Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
  • G01N35/10—Devices for transferring samples or any liquids to, in, or from, the analysis apparatus, e.g. suction devices, injection devices
  • G01N35/1095—Devices for transferring samples or any liquids to, in, or from, the analysis apparatus, e.g. suction devices, injection devices for supplying the samples to flow-through analysers
  • G01N35/1097—Devices for transferring samples or any liquids to, in, or from, the analysis apparatus, e.g. suction devices, injection devices for supplying the samples to flow-through analysers characterised by the valves
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L7/00—Heating or cooling apparatus; Heat insulating devices
  • B01L7/52—Heating or cooling apparatus; Heat insulating devices with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples
• • 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
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
  • C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor

Definitions

• the present invention relates generally to the treatment of a sample in a liquid medium, in particular an aqueous medium, in order to release and separate a predetermined fraction of interest from said sample.
• the invention relates to the treatment of a biological material, for example cellular, to release and separate a constituent or a predetermined, sub-cellular or intra-cellular fraction of l 'processed sample, for example a nucleic acid or protein fraction.
• a biological material for example cellular
• a constituent or a predetermined, sub-cellular or intra-cellular fraction of l 'processed sample for example a nucleic acid or protein fraction.
• the subject of the present invention is a set for processing a sample, said set being sterilizable and making it possible to avoid, on the one hand any contamination of the constituent or of the predetermined fraction, to be analyzed, due to the prior treatment of a different sample, and on the other hand any contamination by different external sources, likely in particular to subsequently distort the analysis.
• nucleic acid fractions or constituents With regard to the processing of nucleic acid fractions or constituents, such an object is particularly important, when it is known in practice that many analyzes of nucleic material are disturbed, or even falsified, with the same analysis apparatus, on the one hand by remainders or remains of the previously treated sample, and on the other hand by the existence in the immediate environment of the analysis apparatus of nucleic "pollutants". Consequently, the analysis of nucleic acid fractions or constituents imposes constraints, in particular work under particularly severe conditions, to avoid any parasitic contamination of the constituent or of the fraction to be analyzed.
• a processing unit comprises, on the one hand, an active, permanent, essentially static module, closed with respect to the outside, gathering in a processing circuit in series, between a main input for the sample to be process, and a main output for the predetermined fraction or constituent of this same sample:
• a disposable external container for the sample to be analyzed, connectable to the main input of the treatment circuit, and an disposable external analysis container for the predetermined and sought fraction or constituent, in particular a card connectable to the main output of the treatment circuit, this card comprising different reagents and means for analyzing the predetermined fraction; optionally the disposable outer container and the disposable outer card are combined according to the same component or
• At least several control valves of the treatment circuit are each a so-called “static freezing” valve of the liquid of the sample, and / or of any other liquid agent circulating in the treatment circuit.
• Each valve comprises for this purpose, a conduit element arranged to receive the liquid medium in its passage section, the wall of which conducts heat, and a cold source disposed in thermal contact, in particular in exchange for heat with said element. conduit, the refrigerating temperature and power of which are adapted to the freezing of the liquid medium, in said passage section.
• the duct element is dimensioned in relation to the cold source, and the hot source generated by passage of the electric current therein, to quickly pass, for example in an elementary time less than 10 seconds, from a cold state, in which the liquid medium is likely by freezing to seal the passage through the duct element, including with respect to relatively high pressures, for example at least equal to 50 bars, in particular 150 bars, in a hot state in which the liquid medium is liquefied by releasing the aforementioned passage.
• FIG. 1 shows schematically a static freezing valve, belonging to the analysis assembly shown in Figure 1.
• a treatment assembly according to the invention has three parts, namely:
• an active module A permanent, essentially static, closed by an enclosure 26 with respect to the outside, grouping together a processing circuit 6 described below
• an external container B disposable, for the sample to be treated, for example an inoculum of a cell culture
• this container is connectable to the main input 7 of the processing circuit 6 - and another external container C, disposable, for analyzing the nucleic fraction obtained in the circuit 6 processing, connectable to the main output of the processing circuit;
• this container comprises, in a traditional manner and not described below, various reagents and means for analyzing the nucleic fraction, for example for the amplification and / or detection of a particular nucleic fragment.
• the processing circuit 6 gathers between the main input 7 and the main output 8:
• first conduit 3 forming a first enclosure 1 for dissociation or lysis of the biological material treated, in this case the cell culture analyzed
• second conduit 4 forming a second enclosure 2 for separation of the nucleic fraction to be analyzed - different conduits connection, one of which 9 between the first conduit 3 and the second conduit 4, and the other 10 between the second conduit 2 and the main outlet 8
• control valves 15 to 25 and 27 of the liquid or gaseous flows circulating in the treatment circuit preferably, the control valves 15, 16, 17, 18, 21, 22, 23, 24 and 25 are so-called static freezing valves, as described below; the valve 27 is either a mechanical or traditional valve, or a static freezing valve; in the latter case, it is necessary to provide means for supplying liquid, not shown in Figure 1 attached.
• All the components of the treatment circuit 6, including the first 3 and second 4 elements of conduits of the first 1 and second enclosures 2 respectively, are obtained from a material, in particular metallic, mechanically resistant, capable of dissipating from the heat by ohmic effect, to constitute a hot source directly in their mass.
• an electrical source represented diagrammatically under the reference numeral 5 is connected to all of these components, including the first and second elements 3 and 4 of conduits of the first 1 and second 2 enclosures respectively, for passing within them, and in a controlled manner, an electric current generating a hot source.
• each static freezing valve comprises, for freezing the liquid medium of the treated sample, and / or any other liquid agent circulating in the treatment circuit 6: - an element duct 29, arranged to receive the liquid medium in its passage section 29a, the wall of which is heat conductive,
• a cold source 28 shown globally and common in Figure 1, arranged in thermal contact, in particular in exchange for heat with the duct element 29, the temperature and the cooling capacity of which are adapted to the freezing of the liquid medium , in the passage section 29a
• the duct element 29 being dimensioned in relation to the cold source 28 and the hot source generated by the passage through it of the electric current, coming from the electric source 5, for passing quickly, for example in an elementary time less than 10 seconds, from a cold state in which the liquid medium is capable, by freezing, of sealingly blocking the passage through said conduit element, including with respect to relatively high pressures, for example at least equal to 50 bars, in particular 150 bars, in a hot state in which the liquid medium is liquefied by releasing said passage. Consequently, it follows from the preceding description that practically the entire processing circuit 6, and consequently of the active module A, is arranged for complete internal incineration, by simple passage of an electric current through the materials constituting said circuit.
• the circulation of liquids in the first enclosure 1, or first conduit element 3, is controlled by the static freezing valves 16 and 17, respectively at the inlet la and at the outlet lb of the conduit element 3.
• the circulation liquids in the second enclosure 2 is controlled by the static valves 22 and 23, respectively at the inlet 2a and at the outlet 2b of the second conduit element 4.
• Three secondary inlets 11 to 13, respectively for three washing liquids 30 to 32, namely guanidium thiocyanate, ethanol and acetone respectively, are arranged on the treatment circuit 6, between the outlet lb of the first enclosure and the inlet 2a of the second enclosure.
• a secondary inlet 33 for an elution liquid 34 is disposed on the treatment circuit, between the outlet lb of the first enclosure and the inlet 2a of the second enclosure 2, downstream of the secondary inlets 11 to 13.
• the secondary inputs 11 to 13 and 33 are controlled respectively by the valves 18 to 21, and the latter are static freezing valves.
• the outlet 2b of the second enclosure 2 communicates with a container 35 for collecting liquid effluents, by means of a static freezing valve 24.
• the input la of the first enclosure 1 communicates with a reservoir 36 of an aqueous medium, by means of a static freezing valve 15.
• This same reservoir 36 communicates with the collection container 35, via a duct element 9 'controlled by the static freezing valve 25.
• An intermediate storage capacity 37 is disposed in the processing circuit 6, between the outlet 2b of the second enclosure 2 and the main outlet 8.
• Two secondary outlets 38 and 39 possibly in the liquid and / or gaseous phase, for depressurization of the treatment circuit 6, and / or sweeping of the same circuit with a gas, communicate with the circuit 6, downstream of the outlet 2a of the second enclosure 2.
• the secondary outlet 38 is disposed on the effluent collection container 35, and the secondary outlet 39 is disposed above the intermediate storage capacity 37, being controlled by a static freezing valve 27.
• the reservoir 36 for the aqueous medium, the containers for storing washing liquids 30 to 32, the container for storing the elution liquid 34, and the container 35 for collecting liquid effluents are arranged outside the enclosure 26 containing the processing circuit 6 proper.
• closing or opening a static freezing valve 16 or 17 means respectively freezing the liquid medium in its passage section, by means of the cold source 28, and passing an electric current through the mass of the 'conduit element, for the purpose of heating and liquefying this same liquid medium, and thus freeing the passage in said conduit element.
• the sample to be treated is transferred to the first enclosure 1, the valve 16 being open, and the valve 17 initially open closing by freezing on contact with the liquid sample.
• the interior of the enclosure 1 is isolated and filled with the sample to be treated, by closing the valve 1a.
• the valves 16 and 17 By passing an electric current through the first conduit element 3, the valves 16 and 17 being always closed, the liquid medium is vaporized on the one hand, and the pressure of the same medium is raised inside the conduit element 3, at relatively large values, on the other hand, so that the cellular material is lysed, releasing the nucleic acid fraction (s) to be analyzed, but in admixture with other residual fractions, in particular protein, originating from the same cellular material.
• the second enclosure is provided with means for fixing or retaining the desired nucleic fraction, arranged in the second conduit element 4, and consisting for example of an adsorbent material, in particular silica.
• the lysed sample comprising the nucleic fraction of interest and the residual fractions, is transferred, for example by aspiration, into the second element of conduit 2.
• the nucleic fraction of interest is adsorbed on said adsorbent material of the second enclosure 2.
• valves 18 to 20 and the valves 22, 23 and 24, for example by suction the residual fractions of the lysed sample are transferred to the collection container 35, it being understood that the valve 25 is closed .
• the second enclosure is rinsed by the rinsing liquids 30 to 32 respectively, the effluents being discharged towards the collection container 35. In this way, the non-nucleic fractions are eliminated from the second enclosure 2.
• the elution liquid 34 is circulated in the second enclosure 2, which takes with it during its passage into the second enclosure 2, specifically and only the nucleic fraction, which is found in suspension in the intermediate storage capacity 37. Then, this same elution fraction can be transferred, still by aspiration for example, into the external container or analysis card C, by the main outlet 8. And it is in this card that is carried out by appropriate means not being part of the present invention, the actual analysis of the nucleic fraction, for example by amplification and / or detection, using reagents already available or added to the external analysis container C.
• valve 27 being opened by any appropriate means, either by suction or by discharge, the entire sterile air treatment circuit is circulated, which is evacuated by the secondary outlets 38 and 39.
• the elution liquid can be transferred, in the same way and with the same means, through the second enclosure 2 and the capacity 37, to the external analysis container C.
• the entire treatment circuit 6 is washed by means of the aqueous medium 36.
• the secondary outlets 38 and 39 all of the valves of the circuit being open, drying air is circulated in said circuit.
• the capacity 37 is decontaminated by any suitable thermal means (not shown).
• the active module A is then ready for a new analysis, from a new sample in the container B, and with a new external analysis container C.

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• 1995
  • 1995-10-23 FR FR9512669A patent/FR2740222B1/fr not_active Expired - Fee Related
• 1996
  • 1996-10-18 EP EP96934938A patent/EP0803056A1/de not_active Withdrawn
  • 1996-10-18 AU AU73065/96A patent/AU7306596A/en not_active Abandoned
  • 1996-10-18 US US08/849,515 patent/US5902746A/en not_active Expired - Fee Related
  • 1996-10-18 WO PCT/FR1996/001635 patent/WO1997015815A1/fr not_active Ceased
  • 1996-10-18 JP JP9516348A patent/JPH10511862A/ja active Pending
  • 1996-10-18 CA CA002209267A patent/CA2209267A1/fr not_active Abandoned

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