CN218596391U

CN218596391U - Nucleic acid extraction and purification device based on paramagnetic particle method

Info

Publication number: CN218596391U
Application number: CN202221954191.1U
Authority: CN
Inventors: 吴尧; 张继超; 李秋实
Original assignee: Suzhou Jingrui Biotechnology Co ltd
Current assignee: Suzhou Jingrui Biotechnology Co ltd
Priority date: 2021-07-30
Filing date: 2022-07-27
Publication date: 2023-03-10
Anticipated expiration: 2032-07-27

Abstract

The application discloses nucleic acid extraction purification device based on paramagnetic particle method includes: a reaction zone, a sample zone, a waste zone, and at least one reagent zone, wherein the sample zone, the waste zone, and the reagent zone are independent of each other and are each connected to the reaction zone by a channel, and wherein the device is configured to flow fluid from the sample zone or the reagent zone to the reaction zone and from the reaction zone to the waste zone; the device comprises magnetic particles and reagents for the preparation and recovery of nucleic acids. The nucleic acid extraction and purification device based on the magnetic bead method is simple in structure, simple and convenient to operate, high in nucleic acid extraction efficiency and capable of meeting the requirements of low cost and portability.

Description

Nucleic acid extraction and purification device based on paramagnetic particle method

Technical Field

The application relates to a nucleic acid extraction technology, in particular to a novel magnetic bead method nucleic acid extraction and purification liquid bag reactor.

Background

With the rapid development of gene diagnosis, transgenic food detection, personalized medicine and the like, the current nucleic acid extraction technology cannot meet the requirements of the current biotechnology, and a high-throughput and automatic nucleic acid extraction method is urgently needed. In this context, the magnetic bead method is used for nucleic acid extraction. According to the same principle as a silica gel membrane centrifugal column, the surface of the superparamagnetic nano-particles is modified and surface-modified by a nanotechnology to prepare superparamagnetic silicon oxide nano-magnetic beads. The magnetic beads can be specifically identified and efficiently combined with nucleic acid molecules on a micro interface. By utilizing the superparamagnetism of the silicon oxide nano-microspheres, DNA and RNA can be separated from samples such as blood, animal tissues, food, pathogenic microorganisms and the like under the action of Chaotropic salts (guanidine hydrochloride, guanidine isothiocyanate and the like) and an external magnetic field, and the silicon oxide nano-microspheres can be applied to various fields such as clinical disease diagnosis, transfusion safety, forensic identification, environmental microorganism detection, food safety detection, molecular biology research and the like. The magnetic bead method for extracting nucleic acid has the advantages that the traditional DNA extraction method is incomparable, and is mainly characterized in that (1) the automatic and large-batch operation can be realized, the existing 96-hole automatic nucleic acid extractor can realize the treatment of 96 samples by using the extraction time of one sample, the biological high-flux operation requirement is met, the rapid and timely response can be carried out during the outbreak of infectious diseases, and the characteristic enables the traditional method to look at dust; (2) the operation is simple, the time consumption is short, the whole extraction process only comprises four steps, and most of the extraction process can be completed within 36-40 minutes; (3) the method is safe and nontoxic, toxic reagents such as benzene and chloroform in the traditional method are not used, the harm to experiment operators is reduced to the minimum, and the method completely conforms to the modern environmental protection concept; (4) the specific combination of the magnetic beads and the nucleic acid ensures that the extracted nucleic acid has high purity and high concentration.

The existing magnetic bead method automatic nucleic acid extraction and purification scheme mainly comprises two types, one type is represented by a filmarrray product of biological Merriean company, a thin film liquid bag is simply used as a reactor, all reaction liquid including magnetic beads is added into the liquid bag in subsequent experiments, the reaction liquid moves among different reaction cavities under external extrusion, and the on-off of the reaction cavities is controlled by openable valves. This scheme stability is better, but involves dozens of valve switching in the reaction process, and solution can not prestore in the liquid bag moreover, and consumptive material volume and cost are higher. In another purification scheme, represented by the cobas coat product of roche, all solutions including magnetic beads are pre-encapsulated in small liquid bags, and in operation, lysate washing solution and eluent flow over the magnetic beads in sequence by crushing the liquid bags in sequence. However, in this scheme, the solution passes through the surface of the magnetic beads, so that impurities in the solution are seriously remained, which greatly affects the subsequent amplification reaction and nucleic acid purification effect and limits the system of the subsequent reaction.

SUMMERY OF THE UTILITY MODEL

In view of the above-mentioned shortcomings of the related art, the present application aims to provide a novel capsule reactor for nucleic acid extraction and purification by magnetic bead method, wherein firstly, all reaction solutions are connected to a main reaction chamber containing magnetic beads through independent channels, and after the reaction step is finished, the solution is completely absorbed by a water absorbing material and then the subsequent steps are performed, thereby effectively avoiding the solution residue. And secondly, the solution and the main reaction cavity are separated by a breakable prepackage isolation belt, so that the solution can play an isolation role in transportation and other non-working states, but the prepackage isolation belt is broken under the extrusion of controllable external force, and the solution enters the reaction area to react. All solutions are integrated on the liquid bag, so that the material cost and the volume of the consumable liquid bag are reduced, and the requirement of low cost and portability is met. And thirdly, the external mechanical structure for extruding the pre-stored solution into the main reaction zone is changed into a roller pressing type or a sliding block type from a conventional flat plate extrusion type, the solution is extruded to move in a single direction by controlling the roller or the sliding block, the extrusion device can not only realize extrusion, but also play the role of a one-way valve, the valve in the equipment can be reduced, the equipment cost is saved, and the mechanical structure is simplified.

In one aspect, the present application provides an apparatus comprising: a reaction zone, a sample zone, a waste zone, and at least one reagent zone, the sample zone, waste zone, and reagent zone being independent of each other and connected to the reaction zone by a channel, respectively, the device being configured to flow fluid from the sample zone or reagent zone to the reaction zone, and from the reaction zone to the waste zone; the device comprises magnetic particles and reagents for the preparation and recovery of nucleic acids.

In certain embodiments, wherein the waste zone is filled with and contained within an adsorbent material at the time of device manufacture.

In certain embodiments, wherein a first isolation valve is disposed between the waste zone and the reaction zone.

In certain embodiments, the first isolation valve is a one-way valve that only allows fluid flow from the reaction zone to the waste zone when the first isolation valve is open.

In certain embodiments, wherein the reaction zone is filled with and contained within magnetic particles at the time of device manufacture, the device is sealed by a breakable seal until the device is used by the end user.

In certain embodiments, wherein the reagent zone is filled with a fluid and contained therein at the time of device manufacture, it is sealed by a breakable seal until the device is used by the end user.

In certain embodiments, the reagent zone comprises a lysis buffer zone, a wash buffer zone, and/or an elution buffer zone.

In certain embodiments, the wash buffer zone is a plurality.

In certain embodiments, wherein the sample zone is provided with a sealable port for adding a sample.

In certain embodiments, wherein the sample zone and reagent zone are compressible, when compressed, are substantially free of any fluid.

In certain embodiments, wherein the reaction zone is expandable to facilitate receiving fluid expelled from the sample, quantification, or reagent zone, and compressible so as to be substantially free of any fluid when compressed.

In certain embodiments, wherein a quantification zone is further disposed between the sample zone and the reaction zone, the quantification zone being sized and dimensioned to receive a selected volume of fluid from the sample zone.

In certain embodiments, when the device is present in a dosing zone, which is filled with and contained within magnetic particles at the time of device manufacture, the device is sealed by a breakable seal until the device is used by the end user.

In certain embodiments, the sample flows from the sample zone to the quantification zone, and flows to the reaction zone after quantification in the quantification zone and mixing with the magnetic particles.

In certain embodiments, wherein the reaction zone is provided with a second isolation valve for withdrawing product.

In certain embodiments, wherein the second isolation valve is connected to an external collection device for recovery of the sample.

In certain embodiments, the apparatus further comprises a recovery zone connected to the reaction zone by a second isolation valve.

In certain embodiments, the second isolation valve is a one-way valve that only allows fluid to flow from the reaction zone to the recovery zone when the second isolation valve is open.

In some embodiments, the device comprises a sac formed by two pieces of flexible film materials which are attached together, a frame for supporting the shape of the sac is arranged on the periphery of the sac, and the sac comprises a plurality of hollow cavities and channels for connecting the cavities.

In certain embodiments, the flexible film material comprises PE, PVC, PU, PA or EVA.

In certain embodiments, the sac comprises a reaction area cavity, a waste area cavity, a sample area cavity, and a plurality of reagent area cavities, the reaction area cavity is connected to the waste area cavity, the sample area cavity, and the reagent area cavities, respectively, a first isolation valve is disposed between the reaction area cavity and the waste area cavity, and the reaction area cavity and the sample area cavity are pre-sealed with a breakable seal until the device is used by an end user.

In certain embodiments, wherein the waste zone cavity is filled with and contained within an adsorbent material at the time of device manufacture.

In certain embodiments, wherein the adsorbent material comprises a silica-surfaced porous adsorbent material.

In certain embodiments, wherein the adsorbent material comprises a silica gel sponge or silica-modified filter paper.

In certain embodiments, wherein the reagent zone cavities comprise a lysis buffer zone cavity, an elution buffer zone cavity, and at least one wash buffer zone cavity.

In certain embodiments, wherein the reagent zone cavity is filled with a reagent and contained therein at the time of device manufacture, sealed by a breakable seal until the device is used by the end user.

In certain embodiments, wherein the reaction zone cavity is filled with and contained within magnetic particles at the time of device manufacture until the device is used by an end user.

In certain embodiments, the sac further comprises a quantification region cavity, the sample region cavity, the quantification region cavity, and the reaction region cavity being connected in series, the quantification region cavity being sized and dimensioned to receive a selected volume of sample from the sample region cavity.

In certain embodiments, when present, the dosing zone cavity is filled with and contained within magnetic particles at the time of device manufacture, and sealed by a breakable seal until the device is used by the end user.

In certain embodiments, wherein the sample zone cavity is provided with a sealable port for adding a sample.

In certain embodiments, wherein the reaction area cavity is provided with a second isolation valve for draining the sample.

In certain embodiments, the sac further comprises a recovery area cavity, the reaction area cavity and the recovery area cavity are connected through a second isolation valve, and the recovery area cavity is provided with a sealable outlet for discharging the sample.

In certain embodiments, the apparatus further comprises an external device that mates with the device.

In some embodiments, the external device comprises a compression module comprising: a first extrusion module mated to the reaction zone, and a plurality of second extrusion modules independently mated to the reagent, quantification, or sample zones; wherein actuation of the compression module of the external device with the isolation valve of the apparatus provides directional movement of fluid in the apparatus.

In certain embodiments, the first extrusion module is capable of extruding fluid from the reaction zone into a waste zone or a recovery zone, or out an outlet.

In certain embodiments, the second expression module is capable of expressing fluid in the reagent, quantification, or sample regions into the reaction region while also being capable of acting as a one-way valve to prevent backflow of fluid.

In certain embodiments, the second compression module comprises a sample zone compression module, a quantification zone compression module, or a reagent zone compression module.

In certain embodiments, the reagent zone extrusion module comprises a lysis buffer zone extrusion module, a wash buffer zone extrusion module, or an elution buffer zone extrusion module.

In some embodiments, when a quantification region is present, the sample region and the quantification region can share a second extrusion module, and the fluid can be sequentially extruded from the sample region into the quantification region and then from the quantification region into the reaction region.

In certain embodiments, the second compression module comprises a roll compression module or a slide compression module.

In certain embodiments, the first extrusion module is a flat plate extrusion module or a slide extrusion module.

In certain embodiments, the external device further comprises a magnet module for recovering and separating the magnetic particles in the reaction zone.

In certain embodiments, the magnet module is located below the first extrusion module and above the reaction zone.

In certain embodiments, the external device further comprises a temperature control module for controlling the temperature of the reaction zone.

In certain embodiments, the external device further comprises a shaking module for shaking the reaction zone to generate a lysate from the sample.

In certain embodiments, the external device includes a first sealing module for opening or closing a first isolation valve.

In certain embodiments, the external device comprises a second sealing module for opening or closing a second isolation valve or reaction zone outlet.

In some embodiments, the external device comprises: a first support member and a second support member forming an opening therebetween to receive the sac.

In certain embodiments, wherein the extrusion module, sealing module, magnet module, temperature control module, or vibration module may extend from at least one of the support members.

In certain embodiments, the external device further comprises a control module configured to control the external device.

In another aspect, the present application provides a method for extracting nucleic acid from a sample using the aforementioned device, comprising:

(1) Introducing a sample and a lysis buffer from the sample zone or the lysate buffer zone, respectively, into the reaction zone, generating a lysate in the presence of the magnetic particles, and allowing the nucleic acids to bind to the magnetic particles;

(2) Separating said magnetic particles from said lysate and introducing the lysate from the reaction zone to the waste zone;

(3) Introducing a washing buffer solution into the reaction zone from the washing buffer solution zone, washing the magnetic particles, and then introducing the washing solution into the waste solution zone from the reaction zone;

(4) Introducing an elution buffer from the elution buffer zone into the reaction zone to separate the nucleic acids from the magnetic particles, and then withdrawing the eluate from the reaction zone.

In certain embodiments, further comprising adding a sample to the sample area prior to step (1).

In certain embodiments, wherein step (1) comprises introducing the sample from the sample region into the quantification region, and after mixing the sample and the magnetic particles in the quantification region, introducing the sample and the magnetic particles into the reaction region.

In certain embodiments, wherein step (1) comprises extruding the sample from the sample region into the quantification region using a sample region extrusion module, and extruding the sample and the magnetic particles from the quantification region into the reaction region using a quantification region extrusion module.

In certain embodiments, when the sample region and the quantification region share a second extrusion module, step (1) comprises extruding the sample from the sample region into the quantification region using the second extrusion module, and continuing to extrude the sample and the magnetic particles from the quantification region into the reaction region using the second extrusion module.

In certain embodiments, wherein step (1) comprises adjusting the reaction zone temperature with a temperature control module while generating the lysate.

In certain embodiments, wherein step (1) comprises shaking the sample while generating the lysate.

In certain embodiments, wherein step (2) comprises: and opening the magnet module, separating the magnetic particles from the lysate, opening the first isolation valve, extruding the lysate from the reaction zone into the waste liquid zone by using the first extrusion module, then closing the first isolation valve, closing the magnet module, and resetting the first extrusion module.

In certain embodiments, wherein step (3) comprises: the washing buffer solution is squeezed into the reaction zone from the washing buffer solution zone by the washing buffer solution squeezing module, the magnetic particles are washed, then the magnet module is started, the magnetic particles and the washing solution are separated, the first isolation valve is started, the washing solution is squeezed into the waste solution zone from the reaction zone by the first squeezing module, then the first isolation valve is closed, the magnet module is closed, and the first squeezing module is reset.

In certain embodiments, it further comprises repeating step (3) at least 1 time.

In certain embodiments, wherein step (4) comprises withdrawing the eluent directly from the reaction zone or introducing the eluent from the reaction zone into the recovery zone.

In certain embodiments, wherein said directing the eluent from the reaction zone or directing the eluent from the reaction zone to the recovery zone comprises: and extruding the elution buffer solution into the reaction zone from the eluent buffer zone by using an eluent buffer zone extrusion module, then opening the magnet module, separating the magnetic particles from the eluent, opening a second isolation valve of the reaction zone, extruding the eluent from the reaction zone or into a recovery zone by using the first extrusion module, and closing the second isolation valve.

In certain embodiments, wherein said introducing the eluate from the reaction zone to the recovery zone further comprises: the eluent is led out from the recovery area.

In certain embodiments, wherein said withdrawing eluate from the recovery zone comprises: and opening the sealable port of the recovery area, and extruding the eluent from the recovery area by using the recovery area extrusion module.

In certain embodiments, the method comprises:

(1) Squeezing the sample from the sample area into the reaction area by using a sample area squeezing module, squeezing the lysis buffer from the lysis buffer area into the reaction area by using a lysis buffer area squeezing module, and generating a lysate in the presence of the magnetic particles so as to combine the nucleic acid with the magnetic particles;

(2) Opening a magnet module, separating the magnetic particles from the lysate, opening a first isolation valve, extruding the lysate from the reaction zone into a waste liquid zone by using a first extrusion module, then closing the first isolation valve, closing the magnet module, and lifting the first extrusion module;

(3) Squeezing a washing buffer solution into the reaction zone from the washing buffer solution zone by using a washing solution buffer zone squeezing module, washing the magnetic particles, then opening the magnet module, separating the magnetic particles from the washing solution, opening the first isolation valve, squeezing the washing solution into the waste solution zone from the reaction zone by using the first squeezing module, then closing the first isolation valve, closing the magnet module, and lifting the first squeezing module;

(4) And extruding the elution buffer solution from the lysate buffer area into the reaction area by using an elution buffer solution area extrusion module, then opening the magnet module, separating the magnetic particles from the eluent, opening a sealable port of the reaction area, and extruding the eluent from the reaction area by using the first extrusion module.

Compared with the prior art, the application has at least the following beneficial effects:

1. all reaction solutions are connected with the main reaction cavity containing the magnetic beads through the independent channels, after the reaction step is finished, the subsequent steps are carried out after all the solutions are absorbed through the water absorbing material, and the solution residual condition is effectively avoided.

2. A quantitative area can be designed behind the sample area, and the sample can be quantified for 2 times. Therefore, when a sample is added, the sample can be added not only by a quantitative pipette, such as a dropper, but also by sample adding operation, and the overall use requirement is reduced.

3. The solution and the main reaction cavity are separated by a breakable prepackage isolation belt, which can play the role of isolation in transportation and other non-working states, but the prepackage isolation belt is broken under the extrusion of controllable external force, and the solution enters the reaction area for reaction. All solutions are integrated on the liquid bag, so that the material cost and the volume of the consumable liquid bag are reduced, and the requirement of low cost and portability is met.

4. The outside mechanical structure that solution got into the main reaction zone is prestore in the extrusion, can be changed into roll extrusion formula or slider formula by conventional dull and stereotyped extrusion formula, removes through control running roller/slider and extrudees solution unidirectional movement, and running roller/slider extrusion device not only can realize the extrusion, has also played the effect of check valve simultaneously, utilizes the running roller, can reduce 2 with the valve in the equipment, has saved equipment cost, has simplified mechanical structure.

5. The silica gel sponge is prefabricated in the waste liquid area, so that various waste liquids can be quickly adsorbed, and meanwhile, the silica gel can also adsorb residual nucleic acid in the waste liquids, so that aerosol pollution is avoided to the maximum extent.

6. The nucleic acid purification card box is the whole disposable type, has realized after the sample gets into the card box totally sealed, has not had the material exchange with the external world, after the reaction, after the nucleic acid solution after the purification of collection finishes, other all parts can be directly according to medical waste treatment for a whole disposable part, need not carry out extra disinfection operation.

Drawings

The specific features of the invention to which this application relates are set forth in the appended claims. The features and advantages of the invention to which the present application relates will be better understood by reference to the exemplary embodiments and the accompanying drawings described in detail below. The drawings are briefly described as follows:

FIG. 1 is a schematic diagram showing the structure of a liquid sac reactor for nucleic acid purification according to example 1 of the present application; wherein, 10 is an external collecting pipe, 11 is a liquid bag reactor, 111 is a liquid bag, and 112 is a liquid bag bracket;

FIG. 2 is a schematic plan view showing a liquid sac reactor for nucleic acid purification in example 1 of the present application; wherein 1 is a reaction area cavity, 101 is a first isolation valve, 102 is a second isolation valve, 103 is a breakable seal, 2 is a waste liquid area cavity, 3 is a sample area cavity, 41 is a lysis buffer area cavity, 42 is an elution buffer area cavity, 431 is a cleaning

buffer area cavity

1, and 432 is a cleaning buffer area cavity 2;

FIG. 3 is a schematic diagram showing the construction of a liquid sac reactor and a matching external device for nucleic acid purification in example 1 of the present application; wherein 11 is a liquid bag reactor, and 12 is an external device;

FIG. 4 is a schematic plan view showing a liquid sac reactor and a matching external device for nucleic acid purification in example 1 of the present application; wherein 51-56 are respectively a motor 51-56;

FIG. 5 is a front view showing a liquid sac reactor and a matching external device used for nucleic acid purification in application example 1, in which 11 is the liquid sac reactor, 12 is the external device, 8 is a heating plate, and 9 is a vibration motor.

Fig. 6 is a schematic structural diagram showing a stepping motor and a slider push rod in the external device according to embodiment 1 of the present application; wherein, 5 position stepping motors and 6 position stepping motors are sliding block push rods;

FIG. 7 shows QPCR curves using both liquid bag reactor extraction and manual extraction in example 2 of the present application;

FIG. 8 shows QPCR curves for the liquid bag reactor extraction and manual extraction used in example 2 of the present application.

Detailed Description

Illustrative embodiments are described below with reference to the drawings. Many different forms and embodiments are possible without departing from the spirit and teachings of the disclosure, and therefore the disclosure should not be construed as limited to the illustrative embodiments set forth herein. Rather, these illustrative 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 size and relative sizes of layers and regions may be exaggerated for clarity. Like reference numerals refer to like elements throughout the specification.

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 belongs. It will be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of this application and the relevant art and should not be interpreted in an idealized or overly formal sense unless expressly so defined herein. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of 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.

All publications, patent applications, patents, or other references mentioned herein are incorporated by reference in their entirety. In case of conflict in terminology, the present specification will control.

It should be noted that, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to "a tile" includes one, two or more tiles. Similarly, references to multiple referents should be construed to include a single referent and/or multiple referents unless the content and/or context clearly dictates otherwise. Thus, reference to "tiles" does not necessarily require a plurality of such tiles. Rather, it should be appreciated that no dependency on the word-shape variation of the verb is required; one or more tiles are contemplated herein.

As used throughout this application, the words "may" and "may" are used in an permissive sense (i.e., meaning having the potential to), rather than the mandatory sense (i.e., meaning must). Furthermore, the terms "comprising," "having," "involving," "including," "characterized by," variants thereof (e.g., "comprises," "having," "involves," "including," etc.), and similar terms used herein (including the terms) are intended to be inclusive and/or open-ended, and are intended to have the same meaning as the word "comprising" and variations thereof (e.g., "comprises" and "comprising"), and do not exclude additional, unrecited elements or method steps.

In this application, directional terms and/or any terms, such as "top," "bottom," "left," "right," "upper," "lower," "inner," "outer," "proximal," "distal," "forward," "reverse," and the like, may be used solely to indicate relative direction and/or orientation and may not otherwise limit the scope of the present disclosure.

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

Illustrative embodiments of the present inventive concept are described herein with reference to cross-sectional illustrations that are schematic illustrations of idealized embodiments (and intermediate structures) of the illustrative embodiments. Thus, variations from the shapes of the illustrations as a result, for example, of manufacturing techniques and/or tolerances, are to be expected. Thus, the illustrative embodiments of the present inventive concept should not be construed as limited to the particular shapes of regions illustrated herein but are to include deviations in shapes that result, for example, from manufacturing. Thus, the regions illustrated in the figures are schematic in nature and their shapes are not intended to illustrate the actual shape of a region of a device and are not intended to limit the scope of the illustrative embodiments.

It will be understood that, although the present application may use the terms first, second, etc. to describe various elements, these elements should not be limited by these terms. These terms are only used to distinguish one element from another. Thus, a "first" element may be termed a "second" element without departing from the teachings of the present embodiments.

It should also be understood that various embodiments described herein may be used in conjunction with any other embodiment described or disclosed without departing from the scope of this disclosure. Thus, a product, component, element, device, apparatus, system, method, process, composition, and/or kit according to certain embodiments of the present disclosure may include, consist of, or otherwise include the properties, features, components, elements, steps, and/or the like described in other embodiments disclosed herein (including systems, methods, devices, and/or the like) without departing from the scope of the present disclosure. Thus, references to particular features associated with one embodiment should not be construed as limited to application only in that embodiment.

To facilitate understanding, identical reference numerals have been used, where possible, to designate identical elements that are common to the figures. Further, where possible, like element numbers have been used in the various figures.

In an exemplary embodiment, the material is moved between the cavities by applying pressure (e.g., a compression module) on the sac and the channel. Thus, in embodiments employing pressure, the bag material is illustratively sufficiently flexible to allow the pressure to have the desired effect. The term "flexible" is used herein to describe the physical properties of the material of the sac or cavity. The term "flexible" is defined herein as readily deformable by the pressure levels used herein without cracking, breaking, splitting, etc. For example, thin plastic sheets such as Saran ^TM Packaging and

the bag and the thin metal foil, e.g. aluminum foil, are flexible. However, even in embodiments employing an extrusion module, only certain regions of the cavity and channel need be flexible. For example, it may be that only one side of the cavity and channel need be flexible, and other areas may be made of, or reinforced with, a rigid material. Thus, it should be understood that when the term "flexible bag" or "flexible container" is used, it also includes a portion of the bag or sample container that needs to be flexible.

It is to be understood that terms such as "lyse", "lysing", and "lysate" as used herein are not limited to rupturing cells, but that such terms include the destruction of non-cellular particles such as viruses. In another embodiment, in this embodiment, as well as other embodiments described herein, a paddle whipper using reciprocating or alternating paddles (e.g., the paddle whipper described in PCT/US2017/044333, the entire contents of which are incorporated herein by reference) can be used for lysing.

In this application, the term "about" generally means about, within, about, or about. When the term "about" is used in conjunction with a numerical range, it modifies that range by extending the upper and lower bounds of the numerical value. Generally, the term "about" is used herein to vary values above and below the stated value by a difference 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 will be understood that the particular value forms another embodiment. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint.

In this application, the term "or" generally refers to any one member of a particular list and also includes any combination of members of that list.

In the present application, the terms "sample" and "specimen" are used interchangeably and refer generally to an animal; a tissue or organ from an animal; cells (in a subject, taken directly from the subject, or cells 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 (e.g., polypeptides or nucleic acids) derived from cells, cellular material, or viral material; or a solution containing a non-naturally occurring nucleic acid, which is determined as described herein. A "sample" can also be any bodily fluid or discharge (e.g., without limitation, blood, urine, feces, saliva, tears, bile, or cerebrospinal fluid) that may or may not contain host or pathogen cells, cellular components, or nucleic acids.

In the present application, the term "nucleic acid" generally refers to naturally occurring or synthetic oligonucleotides or polynucleotides, whether DNA or RNA or DNA-RNA hybrids, single or double stranded, sense or antisense, which are capable of hybridizing to complementary nucleic acids by Watson-Crick base pairing. Nucleic acids of the disclosure can also include nucleotide analogs (e.g., brdU) and non-phosphodiester internucleoside linkages (e.g., peptide Nucleic Acids (PNAs) or thiodiester linkages). In particular, nucleic acids may include, but are not limited to, DNA, RNA, cDNA, gDNA, ssDNA, dsDNA, or any combination thereof.

As used herein, the term "temperature control module" refers to a device that heats or removes heat from a sample. Illustrative examples of temperature control elements include, but are not limited to, heaters, coolers, peltier devices, resistive heaters, inductive heaters, electromagnetic heaters, thin film heaters, printing element heaters, positive temperature coefficient heaters, and combinations thereof. The temperature control element may include a plurality of heaters, coolers, peltier elements, etc. In one aspect, a given temperature control element may include more than one type of heater or cooler. For example, an illustrative example of a temperature control element may include a peltier device having a separate resistive heater applied to the top and/or bottom surfaces of the peltier. Although the term "heater" is used throughout the specification, it should be understood that other temperature control elements may be used to regulate the temperature of the sample.

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

In certain embodiments, the first isolation valve is a one-way valve that only allows fluid to flow from the reaction zone to the waste zone when the first isolation valve is open.

In certain embodiments, wherein the reaction zone is filled with and contained within magnetic particles at the time of device manufacture, the sealing is effected by a breakable seal until the device is used by the end user.

In certain embodiments, wherein the reagent zone is filled with fluid and contained within at the time of device manufacture, sealed by a breakable seal until the device is used by the end user.

In certain embodiments, the wash buffer zone is a plurality.

In certain embodiments, wherein the sample zone and reagent zone are compressible, they are substantially free of any fluid when compressed.

In certain embodiments, wherein a quantification region is further disposed between the sample region and the reaction region, the quantification region being sized and dimensioned to receive a selected volume of fluid from the sample region.

For example, the quantification region may be connected to the sample region and the reaction region via channels, respectively, and the device is configured such that fluid flows from the sample region to the quantification region and from the quantification region to the reaction region.

In certain embodiments, when the device is present in a dosing region that is filled with magnetic particles and contained therein at the time of device manufacture, the device is sealed by a breakable seal until the device is used by the end user.

For example, the sample may flow from the sample region to the quantification region, be quantified in the quantification region and mixed with the magnetic particles, and then flow to the reaction region.

For example, the sac may comprise a reaction area cavity, a waste area cavity, a sample area cavity, a lysis buffer area cavity, an elution buffer area cavity, and at least one wash buffer area cavity, the waste area cavity, the sample area cavity, the lysis buffer area cavity, the elution buffer area cavity, and the wash buffer area cavity being unconnected to each other and to the reaction area cavity, respectively; a first isolating valve is arranged between the reaction area cavity and the waste liquid area cavity; the reaction zone cavity and the sample zone cavity are pre-sealed by a breakable seal until the device is used by an end user; the lysis buffer zone cavity, the elution buffer zone cavity and the wash buffer zone cavity are filled with and contained within lysis buffer, elution buffer and wash buffer, respectively, at the time of device manufacture, sealed by a breakable seal until the device is used by the end user.

For example, the sac may comprise a reaction zone cavity, a waste zone cavity, a sample zone cavity, a quantification zone cavity, a lysis buffer zone cavity, an elution buffer zone cavity, and at least one wash buffer zone cavity, the waste zone cavity, quantification zone cavity, lysis buffer zone cavity, elution buffer zone cavity, and wash buffer zone cavity being unconnected to each other and connected to the reaction zone cavity, respectively; a first isolating valve is arranged between the reaction area cavity and the waste liquid area cavity; the sample zone cavity, the quantification zone cavity, and the reaction zone cavity being connected in series, the quantification zone cavity being sized and dimensioned to receive a selected volume of sample from the sample zone cavity, and the quantification zone cavity being filled with and contained within magnetic particles at the time of device manufacture, sealed by a breakable seal, until the device is used by an end user; the lysis buffer zone cavity, the elution buffer zone cavity and the wash buffer zone cavity are filled with and contained within lysis buffer, elution buffer and wash buffer, respectively, at the time of device manufacture, sealed by a breakable seal until the device is used by the end user.

In certain embodiments, wherein the reaction zone cavity is provided with a second isolation valve for venting the sample.

In certain embodiments, the sac further comprises a recovery region cavity, the reaction region cavity and the recovery region cavity are connected through a second isolation valve, and the recovery region cavity is provided with a sealable outlet for discharging the sample.

In certain embodiments, a filter (not shown) is also disposed in the channel between the reaction zone cavity and the waste zone cavity, the choice of filter material depending on the sample type and desired pore size. Typically, the pore size of the filter is selected to be large enough to allow passage of all materials in the liquid except the lysed particles. In one embodiment, the filter has a pore size in the range of about 5 μm to 100 μm (e.g., 50 μm to 90 μm or 7 μm to 12 μm). Preferably, the filter element is made of a material compatible with the material used to form the sac, such that the filter can be heat sealed in the sac without damaging the sac or the filter.

In some embodiments, the apparatus further comprises an external device that is mated to the device.

In certain embodiments, the magnet module is located below the first extrusion module and above the reaction zone. For example, when the first compression module is a slider type compression module, the magnet module may be located below the first compression module and above the reaction zone.

In some embodiments, the magnet module is located above the first extrusion module, for example, when the first extrusion module is a flat plate extrusion module, the magnet module may be located above the first extrusion module.

In certain embodiments, the external device includes a first sealing module for opening or closing a first isolation valve. In some embodiments, the first sealing module may be combined with the first isolation valve as one component, and both located on an external device.

In certain embodiments, the external device comprises a second sealing module for opening or closing a second isolation valve or reaction zone outlet. In some embodiments, the second sealing module may be combined with the second isolation valve as one component, and both located on an external device.

In certain embodiments, wherein the compression module, sealing module, magnet module, temperature control module, or vibration module may extend from at least one of the support members.

In certain embodiments, wherein step (2) comprises: opening the magnet module, separating the magnetic particles from the lysate, opening the first isolation valve, extruding the lysate from the reaction zone into the waste liquid zone by using the first extrusion module, then closing the first isolation valve, closing the magnet module, and resetting the first extrusion module.

In certain embodiments, wherein step (4) comprises withdrawing the eluate directly from the reaction zone or introducing the eluate from the reaction zone into the recovery zone.

In certain embodiments, wherein said directing eluent from the reaction zone directly or directing eluent from the reaction zone to a recovery zone comprises: and extruding the elution buffer solution into the reaction zone from the eluent buffer zone by using an eluent buffer zone extrusion module, then opening the magnet module, separating the magnetic particles from the eluent, opening a second isolation valve of the reaction zone, extruding the eluent from the reaction zone or into a recovery zone by using the first extrusion module, and closing the second isolation valve.

In certain embodiments, wherein said withdrawing the eluate from the recovery zone comprises: and opening the sealable port of the recovery area, and extruding the eluent from the recovery area by using the recovery area extrusion module.

The present application also specifically discloses the following embodiments,

1. an apparatus, comprising: a reaction zone, a sample zone, a waste zone, and at least one reagent zone, the sample zone, waste zone, and reagent zone being independent of each other and connected to the reaction zone by a channel, respectively, the device being configured to flow fluid from the sample zone or reagent zone to the reaction zone, and from the reaction zone to the waste zone; the device comprises magnetic particles and reagents for preparing and recovering nucleic acids.

2. The apparatus of embodiment 1, wherein the waste zone is filled with and contained within an adsorbent material at the time of apparatus manufacture.

3. The apparatus according to any of embodiments 1-2, wherein a first isolation valve is disposed between the waste liquid zone and the reaction zone.

4. The apparatus of embodiment 3, wherein the first isolation valve is a one-way valve that only allows fluid to flow from the reaction zone to the waste zone when the first isolation valve is open.

5. The device according to any of embodiments 1-4, wherein the reaction zone is filled with and contained within magnetic particles at the time of device manufacture, sealed by a breakable seal, until the device is used by the end user.

6. The device according to any of embodiments 1-5, wherein the reagent zone is filled with a fluid and contained therein at the time of device manufacture, sealed by a breakable seal, until the device is used by the end user.

7. The device of any one of embodiments 1-6, the reagent zone comprising a lysis buffer zone, a wash buffer zone, and/or an elution buffer zone.

8. The apparatus according to embodiment 7, wherein the wash buffer zone is a plurality of zones.

9. The device of any of embodiments 1-8, wherein the sample zone is provided with a sealable port for adding a sample.

10. The device of any of embodiments 1-9, wherein the sample and reagent zones are compressible and, when compressed, substantially free of any fluid.

11. The device according to any of embodiments 1-10, wherein the reaction zone is expandable to facilitate receiving fluid expelled from the sample, quantification, or reagent zone and is compressible so as to be substantially free of any fluid when compressed.

12. The device of any of embodiments 1-11, wherein a quantification region is further disposed between the sample region and the reaction region, the quantification region being sized and dimensioned to receive a selected volume of fluid from the sample region.

13. The device of embodiment 12, when present in a dosing region, wherein the dosing region is filled with magnetic particles and contained therein at the time of device manufacture, sealed by a breakable seal until the device is used by an end user.

14. The device of embodiment 13, wherein the sample flows from the sample area to the quantification area, is quantified in the quantification area and mixed with the magnetic particles, and then flows to the reaction area.

15. The apparatus according to any one of embodiments 1-14, wherein the reaction zone is provided with a second isolation valve for withdrawing product.

16. The device of embodiment 15, wherein the second isolation valve is connected to an external collection device for recovering the sample.

17. The apparatus of embodiment 15, further comprising a recovery zone connected to the reaction zone by a second isolation valve.

18. The apparatus of embodiment 17, wherein the second isolation valve is a one-way valve that allows fluid to flow only from the reaction zone to the recovery zone when the second isolation valve is open.

19. The device according to any one of embodiments 1-18, comprising a sac formed by two pieces of flexible film materials, wherein a frame for supporting the shape of the sac is arranged on the periphery of the sac, and the sac comprises a plurality of hollow cavities and channels for connecting the cavities.

20. The device of embodiment 19, wherein the flexible film material comprises PE, PVC, PU, PA, or EVA.

21. The device of any of embodiments 19-20, wherein the sac comprises a reaction area cavity, a waste area cavity, a sample area cavity, and a plurality of reagent area cavities, the reaction area cavity is connected to the waste area cavity, the sample area cavity, and the reagent area cavities, respectively, a first isolation valve is disposed between the reaction area cavity and the waste area cavity, and the reaction area cavity and the sample area cavity are sealed by a breakable seal in advance.

22. The apparatus of embodiment 21 wherein said waste zone cavity is filled with and contained within an adsorbent material at the time of apparatus manufacture.

23. The apparatus of embodiment 22, wherein the adsorbent material comprises a silica-surfaced porous adsorbent material.

24. The apparatus of embodiment 22, wherein the adsorbent material comprises a silica gel sponge or silica-modified filter paper.

25. The device of embodiment 21, wherein the reagent zone cavities comprise a lysis buffer zone cavity, an elution buffer zone cavity, and at least one wash buffer zone cavity.

26. The device of embodiment 25, wherein the reagent zone cavity is filled with a reagent and contained therein at the time of device manufacture and sealed by a breakable seal until the device is used by the end user.

27. The device according to any of embodiments 21-26, wherein the reaction zone cavity is filled with and contained within magnetic particles at the time of device manufacture until the device is used by an end user.

28. The device of embodiments 21-27, wherein the sac further comprises a quantification region cavity, the sample region cavity, the quantification region cavity, and the reaction region cavity being connected in series, the quantification region cavity being sized and dimensioned to receive a selected volume of sample from the sample region cavity.

29. The device of embodiment 28, wherein the dosing zone cavity, when present, is filled with and contained within magnetic particles at the time of device manufacture, sealed by a breakable seal, until the device is used by an end user.

30. The device of embodiment 21, wherein the sample zone cavity is provided with a sealable port for adding a sample.

31. The device of embodiment 21, wherein the reaction zone cavity is provided with a second isolation valve for venting the sample.

32. The device of embodiment 21, the sac further comprising a recovery area cavity, the reaction area cavity and the recovery area cavity connected by a second isolation valve, the recovery area cavity provided with a sealable outlet for draining the sample.

33. The apparatus of any of embodiments 1-32, further comprising an external device mated to the device.

34. The apparatus of embodiment 33, the external device comprising a compression module comprising: a first extrusion module mated to the reaction zone, and a plurality of second extrusion modules independently mated to the reagent, quantification, or sample zones; wherein actuation of the compression module of the external device with the isolation valve of the apparatus provides directional movement of fluid in the apparatus.

35. The apparatus of embodiment 34, wherein the first extrusion module is capable of extruding fluid from the reaction zone into a waste zone or a recovery zone, or out an outlet.

36. The device of any of embodiments 34-35, wherein the second expression module is capable of expressing fluid in a reagent, quantification, or sample region into a reaction region while also being capable of acting as a one-way valve to prevent fluid backflow.

37. The apparatus of embodiments 34-36, the second expression module comprising a sample zone expression module, a dosing zone expression module, or a reagent zone expression module.

38. The device of embodiment 37, wherein the reagent zone compression module comprises a lysis buffer zone compression module, a wash buffer zone compression module, or an elution buffer zone compression module.

39. The device of any of embodiments 34-38, wherein the sample region and the quantification region share a second expression module when present, and fluid can be expressed sequentially from the sample region to the quantification region and from the quantification region to the reaction region.

40. The apparatus of any of embodiments 34-39, the second compression module comprising a roll compression module or a slide compression module.

41. The apparatus of any of embodiments 34-40, the first extrusion module being a flat plate extrusion module or a slide-type extrusion module.

42. The apparatus of any one of embodiments 33-41, the external device further comprising a magnet module for recovering and separating magnetic particles in the reaction zone.

43. The apparatus of embodiment 42, the magnet module is located below the first extrusion module and above the reaction zone.

44. The apparatus of any of embodiments 33-43, the external device further comprising a temperature control module for controlling the temperature of the reaction zone.

45. The apparatus of any one of embodiments 33-44, the external device further comprising a shaking module for shaking the reaction zone to generate a lysate from a sample.

46. The apparatus of embodiment 33, the external device comprising a first sealing module for opening or closing a first isolation valve.

47. The apparatus of embodiment 33, wherein the external device comprises a second sealing module for opening or closing a second isolation valve or a reaction zone outlet.

48. The apparatus of embodiment 33, the external device comprising: a first support member and a second support member forming an opening therebetween that receives the sac.

49. The apparatus of embodiment 48, wherein the compression module, sealing module, magnet module, temperature control module, or vibration module may extend from at least one of the support members.

50. The apparatus of embodiment 33, the external device further comprising a control module configured to control the external device.

51. A method of extracting nucleic acid from a sample using the device of any one of embodiments 1-50, comprising:

52. The method of embodiment 51, further comprising adding a sample to the sample region prior to step (1).

53. The method of embodiment 51, wherein step (1) comprises introducing the sample from the sample region to the quantification region, and after mixing the sample and the magnetic particles in the quantification region, introducing the sample and the magnetic particles to the reaction region.

54. The method of embodiment 51, wherein step (1) comprises extruding the sample from the sample region into the quantification region using a sample region extrusion module, and extruding the sample and the magnetic particles from the quantification region into the reaction region using a quantification region extrusion module.

55. In the method of embodiment 54, wherein the sample region and the quantification region share a second extrusion module, step (1) comprises extruding the sample from the sample region into the quantification region using the second extrusion module, and continuing to extrude the sample and the magnetic particles from the quantification region into the reaction region using the second extrusion module.

56. The method of embodiment 51, wherein the step (1) comprises adjusting the reaction zone temperature with a temperature control module while generating the lysate.

57. The method of embodiment 51, wherein said step (1) comprises shaking said sample while generating said lysate.

58. The method of embodiment 51, wherein the step (2) comprises: opening the magnet module, separating the magnetic particles from the lysate, opening the first isolation valve, extruding the lysate from the reaction zone into the waste liquid zone by using the first extrusion module, then closing the first isolation valve, closing the magnet module, and resetting the first extrusion module.

59. The method of embodiment 51, wherein the step (3) comprises: and squeezing a washing buffer solution into the reaction zone from the washing buffer zone by using a washing solution buffer zone squeezing module, washing the magnetic particles, then opening the magnet module, separating the magnetic particles from the washing solution, opening the first isolation valve, squeezing the washing solution into the waste liquid zone from the reaction zone by using the first squeezing module, then closing the first isolation valve, closing the magnet module, and resetting the first squeezing module.

60. The method of embodiment 59, further comprising repeating step (3) at least 1 time.

61. The method of embodiment 51, wherein step (4) comprises withdrawing eluent directly from the reaction zone or introducing eluent from the reaction zone to the recovery zone.

62. The method of embodiment 61, wherein said withdrawing eluent directly from the reaction zone or withdrawing eluent from the reaction zone to the recovery zone comprises: and extruding the elution buffer solution into the reaction zone from the eluent buffer zone by using an eluent buffer zone extrusion module, then opening the magnet module, separating the magnetic particles from the eluent, opening a second isolation valve of the reaction zone, extruding the eluent from the reaction zone or into a recovery zone by using the first extrusion module, and closing the second isolation valve.

63. The method of embodiment 62, wherein said introducing an eluent from a reaction zone to a recovery zone further comprises: the eluent is led out from the recovery area.

64. The method of embodiment 63, wherein the withdrawing the eluate from the recovery zone comprises: and opening the sealable port of the recovery area, and extruding the eluent out of the recovery area by using the recovery area extrusion module.

65. The method of embodiment 51, comprising:

(1) Squeezing the sample from the sample area to the reaction area by using a sample area squeezing module, squeezing the lysis buffer from the lysis buffer area to the reaction area by using a lysis buffer area squeezing module, and generating a lysate in the presence of the magnetic particles so as to combine the nucleic acid with the magnetic particles;

(3) Squeezing a washing buffer solution into the reaction zone from the washing buffer solution zone by using a washing solution buffer zone squeezing module, washing the magnetic particles, then opening a magnet module, separating the magnetic particles from the washing solution, opening a first isolation valve, squeezing the washing solution into a waste solution zone from the reaction zone by using the first squeezing module, then closing the first isolation valve, closing the magnet module, and lifting the first squeezing module;

(4) And extruding the elution buffer solution from the lysate buffer area into the reaction area by using an elution buffer solution area extrusion module, then opening the magnet module, separating the magnetic particles and the eluent, opening a sealable port of the reaction area, and extruding the eluent from the reaction area by using the first extrusion module.

Example 1

As shown in FIGS. 1-2, this example designs a liquid sac reactor for nucleic acid purification, and the specific embodiment is as follows:

1.1 structural composition

The liquid bag reactor 11 comprises a liquid bag 111 and a liquid bag support 112, wherein the liquid bag 111 is formed by laminating two soft material films, the materials comprise various flexible film materials such as PE, PVC, PU, PA, EVA and the like, and the bag support 112 (such as a plastic frame) is arranged at the periphery and is responsible for supporting the shape of the liquid bag. Illustratively, the film layers of the sac reactor 11 may be formed of flexible plastic films or other flexible materials, similar to the sac 111 depicted in fig. 1. For example, the sac 111 may be made from materials such as, but not limited to, polyester, polyethylene terephthalate (PET), polycarbonate, polypropylene, polymethyl methacrylate, combinations thereof, blends thereof, and laminates thereof by any process known in the art, including extrusion, plasma deposition, and lamination. Other materials may also be used, including metal foils or plastics with aluminum lamination. Other barrier materials that can be sealed together to form bubbles and channels are known in the art. If plastic films are used, the layers may be bonded together, illustratively by heat sealing or laser welding. For example, the material has low nucleic acid binding capacity and low protein binding capacity. If fluorescence detection is used, optionally, a light transmissive material may be used in the appropriate area of the bag.

The liquid sac 111 has a hollow cavity and a reaction channel, and is formed by hot-pressing and attaching a hollow mold, and the liquid sac 111 comprises: a reaction zone cavity 1, which is filled with about 20 μ L of magnetic particles and contained therein at the time of manufacture, provided with a second isolation valve 102, connectable to an external collection device such as a collection tube 10;

a waste zone cavity 2 pre-filled with an adsorbent material during device manufacture, the adsorbent material comprising a silica gel sponge;

a sample zone cavity 3 and a plurality of reagent zone cavities 4, the reagent zone cavities being prefilled with reagent at the time of manufacture and sealed by a breakable seal 103, the breakable seal 103 being openable at an allowable pressure;

the reagent area cavity 4 comprises a lysis buffer area cavity 41, an elution buffer area cavity 42, a washing buffer area cavity 431 and a washing buffer area cavity 432; wherein the lysis buffer zone cavity 41 is pre-filled with about 600 μ L of lysis buffer, the elution buffer zone cavity 42 is pre-filled with about 100 μ L of elution solution, the wash buffer zone cavity 431 is pre-filled with about 1700 μ L of wash solution, and the wash buffer zone cavity 432 is pre-filled with about 2700 μ L of wash solution;

the waste liquid zone cavity 2, the sample zone cavity 3 and the plurality of reagent zone cavities 4 are not connected with each other, and the waste liquid zone cavity 2, the sample zone cavity 3 and the plurality of reagent zone cavities 4 are respectively connected with the reaction zone cavity 1;

wherein the sample area cavity 3 and the plurality of reagent area cavities 4 are connected to the reaction area cavity 1 by a breakable seal 103, wherein the breakable seal 103 is openable upon the sample area cavity 3 and the plurality of reagent area cavities 4 receiving external pressure, and wherein stored fluid, such as sample or reagent, can enter the reaction area cavity 1 through the openable seal;

wherein the reaction zone cavity 1 and the waste zone cavity 2 are connected by a one-way valve 101, when the one-way valve is open, fluid can only flow from the reaction zone cavity 1 to the waste zone cavity 2.

As shown in fig. 3-6, the external device 12 comprises a first support member 121 and a second support member 122, the first support member 121 and the second support member 122 forming an opening 120 for receiving the sac reactor, the stepper motor 5, the slide pusher 6, the electromagnet 7 (not shown) and the heater plate 8 extending from the first support member 121, and the vibrating motor 9 extending from the second support member.

1.2 working procedures

The whole extraction reaction flow is as follows:

a. as shown in fig. 3, after adding a sample in the sac sample chamber by using a pipette, the sac reactor 11 is inserted into the external device 12;

as shown in fig. 4-6, a stepping motor 5 is disposed above each reservoir cavity of the sac, and respectively marked as motor 51, motor 52, motor 53, motor 54, and motor 55, and sequentially corresponds to the wash buffer area cavity 432, the sample area cavity 3, the elution buffer area cavity 42, the wash buffer area cavity 431, and the lysis buffer area cavity 41;

a motor 56 is arranged right above the reaction area cavity 1, the direction of the motor 56 can be vertical to the motors 51-55, a slide block of the stepping motor 5 is connected with a slide block push rod 6 below, slide block push rods 61-65 of the motors 51-55 can extrude the solution in the liquid storage area to enter the reaction area cavity 1, and a slide block push rod 66 of the motor 56 can extrude the solution in the reaction area cavity to enter the waste liquid area cavity 2 or a sample outlet.

Although a slider pusher is discussed herein, it should be understood that other ways of providing pressure to the cavity (or bladder) are contemplated, including various pneumatic brakes, electromechanical actuators, such as linear stepper motors, motorized cams, roller sliders, rigid paddles driven by pneumatic, hydraulic, or electromagnetic forces, rollers, rocker arms, and in some cases, a cock spring. In addition, there are various methods of closing the channel, either reversibly or irreversibly, in addition to applying pressure perpendicular to the channel axis. These include kinking the bag at the channel, heat sealing, rolling actuators, and various physical valves such as butterfly and ball valves that are sealed in the channel. In addition, a small peltier device or other temperature regulator may be placed near the channel and set at a temperature sufficient to freeze the fluid, effectively forming a seal. Moreover, while the design of fig. 4 is suitable for automated instruments having an actuator element located on each bubble and channel, it is also contemplated that the actuators may remain stationary and the sac 11 may be transferred so that a small number of actuators may be used in several processing stations, including those for sample disruption, nucleic acid capture, other applications (e.g., immunoassays and immuno-PCR). While a slider pusher is used in the presently disclosed embodiments, when the term "slider pusher" is used herein, it should be understood that other actuators and other ways of providing pressure may be used, depending on the configuration of the bag and the instrument.

Meanwhile, an electromagnet 7 is arranged below a sliding block push rod 66 of the motor 56 and can adsorb magnetic beads; it should be understood that electromagnets may be used and may be activated and deactivated by controlling the current through the electromagnets. Thus, while this specification discusses withdrawing or retracting a magnet, it should be understood that these terms are broad enough to encompass other ways of withdrawing a magnetic field;

a heating plate 8 is arranged above the reaction area cavity 1, and a vibrating motor 9 is arranged below the reaction area cavity 1 and is respectively used for controlling the temperature of the reaction area and mixing magnetic beads and fluid in the reaction area; it should also be understood that the vibrating motor 9 is merely exemplary and that other devices may be used to grind, shake or vortex the sample. In some embodiments, in addition to or instead of mechanical lysis, chemicals or heat may be used;

b. the motor 52 moves from right to left to extrude the sample from the sample area cavity 3 into the reaction area cavity 1;

c. the motor 55 moves from right to left, and the lysate is extruded into the reaction zone cavity 1 from the lysis buffer zone cavity 41;

d. a vibrating motor 9 positioned below the cavity 1 of the reaction area works to fully mix lysis solution and sample magnetic beads in the reaction area;

e. the electromagnet 7 works to attract and concentrate the magnetic beads in the solution;

f. the slide block push rod 66 moves from top to bottom, the solution in the reaction area cavity 1 is extruded into the waste liquid area cavity 2, and then the solution is reset and the electromagnet 7 is closed;

g. the motor 54 moves from right to left, and the cleaning liquid is squeezed into the reaction zone cavity 1 from the cleaning buffer zone cavity 431;

h. the vibration motor 9 works to fully mix the cleaning liquid in the cavity 1 of the reaction area with the magnetic beads; optionally, the heating plate 8 works to adjust the temperature of the reaction system in the cavity 1 of the reaction zone;

i. the electromagnet 7 works to attract and concentrate the magnetic beads in the solution;

j. the slide block push rod 66 moves from top to bottom, the solution in the reaction area cavity 1 is extruded into the waste liquid area cavity 2, and then the solution is reset and the electromagnet 7 is closed;

k. the motor 51 moves from right to left, and the cleaning liquid is squeezed into the reaction zone cavity 1 from the cleaning buffer zone cavity 432;

l, the vibration motor 9 works to fully mix the cleaning liquid in the cavity 1 of the reaction area with the magnetic beads;

m, the electromagnet 7 works to attract and concentrate magnetic beads in the solution;

n, the slide block push rod 66 moves from top to bottom, the solution in the reaction area cavity 1 is extruded into the waste liquid area cavity 2, and the electromagnet 7 below the slide block push rod 66 is closed;

optionally, the heating sheet 8 works, and after the residual reaction liquid in the reaction area cavity 1 is completely dried, the heating sheet is closed;

p. the motor 53 moves from right to left to extrude the eluent from the cavity 42 of the elution buffer zone into the cavity 1 of the reaction zone;

q. the electromagnet 7 works to attract and concentrate the magnetic beads in the solution;

r, the slide block push rod 66 moves from bottom to top to extrude the solution in the reaction zone into the outlet collection pipe 10;

s. taking the collecting tube 10 off the liquid sac reactor, sealing and storing for later experiments, and discarding the liquid sac reactor as medical waste.

Example 2

2.1 sample Collection and preparation

1mL PBS was added to a 1.5mL centrifuge tube, the oral cavity sample was hung with an oral swab, the swab cotton head was rinsed thoroughly in PBS, and the oral swab cotton head was discarded. Adding 10 μ L of the rinse solution to the solution with the titer of 10 ³ mu.L of packaged adenovirus was used as the sample for extraction.

2.2 nucleic acid extraction:

(1) Automatic extraction of liquid sac:

a200. Mu.L sample was pipetted into the reaction sac and the sac was inserted into the device for automatic extraction. After the extraction is completed, about 50 μ L of sample extract is collected in the collection tube, and two samples are simultaneously extracted for subsequent experiments.

(2) Manual extraction was performed using a commercially available magnetic bead method nucleic acid extraction kit:

a. adding 1mL pbs, scraping the oral swab, putting the oral swab into the pbs solution, soaking for 1min, taking out, adding 10 microliter, and adding ³ mu.L of packaged adenovirus is used as an extraction sample;

b. adding 200 μ L of sample to be extracted (less than 200 μ L of sample can be filled with physiological saline), adding 20 μ L of protease K, slightly swirling or mixing up and down, adding 20 μ L of Magnetic Beads and 600 μ L of lysine Solution, swirling, shaking, mixing for 15sec, and cracking at room temperature for 5min, wherein the period is up and down and mixing twice;

c. performing instantaneous centrifugation, placing the EP tube on a magnetic frame, standing for 1min, and removing the supernatant by using a pipettor;

d. washing: taking the sample off the magnetic frame, adding 700 mu L Washing Buffer 1, uniformly mixing by vortex for 15sec, carrying out instantaneous centrifugation, placing the EP tube on the magnetic frame, standing for 1min, and removing the supernatant;

e. rinsing: taking the sample off a magnetic frame, adding 700 mu L Washing Buffer 2, uniformly mixing by vortex for 15sec, carrying out instantaneous centrifugation, putting an EP tube on the magnetic frame, standing for 1min, and removing a supernatant;

f. placing the magnetic rack again after instantaneous centrifugation, removing residual supernatant, uncovering the magnetic rack, and airing at room temperature for 3-5min until the surfaces of the magnetic beads have no reflection (in order to ensure the purity of nucleic acid, rinse liquid needs to be removed completely, and meanwhile, the final yield is influenced by over-drying (cracking) of the magnetic beads);

g. adding 50 μ L of Elution Buffer, mixing gently for 15sec, standing at room temperature for 3min, shaking and mixing for 2 times;

h. after the sample is instantaneously centrifuged to the bottom of the EP tube, the sample is placed on a magnetic frame again, and after the sample is kept still for 1min, the supernatant is absorbed into a new nucleic-free centrifugal tube (self-prepared) for subsequent detection or is stored at-4 ℃ for waiting for an experiment;

2.3 QPCR comparison of samples:

the extracted samples were subjected to QPCR amplification using a powerup SYBR green master mix from Roche, 4 duplicate wells per sample. A MA-688QPCR instrument of an Rui organism is adopted, a pair of KRAS primers are adopted for amplification, and the result of each extracted nucleic acid is verified.

Reaction Qpcr system (gene kras) was configured:

the reaction conditions were as follows:

the results after extraction were as follows:

	manual extraction	Equipment auto extraction 1	Equipment automatic extraction 2	ntc
					ct value	21.99	23.29	23.48	36.29
ct value	21.51	23.53	23.32	34.08
					ct value	21.93	23.1	23.04	33.28
ct value	21.78	23.12	23.01	33.91
					Mean value of	21.80	23.26	23.21	34.39
Deviation of	0.21	0.20	0.23	1.31
					cv value	0.010	0.009	0.010	0.038

The manually extracted ct value is about 21.8, the automatically extracted ct value of the device is about 22.5, and the extraction result is equivalent. The manual extraction takes about 15 minutes, and the automatic extraction takes about 8 minutes, so that the experimental time can be effectively reduced, and the working efficiency is improved.

Example 3

3.1 sample preparation:

(1) Scraping a mouth swab: preparing 8 tubes of 2mL PBS buffer solution, scraping 20 times at different positions of the oral cavity respectively, and soaking the swabs in 2mL PBS buffer solution for 3 minutes;

(2) Mixing oral liquid: mixing samples of all tubes, and mixing 8 tubes of samples into 1 tube;

(3) Diluting pseudoviruses: mixing V8 (1.5x10) ⁸ copies/. Mu.L) pseudovirus were diluted in V2 gradient with mixed oral fluid. Pseudovirus (packaged virus) v8 (1.5x10) ⁸ μ L) to the following gradient concentrations for use as samples;

3.2 nucleic acid extraction and purification:

dividing 400 μ L sample into two parts, adding one part into sac sample area, automatically extracting on machine, squeezing the extracted nucleic acid solution into new 1.5mL nucleic-free EP tube.

The other part adopts a nucleic acid extraction kit adopting a commercially available paramagnetic particle method, and the operation is carried out according to the following steps:

a. adding 200 microliter of sample to be extracted (less than 200 microliter of sample can be filled with normal saline), 20 microliter of proteinase K,20 microliter of magnetic bead and 500 microliter of lysate into a 1.5mL Nuclear-free EP tube, vortexing, shaking and mixing for 30 seconds, and lysing at 65 ℃ for 2 minutes;

b. performing instantaneous centrifugation, placing an EP tube on a magnetic frame, and removing supernatant by using a pipettor after the liquid is clarified;

c. rinsing: taking the sample off the magnetic frame, adding 700 mu L of rinsing liquid, uniformly mixing by vortex for 30 seconds, instantly centrifuging, putting the EP tube on the magnetic frame, standing for 1 minute, clarifying the liquid, and removing the supernatant;

d. repeating the above steps for 1 time;

e. after instantaneous centrifugation, the magnetic frame is put on again, and residual supernatant is removed completely. And opening the cover and airing at room temperature for 3-5min (or 65 ℃ for 2 min) until the surface of the magnetic bead is not reflected. Note: in order to ensure the purity of nucleic acid, the rinsing liquid is removed completely; also, excessive drying (cracking) of the beads can affect the final yield;

f. adding 50 μ L of the eluate, mixing for 30sec, and standing at room temperature for 2min (or 65 deg.C for 2 min);

g. the solution was centrifuged instantaneously, placed on a magnetic stand, and after the solution was clarified, 45. Mu.L of the supernatant was transferred to a new 1.5mL Nuclear-free EP tube.

3.3QPCR amplification:

and carrying out QPCR amplification on the manually extracted nucleic acid solution and the automatically extracted nucleic acid solution by adopting a pair of primers, verifying each extracted nucleic acid result, and making three multiple holes for each extracted sample.

Reaction Qpcr system (gene kras) was configured:

composition (I)	Volume (mu L)
		PCRmix(360)2X	12.5
Kras-F	1
		Kras-R	1
Kras-Proble	1
		NF water	4.5
Sample(s)	5
		In total	25

qpcr instrument setup program:

3.4 extraction results:

it can be seen that the automatic extraction effect is similar to the manual extraction, and the replacement of the manual nucleic acid extraction can be realized.

The above embodiments are merely illustrative of the principles and utilities of the present application and are not intended to limit the application. Any person skilled in the art can modify or change the above-described embodiments without departing from the spirit and scope of the present application. Accordingly, it is intended that all equivalent modifications or changes which can be made by those skilled in the art without departing from the spirit and technical concepts disclosed in the present application shall be covered by the claims of the present application.

Claims

1. Nucleic acid extraction purification device based on magnetic bead method includes: a reaction zone, a sample zone, a waste zone, and at least one reagent zone, wherein the sample zone, the waste zone, and the reagent zone are independent of each other and are each connected to the reaction zone by a channel, and wherein the device is configured to flow fluid from the sample zone or the reagent zone to the reaction zone and from the reaction zone to the waste zone; the device comprises magnetic particles and reagents for preparing and recovering nucleic acids.

2. The apparatus of claim 1, wherein the waste zone is filled with and contained within an adsorbent material at the time of apparatus manufacture.

3. The device of claim 1, wherein the reaction zone is filled with and contained within magnetic particles at the time of device manufacture, sealed by a breakable seal, until the device is used by an end user.

4. The device of claim 1, wherein the reagent zone is filled with a fluid and contained therein at the time of device manufacture, sealed by a breakable seal, until the device is used by the end user.

5. The device of claim 1, comprising a sac formed by two pieces of flexible film materials, wherein a frame for supporting the shape of the sac is arranged on the periphery of the sac, and the sac comprises a plurality of hollow cavities and channels for connecting the cavities.

6. The apparatus of claim 1, further comprising an external device mated to the apparatus.

7. The apparatus of claim 6, the external device comprising a compression module, the compression module comprising: a first extrusion module mated to the reaction zone, and a plurality of second extrusion modules independently mated to the reagent, quantification, or sample zones; wherein actuation of the compression module of the external device with the isolation valve of the apparatus provides directional movement of fluid in the apparatus.

8. The apparatus of claim 7, the first extrusion module capable of extruding fluid from the reaction zone into a waste zone or a recovery zone, or from an outlet.

9. The device of any one of claims 7-8, wherein the second expression module is capable of expressing fluid in a reagent zone, a quantification zone, or a sample zone into a reaction zone while also being capable of acting as a one-way valve to avoid backflow of fluid.