WO1997032645A1 - Extraction automatisee des acides nucleiques d'echantillons - Google Patents

Extraction automatisee des acides nucleiques d'echantillons Download PDF

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Publication number
WO1997032645A1
WO1997032645A1 PCT/US1997/003533 US9703533W WO9732645A1 WO 1997032645 A1 WO1997032645 A1 WO 1997032645A1 US 9703533 W US9703533 W US 9703533W WO 9732645 A1 WO9732645 A1 WO 9732645A1
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WO
WIPO (PCT)
Prior art keywords
housing
nucleic acid
fluid
elution
solid phase
Prior art date
Application number
PCT/US1997/003533
Other languages
English (en)
Inventor
Benjamin J. Chemelli
Donald Robinson
G. A. P. Borst
Matthew Arthur Jones
Pamela Jean Gonzales
Original Assignee
Akzo Nobel N.V.
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 Akzo Nobel N.V. filed Critical Akzo Nobel N.V.
Priority to JP9531948A priority Critical patent/JP2000507927A/ja
Priority to EP97908045A priority patent/EP0885039A1/fr
Priority to AU19891/97A priority patent/AU1989197A/en
Publication of WO1997032645A1 publication Critical patent/WO1997032645A1/fr

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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
    • 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
    • C12N15/1017—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by filtration, e.g. using filters, frits, membranes
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/40—Concentrating samples
    • G01N1/405—Concentrating samples by adsorption or absorption

Definitions

  • the present invention relates to the isolation of a biological material, for example nucleic acid, from a basic material containing said biological material.
  • a biological material for example nucleic acid
  • An example of a method for the isolation of nucleic acid is known from US Patent 5,234,809 to Boom et al. , the subject matter of which is incorporated herein by reference.
  • the basic material, a chaotropic fluid and silica particles are mixed, with the result that the silica particles are then separated from the fluid and treated with a buffered eluant, in which the nucleic acid is dissolved off the particles.
  • HIV tests for example, can be prepared by isolating the nucleic acid from the basic material, which can be blood or blood products such as serum or plasma.
  • a sample is added to a lysis buffer containing guanidine thiocyanate (GuSCN) and a surfactant. Any viral particles and cells are thereby dissociated. Rnases and Dnases associated with the sample are inactivated. Nucleic acid is isolated by adding silicon dioxide particles to the sample lysis buffer cocktail. These particles act as a solid phase to which the nucleic acid is bound, and are washed several times.
  • GuSCN guanidine thiocyanate
  • the washing steps in the manual Boom method are very labor intensive and time consuming.
  • the silica is centrifuged to form a pellet and the supernatant aspirated. The pellet must be then resuspended in a washing solution and the process repeated.
  • the washing step is repeated five times, two times with wash buffer, twice with ethanol and once with acetone. After the pellet is sufficiently washed, it is then dried.
  • the nucleic acid is eluted from the silica using nuclease-free water as the eluant.
  • the eluate contains nucleic acid, both DNA and RNA, from the starting sample.
  • the nucleic acid in the eluate is concentrated and purified and is suitable for nucleic acid based diagnostics, e.g., direct probes and amplification based assays.
  • sample processor an automated apparatus, hereinafter called the "sample processor” , is provided to perform automated nucleic acid extraction.
  • the sample processor can be used in conjunction with a nucleic acid release kit, an isolation kit, and a disposable filter (called the "device” or the “disposable device” hereinafter) .
  • This combination provides a closed environment for the extraction of nucleic acid to take place.
  • the nucleic acid is stabilized.
  • the release kit is kept separate so samples can be lysed as shortly after collection as possible. Samples can be frozen for later processing with no degradation of the nucleic acid.
  • Homologous control contains the same target sequence as the wild type target. They are distinguishable from the target by either size or the presence of internal sequences. Homologous controls are amplified with the same primers as the wild type target Heterologous control does not contain the same sequences as the target and requires a different set of primers to amplify. Nucleic acid and control bind to the silicon dioxide particles.
  • a system control is a segment of nucleic acid that acts as an internal control to verify that all the steps (extraction, amplification and detection) have been performed correctly. The control is extracted along with the RNA and DNA present in the sample.
  • the disposable device is characterized by a container for holding a mixture of the basic material, a chaotropic fluid and a solid phase which binds the biological material; means for separating the solid phase with the biological material from the fluid bound thereto; and means for connecting the container to an inlet and outlet for washing fluid for washing the biological material bound to the solid phase, to an inlet for an eluant fluid, and to an eluate reservoir for collection of the eluant with the nucleic acid and control.
  • the means for separating the solid phase from the fluid are provided with a filter for allowing passage of the fluid and retaining the particle material.
  • the purpose of the solid phase is to fix or localize the nucleic acid so that debris and contaminants can be washed away.
  • the solid phase does not have to be in suspension with the lysis buffer and sample.
  • the solid phase could be incorporated into the filter membrane itself .
  • the solid phase could have various form factors, such as spun silica fibers filling the chamber in the disposable device, a sintered pellet of silica, a honeycomb structure of silica-based ceramic, etc.
  • the means for connecting the container to the inlet or outlet for eluant fluid and washing fluid and/or to the eluate reservoir are preferably provided with a shut-off element, which can be provided with, for example, a septum and an outlet channel for allowing through fluid from the separating means to the outlet, and also a hollow needle element connecting to the separating means, for piercing the septum in order to connect the separating means to the eluate reservoir.
  • a shut-off element is simple, user-friendly and reliable in operation.
  • the container is provided with two sections lying one above the other and separated by a constriction, and is also provided with a supply element which can be connected to the inlet and is movable between a position above the constriction and a position connecting in a close fit to the constriction.
  • both compressed gas for discharging the sample fluid from the container and the washing fluid and the eluant fluid can be fed into the container by one and the same supply element.
  • the supply element will be in the position above the constriction, following which the supply element is moved to the position connecting suitably to the constriction, so that only the bottom part of the container, containing the solid phase with the biological material bound thereto, need be flushed, with the result that less washing fluid is needed and there is less of a risk of contamination occurring.
  • the shut-off element is a valve element designed with at least one non ⁇ return valve between inlet and container.
  • the valve element is provided with three non-return valves: the first-mentioned non-return valve in the connection to a compressed gas supply, a second non-return valve between the container and the separating means, and a third non-return valve in a connection between the separating means and the inlet for the washing fluid and the eluant.
  • this embodiment has the advantage that the washing fluid is introduced directly into the separating means, and the container therefore does not need to be washed, with the result that only a small amount of washing fluid is needed.
  • Fig. 1 is a vertical longitudinal section of a first embodiment of the disposable device which can be removed from the sample processor;
  • Fig. 2 is a section corresponding to Fig. 1, in which the device according to the invention is placed in a control apparatus ("sample processor") ;
  • Fig. 3 shows on a larger scale the detail III from Fig. 2, with the supply element in the bottom position;
  • Fig. 4 is a vertical section of an alternative embodiment of a part of the device according to Fig. 1, in which the shut-off element is in the washing position;
  • Fig. 5 is a section corresponding to Fig. 4, with the shut-off element in the elution position;
  • Fig. 6 shows on a larger scale the detail VI from Fig. 5; -8- Fig. 7 is a vertical longitudinal section of a second embodiment of the disposable device;
  • Fig. 8 is a vertical longitudinal section of a third embodiment of the disposable device
  • Fig. 9 is a front view of the sample processor for holding a plurality of the disposable devices
  • Fig. 10 is a close-up view of a disposable device being loaded into the sample processor
  • Fig. 11 is a rear view of the sample processor showing the source of fluids used in the automated process
  • Fig. 12 is a top view of the sample processor
  • Fig. 13 is a front view of a second embodiment of the sample processor for holding a plurality of the disposable devices;
  • Fig. 14 is a vertical longitudinal section of one embodiment of a reagent bottle for use in the sample processor
  • Fig. 15 is a vertical longitudinal section of a second embodiment of the reagent bottle for use in the sample processor;
  • Fig. 16 is a front view of the air dryer and sterilizer for the sample processor
  • Fig. 17 is a vertical longitudinal section of one embodiment of the waste disposal system of the invention.
  • Fig. 18 is a sample processor functional block diagram;
  • Fig. 19 is a sample processor system block diagram
  • Fig. 20 is a system controller block diagram
  • Fig. 21 is a sample processor device controller block diagram.
  • the drawings show exemplary embodiments of a sample processor and a disposable device for use in the isolation of a biological material, such as nucleic acid, from a basic material containing said biological material.
  • the basic material can be, for example, blood, blood serum, urine, faeces, cell cultures and the like.
  • the isolation of the biological material, in particular nucleic acid, is necessary for carrying out tests, such as, for example, an HIV test.
  • the detachable/disposable element of the apparatus (the “sample processor") followed by a description of the sample processor itself.
  • the device is made detachable/disposable so that it can be used as the point for isolating the nucleic acid, and can then be separated from the sample processor for removal of the nucleic acid therein.
  • the detachable device can then be disposed of (preferably) or carefully washed for further use. In this way, problems of contamination from one nucleic acid isolation to the next, can be avoided.
  • the disposable device shown in Figs 1 and 2 comprises a container 1 for holding a mixture of the basic material, a chaotropic substance and a solid phase which binds the nucleic acid, in this exemplary embodiment silica particles.
  • chaotropic substance is any substance which is capable of altering the secondary, tertiary and/or quaternary structure of proteins and nucleic acid, but leaves at least the primary structure intact. Examples thereof are guanidine, (iso) thiocyanate and guanidine hydrochloride.
  • the container 1 belongs specifically to the device and the sample to be examined must be placed in the container by pipetting. The container is then sealed with a cover 2.
  • the cover 2 is designed with an inlet connection 3, for connection of the container 1 to an inlet for compressed air, washing fluid and eluant fluid.
  • These inlets for fluids form part of the sample processor, parts of which are shown in Fig. 2 and in which the device according to the invention can be placed for carrying out the isolation of the nucleic acid.
  • Fig. 2 shows, for example, a connecting ring 4 for suspending the device in the apparatus, for which purpose the container 1 is designed with a circular flange 5.
  • Fig. 2 also shows a connecting element 6 for the inlets for the fluids, which connecting element can be connected to the inlet connection 3 of the cover 2.
  • the container 1 forms the top element of the device, which is connected at the bottom end to a bottom element 7.
  • This cylindrical bottom element 7 comprises on the periphery an outlet connection 8 for connecting the device to an outlet for sample fluid and washing fluid, which forms part of the apparatus and is indicated by 9.
  • membrane 10 Clamped between the top end of the bottom element 7 and the container 1 is membrane 10, which serves as a filter and on which the silica particles with nucleic acid adsorbed thereon can settle.
  • a channel 11 connects to the space below the membrane 10.
  • the shut-off element 13 made from, for example, a silicone material, is provided with an outlet channel 14 with a top part lying in line with the channel 11 and a bottom part running radially towards the periphery. At the periphery of the shu -off element 13 the outlet channel 14 opens out into an annular peripheral channel 15 which can be placed in communication with the outlet connection 8 in the bottom element 7.
  • sample fluid and washing fluid can be conveyed out of the container 1, by way of the membrane 10 and the channels 11 and 14 to the outlet 9.
  • a removable sealing plate 16 ensures that the outlet connection 8 is sealed before the device is used.
  • the shut-off element could also be placed initially in a closed position and pushed to a discharge position only when the sample fluid is to be discharged.
  • the disposable device also comprises an eluate reservoir 17 for the collection of an eluant supplied from the inlet connection 3, and containing the nucleic acid dissolved off the silica particles.
  • the eluate reservoir can be a standard cup with a capacity of, for example, 0.5 ml, which is shut off by a septum 18 of silicone material.
  • the eluate reservoir 17 can be placed in a positioning element 19 of the apparatus, and with this positioning element 19 eluate reservoir 17 and shut-off element 13 can be pushed up relative to the bottom element 7 with the needle 12, in such a way that the needle cuts open in a sealed- off manner the septum 20 in line with the top part of the outlet channel 14 in the shut-off element 13 and the septum 18 of the eluate reservoir 17, following which eluate supplied can pass into the eluate reservoir 17 without the risk of leakages.
  • a vent channel 21 together with a vent groove in the periphery of the needle 12 ensure that air can escape from the eluate reservoir 17 for the admission of the eluant fluid.
  • the vent channel 21 in the shu -off element 13 can also be combined with the discharge channel 14, while a second needle can also be disposed in the shut-off element for the venting.
  • a hollow cylindrical-shaped inlet element 23 is formed in line with the inlet connection 3 of the cover 2, which inlet elements projects until it is deep inside the container 1 and is movable in the container 1 due to the construction of the cover with flexible ridges 30.
  • the container 1 is formed in two parts * namely a top part 25 of large volume tapering downwards toward a constriction 24, and a bottom part 26 of small volume and flaring out slightly downwards from the constriction 24, and connecting to the membrane 10.
  • the inlet element 23 can be moved by means of the connecting element 6 of the apparatus between a top position shown in Figs.
  • inlet element 23 opens out above the constriction 24 in the top part 25 of the container 1 and a bottom position, in which the feed element engages in a shut-off manner in the constriction 24 and therefore opens out in the bottom part 26 of the container 1.
  • the inlet element 23 and/or the constriction 24 could be provided with snap means 27 for reliably maintaining the grip.
  • the device according to Figs. 1 - 3 works as follows: First, the mixture of the basic material, the chaotropic substance and the silica particles is placed in the container 1, and the sealed device is then placed in the sample processor in the position shown in Fig. 2. Air is then pumped through the inlet connection 3 and the inlet element 23 into the container 1, in order to build up pressure in the container 1 for promoting the discharge of the sample fluid from it. After this discharge of the sample fluid, only the silica particles with adsorbed nucleic acid material remain behind on the membrane 10, together with residues of the sample fluid. The inlet element 23 is then moved to the bottom position in engagement with the constriction 24, following which a washing buffer (e.g.
  • a mixture of salts), ethanol and acetone are fed in through the inlet element, in order to wash the silica particles and the cavities and passages of the device in question.
  • Air can also be pumped through intermittently, in order to achieve an additional scraping effect.
  • conditioned warm air is passed through.
  • the next step is then to move up the positioning element 19, in order to move the eluate reservoir 17 and the shut-off element 13, so that the septums 18 and 20 can be pierced by the needle 12, as a result of which the container 1 enters into communication with the eluate reservoir 17 by way of the filter channel 11.
  • the eluant fluid for example in the form of TE buffer, double distilled water or PCR buffer, is fed in through the inlet element 23.
  • the eluant fluid is kept in contact with the silica particles for predetermined period, following which the eluant fluid is pumped further and passes by way of the membrane 10 and the channel 11 in a predetermined quantity, for example 100 ⁇ l, into the eluate reservoir 17.
  • a predetermined quantity for example 100 ⁇ l
  • the nucleic acid is dissolved off the silica particles and is ready for testing.
  • the shut-off element 13 and the eluate reservoir 17 are then moved down again, with the result that the needle 12 returns to the discharge position. Remaining eluant fluid is then pumped away to the outlet.
  • the septum 18 closes automatically, so that a sealed reservoir 17 with the fluid to be examined is obtained.
  • Figs. 4 - 6 show a variant of the shut-off element 13 and the eluate reservoir 17, which in this case are combined to a fixed unit and together can be moved between the washing or discharge position shown in Fig. 4 and the elution position shown in Fig. 5, moved upwards relative to the position shown in Fig. 4, in which elution position the needle 12 has pierced through the septum 20 between the outlet channel 14 and the eluate reservoir 17.
  • Fig. 6 shows the above- mentioned vent groove 22 in the needle, which ensures that air can escape from the eluate reservoir 17 when the eluant fluid flows into the reservoir.
  • the other structural elements are comparable to those in Figs. 1 - 3.
  • Fig. 7 shows an exemplary embodiment of the device according to the invention which is different in principle from the aforementioned embodiments.
  • the shut-off element is a valve element 28 on which a standard sample tube can be fitted as container 1.
  • the valve element 28 is this exemplary embodiment contains three commercially available non- return valves: a non-return valve 29 in a connecting channel 30 between a compressed gas inlet and the container 1, a second non-return valve 31 between the container 1 and filter membrane 10 and channel 11 acting as separating means, and a third non-return valve 32 in a connecting channel 33 between the filter membrane 10 and the channel 11 in the inlet for the washing fluid and the eluant fluid.
  • valve element 28 is provided with a rotary valve 24 which is below the membrane 10 and the channel 11 and is for connecting the channel 11 as desired to the outlet connection 8 and the eluate reservoir 17 connected to the valve element 28.
  • washing fluids are then introduced by way of the connecting channel 33 and the non-return valve 32 directly into the valve element 28 with the filter membrane 10, which can be washed with a relatively small volume of washing fluid.
  • the non-return valve 31 ensures that a seal is provided relative to the container 1.
  • FIG. 8 A further embodiment of the detachable/disposable device is illustrated in Fig. 8.
  • This embodiment of the device shares many of the same structural elements as the embodiment illustrated in Fig 1.
  • Below membrane 10 is disposed a rotary valve which is switched by the sample processor to allow fluid flow either to waste 8 or to eluate reservoir 17.
  • the eluate reservoir Prior to loading the device into the sample processor, the eluate reservoir is held in place with needle 12 piercing septum 18 of the cap of the eluate reservoir. In this way, the additional step of moving upwardly the eluant reservoir to pierce the septum thereof as is performed during nucleic acid isolation in relation to the embodiment of Fig. 1, is not required.
  • sample processor which utilizes the detachable/disposable device described above, will now be described.
  • sample processor in one embodiment of the sample processor illustrated in Figs. 9-12, a plurality of disposable devices are utilized.
  • the sample processor in combination with the disposable device and reagents represents a closed system that minimizes contamination. This is important for assays that detect and/or amplify nucleic acid. Minimal contamination is achieved by ensuring one way flow for reagents and samples, allowing no sample in the primary flow path (waste lines only) , keeping contamination within the disposable device, using filtered air for drying and reagent manipulation, and using closed reagent containers and collecting the eluate in a sealed container.
  • a computer controls a collection of pumps, valves and heaters to execute the method of extracting nucleic acid.
  • the user can control the sample processor via a touch screen and a small display.
  • the display provides control and status reporting. Sensors are provided to control and monitor proper execution of the process steps.
  • Many protocols for extracting nucleic acid are possible with a sample processor of the present invention.
  • the sample processor can be utilized for performing procedures that optimize the time it takes to process samples of different starting volumes or sample types .
  • a plurality of disposable devices 1 can be loaded into the sample processor 50.
  • the eluate reservoir 17 of disposable device 1 is placed in a sensing element 19 of the sample processor to ensure correct assembly and installation of the disposable device.
  • a door 55 is provided to be closed to hold the disposable device in place.
  • the door can be held shut with a magnet, which activates a Hall effect sensor that indicates that the disposable device is properly loaded in place.
  • inlet connection 3 of the disposable device must be properly connected to Luer connection 59 of the sample processor.
  • connection 65 of the disposable device must be properly fitted to connection 66 of the sample processor, which provides for a fluid flow path for waste.
  • Element 62 in Fig. 10 is a lower Luer fitting ejector which helps in removal of the disposable device. By pressing the free end of ejector 62 to the left in Fig.
  • Element 60 in Fig. 10 is a valve actuator for actuating the lower valve (e.g. rotary valve 24 in Fig. 8) of the disposable device for directing fluid flow to a waste container (disposed, for example, in a bottom area of the sample processor) or to eluate reservoir 17.
  • a valve actuator for actuating the lower valve (e.g. rotary valve 24 in Fig. 8) of the disposable device for directing fluid flow to a waste container (disposed, for example, in a bottom area of the sample processor) or to eluate reservoir 17.
  • containers 70 holding fluids for use in the nucleic acid extraction process are disposed at the rear of the sample processor.
  • the sample processor can be rotated around its base 79 to allow easy access to fluid containers 70.
  • operator console 51 can be disposed at any convenient location on the sample processor, or alternatively a separate assembly such as a personal computer connected to the sample processor, is envisioned.
  • bottle 1 in Fig. 11 could contain wash buffer
  • bottle 2 could contain ethanol (e.g. 70% ethanol diluted in water)
  • bottle 3 could contain acetone
  • bottle 4 could contain the elution buffer.
  • Fig. 12 is a top view of the sample processor showing, among other things, fluid containers 70, doors 55 at various locations where the disposable devices are to be held, and operator console 51.
  • the circular arrangement of the disposable devices in the sample processor is preferred and has a number of benefits. With a circular cross section, the sample processor can be made rotatable, as described above. Also, the tubing required to make a fluid connection between each fluid container and each disposable device station, is much less in comparison to an apparatus having a linear arrangement of disposable devices.
  • Fig. 13 illustrates an alternate embodiment where the disposable devices are held in a linear array rather than in a circle as in Figs. 9-12.
  • An advantage of this embodiment is that it can more easily be expanded to allow for more stations for holding disposable devices.
  • Fig. 14 is an illustration of a fluid container ("reagent bottle") for use in the sample processor.
  • Reagent bottle 120 has attached thereto a cap 112.
  • Tubing 151 is attached to cap 112 via quick release fitting 92 and makes a fluid connection to internal tubing 170 via gasket 100 and quick release fitting
  • a sinker 132 Disposed at the bottom of the reagent bottle is a sinker 132 with a sinker adapter 145 attached thereto for connecting to internal tubing 170. Within sinker 132 is a filter 157 held in place by retaining ring 167 for blocking passage of impurities into flow tubing 170. As liquid in the reagent bottle flows out during sample processing, filtered air is allowed to pass into the bottle via syringe filter 142, luer adapter 133, and check valve 127 (held by check valve holder 111) .
  • All the reagent bottles need not be the same size.
  • larger bottles can be used for alcohol, acetone and wash buffer, and a smaller bottle (such as that illustrated in Fig. 15) could be utilized for elution buffer, due to the comparatively lower volumes of elution buffer that are used during sample processing.
  • a smaller sinker is disposed within the reagent bottle, such that no sinker adapter is used as in Fig. 14.
  • the external dimensions of the cap are similar to the cap for the large reagent bottle, the cap has a narrow bottom portion 169 for fitting to the smaller diameter neck of the smaller bottle.
  • air passage 178 is disposed to provide an air passageway from check valve 127 to bottle 120.
  • Fig. 16 is an illustration of the inlet air dryer and sterilizer for connecting to the multi-port (e.g. 6-way or 8-way) valve (illustrated in Fig. 18) .
  • a drying tube 200 e.g. from Hammond Drierite/26930
  • a filter for filtering out air impurities preferably a filter made of polytetrafluoroethylene (e.g. 25mm Acrodisc filter from Gelman/4219) .
  • the filter is connected to the air dryer via vacuum tubing (e.g. Tygon tubing from Norton/AAC00020) and a polypropylene male luer lock.
  • female luer lock 205 and connector 207 are illustrated in Fig. 16, with polyethylene lined plastic tubing extending therebetween.
  • a waste container bottle 301 can be provided with a cap 302, both made from, for example, polypropylene.
  • Waste tubing 303 is connected at one end to quick release fitting 315 (which connects to the main portion of the sample processor) , and at the other end to in line quick release fitting 328.
  • O-rings 311 are provided to create a fluid hermetic seal, and a panel mount quick release fitting 327 is provided to connect to internal waste tubing 303 (structural elements 315 and 316 corresponding to structural elements 328 and 327 mentioned above) .
  • Fig. 18 is a block diagram illustrating the closed nucleic acid extraction system. As can be seen at the top left of this diagram, the sample processor can optionally include a separate computer including a keyboard, barcode wand (the disposable devices might optionally include barcode labels) , modem, printer, etc.
  • the sample processor can optionally include a separate computer including a keyboard, barcode wand (the disposable devices might optionally include barcode labels) , modem, printer, etc.
  • An input and display (whether part of the separate PC system or via a touchscreen as illustrated in the figures) allows for a user interface with the system controller.
  • Door sensors communicate to the system controller that the disposable device (s) are properly positioned in the sample controller.
  • a six- way valve controller controls the 6-way valve which controls the flow of air and reagents to the • disposable device station (the valve need not be a six-way valve, but could be 8-way or more) .
  • a pump is controlled by a device controller for pumping air or reagents through the system. Also in communication with the device controller are the prime valve (used in priming the system prior to actual extraction) , an eluate collection actuator and device sensor, and an eluate vial detector.
  • the block diagram of Fig. 18 is only one embodiment of the present invention and is an illustration of a sample processor with two disposable device stations. Of course higher numbers of stations would be possible.
  • a wash cycle is performed with each reagent to prime the system. For example, a predetermined amount of each reagent (e.g. 300 microliters) is displaced into the line that goes to the disposable device.
  • Air is pumped to follow the predetermined amount of each reagent so as to pass the reagent through the system as a fluid "pulse".
  • the nucleic acid extraction is performed.
  • the sample fluid containing the nucleic acid and solid phase is pushed out of the disposable device past the disposable device filter, such that solid phase with nucleic acid (DNA or RNA) bound thereto is caught on the disposable device filter.
  • the disposable device filter could be the solid phase itself, such that nucleic acid binds directly to the filter.
  • wash fluid is pumped from wash reagent source (s) to wash the solid phase and nucleic acid.
  • wash buffer predetermined amounts of wash buffer containing a chaotropic substance followed by filtered air
  • alcohol e.g. ethanol
  • acetone e.g. acetone
  • the upper luer fitting for connecting to the inlet of the disposable device can be fitted with a heater to help dry the acetone during this step.
  • the nucleic acid is removed from the silica with an elution buffer, with a quantity of eluted nucleic acid being caught in the eluate reservoir of the disposable device.
  • the sample processor controls the flow of buffers and reagents through a biological sample in order to elute and collect amplifiable nucleic acid.
  • Solenoid valves route the fluid materials through tubing lines which are pressurized by syringe pumps. Elution is performed in a disposable device which holds solid phase and a filter. This device can be heated to the optimu temperature for elution. Sensors monitor placement of the device and the position of valves within the device.
  • the sample processor comprises ten such devices, each with an associated pump.
  • a microcontroller module termed a device controller manages the syringe pumps, valves, heaters and other device-specific functions.
  • a system controller manages the user interface, initialization and other system-wide functions. This modularity enables system design flexibility and serviceability.
  • the system controller sends commands to device controllers through an internal network.
  • a device controller can also send unsolicited status messages to the system controller.
  • the system controller is assigned the master address on the network.
  • Microcontroller hardware handles message routing and arbitration. When the sample processor is powered on, all microcontrollers perform self tests on their local code and data memory and initialize attached devices. The system controller determines which device controllers are ready to run by sending queries to them and listening for a response. If all device controllers are ready, the system controller enters a system ready state, awaiting a command from the operator.
  • the system can be operated stand-alone or by commands sent to its host interface port.
  • the operator interacts with the stand-alone instrument through an LCD character display with a touch- sensitive overlay.
  • a message window on the display shows status items such as time to completion, warming up, standby, and temperature.
  • the system can be configured to provide messages in English, German, French or a user-defined message set.
  • the host interface port is an RS-232 serial port which can accommodate a terminal, laboratory information system, personal computer, or modem. An expanded level of diagnostics is available from the serial port for long-term error logging and for teleservicing applications.
  • the system controller sends error messages to the status display and the host interface port whenever it detects a problem. The time of occurrence is appended to error messages going to the host interface port .
  • the system controller also maintains in its memory an error history log (up to a limit of 150 errors) . This log may be sent to the interface port on command, and it may be viewed on the display through a service menu command.
  • the system controller sends commands to the device controllers and monitors their status. It accepts commands from and sends status messages to a host interface port. In addition, the system controller is assigned other system-level functions.
  • the system controller sends commands to a device controller to select the position of the multiport valve which is connected to one of the syringe pumps. This selects the working fluid for the entire system.
  • the system controller sends commands to a dedicated motor driver which is connected to the internal network. This driver actuates the "airpen" mechanisms which create a fluid path through the device filter.
  • the controller reads sensors which monitor the position of these mechanisms and verify proper operation.
  • Sensors monitor the instrument cover doors and the supply and waste reservoirs (wash buffer, alcohol, acetone, elution buffer, auxiliary reagent, waste reservoir, and auxiliary reservoir) . If the system controller detects an empty supply bottle or full waste reservoir, it sends an error message to the status display and to the host computer. If a door sensor shows a door to be open while the instrument is running, the system controller sends a status message and lowers the setpoint temperature for the heaters to a safe contact level. The system controller manages one heater for the reagent block. The temperature of the reagent block and the air are maintained at a setpoint of 56°C. The relative humidity of the air is also monitored.
  • the controller sends an error message to the status display and to the host computer.
  • the system controller waits a specific time after the control process begins before it announces temperature out-of-range errors. Two system voltages are monitored. If a voltage is out of range, the system controller sends an error message to the status display and to the host computer.
  • a sequence of device controller commands can be defined and assigned a name. Such a sequence is termed a macro. The sequence can then be invoked by sending a simple macro execution command. Macros may invoke other macros to a nesting of six levels deep. Macros may use variable parameters. If an error occurs during execution of a macro, the macro terminates, and any macros which follow in the sequence are canceled.
  • Macros are defined for common operations which the system performs, such as wash, rinse, displace and clean. This enables the system controller to operate within a high-level command structure which conserves memory and limits traffic on the internal network. Macros are maintained in nonvolatile memory in the device controllers. System operations are associated with macro names. In response to a command from the user interface, the system controller may simply send a run-macro command to the device controllers.
  • the system controller includes a block of battery-backed memory which stores system configuration information. It also contains a clock- calendar circuit which supplies data when an error report is time-stamped.
  • the system controller memory includes its own operating program code as well as a copy of the device controller operating program. This memory can be reprogrammed by sending appropriate commands and data to the host interface port. Similarly, the system controller can reprogram the memory on the device controllers by sending commands and data on the internal network. In this way system code revisions can be made by connecting a personal computer or a modem to the host interface port.
  • Each device controller sends commands to operate two syringe pumps through an RS-232 serial port.
  • One pump connects to the RS-232 pump and routes commands for the second pump to a pump command bus. While the pump is active, pressure sensors check for excess pressure levels in the tubing; high levels can indicate obstructions in the tubing or device.
  • Devices are heated in order to provide proper temperature for the nucleic acid isolation process.
  • the device controller provides current to the heater and monitors the temperature of the device for closed loop control .
  • the device controller supplies current to actuate solenoid valves which select the fluid path through the device for priming, washing or reagent dispensing. It also drives a motor attached to each device which moves a valve for eluate flow to a collection cup. Position sensors in each device verify proper operation of the airpen which controls fluid flow within the device and the eluate removal valve. The device controller also reads a sensor which signals that a device or a shunt is properly inserted in the instrument. 7.
  • the sample processor executes predefined methods which consist of a number of phases.
  • the user selects a method to perform by pressing buttons on the user interface panel .
  • the system controller then sends commands to all device controllers to start each phase.
  • the system controller maintains coordination of device controllers when necessary for use of shared mechanisms, such as the airpen actuator and multiport valve.
  • the system controller monitors and/or controls the following: communications to device controllers program loading for device controllers device controller initialization macro commands and response monitoring for device controllers error reporting and logging system configuration memory real-time clock operator interface panel (touch pad, display and beeper) reservoir level sensors (supply and waste) door unlock solenoid (which can be several solenoid locks wired in parallel) send commands to satellite I/O module to control airpen shuttle one door sensor (which can be several door sensors wired in together) one reagent block heater one air humidity sensor 24 volt supply 5 volt supply communications to host computer or modem (not required for operation) Sensors monitor the instrument cover doors and the supply and waste reservoirs (wash buffer, alcohol, acetone, elution buffer, auxiliary reagent, waste reservoir, and auxiliary reservoir) .
  • system configuration memory real-time clock operator interface panel (touch pad, display and beeper) reservoir level sensors (supply and waste) door unlock solenoid (which can be several solenoid locks wired in parallel) send commands to satellite I/O module to control
  • the system controller communicates with the device controllers through the IIC (inter-integrated circuit) control network. Effective data rate of this network is about 80 Kbps.
  • a device controller can send unsolicited messages to the system controller.
  • the system controller is assigned the master address on the network; network hardware handles message routing.
  • the system controller communicates with a host computer through an RS-232 interface.
  • Serial port parameters may be user-configured; default settings are 19200 bps, even parity, 7 data bits, 1 stop it.
  • the interface includes a DSR line for hardware flow control.
  • the system controller sends error messages to the status display and the host interface port whenever it detects a problem. The time and date of occurrence are appended to the error message.
  • the system controller maintains in its memory an error history log (up to a limit of 32 errors) . This log may be sent to the interface port on command, and it may be viewed on the display through a service menu command.
  • An LED lamp mounted on the system controller provides a base level of status information. When power is applied to the controller and it detects no active faults, the LED will blink at about 2 pulses per second. If a memory fault is detected, the rate will decrease to about once every two seconds. If the LED is on continuously, the flash EPROM or the processor probably needs to be reloaded. If the LED does not turn on at all, a power supply fault is likely.
  • the system controller program On power up, the system controller program first tests its program memory, internal ram memory, and external ram memory. If an error is found, a message is sent to the host interface and to the status display. If these are OK, the device controllers on the internal network are queried for status. If necessary, the system controller will upload the program memory to device controllers across the network. If any device controller fails to initialize, the system controller sends an error message to the status display and host interface, and if possible, the instrument will continue to operate in a partial capacity.
  • the system controller then sends the prompt '/' to the host interface and initializes the status display with the start-up screen.
  • the host can query the current revision level of the program by sending the 'RV command, and if desired, the host can upload a different system controller or device controller program file. While the controller program is running, a continuous program memory checksum test executes to verify that memory is intact. If the test fails, the system controller sends a program memory checksum error and enters a graceful shutdown sequence. In this mode, the controller continues to monitor the host interface and it will accept a program file for download.
  • the device controller monitors and/or controls the following: communications to two syringe pumps communications to one multiport valve pressure sensors for two devices priming solenoid valves for two devices bypass solenoid valves for two devices valve actuator for eluate removal shuttle four device heaters device-in-place sensors for two devices limit switches for airpen and eluate removal shuttles
  • Sensors monitor the pressure in the tubing, device temperature and device presence for two devices. If the device controller detects an excessive pressure, it disables the relevant pump and sends an error message to the system controller. The system controller may send a message to query the status of an individual sensor, or a string of data containing all of the current sensor states can be sent upon request.
  • Device controller firmware consists of two major partitions which are largely independent. The partition in the lowest 16 kilobyte block of memory holds the bootup program and the macros. The remaining 48 kilobytes hold the operating program.
  • the bootup program Normally the bootup program performs a memory test of the EPROM and RAM after power-up, then it jumps to the operating program. If a problem is detected during the memory test, the bootup program remains in control and will allow the system controller to upload a copy of the device controller operating program into its EPROM.
  • the device controller communicates with the system controller through the IIC (inter-integrated circuit) control network. Effective data rate of this network is about 80 Kbps.
  • a device controller can send unsolicited messages to the device controller.
  • the device controller reads the setting of a DIP switch to determine its address on the network; the network hardware handles message routing.
  • the device controller communicates with two syringe pumps through an RS-232 (optional RS-485) serial interface.
  • Serial port parameters may be user- configured; default settings are 9600 pbs, no parity, 8 data bits, 1 stop bit. 3. Command and Error Message Formats
  • the system controller sends error messages to the status display and the host interface port whenever it receives an error message from the device controller.
  • the system controller time stamps each error and maintains in its nonvolatile memory an error history log.
  • An LED lamp mounted on the device controller provides a base level of status information. When power is applied to the controller and it detects no active faults, the LED will blink at about 2 pulses per second. If a memory fault is detected, the rate will decrease to about once every two seconds. If the LED is on continuously, the flash EPROM or the processor probably needs to be reloaded. If the LED does not turn on at all, a power supply fault is likely.
  • the device controller program first tests its program memory, internal ram memory, and external ram memory. If an error is found, a message is sent to the system controller. If the memory test results are OK, the device controller waits for a query from the system controller to report its status. If the system controller does not receive the proper response to its query, it marks the device controller "inactive", reports an error, and continues to run with the other active device controllers.
  • a continuous program memory checksum test executes to verify that memory is intact. If the test fails, the device controller sends a program memory checksum error and enters a graceful shutdown sequence. In this mode, the controller continues to monitor the system network and will accept a program file for download from the network.
  • the controller will announce the error to the system controller.
  • the device controller continuously monitors the pressure in the tubing while fluid is moving. If pressure exceed a programmed threshold, the pump is stopped and an error message is sent.
  • IV. Principle of Operation The process begins by lysing a sample. The appropriate amount of sample is placed in a tube containing lysis buffer (Release Kit) . This must be done a short time after sample collection to prevent degradation of the sample by nucleases. Lysing a sample stabilizes the nucleic acid. Sample lysis cocktail can be processed further or frozen for later testing. The following steps take place in a laminar flow hood. Before starting these steps make certain that the instrument is turned on. Add 100 ul of silicon dioxide to the sample lysis buffer cocktail, mix gently several times during a 10 minute incubation at room temperature.
  • the sample lysis silica cocktail is pumped through a disposable filter. Silica is captured and the lysis buffer passes through to waste. Once all the cocktail has been filtered the silica pellet on the filter is washed with the following: wash buffer, 70% EtOH, acetone and dried. At this point a silica pellet with the bound nucleic acid is on the filter. Flow is diverted from waste to the eluate collection cup. The nucleic acid is eluted using water as an eluant. After elution, air is used to force residue reagents to waste. Open the guard and remove the disposable filters, placing them in a rack for transport to the laminar flow hood.
  • the eluate collection cups can be removed and the rest of the disposable filter thrown out . At this point the eluate is ready for amplification or direct probe testing or it can be frozen for later testing.
  • the computers At power up the computers, six of them, go through a self diagnostic and initialization setting all sensors and actuators to their prescribed states. The six computers are arranged with one supervisor and five slaves. The supervisor controls all slave function error reporting, display functions, keypad functions and all connections to external equipment.
  • base turntable
  • pump housing dual device module
  • reagent housings display pod
  • safety cover waste container
  • the base houses the power supply and distributes power. AC power ranging from 90 to 250 volts and 50 - 60 Hz is received by the power supply and rectified to 24VDC.
  • An umbilical from the base connects to the display pod and another to the waste container. Rising from the center of the base is an axle that has a bearing attached. The turntable is attached to the bearing. Between the base and turntable is a cable management system.
  • the design can support a plurality of cable management approaches. The first is a cable wrap-up technique using a cable guide to spiral wind the wiring and tubing to the waste connection around the axle. The other is a torsional approach where the wiring and tubing is located on the axis of rotation and only exposed to torsional forces.
  • the dual device module is where the disposable filter holder connects to the instrument.
  • the disposable When properly installed the disposable must have its valve handle in the valve actuator slot; its waste connection to the instruments waste connection and the inlet connected to the instruments inlet connection.
  • the door When properly installed, the door will close. If it is not properly installed, the door will not close.
  • the presence of the disposable is sensed along with the connection of the inlets and the door closing. When these conditions are met the instrument reports that the disposable is correctly installed.
  • the inlet and waste fittings have check valve in them to prevent fluids from flowing in the wrong direction.
  • the inlet fitting is heated to facilitate the drying step which is to remove any residual acetone.
  • Lower reagent housing and upper housing have sensors to detect the presence of liquid. These are set to a height that insures there is always enough reagent available to complete a test .
  • the caps for the reagents are designed to provide an as closed as possible environment. This is accomplished by using filters on the connection that allows air to fill the space vacated by the liquid as it is consumed. A filter is on the end of the line that is connected to the suction side of the pump to prevent contaminants from entering the instrument. A self sealing quick disconnect fitting is used to make changing the reagents simple.
  • the pump housing is attached to the turntable. Within the pump housing are 10 pumps and respective pressure sensors, the upper reagent housing and a multiport valve that allow selection of the correct reagent.
  • the pumps force liquid through the disposable filter holder at a preset speed and volume.
  • Pressure sensors monitor the pressure to insure the filter has not ruptured or the pressure is not too high.
  • the pressure sensor is also used to determine when the silica cake on the filter is wetted with elution buffer. An increase in pressure indicates that the silica and membrane are wetted.
  • the pressure sensor is used in a feedback control scheme to optimize the time it takes to displace the sample. Measuring the pressure provides indications on obstructions or leaks in the fluidic path. Small leaks are identified by a sensor that detects acetone and alcohol vapors. It is very sensitive. Approximately 50 PPM, this would be a spill of a few drops within the closed space of the sample processor.
  • the final step in the process described herein is an amplification and/or nucleic acid detection step.
  • an amplification of the nucleic acid can be performed, such as NASBA or PCR.
  • final detection steps for detecting the nucleic acid can further be performed.
  • a hollow plunger could be provided in the essentially cylindrical container, in which case a small channel in the plunger is initially shut off by a seal and through the downward movement thereof in the container, the fluid in the container is pressed out through the filter. In the bottom position, the seal can then be pierced, following which the washing fluids and finally the eluant fluid can be supplied through the channel connected to the inlet in the plunger.
  • a second plunger in the channel in the hollow plunger, which second plunger can be moved up and down and, after the sample fluid has been expelled, ensures that washing fluids are extracted from an inlet which is connected to the valve element by means of a non ⁇ return valve, and that said washing fluids and possibly also the eluant are then passed through the filter and the valve element.
  • the plunger In the lowest position of the hollow plunger the plunger seals off the largest part of the container relative to the shut-off element, so that only a small volume needs to be washed, and a small quantity of washing fluid will therefore suffice.
  • a rotary shut-off element can be used instead of the slidable shut-off element shown in Figs. 1 - 6, a rotary shut-off element can be used.
  • This can be comparable to the rotary valve of Figs. 7 and 8, but it is also possible to form the passage channels as recesses on the periphery of the rotary valve. In one position a first recess provides the passage to the outlet, while this first recess in a second position provides the venting, and a second peripheral recess permits the passage of eluant fluid to the eluate reservoir.
  • the needle elements for eluate passage and venting are formed at a position below the rotary valve in this embodiment.
  • wash fluids are pumped from closed containers, with any air that is allowed into the containers being filtered. Air that is pumped into the sample processing system, such as for drying solid phase, is filtered. Tubing from wash fluid containers or the air drier, allow fluid flow in one direction only, such that there can be no passage of nucleic acid sample (e.g. as an aerosol) into the tubing.
  • nucleic acid sample e.g. as an aerosol
  • the parts of the sample processing system that would be in contact with nucleic acid are the disposable device and the waste tubing.
  • the disposable device is completely removable from the sample processor and can be disposed of after use, and the waste tubing allows fluid flow in only one direction, to waste, and thus will not act as a source of nucleic acid contamination.

Abstract

Système de traitement fermé d'échantillons, dans lequel les fluides de lavage sont pompés dans des récipients fermés, tout l'air autorisé à pénétrer dans les récipipents étant filtré. L'air qui est pompé dans le système de traitement des échantillons, tel que celui utilisé pour sécher la phase solide, est filtré. Un tubage venant des récipients de fluide de lavage ou du sécheur d'air permet au fluide de s'écouler dans un seul sens, de façon qu'il ne puisse y avoir aucun passage d'échantillons d'acide nucléique (par exemple sous forme d'aérosol) dans le tubage. Les parties du système de traitement des échantillons qui seraient en contact avec un acide nucléique (et donc se comporterait en fin de compte comme des sources de contamination) sont le dispositif jetable et le tubage de déchets. Mais le dispositif jetable peut être enlevé totalement du système de traitement et être jeté après usage, et le tubage de rejet ne laisse circuler le fluide que dans un seul sens, vers les déchets, et ne fera donc pas office de source de contamination.
PCT/US1997/003533 1996-03-06 1997-03-05 Extraction automatisee des acides nucleiques d'echantillons WO1997032645A1 (fr)

Priority Applications (3)

Application Number Priority Date Filing Date Title
JP9531948A JP2000507927A (ja) 1996-03-06 1997-03-05 試料からの自動核酸抽出
EP97908045A EP0885039A1 (fr) 1996-03-06 1997-03-05 Extraction automatisee des acides nucleiques d'echantillons
AU19891/97A AU1989197A (en) 1996-03-06 1997-03-05 Automated nucleic acid extraction from samples

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US61192696A 1996-03-06 1996-03-06
US08/611,926 1996-03-06

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WO1997032645A1 true WO1997032645A1 (fr) 1997-09-12

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JP (1) JP2000507927A (fr)
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JP2001002695A (ja) * 1999-06-23 2001-01-09 Hitachi Ltd 核酸抽出装置および核酸抽出処理方法
WO2002097084A1 (fr) * 2001-05-25 2002-12-05 Hitachi, Ltd. Appareil et procede pour la purification d'acide nucleique
EP1851302A1 (fr) * 2005-02-21 2007-11-07 Fujifilm Corporation Mécanisme de retenue de cartouche pour appareil d extraction d acide nucléique
JP2009236933A (ja) * 1997-12-24 2009-10-15 Cepheid 一体型流体操作カートリッジ
EP2206792A1 (fr) * 1998-08-12 2010-07-14 PreAnalytiX GmbH Recipient servant à un prélèvement de sang
EP2402732A1 (fr) * 2010-07-02 2012-01-04 Instrumentation Scientifique de Laboratoire (ISL) Procédé d'injection d'un échantillon à analyser dans le tube d'injection d'une cellule de mesure, en particulier d'un densimètre
EP2640825A2 (fr) * 2010-11-18 2013-09-25 Bioneer Corporation Appareil automatique de purification d'acides nucléiques et procédé de protection d'un aérosol
CZ304743B6 (cs) * 2013-01-18 2014-09-17 Wolf & Danniel S.R.O. Prostředek pro izolaci nukleových kyselin a způsob prováděný pomocí tohoto prostředku
WO2014153629A1 (fr) * 2013-03-27 2014-10-02 Universidade Estadual De Campinas - Unicamp Système automatisé d'extraction en phase solide
EP3009510A1 (fr) * 2014-10-17 2016-04-20 Daniel Lai Procédé et appareil d'extraction rapide d'acide nucléique
WO2020027566A1 (fr) * 2018-08-01 2020-02-06 주식회사 미코바이오메드 Dispositif d'extraction d'acide nucléique et son procédé de fonctionnement
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US6110428A (en) * 1994-08-29 2000-08-29 Akzo Nobel N.V. Device for use in the isolation of a biological material such as nucleic acid
WO1998042874A2 (fr) * 1997-03-24 1998-10-01 Fields Robert E Processeur biomoleculaire
WO1998042874A3 (fr) * 1997-03-24 1998-12-23 Robert E Fields Processeur biomoleculaire
JP2009236933A (ja) * 1997-12-24 2009-10-15 Cepheid 一体型流体操作カートリッジ
JP4522480B2 (ja) * 1997-12-24 2010-08-11 セフィード 一体型流体操作カートリッジ
EP2206792A1 (fr) * 1998-08-12 2010-07-14 PreAnalytiX GmbH Recipient servant à un prélèvement de sang
JP2001002695A (ja) * 1999-06-23 2001-01-09 Hitachi Ltd 核酸抽出装置および核酸抽出処理方法
WO2002097084A1 (fr) * 2001-05-25 2002-12-05 Hitachi, Ltd. Appareil et procede pour la purification d'acide nucleique
US7273702B2 (en) 2001-05-25 2007-09-25 Hitachi, Ltd. Apparatus for purifying nucleic acid and method of purifying nucleic acid
KR100807931B1 (ko) * 2001-05-25 2008-02-28 가부시키가이샤 히타치세이사쿠쇼 핵산 정제 장치 및 핵산 정제 방법
EP1851302A1 (fr) * 2005-02-21 2007-11-07 Fujifilm Corporation Mécanisme de retenue de cartouche pour appareil d extraction d acide nucléique
EP1851302A4 (fr) * 2005-02-21 2014-04-09 Kurashiki Boseki Kk Mécanisme de retenue de cartouche pour appareil d extraction d acide nucléique
FR2962218A1 (fr) * 2010-07-02 2012-01-06 Instrumentation Scient De Laboratoire Isl Procede d'injection d'un echantillon a analyser dans le tube d'injection d'une cellule de mesure, en particulier d'un densimetre
EP2402732A1 (fr) * 2010-07-02 2012-01-04 Instrumentation Scientifique de Laboratoire (ISL) Procédé d'injection d'un échantillon à analyser dans le tube d'injection d'une cellule de mesure, en particulier d'un densimètre
EP2640825A2 (fr) * 2010-11-18 2013-09-25 Bioneer Corporation Appareil automatique de purification d'acides nucléiques et procédé de protection d'un aérosol
EP2640825A4 (fr) * 2010-11-18 2014-08-13 Bioneer Corp Appareil automatique de purification d'acides nucléiques et procédé de protection d'un aérosol
CZ304743B6 (cs) * 2013-01-18 2014-09-17 Wolf & Danniel S.R.O. Prostředek pro izolaci nukleových kyselin a způsob prováděný pomocí tohoto prostředku
WO2014153629A1 (fr) * 2013-03-27 2014-10-02 Universidade Estadual De Campinas - Unicamp Système automatisé d'extraction en phase solide
CN105524915A (zh) * 2014-10-17 2016-04-27 D·赖 快速核酸提取方法、核酸提取方法和核酸提取设备
EP3009510A1 (fr) * 2014-10-17 2016-04-20 Daniel Lai Procédé et appareil d'extraction rapide d'acide nucléique
KR20160045560A (ko) * 2014-10-17 2016-04-27 다니엘 라이 신속한 핵산 추출 방법 및 장치
EP3323891A1 (fr) * 2014-10-17 2018-05-23 Daniel Lai Procédé et appareil d'extraction rapide d'acide nucléique
KR101954218B1 (ko) 2014-10-17 2019-03-05 다니엘 라이 신속한 핵산 추출 방법 및 장치
WO2020027566A1 (fr) * 2018-08-01 2020-02-06 주식회사 미코바이오메드 Dispositif d'extraction d'acide nucléique et son procédé de fonctionnement
US11643645B2 (en) 2018-08-01 2023-05-09 Mico Biomed Co., Ltd. Nucleic acid extraction device and operating method therefor
WO2023241597A1 (fr) * 2022-06-14 2023-12-21 南京金斯瑞生物科技有限公司 Système pipeline pour le tri de cellules, dispositif de tri de cellules et procédé de commande

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JP2000507927A (ja) 2000-06-27
EP0885039A1 (fr) 1998-12-23
AU1989197A (en) 1997-09-22

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