42 cycles, representing an average increase of approximately 1.34-fold in nucleic acid concentration when magnetic particles were included in the lysis compared to the control in which the magnetic particles were added after lysis was complete. Figures 7B and 7C show the average Cp data for the DNA-dependent assay (Figure 7B) and the RNA-dependent assay (Figure 7C). Figure 7B shows that the DNA-dependent assay showed an improvement of approximately 1 Cp, indicating an overall approximately 2-fold increase in DNA recovery from the lysate when a first amount of magnetic particles was added before forming the lysate and a second amount of magnetic particles was added after lysis. Figure 7C shows that the Cp performance of RNA recovered from magnetic particles present during lysis is nearly equivalent to that of the control. Taken together, these data demonstrate that nucleic acids (i.e., DNA and RNA) recovered when a first amount of magnetic particles is included during lysate formation and a second amount of magnetic particles is added after lysate formation are likely to be more concentrated and of higher quality than nucleic acids recovered when silica-coated magnetic particles are added after lysis. Furthermore, while changes in Cp may not significantly alter the number of cycles required to detect a given nucleic acid, the higher nucleic acid concentrations obtained when magnetic particles are included in the lysate likely mean fewer missed organisms at or near the detection limit. This is reflected in Figures 6A and 6B.

[0113]

[0128] These data are surprising and unexpected, as the inclusion of magnetic silica particles in mechanical lysis has traditionally been unpopular with those skilled in the art. It has long been believed that the presence of silica-coated magnetic particles during mechanical lysis damages the magnetic particles by removing the silica coating from the particles. It was thought that the loss of silica from silica-coated magnetic particles reduces the nucleic acid binding capacity of the magnetic particles and may lead to nucleic acid loss by binding to unrecoverable silica fragments that are removed from the magnetic particles. Nevertheless, mechanical lysis of organisms in the presence of magnetic silica particles (e.g., by bead-beating) appears to result in better nucleic acid binding and recovery. Nucleic acid binding to magnetic silica particles may occur during lysis, resulting in more efficient nucleic acid binding and higher recovery rates. Indeed, at least for DNA, nucleic acid binding and recovery appear to be significantly better. RNA assays are not necessarily aided by the inclusion of magnetic particles in lysis, but they are not adversely affected. Furthermore, the inclusion of magnetic silica particles in lysis appears to improve the quality of the recovered nucleic acids. For example, fewer proteins and other inhibitors may be co-isolated with the nucleic acids, leading to a more concentrated nucleic acid. This is evidenced by the fact that the magnetic silica particles require less washing when they are included in the dissolution.

[0114]

[0129] The differences in washes are shown in Figure 8, which compares the Cp of amplification of nucleic acids recovered from magnetic particles included in lysis with no wash, one wash, and two washes. Samples were identical except for the number of washes. The no-wash condition was clearly at a disadvantage in both DNA and RNA assays compared to the one-wash condition. This is likely due to the presence of PCR inhibitors co-isolated with the nucleic acids in the no-wash condition. The one-wash condition improved Cp in both RNA and DNA assays, suggesting that one wash is sufficient to remove most PCR inhibitors without reducing the amount of nucleic acids that can be recovered from the magnetic particles. The two-wash condition appears slightly better for DNA assays but slightly worse for RNA assays, suggesting that some RNA may have been washed away. In any case, the two-wash condition is superior to the no-wash condition for RNA assays. When the same assays were performed with magnetic particles added after lysis, at least three washes were required to wash the nucleic acids and remove PCR inhibitors, compared to one or two washes performed with magnetic particles added during lysis, after which Cp was observed (data not shown). Without being bound by any 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, which may result in fewer washes being required compared to the slower, lengthy binding process when magnetic particles are only added after lysis is complete.

[0115]

[0130] Typically, magnetic silica particles for nucleic acid recovery are added only after bead-beating for lysis is complete. It has long been believed that the presence of silica-coated magnetic silica particles during manual lysis can damage the magnetic particles by removing the silica coating during lysis. If such damage occurs, nucleic acids subsequently bind to the free silica that is no longer bound to the magnetic core, and subsequent isolation of the magnetic particles prevents recovery of the free silica with bound nucleic acids. The present inventors have observed the opposite phenomenon. Nucleic acid recovery appears to be more efficient when magnetic silica particles are included in the lysis formulation, and the recovered nucleic acids are co-purified with fewer contaminants and are more concentrated than nucleic acids obtained from organisms lysed by conventional methods. It has also been thought that bead-beating nucleic acid-bound magnetic particles can result in excessive shearing and damage to the nucleic acids. If such damage occurs, the nucleic acids may be compromised, reducing their availability for amplification. Therefore, the method described herein is atypical and contrary to conventional teachings in the art. The data presented herein (see, e.g., Figures 6A-8) demonstrate that adding magnetic silica particles to lysis improves nucleic acid recovery from samples, and nucleic acids are likely to co-isolate with fewer PCR inhibitors. The data in Figure 9 demonstrate that bead-beating lysis in the presence of magnetic silica particles does not cause shearing of nucleic acids. This means that in a given assay, unknown nucleic acids may be detected more quickly (e.g., after fewer amplification cycles), or the detection limit for a given cell type may be lower (i.e., the assay may be more sensitive for a given cell type due to enhanced nucleic acid recovery from the cells), or both.

[0116]

[0131] Without being bound by theory, we believe that improved recovery in samples in which bead-beating is performed in the partial or complete presence of magnetic particles improves nucleic acid recovery due to better mixing and binding kinetics between magnetic silica particles, recovery of fewer proteins and other PCR inhibitors, recovery of more nucleic acids, and disruption of magnetic particle aggregates, which provide additional surface area for nucleic acid binding. It is also believed that the mechanical action of bead-beating alters the silica surface, increasing the surface area of individual magnetic silica particles (e.g., by creating microscopic scratches on the magnetic particle surface), thereby increasing the likelihood of positive binding interactions between magnetic silica particles and nucleic acids. Furthermore, when magnetic particles are included in the lysis, the magnetic particles appear to co-isolate fewer PCR inhibitors with nucleic acids due to the shorter contact time between the lysate and the magnetic particles. Again, without being bound by theory, we believe that the fast and rigorous motion during bead-beating may alter how nucleic acids or proteins and inhibitors bind to the magnetic particles, resulting in greater nucleic acid capture and less protein and / or inhibitor capture compared to protocols in which magnetic particles are added only after lysis is complete.

[0117]

[0132] Protocols in which some or all of the magnetic particles are present during mechanical lysis also offer certain speed advantages over traditional protocols in which the magnetic particles are added after bead beating. These advantages include, but are not limited to, breaking down aggregates, meaning fewer aggregates settle to the bottom of the vessel, making separation from the lysed particles faster and easier. Similarly, breaking down aggregates is believed to increase the surface area available for nucleic acid binding on the beads. Furthermore, because some or all of the magnetic particles are present during lysis, these protocols may reduce or eliminate the time spent rehydrating or resuspending the magnetic particles. Furthermore, because the magnetic particles are present during lysis and constantly mixed, their interaction with nucleic acids may increase, potentially resulting in more rapid nucleic acid binding. Lysis in the presence of magnetic particles may also increase the likelihood of nucleic acid binding to a given magnetic particle. On the other hand, when magnetic particles are included in the lysis, the binding kinetics are faster and the interaction time is correspondingly shorter, meaning the magnetic particles may be less likely to collect proteins and other inhibitors of downstream nucleic acid amplification. Furthermore, the lysis solution may simply be heated by friction during mechanical bead-beating, and while this heating may aid in faster diffusion kinetics and more efficient lysis, it may be potentially detrimental to some binding kinetics, causing loosely bound particles to detach. Lysis in the presence of magnetic particles leads to shorter bead-beating times and faster recovery, reducing the chance of the sample overheating and affecting recovery.

[0118]

[0133] The methods described herein may suitably include adding a first amount of magnetic particles before mechanical lysis (e.g., bead beating), followed by adding a second amount of magnetic particles after mechanical lysis. It is believed that some fragments (e.g., double-stranded DNA) may bind tightly to magnetic particles or other silica surfaces, and this binding may persist during the lysis and agitation process. These fragments are believed to preferentially bind to magnetic particles during the lysis and mixing steps. Other nucleic acids, such as single-stranded RNA, tend to bind more loosely and may not survive the agitation process. Adding a second amount of magnetic particles with new nucleic acid binding sites can rapidly and efficiently bind unbound nucleic acids after mechanical agitation. It is also believed that magnetic particles are not completely uniform and may have local physical or chemical properties that favor nucleic acid binding in different locations. When a particular type of binding site becomes saturated, the beads may lose their ability to bind nucleic acids to the remaining binding sites. The new amount of magnetic particles also provides a new infusion of more diverse binding sites for any unbound nucleic acids.

[0119]

[0134] Because the lysis solution may simply heat up from friction during mechanical bead-beating, which can be detrimental, a fresh infusion of cooler magnetic particles followed by air flow through the instrument to cool the solution can enhance the binding kinetics of loosely bound nucleic acid molecules. Thus, adding magnetic particles both before and after mechanical lysis advantageously promotes binding of molecules that are tightly bound to the surface as well as more loosely bound molecules.

[0120]

[0135] The methods described herein may preferably further include capturing the magnetic particles with a magnet and transferring the magnetic particles to another container using a magnet. Alternatively, the lysis solution containing the suspended magnetic particles may be flowed to another container, where the magnetic particles can be extracted from the fluid. The spent lysis solution without the magnetic particles may be flowed back to the lysis container or another waste container. The methods described herein may preferably further include washing the nucleic acid-bound magnetic particles with a wash buffer to remove waste from the lysis solution from the magnetic particles. Preferably, the wash buffer can remove residual lysis solution, contaminants, etc. from the magnetic particles without eluting a significant amount of nucleic acid from the magnetic particles. Exemplary washes may include adding a wash buffer to the magnetic particles, gently agitating the magnetic particles for a period of time (e.g., a few seconds), recapturing the magnetic particles with a magnet, and removing the spent wash buffer from the container or transferring the magnetic particles to another container. Depending on the sample type, washes may not be necessary. Thus, the methods described herein may include zero washes. However, typically, the methods described herein may include one to three washes. Washes performed in the methods described herein may not include one or more of heating the wash buffer and / or magnetic particles before or during the wash, vigorously mixing the magnetic particles and wash buffer, or incubating the magnetic particles and wash buffer for more than 1 second, more than 2 seconds, more than 3 seconds, more than 4 seconds, more than 5 seconds, more than 6 seconds, more than 7 seconds, more than 8 seconds, more than 9 seconds, more than 10 seconds, or any time period therebetween. Preferably, a wash cycle may include incubating the magnetic particles and wash buffer for a period of about 2-3 seconds.

[0121]

[0136] After washing, the methods described herein may preferably include releasing the magnetic particles from the capture magnet (either in the container after the wash buffer has been removed or in a new container), adding an elution buffer to the magnetic particles, mixing the magnetic particles with the elution buffer, recapturing the magnetic particles with the magnet, and isolating the elution buffer from the magnetic particles. Optionally, the elution buffer may be heated before adding it to the magnetic particles. This preheating can occur during the lysis and / or washing steps, so that the elution buffer is already heated at the time of elution. Illustratively, the elution buffer may be heated to a temperature between 35°C and 105°C (e.g., 50°C-100°C) before mixing it with the magnetic particles, depending on the sample type and other factors. The inventors have found that heated elution temperatures and durations can rapidly and efficiently elute RNA / DNA while leaving proteins and inhibitors on the magnetic particles. This is shown, for example, in Figure 10A, which shows the improvement in average Cp for DNA and RNA assays when the preheating temperature of the elution buffer was increased from approximately 35°C to approximately 105°C. As can be seen from Figure 10A, increasing the elution buffer temperature from approximately 60°C to approximately 100°C improved the average Cp by up to approximately 1.5-2.5 Cp units. This represents an approximately 3-5-fold increase in the amount of nucleic acid eluted from the magnetic particles. Figures 10B and 10C show the same trend for both the DNA and RNA assays. The best-fit lines for the DNA and RNA data show that the average Cp improved by approximately 2 Cp units for the DNA assay and approximately 1.5 Cp units for the RNA assay. This represents an approximately 4-fold increase in the amount of DNA and approximately 3-fold increase in the amount of RNA eluted from the magnetic particles.

[0122]

[0137] Although various embodiments discussed herein refer to moving the magnetic particles to a second chamber, it is understood that an alternative embodiment is to retain the magnetic particles in the first chamber and remove unbound lysate from this chamber.

[0123]

[0138] 5A-5E, an exemplary method for cell lysis and nucleic acid recovery is shown. As shown in FIG. 5A, sample and lysis buffer 5002 are combined in vessel 5000 along with lysing particles 5006 and magnetic particles 5004. As shown in FIG. 5B, the contents of vessel 5000 can be bead-beating (as depicted diagrammatically by arrow 5009) for a time sufficient to produce lysate 5008. In one embodiment, bead-beating can be more efficient by compressing the vessel against a bead-beating device with an appropriate amount of pressure. The time required to prepare the lysate can vary depending on factors such as, but not limited to, the sample material being lysed, the amount of sample, the speed or frequency of the bead-beating motor, the pressure applied to the bead beater, and the bead lysing equipment. Reducing air from the chamber (e.g., by drawing a vacuum on the vessel or using pressure on the vessel to remove air from the vessel) and bead-beating under pressure can impart more energy to the sample and reduce bubble formation for more efficient lysis. Illustratively, a pressure-regulating feedback control mechanism and an electronically controlled motor can be used to adjust the system's lysis energy to provide more power and achieve shorter bead lysis times. Times ranging from a few seconds to a few minutes are typical. Using the bead-beating device shown in FIG. 4 and the instruments and pouches of FIGS. 1 and 2, bead-beating times of a few seconds may be sufficient (e.g., 1, 5, 10, 20, 25, 30, 40, 45, 50, 55 seconds to 1 minute). It is preferable to bead-beat with a force and / or intensity that the system can tolerate, but for no longer than necessary to lyse the material in the sample. This not only saves time in sample preparation, but excessive bead-beating times can shear nucleic acids, ultimately reducing sample quality. This is illustrated in FIG. 11, which shows the fragment sizes of human genomic DNA detected with and without 120 seconds of bead-beating. Longer bead-beating times may result in a shorter maximum fragment length and higher concentrations of smaller fragments.

[0124]

[0139] The protocols discussed herein do not significantly shear nucleic acids or degrade sample quality. Generally, difficult-to-lyse organisms, such as Cryptosporidium and yeast, prefer high RPMs and long bead-beating durations, while other organisms, such as E. coli and viruses, prefer low RPMs and short bead-beating durations. The optimal combination of RPM and duration varies by assay. Optimizing RPM and duration for easily lysed organisms may leave difficult-to-lyse organisms unlysed, while optimizing for difficult-to-lyse organisms may shear nucleic acids from easily lysed organisms. In addition to shearing, prolonged bead-beating at high speeds can lead to sample heating and reduced yield. Generally, it is best to optimize to balance the lysis needs of all organisms in the panel. Bead-beating at high speeds (e.g., 10,000–12,000 RPM) for the shortest possible time (e.g., 20–60 seconds) maximizes lysis efficiency and sample quality while significantly reducing sample preparation time and overall time to results.

[0125]

[0140] Returning to FIG. 5C, after bead beating, a second amount of magnetic particles 5010 may be combined with the lysate 5008. The second amount may be substantially the same as the first amount, or may be substantially larger or smaller. Illustratively, the first and second amounts of magnetic particles are substantially the same. Furthermore, the first and second amounts may be the same type of silica-coated magnetic particles, or the thickness of the silica may be varied as desired to bias the recovery of RNA, DNA, or both.

[0126]

[0141] The second quantity of magnetic particles 5010 can be combined with the lysis solution 5008, for example, by stirring (as depicted schematically by arrow 5011). The stirring 5011 can be performed by a bead beater used in the lysis step, although the duration of the stirring may be shorter and less intense than the stirring used to generate the lysis solution. The first quantity of magnetic particles 5004 and the second quantity of magnetic particles 5010 can be incubated in the lysis solution 5008 for a selected period of time (e.g., seconds to minutes) sufficient to capture nucleic acids from the solution. As illustrated in Figures 5D and 5E, the lysis particles 5006 are typically much larger than the magnetic particles 5004 and 5010 and may sediment much faster than the magnetic particles. As illustrated in Figure 5E, a magnet 5015 can be used to recover the magnetic particles 5004 and 5010 from the lysis solution 5008. In one embodiment, the magnetic particles 5004 and 5010 may be preferably left in suspension to facilitate recovery by gentle mixing of the vessel. Because the lysed particles are much larger than the magnetic particles, such gentle mixing preferably keeps the magnetic particles in suspension and facilitates separation of the magnetic and lysed particles (not shown), while potentially promoting settling of the lysed particles. [Example]

[0127] Example 1

[0142] We have found that lysis and nucleic acid recovery are more efficient when lysis is performed in the presence of magnetic silica particles. This is shown, for example, in Figures 6A-7C. Figures 6A and 6B are bar graphs showing the detection rates of organisms at various concentrations in the presence (with BB and MB) or absence (without BB and MB) of silica-coated magnetic particles during bead-beating lysis for a set of assays based on amplification from DNA (Figure 7A) and a set of assays based on amplification from RNA (Figure 7B). Figure 7A shows that when bead-beating is performed in the presence of silica-coated magnetic particles, the detection rates of the DNA-based assays are consistently improved at 1x LOD, 0.1x LOD, and 0.01x LOD. However, the RNA-based assays appear to be nearly equivalent when silica-coated magnetic particles are added after lysis or when bead-beating is performed in the presence of silica-coated magnetic particles.

[0128]

[0143] The data in Figure 7A show that the average Cp of DNA and RNA assays (with divided Mag beads) in which a first amount of magnetic particles was added to the sample before forming the lysate and a second amount of magnetic particles was added after lysis was better than the control in which magnetic particles were added after lysate formation. The Cp improvement demonstrates that the "with divided Mag beads" protocol improved Cp by approximately 0.42 cycles, representing an average increase of approximately 1.34-fold in nucleic acid concentration when magnetic particles were included in the lysis compared to the control in which magnetic particles were added after lysis was complete. Figures 7B and 7C show the average Cp data for the DNA-dependent assay (Figure 7B) and the RNA-dependent assay (Figure 7C). Figure 7B shows that the DNA-dependent assay showed an improvement of approximately 1 Cp, indicating an overall approximately 2-fold increase in DNA recovery from the lysate when a first amount of magnetic particles was added before forming the lysate and a second amount of magnetic particles was added after lysis. Figure 7C shows that the Cp performance of RNA recovered from magnetic particles present during lysis was nearly equivalent to the control. These data demonstrate that nucleic acids (i.e., DNA and RNA) recovered when lysis is performed in the presence of magnetic particles are likely to be more concentrated and of higher quality compared to nucleic acids recovered when silica-coated magnetic particles are added after lysis.

[0129]

[0144] This is surprising and unexpected, because the inclusion of magnetic silica particles in mechanical lysis has traditionally been discouraged by those skilled in the art due to the long-held belief that the presence of silica-coated magnetic particles during mechanical lysis could remove the silica coating from the particles and damage the magnetic particles. It was thought that loss of silica from silica-coated magnetic particles would reduce the nucleic acid binding capacity of the magnetic particles and could potentially result in nucleic acid binding and loss to unrecoverable silica fragments that are removed from the magnetic particles. Nevertheless, mechanical lysis of organisms in the presence of magnetic silica particles (e.g., by bead-beating) can result in nucleic acid binding to the magnetic silica particles during lysis, resulting in more efficient nucleic acid binding and greater recovery. Indeed, nucleic acid binding and recovery appear to be significantly superior, at least for DNA. Furthermore, the inclusion of magnetic silica particles in lysis appears to improve the quality of the recovered nucleic acids.

[0130] Example 2

[0145] Figure 8 compares the Cp values for DNA (-●-) and RNA (-*-) recovered from magnetic particles included in lysis with no wash, one wash, and two washes. Samples were identical except for the number of washes. The no-wash condition was clearly at a disadvantage in both DNA and RNA assays compared with the one-wash condition. This is likely due to the presence of PCR inhibitors co-isolated with the nucleic acids in the no-wash condition. The one-wash condition improved Cp values in both RNA and DNA assays, suggesting that one wash is sufficient to remove most PCR inhibitors without reducing the amount of nucleic acids that can be recovered from the magnetic particles. The two-wash condition appears slightly better for DNA assays but slightly worse for RNA assays, suggesting that some RNA may have been washed away. In any case, the two-wash condition outperforms the no-wash condition in RNA assays. When the same assays were performed with magnetic particles added after lysis, at least three washes were required to wash nucleic acids and remove PCR inhibitors, compared with one or two washes performed with magnetic particles added during lysis, after which Cp values were observed (data not shown).

[0131] Example 3

[0146] Referring now to Figure 11, a bar graph showing time savings for several specific examples of FilmArray sample preparation is shown. Over the course of several years, sample preparation time was reduced from approximately 13 minutes to approximately 2 minutes. This represents a reduction of 11 minutes in sample preparation time. The FilmArray is a state-of-the-art system, and the reduction in 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) again shows time savings at every step. The R / ST time represents the time savings achievable using the methods described in this application. Comparing the RP2.1 sample preparation time to the R / ST sample preparation time, the absolute time difference was 2 minutes 44 seconds, representing a 58% time reduction. As can be seen from Figure 11, time savings were achieved at every sample preparation step, particularly in magnetic particle recovery from the lysate and bead-beating times. Nucleic acid release from cells can be accelerated by combining multiple lysis mechanisms with pressure control and motor feedback mechanisms. Tighter control and reduced variability also allow for shorter buffer times, ensuring robust performance across the most easily lysed and most difficult-to-lyse organisms. By performing multiple parallel steps to increase the kinetics and efficiency of nucleic acid binding to magnetic particles and magnetic particle binding to the magnet, the most dramatic reduction in binding and magnetic bead recovery was achieved, from 7 minutes to 30 seconds. Comparing the time for bead beating and magnetic bead recovery alone between the RP2.1 and R / ST, the reduction was from 2:53 in the RP2.1 to 1:13 in the R / ST, representing a 1:40 absolute time reduction—a 57% time reduction for these two steps alone. Bead beating in the presence of magnetic particles allows for faster nucleic acid binding and faster recovery of nucleic acids from the lysate. Wash time was reduced from 40 seconds to approximately 8 seconds, and elution time was reduced from 1:09 to 22 seconds.

[0132]

[0147] The present invention may be embodied in other specific forms without departing from its spirit or essential characteristics. The described embodiments are to be considered in all respects only as illustrative and not restrictive. The scope of the present invention is, therefore, indicated by the appended claims rather than by the foregoing description. While specific embodiments and details have been included in this specification and accompanying invention disclosure for the purpose of illustrating the invention, it will be apparent to those skilled in the art that various modifications of the methods and apparatus disclosed herein can be made without departing from the scope of the invention as defined by the appended claims. All changes that come within the meaning and range of equivalency of the claims are embraced within their scope.

Claims

1. 1. A method for cell lysis and nucleic acid recovery, comprising: providing a sample vessel with a quantity of lysis particles, a sample suspected of containing one or more target nucleic acids, a lysis buffer, and a first quantity of nucleic acid-binding magnetic particles; placing a sample, a quantity of lysis particles, a lysis buffer, and a first quantity of nucleic acid-binding magnetic particles in a container; agitating a vessel containing lysis particles, a sample, a lysis buffer, and a first amount of nucleic acid-binding magnetic particles for a first period of time to produce a lysis solution; dispersing a second amount of nucleic acid-binding magnetic particles in a lysis solution in the container; recovering a first amount of nucleic acid-binding magnetic particles and a second amount of nucleic acid-binding magnetic particles from the lysate; wherein adding a first amount of nucleic acid-binding magnetic particles followed by a second amount of nucleic acid-binding magnetic particles is selected to enhance recovery of nucleic acids from the sample.

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

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

4. 4. The method of claim 3, wherein the chaotropic agent is a guanidinium salt and the non-ionic detergent is one of Triton X-100, polidocanol (Thesit), Triton X-114, NP-40, Arlasolve 200, Brij O10, octyl β-D-glucopyranoside, saponin, nonaethylene glycol monododecyl ether, and combinations thereof.

5. The method of claim 1, wherein the recovering further comprises one of releasing the first amount of nucleic acid-binding magnetic particles and the second amount of nucleic acid-binding magnetic particles recovered from the lysis solution into another sample container, or removing the lysis solution and the lysis particles from the container and returning the first amount of nucleic acid-binding magnetic particles and the second amount of nucleic acid-binding magnetic particles recovered from the lysis solution to the container.

6. 6. The method of claim 5, further comprising washing the first amount of nucleic acid-binding magnetic particles and the second amount of nucleic acid-binding magnetic particles with a wash buffer, recapturing the first amount of nucleic acid-binding magnetic particles and the second amount of nucleic acid-binding magnetic particles using a magnet, and removing the wash buffer.

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

8. 7. The method of claim 6, further comprising releasing the first quantity of nucleic acid-binding magnetic particles and the second quantity of nucleic acid-binding magnetic particles from the magnet, adding an elution buffer to the first quantity of nucleic acid-binding magnetic particles and the second quantity of nucleic acid-binding magnetic particles, releasing the magnet and mixing the magnetic particles with the elution buffer, recapturing the first quantity of nucleic acid-binding magnetic particles and the second quantity of nucleic acid-binding magnetic particles using the magnet, and transferring the elution buffer to another sample container.

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

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

11. The method of claim 1 , wherein the amount of the second amount of nucleic acid-binding magnetic particles is substantially equal to the amount of the first amount of nucleic acid-binding magnetic particles.

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

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

14. 14. The method of claim 1, wherein the agitating step comprises heating the sample and the dispersing step comprises cooling the sample.

15. 1. A method for cell lysis and nucleic acid recovery, comprising: providing a sample container; combining in a sample vessel a quantity of lysing particles, a first quantity of nucleic acid-binding magnetic particles, a sample suspected of containing one or more target nucleic acids, and a lysis buffer; agitating the lysis particles, the sample, the lysis buffer, and the first amount of nucleic acid-binding magnetic particles in the sample container for a period of time sufficient to generate a lysis solution; Dispersing a second amount of nucleic acid-binding magnetic particles in a lysis solution in the sample vessel; Incubating a first amount of nucleic acid-binding magnetic particles and a second amount of nucleic acid-binding magnetic particles in a lysis solution for a period of time; capturing a first amount of nucleic acid-binding magnetic particles and a second amount of nucleic acid-binding magnetic particles from the lysate using a magnet; wherein adding a first amount of nucleic acid-binding magnetic particles followed by a second amount of nucleic acid-binding magnetic particles is selected to enhance recovery of nucleic acids from the sample.

16. 16. The method of claim 15, wherein the period of incubation of the first amount of nucleic acid-binding magnetic particles and the second amount of nucleic acid-binding magnetic particles in the lysis solution ranges from 1 second to 1 minute.

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

18. 16. The method of claim 15, wherein capturing the first quantity of magnetic particles and the second quantity of magnetic particles from the lysate further comprises transferring the magnetic particles to a second sample vessel and releasing the magnetic particles from the magnet into the second sample vessel, or removing the lysate and lysed particles from the first sample vessel and returning the magnetic particles from the magnet to the first sample vessel.

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

20. 20. The method of claim 15, 18, or 19, further comprising releasing the first amount of nucleic acid-bound magnetic particles and the second amount of nucleic acid-bound magnetic particles from a magnet, adding a wash buffer to the magnetic particles, washing the magnetic particles with the wash buffer, recapturing the magnetic particles using 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 the elution buffer, recapturing the magnetic particles using a magnet, and transferring either the magnetic particles or the elution buffer to another sample container.

21. 21. The method of claim 20, wherein washing does not include one or more of heating the wash buffer and magnetic particles before or during washing, vigorously mixing the magnetic particles and wash buffer, or incubating the magnetic particles and wash buffer for a period of more than 10 seconds.

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

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

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

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

26. 1. A method for cell lysis and nucleic acid recovery, comprising: providing a sample vessel including a plurality of fluidly connected reaction chambers, including a sample lysis chamber, a nucleic acid recovery chamber, and at least a first nucleic acid amplification chamber; combining in a sample lysis chamber a quantity of lysis particles, a first quantity of nucleic acid-binding magnetic particles, a sample suspected of containing one or more target nucleic acids, and a lysis buffer; agitating the lysis particles, the sample, the lysis buffer, and the first amount of nucleic acid-binding magnetic particles in the lysis chamber for a period of time sufficient to produce a lysis solution; mixing a second amount of nucleic acid-binding magnetic particles into the lysis solution; transferring at least a portion of the lysate in which the first amount of nucleic acid-binding magnetic particles and the second amount of nucleic acid-binding magnetic particles are suspended to a nucleic acid recovery chamber; capturing a first amount of nucleic acid-binding magnetic particles and a second amount 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, but not removing the first amount of nucleic acid-binding magnetic particles and the second amount of nucleic acid-binding magnetic particles; Releasing the first amount of nucleic acid-binding magnetic particles and the second amount of nucleic acid-binding magnetic particles from the magnet, and washing the first amount of nucleic acid-binding magnetic particles and the second amount of nucleic acid-binding magnetic particles with a wash buffer in the nucleic acid recovery chamber; recapturing the first amount of nucleic acid-binding magnetic particles and the second amount of nucleic acid-binding magnetic particles using a magnet and removing the wash buffer; releasing the magnet and mixing the first amount of nucleic acid-binding magnetic particles and the second amount of nucleic acid-binding magnetic particles with an elution buffer in the nucleic acid recovery chamber to elute the nucleic acids from the magnetic particles; using a magnet to recapture the first amount of nucleic acid-binding magnetic particles and the second amount of nucleic acid-binding magnetic particles, and transferring the elution buffer to the first nucleic acid amplification chamber; wherein adding a first amount of nucleic acid-binding magnetic particles followed by a second amount of nucleic acid-binding magnetic particles is selected to enhance recovery of nucleic acids from the sample.

27. 27. The method of claim 26, wherein capturing the nucleic acid-bound magnetic particles comprises disposing a magnet adjacent to a nucleic acid recovery chamber and containing the magnetic particles within the chamber.

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

29. 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. 27. The method of claim 26, wherein the second amount of nucleic acid-binding magnetic particles is substantially equal to the first amount of nucleic acid-binding magnetic particles.

31. 27. The method of claim 26, wherein the second quantity of nucleic acid-binding magnetic particles is substantially larger than the first quantity of nucleic acid-binding magnetic particles.

32. 27. The method of claim 26, wherein the second quantity of nucleic acid-binding magnetic particles is substantially smaller than the first quantity of nucleic acid-binding magnetic particles.

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

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

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

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

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

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

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