EP2129467A2 - Pulvérisateur et procédé de préparation du traitement d'un échantillon biologique - Google Patents

Pulvérisateur et procédé de préparation du traitement d'un échantillon biologique

Info

Publication number
EP2129467A2
EP2129467A2 EP08735665A EP08735665A EP2129467A2 EP 2129467 A2 EP2129467 A2 EP 2129467A2 EP 08735665 A EP08735665 A EP 08735665A EP 08735665 A EP08735665 A EP 08735665A EP 2129467 A2 EP2129467 A2 EP 2129467A2
Authority
EP
European Patent Office
Prior art keywords
sample
vessel
pulverizer
movable body
cooled
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Granted
Application number
EP08735665A
Other languages
German (de)
English (en)
Other versions
EP2129467B1 (fr
Inventor
Dennis Mertens
Thomas Voit
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Qiagen GmbH
Original Assignee
Qiagen GmbH
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Qiagen GmbH filed Critical Qiagen GmbH
Publication of EP2129467A2 publication Critical patent/EP2129467A2/fr
Application granted granted Critical
Publication of EP2129467B1 publication Critical patent/EP2129467B1/fr
Not-in-force legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B02—CRUSHING, PULVERISING, OR DISINTEGRATING; PREPARATORY TREATMENT OF GRAIN FOR MILLING
    • B02C—CRUSHING, PULVERISING, OR DISINTEGRATING IN GENERAL; MILLING GRAIN
    • B02C17/00—Disintegrating by tumbling mills, i.e. mills having a container charged with the material to be disintegrated with or without special disintegrating members such as pebbles or balls
    • B02C17/14—Mills in which the charge to be ground is turned over by movements of the container other than by rotating, e.g. by swinging, vibrating, tilting

Definitions

  • the invention relates to a method for the preparation of a vegetable or animal sample for processing, ie, for example, for the isolation of nucleic acids or proteins from the sample and a pulverizer.
  • a method for the preparation of a vegetable or animal sample for processing ie, for example, for the isolation of nucleic acids or proteins from the sample and a pulverizer.
  • Such preparations and examinations are carried out in a laboratory by a laboratory assistant or a laboratory technician on the basis of standardized work instructions.
  • Such a manual includes a so-called protocol.
  • An example of such a protocol for the isolation of plasmid DNA from E. coli is apparent from the document DE 1 01 53 957 Al.
  • kits can be obtained commercially, for example the "UltraClean Tissue DNA Isolation Kit” of the company, depending on the sample and the desired result.
  • Qiagen www.Qiagen.com
  • a sample is processed using such a kit according to a given protocol, it must be suitably prepared.
  • an organ is taken from a test animal, such as a rat. It depends on the objective, which organ of an animal is selected.
  • the removed tissue of the animal is washed in a washing buffer solution, for example in PBS (Phosphate Buffered Saline with the following contents: Na 2 HPO 4 (dried), NaH 2 PO 4 (dried), NaCl and distilled water).
  • a washing buffer solution for example in PBS (Phosphate Buffered Saline with the following contents: Na 2 HPO 4 (dried), NaH 2 PO 4 (dried), NaCl and distilled water).
  • tissue with a body temperature of, for example, 37 ° C is immersed in liquid nitrogen. Bubbles develop. The tissue is only removed from the liquid nitrogen when bubbling stops. Subsequently, the tissue is stored at -80 0 C, for example, with the aid of dry ice.
  • RNAIater® is a viscous liquid developed by Ambion (www.ambion.com) for the preservation of fresh tissue.
  • the preservative effect is based primarily on the fact that all enzymes inactivated by dehydration in the tissue and cell activities are stopped.
  • the viscous fluid must quickly diffuse into all cells of the tissue.
  • the size of the fabric pieces is therefore limited to an edge length of a maximum of half a centimeter.
  • the tissue thus treated is also cooled at -80 ° to store it until processing.
  • tissue For processing typically 10 to 100 mg of tissue is needed to perform the desired assay, isolation or the like. It is now separated before starting the processing, the required amount of animal tissue, for example, with a scalpel.
  • the separated sample, ie the separated tissue is now digested, that is, the cell walls must be opened. This can be done mechanically, chemically or enzymatically. Mechanical disruption takes place, for example, with the aid of a "TissueRuptor" from Qiagen, which is known from the TissueRuptor Handbook, July 2006 by Qiagen, in which a rotating knife with 35,000 revolutions per minute shatters cell walls of the tissue.Mechanical digestions are regularly carried out in a buffer. to avoid damage to the ingredients such as nucleic acids.
  • a sample is first blocked in paraffin and then cut into thin tissue layers by means of a microtome. If vegetable samples are to be processed, scalping can only be done with soft materials such as leaves, soft beans, etc. For dried or frozen plant samples, they are cooled in liquid nitrogen and ground in a liquid nitrogen cooled mortar using a pestle.
  • German Patent 738,286 teaches to freeze and grind cells together with a dispersion liquid so as to comminute cells.
  • a solution to the problem comprises the features of claim 1.
  • Other solutions include the features of the dependent claims, which are also directed to a method.
  • An apparatus for carrying out the method comprises the features of the last subsidiary claim.
  • a sample is first washed as described above and treated, for example, with liquid nitrogen or stored in RNAIater.
  • a closable preferably made of metal vessel or plastic existing vessel, which is equipped with a metal inlay, and a container located in the body, movable body cooled to a temperature which is well below 0 0 C.
  • the plastic container is a shell for the inlay.
  • An inlay is a body made separately from the shell. To distinguish this is a double-walled vessel, as it is known for example from the document US 6,235,501 B l, Figure 1 0, which does not include inlay in the sense of the present invention, since the two known vessel walls are integrally connected to each other.
  • the temperature to which the vessel is cooled should be at least -50 0 C.
  • a temperature of approx. - 80 0 C for example, from -70 0 C to -90 0 C to choose, since this is significantly further away from 0 ° C.
  • Dry ice can be provided inexpensively. It has been shown that cooling to approx. -80 0 C for particularly good results. Lower temperatures than -80 0 C up to a certain extent possible. It should be noted, however, that the vessel must not be allowed to cool too much. Thus, a temperature of liquid nitrogen, ie of -1 96 0 C has been found to be too low to get good results.
  • the interior of the vessel is dimensioned and designed in addition to the movable body therein.
  • Particularly suitable is a cylindrical interior with hollow spherical ends.
  • the movable body is then preferably a ball or a bolt with spherical ends.
  • the diameter the ball or the bolt is smaller than the diameter of the interior, so as to ensure the mobility.
  • the movable body is made of a hard, preferably heavy material such as metal in order to be able to crush the sample.
  • the biological sample for example a cooled rat heart, is preferably placed completely in the vessel without first chopping the sample in order to obtain good results.
  • a least -50 0 C cold sample is placed into the vessel.
  • the vessel is shaken for 10 to 30 seconds, in particular by hand, so that the movable body is hurled back and forth.
  • the thus crushed sample is removed and the desired amount is processed using a kit according to a protocol.
  • Seeds of plants can be prepared in this way for further processing. For skin and bone and comparatively hard or viscous samples, however, this method is not suitable.
  • the sample is in powder form, it advantageously has a particularly large surface area, from which subsequently used chemicals can attack.
  • a desired amount of powder can be provided particularly easily for subsequent steps, for example by weighing or even by appropriately sized measuring vessels, such as a measuring spoon.
  • Figure 1 shows in section a vessel 1, on which a cover 2 can be screwed.
  • a ball 3 which has a smaller diameter compared to the diameter of the vessel 1.
  • the diameter of the vessel is in the range of a few centimeters.
  • the interior of the vessel 1 is cylindrical. When closed, the ends are 5 and 6 of the vessel spherically shaped. By appropriate shaking of the vessel 1, the ball 3 is moved back and forth in the interior and strikes the ends 5 and 6.
  • the vessel 1, the lid 2 and the ball 3 are made of metal and that made of stainless steel.
  • the vessel is cooled in dry ice at -80 0 C.
  • a sample 7 is taken from an animal.
  • the sample is washed and immersed in nitrogen until no more bubbles form or stored in RNAIater for 24 hours, for example. Following this, the sample may initially be stored in dry ice at -80 0 C.
  • dry ice In the case of a plant dried plant tissue such as seeds in the vessel 1 is cooled on dry ice. Fresh plant material like leaves are previously cooled to -80 0 C.
  • the vessel l has been cooled to the desired temperature, this is removed by hand.
  • the hand is expediently protected from the cold with a cotton glove and a glove made of latex.
  • the sample 7 is placed in the vessel 1, in which the ball 3 is located.
  • the lid 2 is screwed onto the vessel 1 and this closed so tightly.
  • the vessel 1 is shaken by hand back and forth, so that the ball 3 impinges alternately on the two ends 5 and 6.
  • the sample 7 is thereby smashed.
  • the result is a free-flowing powder that can be initially stored in the vessel 1 further. It is unscrewed immediately or following storage of the lid 2 and removed the crushed sample. A desired amount can now be provided by weighing, for example.
  • a liver was taken from a rat and washed in PBS. Following this, the liver was stored in RNAIater® for 24 hours. Following this, the rat liver was stored at -80 ° C until further use. The liver was crushed in the cooled, closed vessel for 20 seconds as previously described. Using a funnel, the crushed sample was placed in a Falcon tube, and further stored in dry ice at -80 0 C initially.
  • the used DNeasy protocol from Qiagen includes the following steps.
  • the 1 0 mg tissue is incubated with 1 80 ⁇ l of BUFFER 1 + 20 ⁇ l proteinase K at 56 ° C for one hour in a rocking platform.
  • 4 ⁇ l RNase A are pipetted in and incubated for 2 minutes at room temperature.
  • 200 ul AL lysis buffer
  • 200 ⁇ l of ethanol (100%) are pipetted in and mixed by gentle inverting.
  • the column is washed as described in the protocol with 500 ⁇ l of buffer AW 2 for 3 minutes at 1 4000 revolutions per minute. The supernatant is again discarded.
  • the optional drying step described in the protocol is used for 1 minute at 1 4000 rpm.
  • 200 ⁇ l of RNase-free water is pipetted into the center of the column and incubated at room temperature for 1 minute, then centrifuged at 8000 rpm for 1 minute. This eluate is measured by means of a spectrometer. This serves to determine the concentration and to determine the degree of purity.
  • an agarose gel is made for qualitative and quantitative DNA determination. On this basis, the quality of the DNA, the degree of degradation, the size of the molecule and its amount can be determined.
  • the bright upper regions in FIG. 2 characterize the genomic DNA.
  • the bright area above TR is not as high as above TD +, TD- or TD / TR.
  • Figure 2 shows images of DNA agarose gels used as described above to determine the quantity and quality of the DNA.
  • a 1% gel is poured.
  • 1 g of agarose powder long-chain carbohydrate molecules that can polymerize
  • 1 00 ml (1 x) TAE buffer heated in the microwave.
  • the resulting solution is mixed with 5 .mu.l Ethid iumbromid and mixed by shaking the flask and poured into a mold.
  • the hot solution now combs are clamped, which leave as the solution cools, which polymerizes, prints in the gel, which later form the slots in which the sample mixed with 5 ⁇ l Lo ⁇ ding Dye is pipetted into it.
  • a marker is additionally added to the gel for later size comparison.
  • the DNA spreads after application of an electrical voltage due to its different weight and charge, d. H . Small degraded fragments move faster in the gel than high molecular weight DNA.
  • the DNA obtained by the method according to the invention can be seen in the form of a cloud with the naked eye.
  • Stabilization reagent has been treated, but was first frozen in liquid nitrogen. It was obtained with the aid of the method according to the invention, the double concentration of RNA compared to the known from the prior art, the method described above.
  • the supernatant was then pipetted into a new 2 ml reaction tube and 0.5X volume of 96-100% ethanol pipetted in and mixed through the pipette. Subsequently, first 700 ⁇ l of the solution were pipetted into the RNeasy column and centrifuged for 1 5 seconds at 10,000 revolutions per minute. Since the column has only 700 ⁇ l capacity, this step was repeated for the remaining solution so that the entire sample was passed over the column. The supernatant after centrifugation was discarded. Thereafter, the column was washed with 350 ul buffer RWl for 1 5 seconds at 8000 g and the supernatant discarded.
  • the column was then transferred once more to a new 2 ml reaction vessel and dried therein for 1 minute at maximum speed.
  • the column was transferred to a new 2 ml reaction vessel and filled with 30 ⁇ l of RNase-free water and centrifuged for 1 minute at 10,000 revolutions per minute to elute.
  • the eluate was then also measured by spectrometer and evaluated by RNA agarose gel.
  • FIG. 3 shows a particularly preferred embodiment of a closable vessel with which a sample is pulverized.
  • This vessel is called pulverizer below. It comprises an inlay 10, which preferably consists of metal for the reasons stated above.
  • the inlay 10 is comprised of a shell 11 preferably made of plastic.
  • the plastic shell serves primarily as an insulator to maintain the low temperature during Scblinins. Overall, the weight can be advantageously reduced in comparison to a completely made of metal pulverizer, which facilitates handling.
  • the double arrow below the pulverizer shown in FIG. 3 illustrates the preferred direction of movement in order to comminute the sample 7.
  • the buffers and reagents mentioned in the present application can be used in the Fa. Qiagen GmbH, Hilden, Germany, unless otherwise expressly stated.
  • FIG. 4 shows a section through a further improved embodiment of the invention.
  • the pulverizer comprises an inner sealable container 4 made of plastic.
  • the plastic inner container adjacent to so closely to the preferably made of metal inner walls of the pulverizer, so that the plastic inner container can not be destroyed by a ball 3 or a similar means during pulverization.
  • the plastic inner container 4 is removed after pulverization and can now serve as a storage vessel. Since the inner container is made of plastic, this can be made very inexpensive and therefore suitable for single use.
  • the lid of the inner container closes the remaining part of the container preferably by a positive connection, or it can be screwed onto the remaining part.
  • a positive connection is possible because plastic can be sufficiently elastic, so that, for example, an inwardly projecting bead of the lid snaps into a designated annular recess of the rest of the container body or vice versa, when the lid is pressed to the rest of the container body suitable.
  • Such a positive connection is particularly preferable because accidental release of the lid is particularly reliably avoided.
  • a container can be closed faster compared to a closure with a screw cap.
  • a set comprises, in addition to a pulverizer, a multiplicity of inner containers, which are preferably optically different, for example because of differently colored covers or different embossments.
  • the different optics can be used advantageously for the identification of a content.
  • a red-colored container lid can be used to mark a "heart" located therein and another lid, for example a green-colored lid, to identify another organ, such as a lung, for example no longer be labeled separately, which can be problematic due to the low temperatures.
  • the existing plastic plastic inner container can be made of PET, but also made of PP or PE, since such plastics are grown to the intended low temperatures in principle.
  • a kit in one embodiment, includes one or more measuring spoons besides a pulverizer so as to facilitate or accelerate the removal of the required amount of sample from the pulverizer or from an inner container.
  • a measuring spoon is so dimensioned and assigned to certain samples that the measuring spoon is capable of receiving a suitable amount of sample for further processing.
  • a separate weighing of a sample taken from the pulverizer can thus be accelerated or even completely eliminated.
  • a set comprises a plurality of optically labeled measuring spoons associated with different samples.
  • a red-colored measuring spoon can be provided for the removal of a heart sample.
  • the identifications of inner containers or parts of inner containers coincide with the identifications of measuring spoons.
  • Such a set then also includes a predetermined assignment of identifiers to samples, that is, for example, to organs. If, for example, a green-colored measuring spoon is provided for the organ lung, then an inner container is likewise completely or partially dyed green. The dimensioning of the measuring spoon is adapted to the sample "lung.”
  • the set then contains a corresponding assignment rule, which can be that a corresponding sample, for example a lung, is already displayed on the corresponding container and / or the measuring spoon.
  • a set comprises, in addition to a pulverizer, a shaking apparatus to facilitate the opening of an imprinter Automated Pulveris ⁇ tor sample to perform automated.
  • a shaking apparatus to facilitate the opening of an imprinter Automated Pulveris ⁇ tor sample to perform automated.
  • One or more Pulverisatoren can be used in the shaker or attached to this suitable.
  • the shaking apparatus comprises coolable receiving devices for receiving pulverizers.
  • the shaking apparatus does not need to be digested immediately after filling the sample in the pulverizer, if it can be sufficiently cooled. So it can be a variety of

Landscapes

  • Engineering & Computer Science (AREA)
  • Food Science & Technology (AREA)
  • Sampling And Sample Adjustment (AREA)
  • Investigating Or Analysing Biological Materials (AREA)
  • Peptides Or Proteins (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

L'invention concerne un procédé de préparation d'un échantillon végétal ou animal pour un traitement, par exemple, pour isoler des acides nucléiques ou des protéines issus de l'échantillon. L'invention concerne également un pulvérisateur.
EP08735665.5A 2007-04-04 2008-04-02 Pulvérisateur et procédé de préparation du traitement d'un échantillon biologique Not-in-force EP2129467B1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE102007016221A DE102007016221A1 (de) 2007-04-04 2007-04-04 Pulverisator und dazugehöriges Verfahren für die Vorbereitung zur Prozessierung einer biologischen Probe
PCT/EP2008/053896 WO2008122550A2 (fr) 2007-04-04 2008-04-02 Pulvérisateur et procédé de préparation du traitement d'un échantillon biologique

Publications (2)

Publication Number Publication Date
EP2129467A2 true EP2129467A2 (fr) 2009-12-09
EP2129467B1 EP2129467B1 (fr) 2014-07-02

Family

ID=39736155

Family Applications (1)

Application Number Title Priority Date Filing Date
EP08735665.5A Not-in-force EP2129467B1 (fr) 2007-04-04 2008-04-02 Pulvérisateur et procédé de préparation du traitement d'un échantillon biologique

Country Status (5)

Country Link
US (2) US8348183B2 (fr)
EP (1) EP2129467B1 (fr)
JP (1) JP5150880B2 (fr)
DE (1) DE102007016221A1 (fr)
WO (1) WO2008122550A2 (fr)

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AU2012346112B2 (en) * 2011-11-28 2017-03-02 Syngenta Participations Ag DNA extraction from seeds using osmoticum
FR2991305B1 (fr) * 2012-06-01 2015-05-01 Assist Publ Hopitaux De Paris Dispositif pour le recueil, le traitement preanalytique, le transport et le broyage d'echantillons solides.
FR3091987B1 (fr) * 2019-01-29 2021-12-03 Peugeot Saveurs moulin a produits condimentaires
US11774328B2 (en) * 2019-02-26 2023-10-03 SPEX SamplePrep, LLC Homogenizer and method of grinding large sample quantities
JP7416496B1 (ja) * 2023-03-03 2024-01-17 株式会社小泉製作所 粉砕器具
JP2024180257A (ja) * 2023-06-16 2024-12-26 株式会社小泉製作所 粉砕器具
CN117511740B (zh) * 2024-01-08 2024-05-10 山东伯桢生物科技有限公司 组织解离装置及用于组织解离装置的控制方法

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Also Published As

Publication number Publication date
US20130026268A1 (en) 2013-01-31
WO2008122550A3 (fr) 2009-03-19
DE102007016221A1 (de) 2008-10-09
US8348183B2 (en) 2013-01-08
US20100137567A1 (en) 2010-06-03
JP2010523956A (ja) 2010-07-15
EP2129467B1 (fr) 2014-07-02
JP5150880B2 (ja) 2013-02-27
WO2008122550A2 (fr) 2008-10-16

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