JP2018537676A - Integrated sample processing system - Google Patents

Abstract

The integrated sample purification system includes a housing, a sample container rack, a filter holder, and a cylindrical magnet. The sample container rack and filter device holder are withdrawn in the housing. The sample container rack is designed to hold one or more sample containers, and the filter device holder is designed to hold one or more filter devices. The cylindrical magnet is located near and outside the sample container rack, and is rotated around the center and the long axis of the magnet by an electric motor disposed in the housing to lyse the cells. The target molecule in the lysed cell is purified using a filter that specifically binds to the target molecule. The system is easily adapted for automation and rapid purification and analysis of molecules of interest such as nucleic acids and proteins. [Selection] Figure 1

Description

  The present invention generally relates to an integrated sample processing system for separating and / or purifying molecules of interest, such as nucleic acids or proteins, from particularly difficult sample matrices and / or difficult to destroy organisms, Furthermore, it relates to an automated suitable method for separating and / or purifying nucleic acids from a sample using magnetically induced vortexing in combination with a solid monolith filter.

  Molecular tests are emerging as the best standard for some diagnostic tests due to their speed, sensitivity and specificity. Self-preparation testing (LDT) is “one of the fastest growing sectors in the in vitro diagnostics (IVD) market. Sample preparation is critical to the validity of the test, but the clinical molecular biology Frequent hindrances to workflow and diagnostic testing, although there are many molecular detection modalities, there are only a few automated sample preparation workflow strategies, and the cost of existing instruments built around these sample preparation strategies and chemistry Is in the range of $ 17,000 to $ 150,000, but nevertheless difficult sample matrices such as raw silkworms and / or destruction such as Gram-positive bacteria and mycobacteria (ie Mycobacterium) Does not provide an integrated method for processing difficult life forms.

  Mycobacteria, including Mycobacterium strains, are typically isolated from sputum of infected patients. The lipid-rich cell membrane is called “acid-fast bacteria” because it is relatively impermeable to various basic dyes unless combined with phenol. The cocoon is thick and viscous and difficult to process. The vast majority of sputum samples to be analyzed contain varying amounts of organism debris and a variety of contaminating, normal or transient flora. Typically, chemical decontamination / treatment is used to reduce viscosity and disinfect contaminating bacteria while allowing for the recovery of mycobacteria. However, due to its unique cell envelope containing mycolic acid and a high content of lipids, the cells tend to be hydrophobic and aggregate together. This makes it impermeable to normal dyes such as Gram stain. In general, two acid-fast bacteriostatic dyes are used: carbole fuchsin and a fluorescent dye such as auramine or auramine-rhodamine. Once stained, the cells resist decolorization with acidified organic solvents and are therefore referred to as “acid-fast bacteria”. However, it retains fuchsin or auramine staining after sequential or simultaneous treatment with acid and alcohol.

  Mycobacterial smear microscopy is insensitive to Mycobacterium species. Microscopic examination sensitivity includes disease prevalence and severity, specimen type, specimen collection quality, number of Mycobacterium cells present in the specimen, processing method (direct method or concentration method), centrifugation method, staining technique, And is influenced by numerous factors such as inspection quality. It is recommended to report negative results for the first time after examining at least 100 fields (low-income countries) and preferably 300 fields (developed countries) (or equivalent fluorescent fields) by immersion microscopy. Therefore, it may take time and effort to correctly perform the microscopic examination.

  Mycobacterium strains are slow growing bacilli and their normal generation time is 12 to 18 hours. Normally, colonies become visible only after an incubation period of 1 to 8 weeks. Samples containing low concentrations of Mycobacterium cells require several more subcultures. When Mycobacterium is cultured in a dedicated medium, it is possible to identify individual Mycobacterium species contained in a biological sample. However, this is time consuming, especially for patients who are still at the beginning of the infection process.

  Nucleic acid hybridization tests have been developed to detect Mycobacterium strains in biological samples. The first test utilized direct probe hybridization. However, the concentration of Mycobacterium cells contained in samples taken from patients is usually too low to produce a positive hybridization signal. Therefore, tests utilizing PCR amplification have been developed. For example, the Gen-Probe® kit marketed as “Amplified® Mycobacterium tuberculosis detection kit” or the MTD test kit (Gen Probe, San Diego 92121, Calif., USA) can be used to amplify MtbC-specific rRNA. After (transcription-mediated amplification), amplicon detection according to the Gen-Probe HPA method (hybridization protection analysis) is used.

  In view of the above limitations, simple and efficient integration of sample homogenization, difficult-to-destruct microbial lysis, and polynucleotide purification to meet both clinical laboratory and user needs alike There is a need for a system.

  In one aspect, the present application provides an integrated sample purification system that includes a housing, a sample container rack, a filter chip rack, and a cylindrical magnet. The sample container rack and the filter chip rack are disposed in the housing. Sample container racks are designed to hold one or more sample containers, and filter tip racks are designed to hold one or more filter tips. The cylindrical magnet is located near and outside the sample container rack, and is driven by an electric motor disposed in the housing to rotate around the center and long axis of the magnet.

  In some embodiments, the housing includes one or more reagent racks that contain one or more reagents.

  In some embodiments, the system includes a plurality of sample containers, a plurality of filter tips, and one or more reagent racks.

  In certain embodiments, the cylindrical magnet has magnetic poles symmetrically disposed along and around the long axis of the magnet. In another embodiment, the cylindrical magnet has a counter magnetic pole that is disposed at the opposite end of the long axis of the magnet. In still other embodiments, the cylindrical magnet is an electromagnet.

  In one embodiment, the one or more sample containers are designed to be sealed and maintain a closed system after introduction of the one or more reagent solutions.

  In one embodiment, the system further includes one reagent rack that is evacuated within the housing, containing reagents stored in wells that are separated and sealed within the rack.

  The sample container, in use, receives a disorderly mixing of the cell material, when the sample container contains the cell material and the cylindrical magnet rotates about its long axis, the magnetic stirrer rotates to stir the beads, Includes magnetic stirrer and multiple beads designed to provide sample homogenization and cell disruption.

  In one embodiment, the beads comprise glass, plastic, ceramic material, mineral, metal or combinations thereof. In a specific embodiment, the beads are silica beads.

  In one embodiment, the beads have a diameter in the range of 10-1000 μm.

  In one embodiment, the magnetic stirrer comprises a metal or alloy. In a specific embodiment, the magnetic stirrer includes stainless steel. In other embodiments, the magnetic stirrer includes an alloy core coated with a polymer. In a specific embodiment, the magnetic stirrer comprises a polymer coated alloy core whereby the alloy core comprises neodymium iron boron or samarium cobalt and / or the polymer is PTFE or parylene.

  In other aspects, the automated nucleic acid purification system is designed to automatically dispense reagents into one or more sample containers in a predetermined manner and to discard sample material and reagents in an automated pipetting system. In combination with one or more robotic arms, the above functions are included. The automated purification system, in use, includes a plurality of sample containers, each containing a stirrer and a plurality of beads, a plurality of filter chips, and one or more reagent racks.

  In other embodiments, a method for generating a target molecule from a sample comprises (a) providing a sample purification system according to the present disclosure; (b) placing the sample in a sample container with a magnetic stirrer and a plurality of beads; c) placing the sample container on the sample container rack; (d) rotating the cylindrical magnet about its long axis, resulting in homogenizing the sample and destroying the cells in the sample to form a cell lysate Rotating the magnetic stirrer enough to stir the beads; (e) flowing at least a portion of the cell lysate through the first opening of the filter chip, resulting in a target in the cell lysate Binding molecules to the filter in the filter chip; (f) unbound portion of cell lysate Through the filter chip through the first opening, wherein the unbound portion passes through the filter at least twice before exiting the filter chip; and (g) elutes through the filter chip first opening. The target molecules bound to the filter are eluted from the first opening of the filter chip by allowing the buffer to flow in and draining the elution buffer from the filter chip through the first opening, before the elution buffer exits the filter chip. Pass through the filter at least twice.

  In some embodiments, the target molecule is a polynucleotide molecule. In one embodiment, the sample includes sputum. In a specific embodiment, the sputum sample is suspected of containing Mycobacterium Tuberculosis (MTB), and the method comprises amplifying the eluted polynucleotide molecule with an MTB-specific primer, and the polynucleotide molecule is MTB It further includes the step of determining whether or not it contains DNA.

  In other embodiments, a method for purifying a target molecule includes an automated pipetting system and automatically dispensing reagents into one or more sample containers in a predetermined manner and discarding sample material and reagents. Use of an automated purification system further comprising one or more robotic arms designed to be In this case, the above steps are repeated for each of a plurality of sample containers using an equivalent number of filter tips in combination with one or more reagent racks.

The detailed description refers to the following drawings:
2 is a flow chart illustrating an embodiment of an integrated method for lysing cells and purifying nucleic acids therefrom. 1 illustrates an exemplary single channel nucleic acid purification system according to one embodiment. A typical magnet placement position relative to the sample lysis chamber is shown. A representative pipette filter tip is shown. FIG. 3 is a schematic diagram illustrating an automated 8-channel nucleic acid purification system according to another embodiment. Fig. 6 shows a disposable transport device according to another embodiment. A typical sequence of steps for MagVor / filter chip purification of cocoon-derived nucleic acids is shown.

  In describing preferred embodiments of the invention, specific terminology is used for the sake of clarity. However, the present invention is not intended to be limited by the specific terms so selected. It should be understood that each specific element includes all technical equivalents that operate in a similar manner to accomplish a similar purpose.

  FIG. 1 is a flowchart showing exemplary steps in an integrated method for lysing cells and purifying molecules of interest such as nucleic acids or proteins therefrom. Method 10 includes placing a sample tube containing a liquid sample suspension, a magnetic stirrer and cell lysis beads in a sample rack near the magnet (step 11); in the presence of a magnetic stirrer and cell lysis beads. Homogenize the sample suspension by rotating the magnet at a speed sufficient to lyse the cells in the sample suspension (step 13); under conditions where the molecule of interest binds to the filter matrix, Flowing the homogenized sample suspension through the filter matrix (step 15); washing the filter matrix (step 17) and eluting the bound molecules of interest from the filter matrix (step 19). In some embodiments, the sample tube is pre-packaged with a magnetic stirrer, and / or cell lysis beads, and / or reagents that promote cell lysis and / or preserve the integrity of the target molecule.

  A liquid sample suspension is a sample suspended in a liquid dissolution medium. Typical samples can include biological samples, environmental samples, or non-natural samples. Typical biological samples can include tissue samples, biological fluid samples, cell samples, fungal samples, protozoan samples, bacterial samples, and viral samples. A tissue sample includes tissue isolated from any animal or plant. Biological samples are blood, umbilical cord blood, plasma, buffy coat, urine, saliva, sputum, NALC-treated sputum, nasopharyngeal swab (NPS), nasopharyngeal aspirate (NPA), gastric aspirate, concentrated sputum extract, cerebrospinal fluid , Buccals, lavage fluids (such as bronchi), pleural effusions, feces, and leucophoresis samples. Cell samples further include tissue from any cell source including cultured cells, fresh or frozen cells, and fixed paraffin embedded (FFPE) tissue. Bacterial samples include, but are not limited to, cultured bacteria, isolated bacteria, and bacteria contained in any of the aforementioned biological samples. Viral samples include, but are not limited to, cultured viruses, isolated viruses, and viruses in any of the aforementioned biological samples. Environmental samples include, but are not limited to, air samples, water samples, soil samples, rock samples, and any other sample obtained from the natural environment. Artifact samples include any sample that does not exist in the natural environment. Examples of “artificial” samples include, but are not limited to, purified or separated materials, culture materials, synthetic materials and any other artificial material.

  The liquid dissolution medium can be isotonic, hypotonic or hypertonic. In some embodiments, the liquid dissolution medium is aqueous. In certain embodiments, the liquid dissolution medium comprises a buffer and / or at least one salt or combination of salts. In some embodiments, the pH of the liquid dissolution medium ranges from about 5 to about 8, from about 6 to about 8, or from about 6.5 to about 8.5. A variety of pH buffers can be used to achieve the desired pH. Suitable buffers are Tris, MES, Bis-Tris, ADA, ACES, PIPES, MOPSO, Bis-Trispropane, BES, MOPS, TES, HEPES, DIPSO, MOBS, TAPSO, HEPPSO, POPSO, TEA, HEPPS, Tricine, Gly -Including, but not limited to, Gly, bicine, and phosphate buffer (especially sodium phosphate or sodium phosphate-potassium). The liquid dissolution medium may comprise about 10 mM to about 100 mM buffer, about 25 mM to about 75 mM buffer, or especially about 40 mM to about 60 mM buffer. The type and amount of buffer used in the liquid medium can vary depending on the application. In some embodiments, the liquid dissolution medium has a pH of about 7.4 that can be achieved using about 50 mM Tris buffer. In some embodiments, the liquid dissolution medium is water.

Eukaryotic cells, prokaryotic cells, and / or viruses can be suspended at any suitable concentration. The sample preferably comprises cells suspended in a liquid medium at a concentration that does not interfere with the movement of the magnetic stirrer. In some embodiments, eukaryotic cells and / or prokaryotic cells are 1 to 1 × 10 ¹⁰ cells / mL, 1 to 1 × 10 ⁵ cells / mL, or especially 1 × 10 ³ to 1 × 10 ⁴ cells / mL. Suspended at concentrations in the range of mL. In some embodiments, virus particles are suspended at a concentration in the range of 1 to 1 × 10 ¹³ cells / mL, 1 to 1 × 10 ¹⁰ cells / mL, or 1 × 10 ⁵ to 1 × 10 ⁷ cells / mL. It becomes cloudy.

  In certain preferred embodiments, the sample is suspected of containing MTB. In one embodiment, the sample is nasopharyngeal aspirate. In other embodiments, the sample is a nasopharyngeal swab.

  As used herein, the term “cell” refers to a eukaryotic cell, prokaryotic cell, virus, endospore or any combination thereof. Therefore, the cells are bacteria, bacterial spores, fungi, virus particles, unicellular eukaryotes (protozoa, yeast, etc.), isolated or aggregated cells derived from multicellular organisms (primary cells, cultured cells, tissues, whole organisms, etc.) ), Or in particular any combination thereof.

  The term “sample” refers to any material that contains or is suspected of containing a target molecule.

  The term “nucleic acid” refers to an individual nucleic acid or a polymerized strand of nucleic acid, including DNA and RNA, or modifications thereof, in particular any length, whether naturally produced or artificially synthesized (including analogs thereof). Refers to naturally-occurring modifications known to have

  The term “sample container” refers to an elongated, generally tubular container or vial for protecting and / or processing a sample to purify nucleic acids, or for receiving reagents with a treated sample. The sample container need not be cylindrical, but may be slightly conical along its entire length or part thereof.

  The term “lyse” with respect to a cell means a disruption of the integrity of at least a portion of the cell to release intracellular components such as nucleic acids and proteins from the disrupted cell.

  The term “homogenize” means to mix or vortex mix (various elements such as stool, tissue, sputum, saliva, etc.) into a uniform mixture.

  The term “closed system” or “closed container” is used to inhibit or prevent the introduction of exogenous or external substances into (or from) a tube or container being processed, whether sealed or not complete. Refers to a tube or container that operates in a fairly closed manner. The components of the closed system or container can be pre-sterilized at the factory before use, sterilized at the site of use, and / or sterilized after each closed system is assembled and closed before use.

  The term “single use disposable” refers to a component that is not reused. That is, it is discarded after completing its intended use, ie, processing or generation of the target sample.

  The term “monolith adsorbent” or “monolith adsorbent material” as used in the present application is a porous three-dimensional adsorbent material having a small pore structure continuously connected to each other in a single piece, which is hard, self-supporting, almost monolith May contain type structure. The monolith is prepared, for example, by casting, sintering, or polymerizing the precursor in a mold of the desired shape. The term “monolith adsorbent” or “monolith adsorbent material” refers to an individual adsorbent particle that is packed into a bed form or embedded in a porous matrix, the final product comprising individual adsorbent particles. Intended to be identified as a collection. Porous monolithic polymers are a new material category developed in recent decades. In contrast to polymers composed of very small beads, a monolith is a single continuous polymer piece prepared using a simple molding process. The term “monolith adsorbent” or “monolith adsorbent material” is intended to be distinguished from an adsorbent fiber or a collection of fibers coated with an adsorbent, such as filter paper or filter paper coated with an adsorbent.

  In one aspect, the present application provides an integrated sample purification system that includes a housing, a sample container rack, a filter chip rack, and a cylindrical magnet. The sample container rack and the filter chip rack are disposed in the housing. Sample container racks are designed to hold one or more sample containers, and filter tip racks are designed to hold one or more filter tips. The cylindrical magnet is located near and outside the sample container rack, and is driven by an electric motor disposed in the housing to rotate around the center and long axis of the magnet.

  FIG. 2 illustrates an exemplary single channel sample purification system 100 according to one embodiment. The system 100 of FIG. 2 includes a housing 104, a sample container rack 108, a filter chip actuator / rack 112, and a cylindrical magnet 116. The sample container rack 108 and the filter chip rack 112 are discharged into the housing 104. Sample container rack or stand 108 holds sample containers 120. The filter tip actuator / rack 112 of FIG. 2 is a filter tip attached to a syringe 176 designed such that the syringe plunger 174 of the syringe 176 moves up and down and sucks and dispenses liquid through the filter tip 124. Designed to hold 124. The plunger 174 is connected to an actuator of the rack 112 that controls the movement of the plunger. The cylindrical magnet 116 is located near and outside the sample container rack 108, and is rotated around the center and long axis of the magnet 116 by the electric motor 130 evacuated in the housing 104.

  Each sample container 120 may include one or more lysis chambers for lysing cells in the sample. Preferably, the sample container 120 (and other system components) is sealed and designed to maintain a closed system before and after the introduction of the sample 144 and / or one or more reagent solutions. Container 120 may be sealed with a lid, cap or cover. Sample container 120 may be made of any suitable material, size and shape. In certain embodiments, the container 120 is made of plastic. Preferably, the inner surface of container 120 is chemically inert. The sample container 120 may have a shape of, for example, a urine collection cup, a microcentrifuge tube (such as an Eppendorf tube), a centrifuge tube, a vial, or a microwell plate. In some embodiments, the container 120 includes a single compartment / chamber for holding cells 180, beads 160, and stirrer 156, as shown in FIG. In some embodiments, a given container 120 includes a plurality of separate compartments / chambers (each capable of holding a mixture of cells 180, beads 160 and magnetic stirrer 156 separated from each other). Well array, etc.). In some embodiments, the sample container 120 is pre-packaged with magnetic stirrers and / or cell lysis beads, as well as chemicals and / or enzymes that promote cell lysis and maintain the biological activity of the target molecule.

  The system 100 may include a plurality of sample containers 120, a plurality of filter tips 124, one or more reagent racks 132, or a combination thereof. Sample container rack 108 is designed to hold a plurality of sample containers 120 and can be mounted on a support surface of housing 104 for simultaneous processing of a plurality of samples 144. Similarly, the filter chip rack 112 is designed to hold a plurality of filter chips 124 and can be mounted on a support surface of the housing 104 for simultaneous processing of a plurality of samples 144. The sample container rack 108 can also be used as a holder for the sample 144 for storage purposes. For example, a plurality of sample containers 120 may be placed in the sample container rack 108 and stored in a refrigerator or freezer prior to analysis.

  Referring now to FIG. 3, in use, the sample container 120 and the cylindrical magnet 116 rotate when the cylindrical magnet 116 rotates about its long axis, the magnetic stirrer 156 in the sample container rotates, and the cell It is designed to agitate beads 160 with sufficient force to cause 180 breaks and homogenization.

  Cylindrical magnet 116 may have numerous magnet geometries or designs. In one embodiment, the magnet has magnetic poles (i.e., N and S poles) that are symmetrically disposed about the magnet's major axis. The magnet has a plurality of, preferably 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, and 24 even counter electrodes arranged alternately around the major axis. Can have. In another embodiment, the cylindrical magnet has a counter magnetic pole that is disposed at the opposite end of the long axis of the magnet. In still other embodiments, the cylindrical magnet is an electromagnet.

  The magnet 116 can rotate about the axis passing through the center of the magnet 116 above, below or beside the sample container 120. In certain embodiments, the sample container 120 is positioned perpendicular to the surface on which the sample container 120 is present, and the magnet 116 has an axis that is also perpendicular to the surface on which the sample container 120 is present. Rotates as the center. In other embodiments, the sample container 120 is disposed perpendicular to the surface on which the sample container 120 is present, and the magnet 116 is centered about an axis parallel to the surface on which the sample container 120 is present. Rotate. In still other embodiments, the sample container 120 is positioned perpendicular to the surface on which the sample container 120 is present, and the magnet 116 forms an angle with the surface on which the sample container 120 is present. Rotate around an axis. The angle is greater than 0 ° but less than 180 °.

  FIG. 3 shows the relative position of the magnet 116 with respect to the sample container 120. The magnet 116 rotates about the axis A, and rotates the magnetic stirrer 156 in the sample container 120 along the axis B parallel to the axis A in the same direction. Although only one axis B is shown in FIG. 3, those skilled in the art will appreciate that the magnetic stirrer 156 can rotate about another B axis parallel to the other A axis shown in the figure. . The rotating magnetic stirrer 156 collides with the beads 160 and lyses the cells 180 being processed. The magnet 116 can be located on the side of, above, below or diagonally above the chamber (or sample container 120 which is positioned perpendicular to the surface 190 of the holder of the sample container 120).

  The sample container 120 and specifically the sample 144, the beads 160 and the magnetic stirrer 156 are located within the operating range of the changing magnetic field. For example, the sample container 120 can be located within the operating range of the rotating magnetic field, for example by placing the container 120 adjacent to or near the cylindrical magnet 116. The fluctuating magnetic field drives the motion of the magnetic stirrer 156, such as rotational motion, reciprocating motion, or a combination thereof, which further drives the motion of the beads 160, cells and liquid media. In some embodiments, the sample suspension 144 is agitated by the magnetic stirrer 156 at a rotational speed and duration sufficient to lyse the cells inside the container 120. The appropriate rotation speed and duration depends on the application and can be determined empirically by one skilled in the art. Generally speaking, sufficient rotation speed to lyse cells is determined by cell type, concentration of sample suspension 144, volume of sample suspension, size and shape of magnetic stirrer 156, cell lysis bead 160 It is determined by factors such as quantity / number, size, shape and hardness, and the size and shape of the sample container 120.

  In certain embodiments, the magnetic stirrer 156 is rotating at 1000-6000 rpm, preferably at about 5000 rpm for 1-600 seconds, preferably about 90-120 seconds. In certain embodiments, the sample container 120 (in the form of a urinalysis cup or tube) is placed in a rack on a magnetic stirrer and stirred at the highest speed setting (> 1,000 rpm). In other embodiments, the sample container 120 is a well of a microplate, such as an ELISA plate. In other embodiments, the sample container 120 is a cylindrical container having a sample inlet and a sample outlet.

  In certain embodiments, the speed of rotation of the magnetic stirrer 156 is increased to increase dissolution efficiency and reduce the time required to achieve dissolution. In certain other embodiments, the speed of rotation is adjusted so that only certain types of cells are lysed. For example, in a sample suspension 144 containing a plurality of types of cells 180, the stirrer 156 rotates at a first speed to lyse a first cell population and then rotates at a second speed to generate a second suspension. Cell populations can be lysed. In other embodiments, the container 120 is connected to a temperature adjustment module that controls the temperature of the sample suspension 144 before, during and / or after the lysis process. In certain embodiments, the temperature of the sample suspension 144 is maintained between 2 ° C and 8 ° C. In some embodiments, the sample suspension 144 is 40 ° C.-80 ° C., 50 ° C.-70 ° C., or about 60 ° C. before, during and / or after the lysis process (such as during rotation of a magnetic stirrer). To be heated.

  The magnetic stirrer 156 may be made of metal or metal alloy. In one embodiment, the magnetic stirrer 156 is made of stainless steel. In other embodiments, the magnetic stirrer 156 is made from an alloy core coated with a chemically inert material such as a polymer, glass or ceramic (such as porcelain). Typical alloy core materials include neodymium iron boron and samarium cobalt. Typical coating polymers include biocompatible polymers such as PTEE and parylene.

  The magnetic stirrer 156 can be of any shape and must be small enough to move, rotate or agitate within the sample container 120 in the sample container 120. The magnetic stirrer 156 can be a rod-shaped, cylindrical, cross-shaped, V-shaped, triangular, rectangular, bowl-shaped or especially a disk-shaped stirrer 156. In some embodiments, the magnetic stirrer 156 has a rectangular shape. In some embodiments, the magnetic stirrer 156 has a bifurcated tuning fork shape. In some embodiments, the magnetic stirrer 156 has a V-like shape. In some embodiments, the magnetic stirrer 156 has a trapezoidal shape. In certain embodiments, the longest dimension of stirrer 156 is slightly smaller than the diameter of the container (eg, about 75-95% of the diameter of the container).

  The cell lysis bead 160 can be any particle-like and / or bead-like structure having a hardness greater than that of the cells. Bead 160 may be made of plastic, glass, ceramic, mineral, metal and / or any other suitable material. In certain embodiments, the beads 160 can be made of a non-magnetic material. The beads 160 may be rotationally symmetric about at least one axis (such as spherical, round, elliptical, oval, egg-shaped, and water droplet-shaped particles). In certain embodiments, the beads 160 have a polyhedral shape. In other embodiments, beads 160 are irregularly shaped particles. In some embodiments, beads 160 are particles with protrusions. The beads 160 may have a diameter of 10 to 1,000 μm, 20 to 400 μm, or especially 50 to 200 μm. The range of the amount of beads 160 added to each dissolution vessel can be about 1 to 10,000 mg, 1 to 1000 mg, 1 to 100 mg, especially 1 to 10 mg.

  After the cells are lysed, the cell lysate is taken into an appropriate filter chip 124, allowing nucleic acids to bind to the filter matrix 126 therein (see FIG. 4). The lysate is typically passed through the filter matrix 126 at least twice before discharging unbound portions from the same end of the filter tip 124. At this point, the nucleic acid bound in the filter chip 124 can be stored in a sealed container for further analysis at another time. Alternatively, the bound nucleic acid can be eluted from the filter chip using a suitable elution buffer, as further described below.

  FIG. 4 shows a typical filter chip. Filter tip 124 includes a porous monolithic filter matrix 126 inserted into pipette tip 127. The monolith binding filter matrix 126 includes a monolith adsorbent or a monolith adsorbent material. Porous monolithic materials bind specifically to nucleic acids, are rigid, self-supporting, and are composed of a nearly monolithic structure. In some embodiments, the porous monolith material does not include additional materials that provide nucleic acid affinity. In some embodiments, the porous monolith material is a glass-based monolith material such as a glass frit. In certain embodiments, the glass frit is a sintered glass frit. The porosity of a porous monolith material such as a glass frit or a sintered glass frit depends on the application. The porous monolithic material should generally have a porosity that allows the desired sample flow rate for a particular application and can retain the desired size range of nucleic acids. In some embodiments, the monolith bonded filter matrix 126 is a glass frit that consists of two portions (126a and 126b) that differ in porosity.

  In some embodiments, the porous monolith material is 2-400 microns, 2-300 microns, 2-220 microns, 2-200 microns, 2-180 microns, 2-160 microns, 2-140 microns, 2-140 120 microns, 2-100 microns, 2-80 microns, 2-60 microns, 2-40 microns, 2-20 microns, 2-16 microns, 2-10 microns, 2-5.5 microns, 4-400 microns, 4-300 microns, 4-220 microns, 4-200 microns, 4-180 microns, 4-160 microns, 4-140 microns, 4-120 microns, 4-100 microns, 4-80 microns, 4-60 microns, 4 to 40 microns, 4 to 20 microns, 4 to 16 microns, 4 to 10 microns, 4 to 5.5 microns, 10 400 microns, 10-300 microns, 10-220 microns, 10-200 microns, 10-180 microns, 10-160 microns, 10-140 microns, 10-120 microns, 10-100 microns, 10-80 microns, 10 60 microns, 10-40 microns, 10-20 microns, 10-16 microns, 16-400 microns, 16-300 microns, 16-220 microns, 16-200 microns, 16-180 microns, 16-160 microns, 16- 140 micron, 16-120 micron, 16-100 micron, 16-80 micron, 16-60 micron, 16-40 micron, 40-400 micron, 40-300 micron, 40-220 micron, 40-200 micron, 40- 180 microns, 40 160 microns, 40-140 microns, 40-120 microns, 40-100 microns, 40-80 microns, 40-60 microns, 100-400 microns, 100-300 microns, 100-220 microns, 100-200 microns, 100- 180 microns, 100-160 microns, 100-140 microns, 100-120 microns, 160-400 microns, 160-300 microns, 160-220 microns, 160-200 microns, 160-180 microns, 200-400 microns, 200- A glass frit or sintered glass frit having a porosity (ie, average pore size) in the range of 300 microns, or 200-220 microns. In other embodiments, the porous monolithic material is a glass frit or sintered glass frit having two portions (126a and 126b) with different porosities. Each portion may have a porosity in the above range (such as a 4-10 micron portion and a 16-40 micron portion, or a 16-40 micron portion and a 100-160 micron portion, etc.).

  In some embodiments, the filter is 1-30 mm, 1-25 mm, 1-20 mm, 1-15 mm, 1-10 mm, 1-8 mm, 1-6 mm, 1-4 mm, 2-30 mm, 2-25 mm, 2-20 mm, 2-15 mm, 2-10 mm, 2-8 mm, 2-6 mm, 2-4 mm, 4-30 mm, 4-25 mm, 4-20 mm, 4-15 mm, 4-10 mm, 4-8 mm, 4- 6mm, 6-30mm, 6-25mm, 6-20mm, 6-15mm, 6-10mm, 6-8mm, 8-30mm, 8-25mm, 8-20mm, 8-15mm, 8-10mm, 10-30mm, 10-25mm, 10-20mm, 10-15mm, 15-30mm, 15-25mm, 15-20mm, 20-30mm, 20-25mm, or 25-30mm range Having a thickness.

  In some embodiments, the porous monolith material may be modified with one or more materials that have an affinity for the molecule of interest, such as a polynucleotide, protein, lipid or polysaccharide. In some embodiments, the porous monolith material may be modified with one or more materials that have an affinity for nucleic acids.

  In some embodiments, the filter is made of a porous glass monolith, a porous glass-ceramic, or a porous monolith polymer. In some embodiments, the porous glass monolith is a sol-gel process as described in US Pat. Nos. 4,810,674 and 4,765,818, which are incorporated herein by reference. Can be used. Porous glass-ceramics can be produced by controlled crystallization of a porous glass monolith. In a preferred embodiment, the porous glass monolith, porous glass-ceramic or porous monolith polymer is coated with any additional material, such as a polynucleotide or antibody, for the purpose of improving its binding affinity with nucleic acids. Or it is not embedded.

  In some preferred embodiments, the filter is made of a porous porous glass frit through which a liquid sample can pass. The porous glass frit is not coated or embedded with any additional material, such as a polynucleotide or antibody, for the purpose of improving its affinity with nucleic acids or other molecules of interest. Suitable substrates for purifying nucleic acids include porous glass frit made by sintered glass formed by grinding the beads with a hot press to form a single monolith structure. The uniform structure of the frit provides predictable liquid flow within the frit and allows the eluent to have a fluid dynamics similar to sample flow. Predictable liquid flow allows high recovery in the elution process.

  The filter matrix 126 is typically disposed on the pipette tip 127, but can also be fitted to columns, syringes or other housings of different volumes and shapes. Liquid solutions can be passed through the filter matrix 126 using manual or automatic pipettes, syringes, syringe pumps, hand held syringes, or other types of automated or manual methods for passing liquids through the filter matrix 126.

  As shown in FIGS. 5A and 5B, the system may further include one or more reagent racks 132 that are disposed within the housing. Reagent rack 132 is designed to hold one or more reagents. The reagent rack 132 may be in the form of a tray into which reagents can be poured when ready for use. In this case, in order to deliver the sample 144 to a plurality of sample wells during processing, the reagent is injected into the tray to facilitate receiving the reagent with a plurality of pipette tips. Alternatively, the reagent rack 132 may be in the form of a block or multi-well plate (24-well, 96-well, etc.) that includes a plurality of wells 152, whereby each of the plurality of wells processes a separate sample 144. In order to hold any of the reagents. In certain embodiments, well 152 can be pre-filled with reagents and sealed in rack 132. In some embodiments, the rack 132 is located near the sample container rack 108 and / or the filter tip rack 112 (FIG. 5B).

  The sample purification system 100 may be operated manually or may be designed to run in a semi-automated or fully automated state with programmable logic. In certain embodiments, the system automatically dispenses reagents from one or more reagent racks 132 to a plurality of sample containers 120 and automatically samples by an automated pipetting system 136 (FIG. 5A) and a predetermined computer controlled manner. Designed to dispose of materials and spent reagents in a suitable disposal receptacle 172 (FIG. 5B), it further includes one or more robotic arms (not shown).

  In one mode of operation, reagent rack 132 is in the form of a multi-well plate (24-well, 96-well, etc.). Preferably, the automated liquid handling reduces the amount of work that needs to be done to prepare the mixture to be tested, so that the mixture is used to mix. Automatic sampling protocols can also be implemented by robotics using equipment and methods known in the art.

  Any suitable instrument or device can be used to move the sample 144 through the automated purification system 100 and its various processing steps. For example, the system 100 used in the present application can automate the movement of samples 144, reagents and other system components using a variety of robotics known in the art. A typical robotics system has the ability to move a sample on one, two or three axes and / or rotate about one, two or three axes. Typical robotics move on a track that can be located above, below or beside the workpiece. Robotics components typically include functional components such as robotic arms that can grip and / or move a workpiece, insert pipettes, dispense reagents, aspirate, and the like. As used herein, a “robotic arm” is a sample 144, container 120, filter chip 124, sample container rack 108, filter chip rack 112, and reagent rack 132 that are preferably controlled by a microprocessor from one location to another. Means a device that moves physically. Each location can be a unit in the automated purification system 100. Software for control of the robotic arm is generally available from the arm manufacturer.

  The robotics may translate, for example, on tracks that are above, below or beside the work area and / or may include joints that allow the arms to reach different positions in the work area. The robotics can be driven by motors known in the art, which can be powered, for example, by electricity, air pressure or water pressure. The robotics can be controlled using any suitable drive control system, such as standard PLC programming or other art known methods. Optionally, the robotics includes a position feedback system that measures position and / or force optically or mechanically and allows the robot to be guided to the desired position. Optionally, the robotics also includes a positioning mechanism such as a mechanical stop, optical marker or laser guidance that allows a specific position to be taken repeatedly.

  Typical automatic sampling protocols utilize, for example, Eppendorf epMotion 5070, epMotion 5075, Hamilton STARlet, STAR and STARplus liquid material handling robots. Such protocols are adapted for RNA isolation, isolation of genomic DNA from whole blood, tissue, saliva, swabs, as well as extraction of cell-free DNA such as circulating tumor DNA and circulating fetal DNA and enrichment from maternal plasma. sell.

(Method for purifying nucleic acid)
In other embodiments, a method for purifying nucleic acid from a sample includes: (a) providing a nucleic acid purification system according to the present disclosure; (b) introducing the sample, a magnetic stirrer, and a plurality of beads into a sample container. (C) rotating the cylindrical magnet about its long axis, so that the magnetic stirrer rotates enough to homogenize the sample and destroy the cells in the sample to form a cell lysate; And the beads are agitated and subjected to random mixing of the cell contents; (d) at least a portion of the cell lysate is flowed through the first opening of the filter chip, thereby filtering the nucleic acid in the cell lysate Binding to a filter in the chip; (e) a first opening of the unbound portion of the cell lysate from the filter chip Passing through the filter at least twice before the unbound portion exits the filter tip; and (f) allowing the elution buffer to flow from the first opening of the filter tip and allowing the elution buffer to pass through the filter tip. Elution of the nucleic acid bound to the filter by discharging it through the first opening, wherein the elution buffer passes through the filter at least twice before leaving the filter chip.

  Any manner of performing the methods described herein can be used, including fully manual, semi-automated, or fully automated protocols. However, the characteristics, suitability, simplicity, and workflow of the filter tip can be easily adapted, automated, and enabled for numerous clinical sample matrices, input sample volumes, and liquid handling systems. Thus, in a preferred embodiment, the mode of operation includes some sort of automation. In one embodiment, a method for purifying a nucleic acid includes an automated pipetting system, automatically dispensing reagents into one or more sample containers in a predetermined manner, and suitable sample materials and reagents. Includes one or more robotic arms designed to be disposed of in a disposable receptacle. In this case, the above steps are repeated for each of a plurality of sample containers using an equivalent number of filter tips in combination with one or more reagent racks.

  Samples suspected of containing MTB present a potential risk to the user. Thus, the sample can be pretreated to reduce this risk by heating and / or containing reagents suitable to inactivate microorganisms present in the sample. Inactivation of microorganisms such as MTB includes heating to denature active protein (90 ° C., 5 minutes, etc.), enzymatic digestion of cell wall structure, mechanical destruction to physically disrupt or inactivate cells, chemical treatment or its Can be accomplished by a combination.

  Chemical inactivation offers the potential to reduce or eliminate the need for heating. When processing samples for culture purposes, digest the sputum using a simple reagent to disinfect the sample. For culture, it is important to inactivate other bacterial flora present in the sputum sample so that MTB can grow without overtaking other faster growing bacteria. The decontamination or inactivation step may use reagents such as sodium hydroxide (such as 3-5%) or cetylpyridinium chloride and preferably inactivate all other bacteria, but the MTB cells are thicker Selected to remain intact and viable due to the robust cell wall. However, depending on the method used, 20-90% of MTB cells may be killed during this treatment. However, with respect to nucleic acid purification, MTB cells need not be alive or intact as long as the cellular genomic DNA can still be amplified.

Preferably, the inactivation reagent is selected to allow limited sample dilution and / or low pH for compatibility with binding to silica. These reagents can be added to the sample at the time of collection. In some embodiments, alcohols such as hydrogen peroxide, ethanol and o-phenylphenol (0.2-0.5%) may be used as the main active ingredients. Hydrogen peroxide (H ₂ O ₂ ) can be used as a chemical sterilant from 6-25% concentration and is very stable in solution. H ₂ O ₂ is active at low pH when mixed with 0.85% phosphoric acid. Ethanol alone (such as 95%) can inactivate MTB in sputum or water in 15 seconds. The agricultural fungicide o-phenylphenol is used at 0.1-0.41% in ethanol or isopropanol in PHENO-CEN, SRAYPAK, and CLIPPERCIDE spray disinfectants. Furthermore, o-phenylphenol may be used for 15 minutes at room temperature as a low reagent to sample ratio and in Low pH Phenolic 256 (50% -100%) ethanol or isopropanol or 6.65% 2-benzyl-4 -Can be used in combination with chlorophenol.

  The volume ratio of inactivation reagent to sample volume typically ranges from about 0.1: 1 to 3: 1.

  Inactivation of microorganisms in the primary specimen container is important to provide a BSL-1 compatible workflow (ie, the workflow does not require a biosafety cabinet). Many protocols involve sample transfer prior to disinfection, which is an action that can generate an aerosol and infect a user. Therefore, adapting to BSL-1 requires careful attention to sample movement prior to disinfection, particularly movement that can generate aerosols and infect users.

  Depending on the nature of the sample, the sample can first be liquefied to reduce its viscosity and non-uniformity for consistent sample processing. The sputum sample is particularly difficult. MTB in sputum is one of the most difficult cell and sample types to handle due to the lipid-rich hydrophobic cell walls of mycobacteria and the viscous and heterogeneous nature of sputum. Standard extraction methods for sputum typically include precipitation, often involving centrifugation, decanting and resuspension after treatment with N-acetyl-L cysteine (NALC) and sodium hydroxide (NaOH). Begin with the process. Thus, when processing a highly viscous sample such as cocoons, the sample can be subjected to chemical treatment to reduce the viscosity so that subsequent processing steps (MagVor) are not inhibited. Typical mucolytic reagents for addition to samples include NALC, Zephyran-Trisodium Phosphate (Z-TSP), Benzalkonium, and specialized formulations that lyse bacteria to stabilize RNA and DNA Primestore (R) (Longhorn Vaccines & Diagnostics, San Antonio, TX), San Antonio TX). In one embodiment, liquefaction of the sample by chemical treatment with a mucolytic agent is performed at 60 ° C. for 20 minutes. Patient sputum samples are typically taken in volumes between 1-10 mL, 5-10 mL or more.

The soot has a viscosity in the range of about 100 to 6,000 cP (mPa) at a shear rate of 90 s ⁻¹ . The viscosity measured in mPa is calculated by dividing the shear strength by the shear rate. Preferably, the sample is liquefied to reduce the viscosity of the wrinkles by at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or at least 99%.

  When processing a sample, there is at least one magnetic stirrer and a plurality of cell lysis beads in the sample container. The user simply places the sample suspension in the sample container, places the sample container in the vicinity of the cylindrical magnet, and rotates the magnetic field in the sample container in a manner sufficient to homogenize and lyse the cells. The sample suspension can be agitated by rotating the stirrer and rotating the magnet at a speed sufficient to agitate the cell lysis beads.

  The sample suspension, cell lysis beads and magnetic stirrer can be placed in the sample container in any order. In some embodiments, the sample suspension is added to the sample container before the cell lysis beads and magnetic stirrer. In other embodiments, the cell lysis beads and / or magnetic stirrer are placed in a sample container prior to sample collection.

  In certain embodiments, lysis of certain types of cells can be facilitated by adding additives to the sample suspension before and / or during the agitation step. Examples of additives include enzymes, detergents, surfactants and other chemicals such as bases and acids. For certain types of cells, it has been confirmed that the dissolution efficiency during stirring can be increased by alkaline conditions (10 mM NaOH). Again or alternatively, the sample suspension may be heated during agitation to increase dissolution efficiency. However, additives should be excluded where possible to simplify the process because they can be detrimental to downstream steps including nucleic acid amplification and detection.

  The stirrer / bead combination provides a number of advantages over conventional dissolution methods. The stirrer / bead method is much faster than chemical and enzymatic methods and provides improved cell or viral lysis compared to many other types of physical lysis methods. The stirrer / bead method is also amenable to automation using robotics and / or microfluidic techniques. Cylindrical magnets can be reused and do not need to be precisely placed with respect to the container, and more than one container can be driven. Magnetic stirrers are low in cost and can be used in a single use disposable.

  After the MagVor step, an appropriate binding buffer containing one or more chaotropic agents is added to the sample container to facilitate binding of the nucleic acid to the filter matrix 126. Boom chemistry, or chaotropic binding of nucleic acid and silica, is most efficient when the pH of the solution is below 7. In this case, a high ionic strength solution containing lithium chloride or sodium chloride or guanidine base ions is typically combined with an aliphatic alcohol such as ethanol or isopropanol to “salt out” the DNA and promote nucleic acid binding, respectively. To do. When added to the treated sample, an appropriate binding buffer is used at a constant concentration so that the volume produced is within the filter chip volume range. This reduces the number of suction and dispensing cycles, thus reducing the total processing time.

  In certain embodiments, binding buffer is added to the sample after MagVor and incubated at 60 ° C. for 10 minutes. In other embodiments, a chaotropic agent and an aliphatic alcohol are included in the liquefaction step prior to the MagFor step. In other embodiments, the inactivation, homogenization and lysis steps are performed in a single step as short as 15 minutes.

  Typical chaotropic agents are guanidinium thiocyanate, guanidine isothiocyanate, guanidine hydrochloride, guanidinium chloride urea, thiourea, sodium dodecyl sulfate (SDS), cetylpyridinium chloride, sodium chloride, lithium chloride, potassium chloride, sodium perchlorate, perchlorate. Including but not limited to chaotropic salts such as lithium chlorate, sodium iodide, and potassium iodide; aliphatic alcohols such as butanol, ethanol, propanol and isopropanol; phenol and other phenolic compounds.

  In some embodiments, an aliphatic alcohol such as isopropanol is present between about 0% and about 10%, preferably about 4%, to facilitate selective binding of high molecular weight (HMW) nucleic acids to the filter matrix. To about 6% (optimum = 4.7%), and a chaotropic salt such as guanidine isothiocyanate and / or guanidine hydrochloride, from 1.0 M to 4.0 M, preferably from about 3.0 M to about Provide in the range between 4.0M.

  In some embodiments, fatty alcohols such as isopropanol are present between about 10% and about 25%, preferably about 15 to facilitate binding (and enrichment) of low molecular weight (LMW) nucleic acids to the filter matrix. % To about 20% (optimum = 17.7%).

  In other embodiments, fatty alcohols such as ethanol are used between about 20% and about 60%, preferably between about 30% and about 50% (optimal =) to facilitate the separation of MTB DNA from sputum. 44%).

  After the inactivation and homogenization steps described above, the pH of the solution must be adjusted as necessary to achieve a pH below 7. If the pH is above 7, the solution can be neutralized with a weak acid such as potassium acetate or sodium phosphate. This is necessary when using NaOH or o-phenylphenol having a pH ≧ 11. Low pH phenol and hydrogen peroxide reagents are acidic in nature and most likely do not require additional buffers.

  In one embodiment, binding of the nucleic acid to the filter matrix may be accomplished by attaching the filter chip 124 to it via a luer lock connection with the syringe 176. In other embodiments, the filter matrix is in the syringe 176. A typical filter chip 124 is shown in FIG. The filter chip 124 includes a porous silica matrix 126 embedded within the chip body 127, an aerosol filter 128 that prevents contamination and exposure to the user, and a chip cap 129 useful for maintaining a closed system. In one embodiment, the cap 129 is connected to the filter tip 124 with an insert. In some embodiments, the cap is a regular falcon tube cap 208. The filter tip 124 is designed so that a liquid sample can flow through the matrix for each suction and dispensing cycle of the filter tip 124. Using a syringe 176 or other suitable device, the cell lysate in the container 120 is passed up through the distal end of the filter tip 124 so that the nucleic acids in the cell lysate are in the filter matrix in the pipette tip 127. 126. Typically, the cell lysate is lifted and lowered through the filter matrix 126 so that the unbound portion is discharged through the distal end of the pipette tip 127 to the appropriate waste receptacle 172. The lysate and unbound portions pass through the filter matrix 126 at least twice.

  The combination of the sample and the chaotropic reagent dehydrates DNA and silica and promotes adsorption to the porous silica matrix 126. Thereafter, impurities are removed from the matrix 126 by washing buffer aspiration and dispensing cycles (2-3 times). At this point, the filter chip 124 containing the nucleic acid bound to the filter matrix 126 can be stored in the sealed container 120 for further analysis at another time. Alternatively, the bound nucleic acid can be eluted from the filter chip using a suitable elution buffer, as further described below.

  Nucleic acids have been shown to be very stable on solid supports containing silica, especially when stored under dehydrating conditions without the use of other stabilizers. Accordingly, the present application, in another aspect, includes a luer lock adapter 204 attached to the upper surface of the tube cap 208 so that the filter tip 124 contacts the lower surface of the cap 208 of a suitable holding tube 212 (such as a 50 mL conical tube). Means are provided for stabilizing purified nucleic acids for transport in the form of a single use disposable transport device 200 (FIG. 6).

  As shown in FIG. 2, the filter chip 124 attached to the cap 208 is removed from the holding tube 212 and attached to the syringe 176 before use (ie, elution of the nucleic acid for analysis). In this case, the user can easily insert and remove the filter tip 124 via the luer lock adapter 204 while supporting on the tube cap 208, so that the holding tube 212 contaminates the user and the filter tip 124. Shield from. When this continuous operation is completed, the porous silica filter matrix 126 to which the nucleic acid is bonded is dried, and the capped filter chip 124 is screwed into the empty holding tube 212 and transported. During transport, the holding tube 212 protects the filter tip from contamination. The stabilized nucleic acid is later rehydrated using an elution buffer and eluted into a storage tube using an automated system similar to that used in the clinic or simply using a disposable syringe 176 for long periods of time. It can be stored frozen or eluted directly into a detection measurement device or sample tube. In the latter case, the syringe 176 serves as a mechanism for elution buffer aspiration and dispensing cycles across the silica matrix 126.

  In preparation for molecular analysis, bound impurities are removed from matrix 126 by elution buffer aspiration and dispensing cycles (2-3 times). When the process is complete, purified nucleic acid is obtained in a buffer suitable for PCR. This approach allows flexibility in format and can therefore be used with liquid handling systems for high throughput applications or with simple pipette tips for low throughput applications.

  FIG. 7 shows a typical sequence of steps aimed at MagVor / filter chip purification of nucleic acids from sputum. Take a sputum sample suspected of containing MTB in a sample container. A chemical reagent mixture containing an inactivator and a mucolytic agent is added (step 1). Next, the sample container is placed on an extraction stand (or rack), and the sample contents are subjected to vortex stirring (MagVor) by magnetic induction for 2 to 15 minutes, preferably 10 minutes (step 2). After cell lysis, the beads are allowed to settle for 1-2 minutes and binding buffer is added to the container (step 3). The user attaches the filter tip to the underside of the tube cap / luer lock adapter of FIG. 6 and uses a luer lock connection to connect the syringe to the top surface of the tube cap / luer lock adapter (step 4). The user penetrates the filter chip from the cap over into the container, draws the cell lysate into the chip, moves the syringe lever up and down to pass the filter matrix 2 to 3 times, and promotes binding with the filter matrix. As a result, the unbonded portion is returned into the tube (step 4). After this binding step, the sample container is replaced with a new tube containing the washing reagent, and the filter matrix is washed with a washing buffer, which is collected in the tube (step 5). After the washing step, the tube is pulled up from the liquid. In some embodiments, the filter tip is further dried by dispensing air through the filter matrix (step 6). In some embodiments, several rounds of air drying are performed to reduce residual wash reagent. The user then removes the filter tip / cap adapter from the syringe, returns the filter tip to a new holding tube containing the desiccant, connects the filter tip / cap adapter and the holding tube and stores (step 7). The nucleic acid in the filter chip is stable for transportation, or the purified nucleic acid can be eluted from the filter chip for the purpose of PCR analysis or the like. Nucleic acid elution may be accomplished by passing the elution buffer through the filter matrix 2-3 times before collection.

  Trehalose, 0.1% Triton-X-100 or DNAstable® Plus reagent (Biomatrica) may be included with the elution buffer or added to the eluted nucleic acid to enhance stability.

  In some embodiments, the method further comprises eluting the nucleic acid, amplifying the eluted nucleic acid with a primer specific for a given target, and determining whether the sample contains nucleic acid corresponding to the target. Preferred targets for detection MTB, Staphylococcus aureus, methicillin-resistant Staphylococcus aureus (MRSA), Streptococcus pyogenes, Streptococcus pneumoniae, Streptococcus agalactiae, Haemophilus influenzae, Haemophilus parainfuluezae, Moraxella catarrhalis, Klebsiella pneumoniae, Escherichia coli, Pseudomonas aeruginosa, Acinetobacter species, Bordetella pertussis, Neisseria meningitid s, Bacillus anthracis, Nocardia spp., Actinomyces spp., Mycoplasma pneumoniae, Chlamydia pneumoniae, Legionella spp., Pneumocystis jiroveci Contains pathogens.

  The system 100 described herein can detect MTB at a level of less than 1,000 / mL, preferably less than 100 / mL, more preferably less than 50 / mL, and most preferably less than 10 / mL. it can. Assuming that one colony forming unit (cfu) is approximately equal to 10 cells, the above system can be used to provide detection of at least 100 cfu / mL, 10 cfu / mL, 5 cfu / mL, or even 1 cfu / mL. it can.

  However, it should be recognized that all clinical samples are unique and differ one by one in viscosity, particles, mucus, surface contaminants, microorganisms and / or human genetic background. Therefore, given the expected diversity of clinical sample composition and the intention to use an automated filter tip sample preparation protocol, it is necessary to modify several steps of the filter tip procedure to achieve the desired results. It is possible.

  For example, while the filter tips described herein have a relatively large pore size, sample homogenization and liquefaction are very important for efficient cell lysis and subsequent binding steps with the filter matrix. If the lysate is uniform and sufficiently liquefied, the sample can be passed through the filter tip at a higher flow rate, thereby reducing the overall sample processing time. As shown in the high volume plasma protocol below, a large sample input volume can be effectively processed by the filter tip, which allows difficult samples (online or online) with little concern about the input sample volume. The user is provided with the opportunity to fully homogenize and liquefy (offline).

  Furthermore, it should be appreciated that lower flow rates during nucleic acid binding or elution typically result in higher nucleic acid yields, at the expense of total processing time. The shearing degree of DNA decreases even when the flow rate is decreased.

  Complete drying of the filter matrix is recommended to prevent residual organic solvents from co-eluting with the purified nucleic acid sample and hinder downstream processing or testing. Since filter chips are not dried by centrifugation or vacuum filtration, it is important to maximize the flow rate and the number of cycles in the drying step.

  Since the geometry, the filter tip material and the attachment method with the robotic channel arm are specific to each manufacturer, different filter tip constructions are required for each liquid handling system. Filter matrix dimensions (diameter, thickness and pore size) correlate with nucleic acid binding capacity (and elution efficiency) as expected for any solid phase extraction technique. Embedding a thick (> 4 mm) matrix in a 1 mL filter chip can enhance the nucleic acid binding capacity of large volume samples and / or equalize the matrix binding capacity among individual filter chip formats, while the thickness of the filter chip There is a trade-off between the flow rate in the initial binding step (in the presence of the crude lysate). Therefore, embedding a large diameter matrix in a large volume filter chip is sometimes advantageous for the first step of the automated protocol (such as 5 mL Hamilton / Aconi TruTips® for large volume extraction). However, assuming that the specific filter chip structure is instructed by the manufacturer of the liquid material handling robot, the filter chip nucleic acid yield is the same between the liquid material manipulation platforms by different manufacturers or between different filter chip sizes. It is not reasonable to expect to be. A clinical evaluation of the automated filter chip protocol and a direct comparison with a commercial automation system will be reported in detail elsewhere.

  The Mag / For filter chip process has a number of advantages over conventional methods. First, the process is compatible with automation. Second, by enclosing the filter matrix in a pipette tip, it is less susceptible to cross contamination. On the other hand, the fibrous silica matrix on the spin disk can easily rupture, releasing fine particles and becoming a source of contamination. Similarly, techniques that rely on the mobility of magnetic beads for purification pose similar risks. Since the pore size of the filter matrix is relatively large, a highly viscous sample can be passed through the matrix without clogging. Multiple aspiration and dispensing cycles allow for increased target nucleic acid binding compared to centrifugation methods in which the sample is passed through the matrix only once. In addition, established methods of removing inhibitors and nucleases by chaotropic chemical reactions are provided, which tend to be stable over time.

  The invention is further illustrated by the following examples which should not be construed as limiting. The contents of all cited references, patent specifications and published patent specifications cited throughout this application, as well as drawings and tables, are incorporated herein by reference.

[Example 1]
(MagVor homogenization and dissolution)
The effectiveness of the MagVor system on Bacillus thuringiensis spores, Streptococcus pyogenes, and MTB in ginger using glass beads to sample volume v: v ratio 1: 1, total sample volume 1 mL, and MagVor lysis for 30-120 seconds Tested. Extraction of MTB DNA from ginger is particularly difficult due to its mycobacterial cell wall and low infectious dose (10 cells). The effectiveness of lysis was estimated by quantitative real-time PCR of comparable ginger samples before and after MagVor treatment. MagVor treatment was confirmed to improve nucleic acid detection on average by 2.5 cycles (approximately log1) compared to untreated samples (ie, some of the sputum samples that were identical but not treated with the MagVor system). It was.

  To investigate the degree to which physical lysis kit components (particles, stir discs, coatings) interfere with nucleic acid extraction and purification, and to analyze 4 types of beads and 3 types of magnetic discs, ultrafine particles after MagVor lysis Were identified for dissolved beads and magnetic disks that did not form in solution. After NPA samples were pooled and confirmed to be negative for the target DNA of interest by real-time PCR, untreated methicillin MRSA or MTB target cells were added. Two sets of specimens (0.5 mL) were treated and dissolved by MagVor treatment at 5000 rpm for 10 minutes. Nucleic acids were purified by manual MagVor / filter chip procedure using guanidine-based binding buffer and then analyzed by quantitative real-time PCR (or RT-PCR). Glass beads readily settled to the bottom of the lysis tube and showed no apparent inhibition or degradation of DNA or RNA during these tests.

  As shown in Table 1, when using MagVor treatment, a consistent improvement in DNA recovery was obtained, especially at high titers, compared to the case without treatment.

(MRSA with MagVor / filter chip combination and its recovery from MTB DNA added NPA)

[Example 3]
(Comparison with the integrated MagVor / filter chip prototype Qiagen nucleic acid purification kit)
The nucleic acid lysis and purification efficiency of the integrated system was evaluated relative to an equivalent Qiagen nucleic acid purification kit. Model sample types included MRSA in NPA, influenza A in NPS, human genomic DNA from whole blood, and MTB in NPA. The Qiagen kit consists of a DNA mini kit (treated with proteinase K for 10 minutes without mechanical lysis), a viral RNA mini kit (without mechanical lysis and using an RNA carrier) and a mini blood kit (incubated with proteinase K for 10 minutes). Inclusive. Since Qiagen has no kit specialized for MTB extraction, the BD GeneOhm lysis kit was used in conjunction with the Qiagen mini DNA extraction kit. Because the BD lysis and Qiagen kits also had input sample volume limitations, NPA and NPS samples were processed in a volume of 200 μL and whole blood was processed in volumes of 100, 10 and 1 μL. Repeat reagent plates are prepared, sealed, processed for each sample type and titer (n = 24 extractions per sample), purified nucleic acids are analyzed by quantitative real-time PCR, and average cycle thresholds (Ct) are comparable. Comparison with that obtained from Qiagen extraction (n = 8 extraction per sample). Positive and negative controls were run with each plate to test for potential cross contamination.

The results of this analysis are summarized in Table 2 and demonstrated performance and effectiveness comparable to the Qiagen kit. The limit of detection of MRSA in NPA and influenza A in NPS was about 10 ³ cells / mL for both systems or virions. Human DNA was easily recovered from 1 μL of whole blood and the template control showed no evidence of nucleic acid cross-contamination. These data demonstrate the scalability and effectiveness of the integrated sample preparation prototype compared to commercially available high quality sample preparation kits.

(Nucleic acid recovery from MagVor filter chip system (n = 24) compared with other DNA extraction kits)

[Example 4]
(喀 痰 liquefaction)
Because most specimens subjected to mycobacterial cultures are contaminated with a variety of microorganisms that can grow faster than mycobacteria, respiratory specimens are typically pre-digested before analysis. It is attached to. Thus, mycobacteria are optimally recovered from clinical specimens through the use of procedures that reduce or eliminate contaminating bacteria while releasing mucin and mycobacteria trapped by the cells.

  NALC-NaOH precipitation has become the standard for decontamination and digestion of non-tuberculous mycobacteria (NTM) sputum specimens. However, extraction of MTB DNA from ginger is particularly difficult due to the high viscosity and heterogeneity of the cocoon on the one hand and the MTB's difficult to destroy cell wall on the other. Transport of the DNA extract reduces transport complexity (in terms of refrigerated transport) compared to ginger. NALC-NaOH is actually a digestion procedure, while NALC quickly loses its activity and requires daily re-dissolution of fresh reagents. Furthermore, the procedure requires centrifugation, which adds additional complexity and equipment. In addition, NaOH exposure causes MTB cell death and DNA degradation.

  Therefore, it was interesting for users interested in processing ginger to develop liquefaction methods that could serve as alternatives. Since ginger is a type of specimen that is difficult to transfer to a sample container, a single-step liquefaction procedure using an enzyme solution has been developed. By adding liquid enzyme 1 to ginger 10 and incubating at 56 ° C. for 15 to 20 minutes, even non-uniform and viscous sputum specimens are liquefied, and 5 to 10% glycerol An equivalent viscosity was obtained. These liquefied sputum specimens were easily pipetted and processed with manual MagVor and filter tip protocols without stopping the rotation of the magnetic disk in the lysis tube or clogging the filter tip.

[Example 5]
(Extraction of MTB DNA from ginger)
The feasibility of DNA extraction from live TB positive sputum was demonstrated using the automated 8-channel prototype system shown in FIGS. 5A and 5B. Patient specimens that were unidentifiable by smear microscopy were identified as either smear 2+ or 4+ using Truant TB fluorescent staining. Four smear 2+ ginger specimens and four smear 4+ rabbit specimens were processed according to the liquefaction protocol of Example 4 and then added to the MagVor tube. Next, an integrated MagVor / filter chip protocol was performed by automated extraction / purification. When the eluent was analyzed by IS6110qPCR analysis, the concentration of the extract was confirmed to be 3.6 ± 0.7 pg / μg for smear 2+ and 49 ± 8 pg / μL for smear 4+. This data confirms the feasibility of an automated nucleic acid separation device for extracting DNA from TB positive specimens.

  Table 3 shows the results of serial dilution tests comparing real-time detection of MTB using an automated MagVor / filter chip system and a manual MagVor / filter chip system. MTB cells are added to 500 μL of TB negative 喀 痰 to precipitate (NALC-NaOH-treated 喀 痰). At this time, 10 cells are approximately equal to 1 cfu / mL. For comparison purposes, corresponding cell levels corresponding to acid-fast bacilli (AFB) smear positive and AFB smear negative were included.

(Serial dilution test showing real-time detection of MTB DNA with manual and automated MagVor / filter chip systems)

  The foregoing is intended to teach those skilled in the art how to practice the present invention and is intended to detail all of the obvious modifications and variations that will become apparent to those skilled in the art upon reading this description. Absent. However, all such obvious modifications and variations are intended to be included within the scope of the present invention as defined in the following claims. The claims are intended to cover the claimed components and steps in any order effective to fulfill their intended purpose, unless the context indicates otherwise.

Claims (20)

A sample purification system:
housing;
A sample container rack disposed within the housing, wherein the sample container rack is designed to hold one or more sample containers;
A filter device holder disposed within the housing, wherein the filter device holder is designed to hold one or more filter devices including a filter for binding the molecule of interest; and A cylindrical magnet in the vicinity of and outside the sample container rack, wherein the magnet includes the cylindrical magnet that rotates about the center and the major axis of the magnet by an electric motor that is disposed inside the housing. Purification system.   The sample purification system of claim 1, further comprising a sample container disposed within the sample rack, wherein the sample container comprises a sample suspension, a magnetic stirrer, and a plurality of beads. Purification system.   2. The sample purification system according to claim 1, further comprising one or more reagent racks disposed in the housing, wherein the reagent racks are individually stored in sealed containers in each rack. The sample purification system comprising a plurality of reagents.   The sample purification system of claim 1, further comprising an automated pipetting system.   5. The sample purification system of claim 4, wherein the one or more designed to automatically dispense reagents into the one or more sample containers and to discard sample material and reagents in a predetermined manner. The sample purification system further comprising a robotic arm.   6. The sample purification system of claim 5, wherein the system further comprises a plurality of sample containers, the plurality of filter chips, and one or more reagent racks.   2. The sample purification system according to claim 1, wherein the cylindrical magnet has magnetic poles disposed symmetrically about and around the major axis of the magnet.   2. The sample purification system according to claim 1, wherein the cylindrical magnet has a counter magnetic pole that is disposed at the opposite end of the long axis of the magnet.   3. The sample purification system of claim 2, wherein the beads are silica beads having a diameter in the range of 10 to 1000 [mu] m.   3. The sample purification system of claim 2, wherein the magnetic stirrer includes an alloy core coated with a polymer.   11. The sample purification system of claim 10, wherein the alloy core comprises neodymium iron boron or samarium cobalt and the polymer is PTEE or parylene. A method for purifying a molecule of interest from a sample using the sample purification system of claim 1 comprising:
Placing the sample tube containing the liquid sample suspension, magnetic stirrer and cell lysis beads on the sample rack;
Homogenizing the sample suspension by rotating the cylindrical magnet at a speed sufficient to lyse the cells in the sample suspension in the presence of the magnetic stirrer and cell lysis beads;
Flowing the homogenized sample suspension through the filter device containing the filter matrix under conditions in which a filter device is attached to the filter device holder and the molecules of interest bind to the filter matrix;
Washing said filter matrix; and eluting bound molecules of interest from said filter matrix.   13. The method according to claim 12, wherein the sample tube is selected from the group consisting of a magnetic stirrer, a cell lysis bead, a reagent that promotes cell lysis, and a reagent that maintains the integrity of the molecule of interest. Said method prepackaged with the above items.   13. The method of claim 12, wherein the molecule of interest is a nucleic acid.   15. The method of claim 14, wherein the liquid sample suspension includes sputum.   16. The method of claim 15, further comprising the step of amplifying the eluted nucleic acid with a primer specific for Mycobacterium tuberculosis and determining whether the nucleic acid contains Mycobacterium tuberculosis DNA. A method for purifying nucleic acid from a sample comprising:
Placing the sample tube in the sample rack, wherein the sample tube contains a liquid sample suspension, a magnetic stirrer and cell lysis beads, and the sample rack is located near a cylindrical magnet;
The cylindrical magnet is moved so that the magnetic stirrer in each sample tube rotates and stirs the cell lysis beads to an extent sufficient to destroy the cells in the sample suspension and produce a cell lysate. Rotating around the long axis;
Flowing at least a portion of the cell lysate through the first opening of the pipette tip so that the nucleic acid from the cell lysate binds to the filter matrix of the filter pipette tip:
Discharging the unbound portion of the cell lysate that has passed through the filter matrix in the step of flowing from the first opening of the filter chip;
The elution buffer is taken in through the first opening of the filter chip, the elution buffer flows through the filter matrix, and the elution buffer that has flowed through the filter matrix passes from the filter chip to the first opening. Eluting the nucleic acid bound to the filter matrix by draining through the process.   18. The method of claim 17, wherein the sample includes sputum.   18. The method of claim 17, further comprising the step of amplifying the eluted nucleic acid with a primer specific for Mycobacterium tuberculosis and determining if the nucleic acid contains Mycobacterium tuberculosis DNA.   18. The method of claim 17, wherein the cylindrical magnet has magnetic poles disposed symmetrically about and about the major axis of the magnet.

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