WO2014111064A1 - Procédé de manipulation d'échantillons de matériau biologique et dispositif pour une telle manipulation - Google Patents

Procédé de manipulation d'échantillons de matériau biologique et dispositif pour une telle manipulation Download PDF

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Publication number
WO2014111064A1
WO2014111064A1 PCT/CZ2013/000005 CZ2013000005W WO2014111064A1 WO 2014111064 A1 WO2014111064 A1 WO 2014111064A1 CZ 2013000005 W CZ2013000005 W CZ 2013000005W WO 2014111064 A1 WO2014111064 A1 WO 2014111064A1
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WO
WIPO (PCT)
Prior art keywords
chamber
section
manipulation
mixing
biological material
Prior art date
Application number
PCT/CZ2013/000005
Other languages
English (en)
Inventor
Jan WOLF
Jaroslav Beran
Jiri KOMAREK
Jozef KANIOK
Martin BILEK
Martin Konecny
Petr Zabka
Original Assignee
Wolf & Danniel S.R.O.
Technicka Univerzita V Liberci
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 Wolf & Danniel S.R.O., Technicka Univerzita V Liberci filed Critical Wolf & Danniel S.R.O.
Priority to EP13709303.5A priority Critical patent/EP2945739A1/fr
Publication of WO2014111064A1 publication Critical patent/WO2014111064A1/fr

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Classifications

    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/06—Auxiliary integrated devices, integrated components
    • B01L2300/0672—Integrated piercing tool
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/06—Auxiliary integrated devices, integrated components
    • B01L2300/0681—Filter
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0832—Geometry, shape and general structure cylindrical, tube shaped
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0861—Configuration of multiple channels and/or chambers in a single devices
    • B01L2300/0867—Multiple inlets and one sample wells, e.g. mixing, dilution
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0861—Configuration of multiple channels and/or chambers in a single devices
    • B01L2300/087—Multiple sequential chambers
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00—Moving or stopping fluids
    • B01L2400/04—Moving fluids with specific forces or mechanical means
    • B01L2400/0403—Moving fluids with specific forces or mechanical means specific forces
    • B01L2400/0409—Moving fluids with specific forces or mechanical means specific forces centrifugal forces
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00—Moving or stopping fluids
    • B01L2400/06—Valves, specific forms thereof
    • B01L2400/0622—Valves, specific forms thereof distribution valves, valves having multiple inlets and/or outlets, e.g. metering valves, multi-way valves
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00—Moving or stopping fluids
    • B01L2400/06—Valves, specific forms thereof
    • B01L2400/0633—Valves, specific forms thereof with moving parts
    • B01L2400/0644—Valves, specific forms thereof with moving parts rotary valves

Definitions

  • the invention relates to the method of manipulation with samples of biological material, especially for molecular diagnostics or for isolation of nucleic acids and from them possible consequent detection of a certain genome, for example a pathogenic one, and/or diagnostics of genetically determined diseases of living organisms.
  • the invention relates to the device for manipulation with samples of biological material, especially for molecular diagnostics or for isolation of nucleic acids and from them possible consequent detection of a certain genome, for example a pathogenic one, and/or diagnostics of genetically determined diseases of living organisms.
  • molecular in vitro diagnostics based on a direct proof of the targeted DNA sequencing is one of the most progressively increasing segments in market of laboratory in vitro diagnostics.
  • diagnostics of tuberculosis where it superseded a lengthy (3-9 weeks) and less sensitive (104 bacteria / 1ml of sample) microscopic examination, further in diagnostics of sexually transmitted diseases (chlamydial infections, syphilis) or in diagnostics of virus diseases with a low antibody response or too fast progression (viral hepatitis B and C, herpetic meningoencephalitis, cytomegalovirus).
  • the goal of the invention is to propose and create a method of manipulation and device for manipulation with samples of biological material, which would totally prevent a possible contact of an operating person with the sample or the reagent chemicals and would prevent whatever leakage of reactive agents outside the device for manipulation.
  • the invention should enable transfer of molecular diagnostics from laboratories directly to the site where the patient is present, this is the Point of care testing (POCT) as a result of simplified manipulation with samples, when it is possible to process each sample independently upon preserving a laboratory quality and shortening of time till achieving the result to a possible minimum, which at present represents 2-4 hours.
  • POCT Point of care testing
  • the goal of the invention has been achieved by the method according to the invention, whose principle consists in that the sample of biological material at the beginning of the process is inserted into one chamber of a manipulation device, into whose other chambers all reagent chemicals necessary for the respective process separately one from another were deposited still before, while the following phases of the process are running inside the manipulation device without a direct manipulation of operating person with reagent chemicals, which are gradually mixed and react, at the same time the unwilling residues from reactions are deposited into the waste area, which is also situated inside the manipulation device and it is safely separated from other areas of manipulation device, operating persons as well as environment.
  • This facilitates method of manipulation with samples of biological material and enables to perform such manipulation directly on site where the patient is present - point of care testing (POCT), instead in laboratories.
  • POCT point of care testing
  • the sample of biological material inside the manipulation device in its mixing chamber mixes with lysing enzyme and simultaneously or subsequently from the respective storage chamber of the manipulation device the lysis buffer is added and after the next mixing the mixture in the mixing chamber is subject to incubation, during which the solution of mixture is repeatedly mixed and lysis in the solution is running till a lysed mixture is created, subsequently to the lysed mixture from another storage chamber of the manipulation device a precipitation agent is brought, preferably ethanol or isopropanol, after which the lysed mixture with precipitation agent is subject to homogenisation and during reaction with precipitation agent the free nucleic acid agglutinates, i.e. its precipitation occurs.
  • a precipitation agent is brought, preferably ethanol or isopropanol
  • the mixture is brought to a separating membrane arranged inside the manipulation device, through which it is filtrated into the waste area, created inside the manipulation device, where the separating membrane retains residues of cell structures and the precipitate nucleic acid, after which the residues of cell structures from separating membrane are removed by flushing with a washing buffer, being brought from another storage chamber of the manipulation device, after then the waste area is closed and to separating membrane from another storage chamber of the manipulation device the elution buffer is brought, which is left to act to the separating membrane and subsequently passes through the separating membrane into the reaction chamber of the manipulation device, while in the solution the corresponding nucleic acid from the examined sample of biological material is isolated. That device, that all reactions necessary to isolate the corresponding nucleic acid from the examined sample of biological material are running inside the manipulation device.
  • the incubation of the mixture is running at a temperature higher than the ambient temperature, preferably 70 °C for a period of 10 to 30 minutes.
  • the sample of biological material is brought into a mixing chamber, into which the lysing enzyme was situated before.
  • the lysing enzyme is a part of the lysis buffer.
  • Accelerated passage of mixture through the separating membrane is achieved by action of a centrifugal force.
  • the separating membrane is formed of an ion exchange membrane.
  • a polymerase chain reaction may be running, while its results can be easily detected through the wall of reaction chamber, which is preferably transparent. In case of a non- transparent wall of reaction chamber the detection can be performed using radioactive radiation.
  • the goal of the invention has also been achieved through the device for manipulation with samples of biological material according to the invention, whose principle consists in that, in one enclosed unit it contains the storage chambers for mutually separated releaseable deposition of reagent chemicals, loading chamber for inserting the sample of biological material, to which there is assigned the mixing chamber, to whose outlet the filtration chamber can be assigned, to whose outlet through the separating membrane the waste chamber or reaction chamber can be assigned, while only the loading chamber is provided with openable and lockable lid for operating person.
  • the device for manipulation with samples of biological material whose principle consists in that, in one enclosed unit it contains the storage chambers for mutually separated releaseable deposition of reagent chemicals, loading chamber for inserting the sample of biological material, to which there is assigned the mixing chamber, to whose outlet the filtration chamber can be assigned, to whose outlet through the separating membrane the waste chamber or reaction chamber can be assigned, while only the loading chamber is provided with openable and lockable lid for operating person.
  • the storage and loading chambers are arranged in the storage section, which extends to the mixing section equipped with mixing chamber, to whose inlet individual chambers of the storage section may be optionally assigned, which can be coupled with opening means for releasing of its content into the mixing chamber, while to outlet of mixing chamber there can be assigned the inlet of filtration chamber of filtration section, to whose outlet there can be optionally assigned the waste chamber or reaction chamber of reaction section, the storage section, while the mixing section, filtration section and reaction section are formed of independent bodies, which are mutually connected, at the same time the connection enables mutual motion and secures tightness.
  • the separating membrane is formed of ion exchange membrane.
  • the storage chambers and the loading chamber in the lower part are equipped with protrusions, to which there is assigned the opening means, which is preferably formed of knife-edge firmly mounted in the mixing chamber. Cutting of the lower part of the protrusion of corresponding chamber secures releasing of its content and its transport into the mixing chamber and in a case this is already opened, transport into further parts of the device.
  • FIG. 1 shows a longitudinal section through the first exemplary embodiment of device for manipulation with samples of biological material
  • Fig. 2 section A-A from the Fig. 1 Fig. 3 section B-B from the Fig. 1
  • Fig. 4 section C-C from the Fig. 1
  • Fig. 5 view to the first exemplary embodiment of the device
  • Fig. 6 section through the second exemplary embodiment of the device.
  • the device for manipulation with samples of biological material especially for molecular diagnostics or for isolation of nucleic acids and from them possible consequent detection of a certain genome, for example a pathogenic one, and/or diagnostics of genetically determined diseases of living organisms will be described on examples of embodiment, which serve to explain the structure and function of the device, not restricting the solution to such embodiments only.
  • the device for manipulation is illustrated and described in a vertical position, which does not preclude a possibility to be in a different position during its function.
  • the device for manipulation comprises four basic sections, which in the represented embodiment are mutually rotatably connected.
  • the storage section 1 which in direction downwards extends to the mixing section 2, which in direction downwards extends to the filtration section 3, under which the waste and reaction section 4 is positioned.
  • Individual sections 1, 2, 3, 4 are mutually rotatably connected by means of locks 5, formed of protrusion 51 on the inner from sections being connected and a groove 52 in the outer from sections being connected.
  • Individual sections 1 , 2, 3, 4 are simultaneously mutually sealed, e.g. by means of o-rings 6 represented in Fig. 1 or by means of other suitable manner. This prevents the reaction mixture to leak outside the specified space of the device or to surroundings and unwilling mixing of residues from reactions and a result of reactions.
  • the storage section i is positioned in a sleeve created in the mixing section 2, so that the protrusion 51 of the lock 5 is created on circumference of the storage section 1_ and the groove 52 of the lock is created in the mixing section 2.
  • the mixing section 2 on its outlet side is provided with a sleeve in which the filtration section 3 is positioned, so that the protrusion 51 of the lock is created on circumference of the filtration section 3 and the groove 52 of the lock is created in the mixing section 2.
  • Filtration section 3 on its outlet side is provided with a sleeve, in which the waste and reaction section 4 is positioned, so that the protrusion 5j of the lock 5 is created on circumference of the waste and reaction section 4 and the groove 52 of the lock is created in the filtration section 3.
  • the device for manipulation with samples of biological material during manipulation are subjected to action of centrifugal force in direction from the storage section ⁇ towards the waste and reaction section 4, advantageous is an opposite mutual arrangement of individual sections 2, 3, 4. That means that upon connection of the filtration section 3 of the waste and reaction section 4 the sleeve is created in waste and reaction section 4 and in this sleeve the filtration section 3 is positioned, so that the protrusion 51 of the lock is created on circumference of the filtration section 3 and the groove 52 of the lock is created in the waste and reaction section 4.
  • Filtration section 3 on its inlet side is provided with the sleeve, in which the mixing section 2 is positioned, so that the protrusion 51 of the lock is created on circumference of the mixing section 2 and the groove 52 of the lock is created in the filtration section 3.
  • Connection of the mixing section 2 and the storage section in this manner is already performed in the represented embodiment. Nevertheless this connection may be created also in opposite manner, that means the sleeve may be created on the storage section 1 in which the mixing section 2 is positioned. Protrusion 51 of the lock is then created on circumference of the mixing section 2 and the groove 52 of the lock is created in the storage section 2.
  • the storage section 1 comprises a loading chamber 11 for inserting of the examined sample of biological material, which is provided with an openable lid 110.
  • the storage section 1 further comprises the storage chambers 12, of which in the represented embodiment are seven pieces and which serve for mutually separated releaseable deposition of reagent chemicals and after inserting reagent chemicals they are closed with a common fixed lid 120.
  • the storage section 1_ in the represented embodiment is formed of upper cylindrical section, which downwards narrows into a lower cylindrical section, under which the loading chamber H is terminated with a drop-like protrusion 111 and individual storage chambers 12 are terminated with drop-like protrusions 1210 to 1270, whose wall in the preferable embodiment is made thinner.
  • two positioning protrusions 13 are performed, which serve for positioning the device for manipulation with samples of biological material during diagnostics, for example in centrifuge.
  • Inlet area of the mixing section 2 is formed of a cylindrical cavity 21 , in which there is positioned a sieve • 22, in which the knife 23 is positioned extending with its edge between the droplike protrusions 112, 122 of chambers 11, 12 of the storage section 1 and serving for cutting their ends.
  • a sieve • 22 in which the knife 23 is positioned extending with its edge between the droplike protrusions 112, 122 of chambers 11, 12 of the storage section 1 and serving for cutting their ends.
  • the pinion 24 is performed around the cylindrical cavity 21 on circumference of the mixing section.
  • the mixing chamber 25 is performed, which in direction downwards narrows up to outlet opening 26, which is performed in a bottom 27 of the mixing section 2.
  • a sealing in the represented embodiment formed of an o-ring 6, which fits the lid 31 of filtration section 3 and prevents leakage of reagents from the mixing chamber 25, until its outlet opening 26 upon mutual rotating the mixing section 2 and filtration section 3 gets above the inlet opening 321 of filtration chamber 32.
  • Filtration section 3 in its principle features a cylindrical shape, while the filtration chamber 32 in the represented embodiment is formed of a cylindrical cavity and the remaining inner space is formed of a lightening cavity 33.
  • filtration chamber 32 at least one separating membrane 322 is positioned.
  • two separating membranes 322 are positioned, among which a separating annulus 323 is mounted. Separating annulus 323 according to the represented embodiment is also arranged above the upper separating membrane 322.
  • an outlet opening 324 is performed in bottom of filtration chamber 32.
  • Waste and reaction section 4 comprise a waste chamber 41 , which in the represented example of embodiment features a shape of an enlarged crescent, and the reaction chamber 42, which is formed of a cylindrical cavity narrowing in downwards direction, while the lower part of reaction chamber 42 protrudes from a system of the device for manipulation downwards, its wall is made thinner and from all sides is freely accessible both for optical and for other evaluation means.
  • waste and reaction section 4 On circumference of waste and reaction section 4 two protrusions 13 are created which serve for positioning the device for manipulation with samples of biological material during diagnostics, for example in a centrifuge.
  • the pinion 24 on the mixing section which in a known manner may be coupled with a not represented control tooth wheel or worm gear, while the storage section 1_ and waste and reaction section 4 feature a stable position, in the represented embodiment given by engaging their circumferential protrusions 13 into corresponding recesses in the not represented bushing.
  • the mixing section 2 is provided with protrusion 28, in embodiment according to Fig.
  • a circumferential protrusion which extends into the groove 34 finished with a stop 341 , which specifies angle of rotation of the mixing section 2 towards the filtration section 3.
  • the groove 34 is created on circumference of the filtration section 3.
  • a basic position of filtration section 3 towards the waste and reaction section 4 is specified by the protrusion 35, which extends into a recess between a pair of stops 44, which at the embodiment according to Fig. 1 to 5 are created on circumference of the waste and reaction section 4 similarly like between them extending protrusion 35 created on circumference of filtration section 3.
  • Embodiment of positioning protrusions, grooves and stops represented in Fig. 1 to 5 is exemplary and serves to explain the structure and function of the device, and not to its restriction. Protrusions, grooves and stops may be performed in any other suitable manner.
  • the device for manipulation with samples of biological material differs from the previous embodiment by another particular connection of the mixing section 2, filtration section 3 and waste and reaction section 4.
  • the mixing section 2 on its outlet side is provided with a sleeve, in whose cylindrical cavity the filtration section 3 is rotatably mounted by its inlet section.
  • Outlet side of filtration section 3 is rotatably mounted in a sleeve created on inlet side of the waste and reaction section 4, while this sleeve of filtration section 3 outreaches up to the mixing section 2, which it embraces and which is mounted inside it with its outer circumference.
  • Protrusion 51_ of the lock is created on circumference of the mixing section 2 and the groove 62 of the lock is created in the waste and reaction section 4.
  • the filtration section 3 is mounted inside the mixing section 2 and the waste and reaction sections 4 without locks. This embodiment is more simple and less expensive as to manufacture.
  • waste and reaction section 4 is provided with auxiliary waste groove 43, which serves for trapping of possible residues from reactions, in case they get between bottom of the filtration section 3 and upper surface of waste and reaction section 4.
  • the device for manipulation with samples of biological material has been designed for carrying out a quick and effective diagnostics of infectious and genetically determined diseases of a human being in the POCT (Point of care testing) mode in premises with fully automatic operation in all phases of diagnostics, simple attendance and high safeness for operating personnel as well as environment.
  • the device for manipulation with samples of biological material is in advance filled with all necessary reagents, which are enclosed in individual storage chambers 12 of the storage section ⁇ .
  • the below mentioned example describes a device for manipulation with samples of biological material with seven storage chambers 12 and one loading chamber 1_1 in the storage section
  • the operating person inserts a sample of biological material into the loading chamber 11. and closes the loading chamber with the openable lid 111.
  • the lysing enzyme e.g. proteinase K is stored.
  • the lysis buffer that is formed of sodium dodecylsulphate and chelating agents.
  • a precipitation agent in this particular case, ethanol is stored.
  • the fourth storage chamber 124 in the described example of embodiment is empty.
  • the first washing buffer in the described exemplary embodiment formed of seventy percent ethanol and stabilising ions.
  • the second washing buffer in the described exemplary embodiment formed of seventy percent ethanol and stabilising ions.
  • elution buffer which is formed of solution with a low concentration of ions Tris- hydroxymethylaminomethane + chelating agent EDTA (ethylenediaminetetraacetic acid).
  • Position of the device for manipulation with samples of biological material is given by position of positioning protrusions 13 of the storage section and positioning protrusions 13 of waste and reaction section into a respective recesses in the not represented centrifuge, while in the represented and described embodiment the positioning protrusions 13 prevent rotation of the storage section 1 and rotation of the waste and reaction section 4.
  • the mixing section 2 and filtration section 3 upon manipulation with samples of biological material rotate, at the same time the torque is transferred to them by means of a pinion 24.
  • the device for manipulation with samples if biological material is positioned into an apparatus, in which the whole diagnostic process is running and which usually is centrifuge.
  • the pinion 24 is coupled with a known driving member of a not represented drive, e.g. with worm gear or toothed wheel.
  • Individual sections of the device for manipulation with samples of biological material are in initial position, in which the knife is to be found before the protrusion 111 of loading chamber 11_, the outlet opening 26 of mixing chamber 25 is closed by the lid 3J. of filtration section 3, and the outlet opening 324 of filtration chamber 32 is to be found above the waste chamber 41.
  • the mixing section 4 is turned by 45°, while the other sections are not moving. Thereby the lower part of the drop-like protrusion 110 of loading chamber 11. is cut off and its content formed of an examined sample flows into the mixing chamber 25, while the cut off lower part of the drop-like protrusion 110 is caught on the sieve 22.
  • the outlet opening 26 has been shifted by 45° above the full part of the lid 31 of filtration section 3_and it is still closed.
  • the mixing section 2 is turned by another 45°, while the other sections are not moving.
  • the drop-like protrusion 1210 of the first storage chamber 121 in which the lysing enzyme is positioned e.g. proteinase K
  • Outlet opening 26 is shifted by another 45° above the full part of the lid 31 of filtration section 3_and its is still closed.
  • Lysing enzyme e.g. proteinase K is transferred into the mixing chamber 25, preferably under action of centrifugal force, and the device for manipulation with samples of biological material is heated to temperature higher than the ambient temperature, preferably to 70 °C, through which possibly precipitated components are dissolved.
  • the mixing section 2 is turned by another 45°, while the other sections are not moving. Total turning of the mixing section 2 is 135°. Thereby the drop-like protrusion 1220 of second storage chamber 122 in which the lysis buffer is positioned, is cut off. Outlet opening 26 is shifted by another 45° above the full part of the lid 31 of filtration section 3 and it is still closed. Lysis buffer is transferred into the mixing chamber 25, preferably under action of centrifugal force, and after a complete mixing of examined sample of biological material with enzyme and lysis buffer the lysis process is started, this is degradation of cell walls and releasing of DNA into a solution.
  • the solution is repeatedly mixed.
  • the mixing section 2 is turned by another 45° to total 180°.
  • the drop-like protrusion 1230 of the third storage chamber is cut off and the precipitation agent, in the described example of embodiment ethanol, is transported from the third storage chamber 1230.
  • Quality and speed for transporting the precipitation agent into the mixing chamber 25 is increased by rotating the centrifuge to cca 500 g.
  • the mixture is thoroughly mixed.
  • a thorough homogenisation and mixing secures a correct bond of DNA to separating membrane 322, in described exemplary embodiment to ion exchange membrane.
  • Outlet opening 26 has been shifted by another 45° above the full part of the lid 31 of filtration section 3_and it is still closed.
  • the mixing section turns by another 45°to total 225° and the knife cuts off the drop-like protrusion 1240 of the fourth storage chamber 124, which in the exemplary embodiment is empty.
  • Outlet opening 26 of the mixing chamber 25 has been shifted by another 45° and it is positioned above the inlet opening 321 of filtration chamber 32, whose outlet opening 324 still is positioned above the waste chamber 4_1 of waste and reaction section 4, as the filtration section 3 was not moving till this time.
  • Protrusion 28 on the mixing section 2 engaged the stop 341 at the end of the groove 34 in filtration section 3, through which these sections are connected and then they will be turning together. Lysing mixture homogenised with ethanol transfers from the mixing chamber 25 into filtration chamber to the separating membrane 322, under which the waste chamber 41 is to be found.
  • the lysing mixture is filtered through a separating membrane 322 into the waste chamber 41 of waste and reaction section 4, while the separating membrane 322 retains residues of cell structures and DNA.
  • the mixing section 2 is turned again by another 45° to total displacement of 270°, and due to this the filtration section 3 turns by 45°, as it is carried away by the protrusion 28 and by the stop 341.
  • the protrusion 35 on filtration section overcomes a force of a pair of stops 44, which till this time specified its position.
  • Outlet opening 26 of the mixing chamber 25 is still to be found above the inlet opening 321 of filtration chamber 32 and the outlet opening of filtration chamber 32 is above the waste chamber 41 of waste and reaction section 4.
  • the knife 23 cuts off the drop-like protrusion 1250 of the fifth storage chamber 125 and from it into the mixing chamber 25 the first washing buffer runs out, which flows through into the filtration chamber 32 above the separating membrane 322.
  • the first washing buffer flows through the separating membrane 322 into the waste chamber 41 of waste and reaction section 4. Thereby the separating membrane 322 is washed and residues of cell structures except for DNA are eliminated.
  • the mixing section 2 is turned by another 45°, simultaneously the mixing section turns also the filtration section 3 by 45°.
  • Outlet opening 26 of the mixing chamber 25 is still above the inlet opening 321 of filtration chamber 32, because the filtration section 3 is carried away by the mixing section 2 ⁇ Outlet opening 324 of filtration chamber 32 though turned by 45°still is to be found above the waste chamber 4JL
  • the knife 23 cuts off the drop-like protrusion 1260 of the sixth storage chamber 126, from which the second washing buffer is released, which also flows through into the filtration chamber 32 above the separating membrane 322.
  • the second washing buffer flows through the separating membrane 322 upon centrifugation of 5000 to 15000 g. Thereby the separating membrane 322 is washed once again, and the residues of cell structures except for DNA are definitely eliminated.
  • At least the middle section of the device for manipulation with samples of biological material is heated above the ambient temperature, preferably to the temperature of 70 °C.
  • the outlet opening 324 of filtration chamber shifts above the waste chamber 41 and the reaction chamber 41 closes, so that the polymerase chain reaction (PCR) may run inside the chamber or the isolated DNA may be taken out of it for another use.
  • PCR polymerase chain reaction runs in a standard mechanism of cyclic cooling and heating the mixture in reaction space.
  • RNA in the same method isolation of RNA as well as general molecular diagnostics is performed.
  • Lysing enzyme may be added into the loading chamber 11_ together with sample of biological material or vice versa the sample may be added into the loading chamber H to lysing enzyme.
  • the first storage chamber is empty.
  • the knife 23 in one step turns by 90°, so as to cut off protrusion of the loading chamber V ⁇ with lysing enzyme and a sample and simultaneously a protrusion of empty storage chamber.
  • Operating person inserts into the loading chamber 11 a sample of biological material together with lysing enzyme, e.g. proteinase K, and closes the loading chamber with the openable lid 111.
  • lysing enzyme e.g. proteinase K
  • lysis buffer is stored, which is formed of a mixture of sodium dodecylsulphate and chelating agents.
  • the precipitation agent is stored, in particular case ethanol.
  • the first washing buffer is stored, in the described exemplary embodiment formed of seventy percent ethanol and stabilising ions.
  • the second washing buffer is stored, in the described exemplary embodiment formed of seventy percent ethanol and stabilising ions.
  • the sixth storage chamber 126 there is stored inert liquid and/or elution buffer in a quantity corresponding to residual content of liquids in separating membrane.
  • elution buffer which is formed of a solution with low concentration of ions Tris- hydroxymethylaminomethane + chelating agent EDTA (ethylenediaminetetraacetic acid).
  • the mixing section 4 is turned by 45°, while the other sections are not moving. Thereby the lower part of drop-like protrusion 110 of loading chamber H is cut off and its content formed of an examined sample and proteinase K flows into the mixing chamber 25.
  • the outlet opening 26 of mixing chamber 25 has been shifted by 45° above a full part of the lid 3_1 of filtration section 3_and it is still closed.
  • the centrifuge is shortly brought to rotation to cca 500 g.
  • the device for manipulation with samples of biological material is heated to temperature higher than the ambient temperature, preferably to 70 °C, through which possibly precipitated components are dissolved.
  • the mixing section 2 is turned by another 45°, while the other sections are not moving. Thereby the drop-like protrusion 1210 of the first storage chamber 121 in which the lysis buffer is stored, is cut off. The outlet opening 26 is still closed. Lysis buffer is transferred into the mixing chamber 25, preferably under action of centrifugal force, and after a complete mixing of examined sample of biological material with lysing enzyme and lysis buffer the lysis process is started, this is degradation of cell walls and releasing of DNA into a solution.
  • the solution is repeatedly mixed. After incubation the temperature, at least in the middle section of the device for manipulation with samples of biological material, is reduced to laboratory temperature (20 to 25 °C).
  • the mixing section 2 is turned by another 45°, while the other sections are not moving. Total turning of the mixing section 2 is 135°. Thereby the drop-like protrusion 1220 of second storage chamber 22 in which the precipitation agent is stored, is cut off. Outlet opening 26 is shifted by another 45° above the full part of the lid 31 of filtration section 3 and it is still closed. Quality and speed of transport of the precipitation agent into the mixing chamber 25 is increased by rotating the centrifuge to cca 500 g. The mixture is thoroughly mixed. Subsequently a thorough homogenisation and mixing secures a correct bond of DNA to separating membrane 322, in described exemplary embodiment to ion exchange membrane. Outlet opening 26 has been shifted by another 45° above the full part of the lid 3J. of filtration section 3_and it is still closed.
  • the mixing section 2 is turned by another 45° to total 180°. Thereby the drop-like protrusion 1230 of the third storage chamber is cut off, in the exemplary embodiment this chamber is empty.
  • Outlet opening 26 of the mixing chamber 25 has been shifted by another 45° and it is to be found above the inlet opening 321 of filtration chamber 32, whose outlet opening 324 is still positioned above the waste chamber 41 of waste and reaction section 4, as the waste and reaction section 4 was not moving till this time.
  • Protrusion 28 on the mixing section 2 engaged the stop 341 at the end of the groove 34 in filtration section 3, through which these sections are connected and then they will be turning together. Lysing mixture homogenised with ethanol transfers from the mixing chamber 25 into filtration chamber to the separating membrane 322, under which the waste chamber 41 is to be found.
  • the lysing mixture is filtered through a separating membrane 322 into the waste chamber 41 of waste and reaction section 4, while the separating membrane 322 retains residues of cell structures and DNA.
  • the mixing section turns by another 45°to total 225° due to which the filtration section 3 turns by 45°, as it is carried away by the protrusion 28 and by the stop 341.
  • Outlet opening 26 of the mixing chamber 25 is still to be found above the inlet opening 321 of filtration chamber 32 and the outlet opening of filtration chamber 32 is above the waste chamber 41 of waste and reaction section 4.
  • the knife 23 cuts off the drop-like protrusion 1240 of the fourth storage chamber 124 and from it into the mixing chamber 25 the first washing buffer runs out, which flows through into the filtration chamber 32 above the separating membrane 322.
  • the first washing buffer flows through the separating membrane 322 into the waste chamber 41 of waste and reaction section 4. Thereby the separating membrane 322 is washed and residues of cell structures except for DNA are eliminated.
  • the mixing section 2 is again turned by another 45° to total displacement of 270° and the mixing section turns also the filtration section 3 by 45°.
  • Outlet opening 26 of the mixing chamber 25 is still above the inlet opening 321 of filtration chamber 32, because the filtration section 3 is carried away by the mixing section 2.
  • Outlet opening 324 of filtration chamber 32 though turned by 45°still is to be found above the waste chamber 41.
  • the knife 23 cuts off the drop-like protrusion 1250 of the fifth storage chamber 125, from which the second washing buffer is released, which also flows through into the filtration chamber 32 above the separating membrane 322.
  • the second washing buffer flows through the separating membrane 322 upon centrifugation of 5000 to 15000 g. Thereby the separating membrane 322 is washed once again, and the residues of cell structures except for DNA are definitely eliminated.
  • the mixing section 2 is turned by another 45°, simultaneously the mixing section turns also the filtration section 3 by 45°.
  • Outlet opening 26 of the mixing chamber 25 is still above the inlet opening 321 of filtration chamber 32, because the filtration section 3 is carried away by the mixing section 2.
  • Outlet opening 324 of filtration chamber 32 though turned by 45°still is to be found above the waste chamber 41.
  • the knife 23 cuts off the drop-like protrusion 1260 of the sixth storage chamber 126, from which inert liquid and/or elution buffer in a quantity corresponding to residual content of liquids in the separating membrane 322 flows out and flows through into the filtration chamber 32 above the separating membrane 322.
  • This inert liquid and/or elution buffer flows through the separating membrane 322 at centrifugation of 5000 to 15000 g and expels the residual liquids in separation membrane 322, which after then cannot affect further reactions. Thus residues of cell structures except for DNA are completely eliminated.
  • At least the middle section of the device for manipulation with samples of biological material is heated above the ambient temperature, preferably to the temperature of 70 °C.
  • Method and device for manipulation with samples of biological material according to the invention are designed for molecular diagnostics or for isolation of nucleic acids and from them possible consequent detection of a certain genome, for example a pathogenic one, and/or diagnostics of genetically determined diseases of living organisms, for example for automatic isolation of DNA/RNA from a sample, automatic detection by qPCR method and automatic evaluation of the result of reaction outside specialised laboratories, this is on site directly - Point of care testing (POCT).
  • POCT Point of care testing

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  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Analytical Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Hematology (AREA)
  • Clinical Laboratory Science (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)

Abstract

L'invention concerne un procédé de manipulation d'échantillons de matériau biologique, en particulier pour des diagnostics moléculaires ou pour l'isolation d'acides nucléiques et, à partir de ceux-ci, l'éventuelle détection résultante d'un certain génome, par exemple pathogène, et/ou des diagnostics de maladies déterminées génétiquement d'organismes vivants. L'échantillon de matériau biologique, au début du processus, est inséré dans la chambre (11) de chargement du dispositif pour manipulation d'échantillons de matériau biologique, dans les chambres (12) de stockage de laquelle tous les produits chimiques réactifs nécessaires au processus en question ont auparavant été déposés séparément les uns des autres, les phases suivantes du processus se déroulant à l'intérieur du dispositif sans manipulation directe par l'opérateur des produits chimiques réactifs, qui sont progressivement mélangés et qui réagissent, en même temps que les résidus indésirables des réactions sont déposés dans la chambre (41) à déchets, qui est également située à l'intérieur du dispositif de manipulation et est séparée de façon sûre des autres zones du dispositif, des opérateurs ainsi que de l'environnement.
PCT/CZ2013/000005 2013-01-18 2013-01-24 Procédé de manipulation d'échantillons de matériau biologique et dispositif pour une telle manipulation WO2014111064A1 (fr)

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CZ2013-39A CZ304743B6 (cs) 2013-01-18 2013-01-18 Prostředek pro izolaci nukleových kyselin a způsob prováděný pomocí tohoto prostředku
CZPV2013-39 2013-01-18

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2017146744A1 (fr) 2016-02-27 2017-08-31 Hewlett-Packard Development Company, L.P. Système de préparation d'échantillons
CN109070081A (zh) * 2016-02-23 2018-12-21 比格泰克私人有限公司 用于净化样品和分析的套筒

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20030012694A1 (en) * 2001-03-15 2003-01-16 Bernd Roesicke System for the analysis of biological liquids
WO2005106040A2 (fr) * 2004-04-28 2005-11-10 Cepheid Procede et dispositif pour controler une preparation d'echantillon
WO2007047814A2 (fr) * 2005-10-19 2007-04-26 Progentech Limited Cassette pour une préparation d'échantillon
EP2187221A2 (fr) * 2008-07-16 2010-05-19 Millipore Corporation Procédé et unité de préparation d'un échantillon pour l'analyse microbiologique d'un liquide
WO2010115192A2 (fr) * 2009-04-03 2010-10-07 Integrated Nano-Technologies, Inc. Soupape rotative à plusieurs chambres
US7851207B1 (en) * 2007-02-07 2010-12-14 The United States Of America As Represented By The Secretary Of The Army Multiplex field device to detect and identify a variety of microbial agents simultaneously

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH04505848A (ja) * 1989-06-02 1992-10-15 ユーレン、マチアス 細胞懸濁液から染色体外ｄｎａを自動精製する装置と方法
JP2000507927A (ja) * 1996-03-06 2000-06-27 アクゾ・ノベル・エヌ・ベー 試料からの自動核酸抽出
EP2453219A4 (fr) * 2009-07-09 2016-06-15 Toppan Printing Co Ltd Trousse d'extraction d'acide nucléique, procédé d'extraction d'acide nucléique et appareil d'extraction d'acide nucléique

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20030012694A1 (en) * 2001-03-15 2003-01-16 Bernd Roesicke System for the analysis of biological liquids
WO2005106040A2 (fr) * 2004-04-28 2005-11-10 Cepheid Procede et dispositif pour controler une preparation d'echantillon
WO2007047814A2 (fr) * 2005-10-19 2007-04-26 Progentech Limited Cassette pour une préparation d'échantillon
US7851207B1 (en) * 2007-02-07 2010-12-14 The United States Of America As Represented By The Secretary Of The Army Multiplex field device to detect and identify a variety of microbial agents simultaneously
EP2187221A2 (fr) * 2008-07-16 2010-05-19 Millipore Corporation Procédé et unité de préparation d'un échantillon pour l'analyse microbiologique d'un liquide
WO2010115192A2 (fr) * 2009-04-03 2010-10-07 Integrated Nano-Technologies, Inc. Soupape rotative à plusieurs chambres

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109070081A (zh) * 2016-02-23 2018-12-21 比格泰克私人有限公司 用于净化样品和分析的套筒
JP2019506615A (ja) * 2016-02-23 2019-03-07 ビッグテック プライベート リミテッドＢｉｇｔｅｃ Ｐｒｉｖａｔｅ Ｌｉｍｉｔｅｄ 試料の精製および分析のためのカートリッジ
WO2017146744A1 (fr) 2016-02-27 2017-08-31 Hewlett-Packard Development Company, L.P. Système de préparation d'échantillons
CN108431577A (zh) * 2016-02-27 2018-08-21 惠普发展公司，有限责任合伙企业 样品制备系统
EP3353523A4 (fr) * 2016-02-27 2019-07-03 Hewlett-Packard Development Company, L.P. Système de préparation d'échantillons
US11060958B2 (en) 2016-02-27 2021-07-13 Hewlett-Packard Development Company, L.P. Sample preparation system

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EP2945739A1 (fr) 2015-11-25
CZ201339A3 (cs) 2014-07-30

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