Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
  • C12N1/06—Lysis of microorganisms
  • C12N1/066—Lysis of microorganisms by physical methods
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
  • C12M1/00—Apparatus for enzymology or microbiology
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
  • C12N1/20—Bacteria; Culture media therefor
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N13/00—Treatment of microorganisms or enzymes with electrical or wave energy, e.g. magnetism, sonic waves
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay

Definitions

• the technical field is biotechnology and, more specifically, methods and apparatus for lysing cells.
• Cell lysis is the destruction, or disruption, of a cell's membrane or wall, which breaks open the cell and exposes its contents.
• Many techniques are available for the disruption of cells, including physical, chemical (e.g., detergent-based methods, chaotropic salts) and biochemical (e.g., enzymes such as lysozyme).
• Mechanical lysis such as vortexing and bead-beating, is one form of physical lysis.
• Sonication is another form of physical lysis, which uses pulsed, high frequency sound waves to agitate and lyse cells, bacteria, spores, and finely diced tissue.
• a method for lysing cells, virus particles and spores comprises stirring cells with a magnetic stir element in a container in the presence of a plurality of cell lysis beads, wherein the stir element rotates at a speed sufficient and duration to lyse the cells.
• the cell lysis beads are selected from the group consisting of polymer beads, glass beads, ceramic beads and metal beads.
• the cell lysis beads have diameters within the range of 10-1000 ⁇ .
• lmg-lOg of cell lysis beads are added to the lysis chamber.
• lul- lOml of an aqueous liquid is added to the lysis chamber.
• a sample or specimen is added directly to the lysis chamber alone.
• a sample is added directly to the lysis chamber together with an aqueous liquid (e.g. a solid specimen that is homogenized and lysed to yield an aqueous form).
• the magnetic stir element has a rectangular shape, a trapezoidal shape, a two- pronged tuning fork shape, a rod shape, and a bar shape.
• the cells are eukaryotic cells, prokaryotic cells, endo-spores, or a combination thereof, and are suspended in a liquid medium at a concentration ranging from 1 to lxlO 10 cells/ml.
• the cells are virus particles and are suspended in a liquid medium at a concentration ranging from 1 to lxlO 13 particles/ml.
• the method comprises suspending cells or virus particles in a liquid medium to form a suspension, and stirring the suspension with a magnetic stir element at high speed, between 1000-5000 rpm, preferably closer to 5000 rpm, in the presence of a plurality of cell lysis beads for a time period between 1-600 seconds, preferably about 90-120 seconds.
• the device includes a chamber having one or more magnetic stir elements and a plurality of cell lysis beads disposed therein.
• the chamber is dimensioned to allow rotation of the one or more magnetic stir elements inside the chamber.
• the device further includes a magnetic stirrer that produces a rotating magnetic field, wherein the one or more magnetic stir elements rotate inside the chamber when placed within the operational range of the rotating magnetic field.
• the device further includes a chamber holder configured to hold the chamber within the rotating magnetic field produced by the magnetic stirrer.
• the method comprises applying a magnetic field to a vessel containing a cell, a stir element, and a plurality of beads, wherein the magnetic field causes the stir element to collide with the plurality of beads and produce a cell lysate; and isolating nuclei acid from the cell lysate.
• the method comprises applying a magnetic field to a vessel containing a cell, a stir element, and a plurality of beads, wherein the magnetic field causes the stir element to collide with the plurality of beads and produce a cell lysate, and amplifying a polynucleotide from the cell lysate.
• the method comprises applying a magnetic field to a vessel containing a cell, a stir element, and a plurality of beads, wherein the magnetic field causes the stir element to collide with the plurality of beads and produce a cell lysate, and detecting a polynucleotide from the cell lysate.
• the detecting step comprises isolating nuclei acids from the cell lysate, amplifying the polynucleotide from isolated nuclei acids, and detecting the amplified polynucleotide.
• FIG. 1 is a flow chart showing an embodiment of a method for lysing cells.
• FIG. 2 is a flow chart showing another embodiment of a method for lysing cells.
• FIG. 3 is a graph showing relative positions of a magnet and a lysis chamber.
• FIG. 4 is a graph showing real-time PCR amplification of a specific genomic region from unlysed E. coli cells (unlysed), E. coli cells lysed by bead beater for 2 minutes (2m beat), and E. coli cells lysed by beads/stirrer for 30 seconds (30s blend) or 2 minutes (2m blend).
• FIG. 5 is a graph showing real-time PCR amplification of a specific genomic region from unlysed E. coli cells (unlysed), E. coli cells lysed by a bead beater for 2 minutes (2m beat), and E. coli cells lysed by beads/stirrer for 1 minute (lm blend) or 2 minutes (2m blend).
• FIG. 6 is a graph showing real-time PCR amplification of a specific genomic region from unlysed Bacillus thuringiensis spores (unlysed), Bacillus thuringiensis spores lysed by a bead beater for 2 minutes (2m beat), and Bacillus thuringiensis spores lysed by beads/stirrer for 2 minutes (2m blend).
• FIG. 7 is a graph showing real-time PCR amplification of a specific genomic region from unlysed Bacillus thuringiensis spores (unlysed), Bacillus thuringiensis spores lysed by bead beater for 2 minutes (2m beat), and Bacillus thuringiensis spores lysed by beads/stirrer for 1.5 minutes (1.5m blend) or 2 minutes (2m blend).
• FIG. 8 is a graph showing real-time PCR amplification of a specific genomic region from unlysed Bacillus thuringiensis spores (unlysed), Bacillus thuringiensis spores lysed by bead beater for 2 minutes (2m beat), and Bacillus thuringiensis spores lysed by beads/stirrer for 1.5 minutes (1.5m blend).
• FIG. 9 is a graph showing the effect of lysis time and bead blender speed on the realtime PCR response for Staphylococcus aureus cells. Efficient lysis of S. aureus cells was achieved in 2 minutes when the bead blender was operated at high-speed. Lower speeds required longer lysis times. Unlysed spores have low-levels of extracellular DNA that are detectable without lysis (0 min). A small amount of free DNA binds to the glass beads. Bead blender speed of 100 corresponds to 5000rpm.
• FIG. 10. is a graph showing the effect of lysis time and bead blender speed on the real-time PCR response for Bacillus thuringiensis spores. Efficient lysis of Bacillus thuringiensis spores was achieved in 2 minutes when the bead blender was operated at highspeed. Lower speeds required longer lysis times. Unlysed spores have low-levels of extracellular DNA that are detectable without lysis (0 min). A small amount of free DNA binds to the glass beads. Bead blender speed of 100 corresponds to 5000rpm.
• the method 100 includes adding a cell sample to a chamber (step 1 10), adding a plurality of cell lysis beads to the container (step 120), adding a magnetic stirring element to the container (step 130), and stirring the cell sample with the magnetic stir element at a rotation speed (between 1000 and 5000 rpm) sufficient to lyse the cells (step 140).
• the method 200 includes forming a mixture that includes cells, a liquid medium, cell lysis beads, and a magnetic stir element (step 210), and driving motion of the magnetic stir element in the mixture using a varying magnetic field to lyse the cells (step 220).
• cells refers to eukaryotic cells, prokaryotic cells, viruses, endo-spores or any combination thereof.
• Cells thus may include bacteria, bacterial spores, fungi, virus particles, single-celled eukaryotic organisms (e.g., protozoans, yeast, etc.), isolated or aggregated cells from multi-cellular organisms (e.g., primary cells, cultured cells, tissues, whole organisms, etc.), or any combination thereof, among others.
• cell sample refers to any cell-containing samples.
• cell samples include specimens of human, plant, animal, food, agriculture or environmental origin, such nasal swab, blood, stool, plasma, tissue, leaves, water and soil samples.
• the cell samples contain cells suspended in a liquid medium at a concentration that does not interfere with the movement of the magnetic stir element.
• the term "lyse” with respect to cells means disruption of the integrity of at least a fraction of the cells to release intracellular components, such as nuclei acids and proteins, from the disrupted cells.
• Eukaryotic cells, prokaryotic cells, and/or viruses may be suspended at any suitable concentration.
• eukaryotic cells and/or prokaryotic cells are suspended at a concentration ranging from 1 to lxlO 10 cells/ml, 1 to lxlO 5 cells/ml, or lxlO 3 to lxlO 4 cells/ml, among others.
• virus particles are suspended in a concentration ranging from 1 to lxlO 13 particles/ml, 1 to lxlO 10 particles/ml, or lxlO 5 to lxlO 7 particles/ml.
• the liquid medium can be isotonic, hypotonic, or hypertonic.
• the liquid medium is aqueous.
• the liquid medium contains a buffer and/or at least one salt or a combination of salts.
• the pH of the liquid medium ranges from about 5 to about 8, from about 6 to 8, or from about 6.5 to about 8.5. A variety of pH buffers may be used to achieve the desired pH.
• Suitable buffers include, but are not limited to, Tris, MES, Bis-Tris, ADA, ACES, PIPES, MOPSO, Bis-Tris propane, BES, MOPS, TES, HEPES, DIPSO, MOBS, TAPSO, HEPPSO, POPSO, TEA, HEPPS, Tricine, Gly-Gly, Bicine, and a phosphate buffer (e.g., sodium phosphate or sodium- potassium phosphate, among others).
• the liquid medium may comprise from about 10 mM to about 100 mM buffer, about 25 mM to about 75 mM buffer, or from about 40 mM to about 60 mM buffer, among others.
• the type and amount of the buffer used in the liquid medium can vary from application to application.
• the liquid medium has a pH of about 7.4, which can be achieved using about 50 mM Tris buffer.
• the liquid medium in water is not limited to, Tris, MES, Bis-Tris, ADA, ACES, P
• the chamber can be a container of any suitable material, size, and shape.
• the chamber is a plastic container.
• the chamber may, for example, be in the shape of a micro centrifuge tube (e.g., an Eppendorf tube), a centrifuge tube, a vial, microwell plate.
• the chamber can define only one compartment/chamber for holding cells, beads, and a stir element, or a plurality of discrete compartments/chambers (e.g., an array of wells) for holding mixtures (cells, beads, and stir elements) in isolation from one another.
• the chamber may be a sealed or sealable container, such as a container with a lid, cap, or cover.
• the interior surface may be chemically inert to preserve the integrity of the analyte of interest.
• the cell lysis beads can be any particle-like and/or bead-like structure that has a hardness greater than the hardness of the cells.
• the cell lysis beads may be made of plastic, glass, ceramic, metal and/or any other suitable materials.
• the cell lysis beads may be made of non-magnetic materials.
• the cell lysis beads are rotationally symmetric about at least one axis (e.g., spherical, rounded, oval, elliptic, egg-shaped, and droplet-shaped particles).
• the cell lysis beads have polyhedron shapes.
• the cell lysis beads are irregularly shaped particles.
• the cell lysis beads are particles with protrusions.
• the amount of beads added to each lysis container is in the range of 1- 10,000mg, 1- l OOOmg, 1- lOOmg, 1- lOmg, among others.
• the cell lysis beads have diameters in the range of 10-1 ,000 ⁇ , 20-400 ⁇ , or 50-200 ⁇ , among others.
• the magnetic stir element can be of any shape and should be small enough to be placed into the container and to move or spin or stir within the container.
• the magnetic stir element can be a bar-shaped, cylinder-shaped, cross-shaped, V-shaped, triangular, rectangular, rod or disc-shaped stir element, among others.
• the magnetic stirring element has a rectangular shape.
• the magnetic stirrer has a two- pronged tuning fork shape.
• the magnetic stirrer has a V-like shape.
• the magnetic stirrer has a trapezoidal shape.
• the longest dimension of the stir element is slightly smaller than the diameter of the container (e.g. about 75-95% of the diameter of the container).
• the magnetic stir element is coated with a chemically inert material, such as polymer, glass, or ceramic (e.g., porcelain).
• a chemically inert material such as polymer, glass, or ceramic (e.g., porcelain).
• the polymer is a biocompatible polymer such as PTFE and paralyne.
• the cell suspension, cell lysis beads and the magnetic stir element may be placed into the chamber in any order.
• the cell suspension is added to the chamber before the cell lysis beads and the magnetic stirring element.
• the cell lysis beads and/or the magnetic stirring element are placed into the chamber before the cells (e.g., before a cell suspension).
• the chamber, and particularly the cells, beads, and magnetic stir element are located within an operational range of a varying magnetic field.
• the chamber may be located with an operational range of a rotating magnetic field (e.g., by placing the container on or adjacent to a magnetic stirrer).
• the varying magnetic field drives motion of the stir element, such as rotational motion, reciprocation, or a combination thereof, among others, which in turn drives motion of the beads, the cells, and the liquid medium.
• the cell suspension is stirred with the magnetic stirring element at a rotation speed and for durations sufficient to lyse the cells inside the container.
• the appropriate rotation speed and duration are application dependent and can be empirically determined by a person of ordinary skill in the art.
• the rotation speed sufficient to lyse the cells is determined by factors such as the type of cells, the concentration of cell suspension, the volume of the cell suspension, the size and shape of the magnetic stirring element, the amount/number, size, shape and hardness of the cell lysis beads, and the size and shape of the chamber.
• the magnetic stirring element is rotating at a speed between 1000-6000 rpm, preferably about 5000 rpm, for a time period between 1-600 seconds, preferably about 90- 120 seconds.
• a chamber e.g., in the shape of a test tube or micro centrifuge tube
• a magnetic stirrer e.g., VP 710C 1 Rotary Magnetic Tumble Stirrer, 5000 RPM, 14cm Long Usable Stirring Deck, With Motor Housing And Plate Holder, Stirs 2 Deep Well Microplates Or 6 Standard Microplates- 1 15 Volts AC - 60 Hz, V&P Scientific
• the chamber is a well in a microplate, such as an ELISA plate.
• the chamber is a cylinder shaped chamber with a cell inlet and a cell outlet.
• the varying magnetic field is generated by rotating a magnet, preferably a permanent magnet, in the proximity of magnetic stir element.
• the magnet may be rotated above, below or by the side of the lysis chamber about an axis that passes through the center of the magnet.
• the chamber or chambers are placed at a position that is vertical to the surface the chamber or the chambers reside on and the magnet is rotated about an axis that is also vertical to the surface the chamber or the chambers reside on.
• the chamber or chambers are placed at a position that is vertical to a the surface the chamber or the chambers reside on and the magnet is rotated about an axis that is parallel to the surface the chamber or the chambers reside on.
• the chamber or chambers are placed at a position that is vertical to a the surface the chamber or the chambers reside on and the magnet is rotated about an axis that forms an angle with the surface the chamber or the chambers reside on. The angle is greater than 0 degree but smaller than 180 degrees.
• the magnet also has an elongated shape and rotates about an axis that extends along the longest dimension of the magnet.
• Figure 3 shows the relative positions of a cylinder shaped magnet 301 and a lysis chamber 303.
• the magnet 301 rotates about an axis A and causes a magnet stir element 305 in the chamber 303 to rotate in the same direction along an axis B.
• the rotating magnet stir element 305 collides with beads 307 and lyse cells 309 in the process.
• the magnet 301 may be positioned alongside, above, below or diagonally from the chamber 303.
• the chamber 303 has an inlet 311 and an outlet 313 to facilitate loading and unloading of the chamber.
• the speed of rotation of the stirrer element is increased to increase lysis efficiency and reduces the time required to achieve lysis.
• the speed of rotation is regulated so that only certain types of cells are lysed.
• the stir element may rotate at a first speed to lyse a first set of cells and then rotate at a second speed to lyse a second set of cells.
• the container is coupled to a temperature regulation module that controls the temperature of the cell suspension before, during and/or after the lysing process. In certain embodiments, the temperature of the cell suspension is maintained at 8-2°C.
• lysing of particular cell types can be facilitated by adding additives to the cell suspension prior to and/or during the stirring step.
• additives include enzymes, detergents, surfactants and other chemicals such as bases and acids. It has been found that alkaline conditions (e.g., 10 mM NaOH) may enhance the lysis efficiency during stirring for certain types of cells.
• the cell suspension may also or alternatively be heated during stirring to enhance the lysis efficiency.
• Additives can be detrimental to downstream processing steps including nucleic acid amplification and detection. Eliminating the need for additives to achieve efficient lysis is desirable as specimen processing can be greatly simplified.
• the stirrer/beads combination provides many advantages over conventional lysing methods.
• the stirrer/beads method is much faster than chemical and enzymatic approaches, and provides improved cell or virus lysis over many other types of physical lysis methods.
• the stirrer/beads method is also amenable to automation using robotics and/or microfluidics.
• the magnetic source is reusable and doesn't require precise alignment with the vessel, can drive a plurality of chambers.
• the magnetic stirrer element is low-cost to enable it to be single-use disposable.
• the device includes a chamber having a magnetic stir element and a plurality of cell lysis beads disposed therein.
• a user may simply add a cell suspension into the chamber, place the chamber on a magnetic stirrer, and stir the cell suspension with the magnetic stir element at a speed sufficient to lyse the cells.
• the system includes a chamber having a magnetic stir element and a plurality of cell lysis beads disposed therein, and a magnetic stirrer that produces a rotating magnetic field, wherein the magnetic stir element rotates inside the chamber when placed within an operational range of the rotating magnetic field.
• the system further contains a rack configured to hold the chamber.
• the rack may be configured to hold multiple chambers and can be placed on a support surface of a magnetic stirrer for simultaneous processing of multiple samples.
• the rack may also be used as holder of the chamber for storage purpose. For example, multiple chambers may be placed on the rack and stored in a refrigerator or freezer for further analysis.
• the chamber may be interfaced with an external instrument (e.g. liquid handling robot, microfluidic device, analytical instrument).
• the method comprises applying a magnetic field to a vessel containing a cell, a stir element, and a plurality of beads, wherein the magnetic field causes the stir element to collide with the plurality of beads and produce a cell lysate; and isolating nuclei acid from the cell lysate.
• the method comprises applying a magnetic field to a vessel containing a cell, a stir element, and a plurality of beads, wherein the magnetic field causes the stir element to collide with the plurality of beads and produce a cell lysate, and amplifying a polynucleotide from the cell lysate.
• the method comprises applying a magnetic field to a vessel containing a cell, a stir element, and a plurality of beads, wherein the magnetic field causes the stir element to collide with the plurality of beads and produce a cell lysate, and detecting a polynucleotide from the cell lysate.
• the detecting step comprises isolating nuclei acids from the cell lysate, amplifying the polynucleotide from isolated nuclei acids, and detecting the amplified polynucleotide.
• Example 1 Lysis of E. coli cells
• E. coli cells were suspended in Tris-EDTA buffer at a concentration of 10 4 cells/ml
• Tris-EDTA buffer at a concentration of 10 4 cells/ml
• 800mg glass beads 106 ⁇ or finer, Sigma G8893
• a magnetic stir disc VP- 7195 Super Tumble Stir Disc, V&P Scientific
• the plastic vial was then placed on a magnetic stirrer (VP 710C1 Rotary Magnetic
• the beads/stirrer method (30s blend, 1 m blend and 2m blend) provides better cell lysis than the bead beater method (2m beat).
• Example 2 Lysis of Bacillus thuringiensis spores
• Bacillus thuringiensis spores were suspended in water at a concentration of 10 4 cells/ml. 500 ⁇ of the cell suspension was added to a 2 ml plastic vial (Wheaton) containing 800 mg of glass beads (106 ⁇ or finer, Sigma) and a magnetic stir disc (VP-7195 Super Tumble Stir Disc, V&P Scientific).
• the plastic vial was then placed on a magnetic stirrer (VP 710C1 Rotary Magnetic
• the beads/stirrer method (1.5 m blend and 2m blend) provides better cell lysis than the bead beater method (2m beat).
• Example 3 Effect of lysis time and bead blender speed on the real-time PCR response for Staphylococcus aureus cells
• Staphylococcus aureus cells were suspended and lysed at various blender speeds for various time periods using the same equipment and procedures described in Example 2.
• FIG. 9 is a graph showing the effect of lysis time and bead blender speed on the realtime PCR response for Staphylococcus aureus cells. Efficient lysis of S. aureus cells was achieved in 2 minutes when the bead blender was operated at high-speed. Lower speeds required longer lysis times. Unlysed spores have low-levels of extracellular DNA that are detectable without lysis (0 min). A small amount of free DNA binds to the glass beads. Bead blender speed of 100 corresponds to 5000rpm.
• Example 4 Effect of lysis time and bead blender speed on the real-time PCR response for Bacillus thuringiensis spores Bacillus thuringiensis spores were suspended and lysed at various blender speeds for various time periods using the same equipment and procedures described in Example 2.
• FIG. 10. is a graph showing the effect of lysis time and bead blender speed on the real-time PCR response for Bacillus thuringiensis spores. Efficient lysis of Bacillus thuringiensis spores was achieved in 2 minutes when the bead blender was operated at highspeed. Lower speeds required longer lysis times. Unlysed spores have low-levels of extracellular DNA that are detectable without lysis (0 min). A small amount of free DNA binds to the glass beads. Bead blender speed of 100 corresponds to 5000rpm.

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• 2010
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