• 专利附录
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    • 专利摘要:
    This extraction system (2) comprises a support bar (3) comprising a sample well (4), a reagent well (5), at least one washing well (6.1, 6.2), and at least one protective and retention housing (9); a pipetting element (10) removably mounted on the support bar (3) and extending at least partially in the at least one protective and retaining housing (9); and a filtering and transfer device (10) for capturing and accumulating microorganisms contained in the sample and for transferring captured and accumulated microorganisms, the filtering and transfer device (10) being removably mounted on the support bar (3) and extending at least partly in the at least one protective and retaining housing (9). The filtration and transfer device (11) comprises a hollow body (12) having an elongated shape and having a proximal end portion (12.1) and a distal end portion (12.2); and a filter element (13) attached to the distal end portion (12.2) of the hollow body (12).
    公开号:FR3058734A1
    申请号:FR1661015
    申请日:2016-11-15
    公开日:2018-05-18
    发明作者:Patrick Broyer；Marie-Helene Charles；Jerome Blaze；Nadine Perrot；Frederic PINSTON；Herve Rostaing
    申请人:Biomerieux SA；
    IPC主号:
    专利说明:

    © Publication number: 3,058,734 (to be used only for reproduction orders) (© National registration number: 16 61015 ® FRENCH REPUBLIC
    NATIONAL INSTITUTE OF INDUSTRIAL PROPERTY
    COURBEVOIE © IntCI 8
    C 12 Q 1/04 (2017.01), C12M 1/00
    A1 PATENT APPLICATION
    ©) Date of filing: 15.11.16. (© Applicant (s): BIOMERIEUX Société anonyme - (© Priority: FR. @ Inventor (s): BROYER PATRICK, CHARLES MARIE-HELENE, JEROME BLAZE, PERROT (43) Date of public availability of the NADINE, PINSTON FREDERIC and ROSTAING HERVE. request: 18.05.18 Bulletin 18/20. ©) List of documents cited in the report preliminary research: Refer to end of present booklet (© References to other national documents ©) Holder (s): BIOMERIEUX Société anonyme. related: ©) Extension request (s): © Agent (s): CABINET GERMAIN & MAUREAU.
    EXTRACTION SYSTEM AND METHOD FOR EXTRACTING MICROORGANISMS CONTAINED IN A SAMPLE.
    FR 3 058 734 - A1 (6 /) This extraction system (2) comprises a support strip (3) comprising a sample well (4), a reagent well (5), at least one washing well (6.1,6.2), and at least one protective and retention housing (9); a pipetting element (10) removably mounted on the support strip (3) and extending at least partially in the at least one protection and retention housing (9); and a filtration and transfer device (10) for capturing and accumulating microorganisms contained in the sample and for transferring the captured and accumulated microorganisms, the filtration and transfer device (10) being removably mounted on the support bar (3) and extending at least partially in the at least one protection and retention housing (9). The filtration and transfer device (11) comprises a hollow body (12) having an elongated shape and comprising a proximal end portion (12.1) and a distal end portion (12.2); and a filter element (13) attached to the distal end portion (12.2) of the hollow body (12).
    12.3
    i
    The present invention relates to an extraction system and method for extracting microorganisms contained in a sample, in particular for the purpose of identifying and / or characterizing the microorganisms extracted, for example by mass spectrometry analysis on MALDI-TOF MS analysis plate or an antibiogram analysis.
    A first known method for extracting microorganisms contained in a positive sample, such as a positive blood culture, consists in:
    -deposit manually several drops of the sample on one or more culture media in petri dishes,
    - incubate the Petri dish (s) for 18 to 48 hours depending on the type of microorganism,
    - manually remove part of the colonies from the Petri dishes,
    manually depositing the colonies taken from different locations on a MALDI-TOF MS analysis plate, also called a MALDI-TOF MS mass spectrometry analysis plate,
    - carry out an identification in mass spectrometry on the MALDI-TOF MS analysis plate.
    A second known method for extracting microorganisms contained in a positive sample, such as a positive blood culture, consists in:
    -deposit manually several drops of the sample on one or more culture media in petri dishes,
    - incubate the Petri dish (s) for 18 to 24 hours,
    - manually remove part of the colonies from the Petri dishes,
    - prepare a concentrated suspension calibrated in optical density of microorganisms from the colonies collected,
    - perform an antibiogram analysis on the suspension taken.
    These first and second known extraction methods are long and require in particular the presence of an experienced operator to perform the many manual steps.
    Automata have thus been developed to carry out in particular the sampling and deposition steps implemented for the processes described above. However, such automata require a very large pecuniary investment for an analysis laboratory without significantly reducing the duration of these extraction processes.
    Document W02013 / 016211 describes a method for extracting microorganisms contained in a positive sample, comprising in particular the use of a filtration and transfer device to isolate the microorganisms contained in the positive sample and to transfer them in particular on a MALDI-TOF MS analysis plate.
    Such a method makes it possible to greatly reduce the duration of extraction of microorganisms contained in a positive sample. However, such an extraction process still requires the prolonged presence of an experienced operator to perform the various manual steps throughout the duration of the process. Furthermore, the deposit on MALDI-TOF MS analysis plate is not standardized in terms of deposit, and does not easily ensure the traceability of the results.
    The present invention aims to remedy these drawbacks.
    The technical problem underlying the invention therefore consists in providing an extraction system which is simple and compact in structure, while allowing rapid and automated extraction of microorganisms contained in a positive sample for analysis on a plate. MALDI-TOF MS analysis or for an antibiogram from a concentrated suspension of microorganisms.
    To this end, the present invention relates to an extraction system for extracting microorganisms contained in a sample, the extraction system comprising:
    - a support strip comprising:
    a sample well intended to receive a sample containing microorganisms, such as a positive blood culture,
    - a reagent well intended to contain a selective lysis buffer,
    at least one washing well intended to contain a washing reagent, and
    - at least one protective and retention housing,
    a pipetting element removably mounted on the support bar and extending at least partially in the at least one protection and retention housing,
    a filtration and transfer device intended to capture and accumulate microorganisms contained in the sample and to transfer the captured and accumulated microorganisms, the filtration and transfer device being removably mounted on the support strip and s 'extending at least partially into the at least one protection and retention housing, the filtration and transfer device comprising:
    a hollow body having an elongated shape and comprising a proximal end portion and a distal end portion, and
    - a filtration element fixed to the distal end portion of the hollow body.
    Such a configuration of the extraction system provides it with great compactness, and also makes it possible to easily load it, via a rack, into an automatic device capable in particular of handling the pipetting element and the filtration and transfer device with a view to automatically carry out the various steps of extracting the microorganisms contained in a positive sample introduced into the sample well.
    In addition, the use of the extraction system according to the present invention does not require training or special skills. In fact, the only manual steps to be implemented by an operator are the introduction of the sample into the sample well, the positioning of the support strip on a rack and the loading of the rack into the automated system. The simplicity of use of the extraction system according to the present invention also makes it possible to treat urgent blood cultures in night service where the level of technicality is greatly reduced.
    In addition, associated with the automat, the extraction system according to the present invention makes it possible not to immobilize a technician during all of the manual extraction operations making it possible to deliver a MALDI-TOF MS analysis plate with deposits. reproducible and standardized in terms of quantity deposited and directly usable in the MALDI-TOF MS analysis system to identify or characterize the pathogens present in the sample.
    Finally, the use of a filtration and transfer device similar to that described in document W02013 / 016211 ensures rapid extraction of the microorganisms contained in a sample loaded in the sample well, and a reliable deposit of the micro -organisms on a MALDI-TOF MS analysis plate.
    Consequently, the extraction system according to the present invention allows rapid and automated extraction of the microorganisms contained in a positive sample for analysis on a MALDI-TOF MS analysis plate or for an antibiogram.
    It should also be noted that the configuration of the extraction system makes it possible to greatly limit the costs of manufacturing the automaton capable of automatically carrying out the extraction of microorganisms, in particular because the various reagents required are already present on each support strip and therefore do not have to be stored in the controller.
    The extraction system can also have one or more of the following characteristics, taken alone or in combination.
    According to one embodiment of the invention, the sample well, the reagent well and the at least one washing well are substantially aligned in the direction of extension of the support strip.
    According to one embodiment of the invention, the distal end portion of the hollow body and the filter element extend in the at least one protection and retention housing.
    According to one embodiment of the invention, the pipetting element comprises a distal end portion extending at least partially in the at least one protection and retention housing.
    According to one embodiment of the invention, the at least one protection and retention housing is configured to retain drops of liquid, such as drops of sample or of reagents, capable of flowing by gravity from the pipetting element and the filtration and transfer device.
    According to one embodiment of the invention, the filtration element is configured to retain at least part of the microorganisms contained in the sample and to allow other elements contained in the sample to pass through, such as elements non-microorganic, for example cellular debris, or lysed blood compounds, such as red blood cells.
    According to one embodiment of the invention, the filtration element is a filtration membrane, such as a membrane of the Pall SuporR type (PES material), for example in the form of a disc or dome.
    According to one embodiment of the invention, the filtration element consists of a material selected from the group comprising in particular polyethersulfone, glass fibers, cellulose acetate and regenerated cellulose.
    According to one embodiment of the invention, the filtration element has a pore size of between 0.1 and 10 μm, preferably from 0.2 to 0.8 μm and very preferably from 0.45 μm.
    According to one embodiment of the invention, the filtration element has a thickness of between 0.08 and 0.14 mm.
    According to one embodiment of the invention, the filtration element has a cylindrical shape of diameter adapted to the diameter of a location of a MALDI-TOF MS analysis plate, and has a flat or hemispherical distal part.
    According to one embodiment of the invention, the filtration element is of convex shape.
    According to one embodiment of the invention, the hollow body has a tapered portion converging towards the distal end portion of the hollow body.
    According to an embodiment of the invention, the distal end portion of the hollow body is cylindrical, flared or flattened.
    According to one embodiment of the invention, the proximal end portion of the hollow body is configured to be connected to a source of vacuum.
    According to one embodiment of the invention, the proximal end portion of the hollow body comprises a retaining part. The retaining part is for example a retaining flange.
    According to one embodiment of the invention, the filter element extends at least partially outside the hollow body. For example, the filter element extends or projects from the distal end portion of the hollow body.
    According to one embodiment of the invention, the filtration and transfer device comprises an absorbent material placed in the hollow body.
    According to one embodiment of the invention, the absorbent material is at least partly in contact with the filter element.
    According to one embodiment of the invention, the absorbent material is selected from the group comprising cotton, cellulose fiber, polyester, polyethylene agglomerate, a porous absorbent resin, a silica gel, a hydrogel, a molecular sieve, a zeolite or other absorbent materials.
    According to one embodiment of the invention, the filtration and transfer device comprises glass beads resting on the absorbent material.
    According to a particular embodiment of the invention, the absorbent material consists of a monolithic block made of agglomerate of plastic beads (for example of polyethylene or polyester type) advantageously replacing the assembly of glass beads and absorbent material at the rear of the filtration membrane.
    According to one embodiment of the invention, the extraction system further comprises a release well, also called recovery well, mounted, for example removably, on the support bar or inserted directly into the rack.
    According to one embodiment of the invention, the release well has an internal layer of crosslinked silicone covering at least partially the internal surface of the release well, and contains a predetermined amount of saline solution, for example 0.45% . The release well may for example contain from 250 to 400 μL of saline solution, preferably 320 μL of 0.45% saline solution.
    According to one embodiment of the invention, the at least one washing well contains a washing reagent and the reagent well contains a selective lysis buffer.
    According to one embodiment of the invention, the reagent well and the at least one washing well are each closed by a closure element.
    According to one embodiment of the invention, each shutter element is a shutter film, such as a shutter seal. Each closure element can for example be made of aluminum.
    According to one embodiment of the invention, the extraction system further comprises a shutter member movable or deformable between a shutter position in which the shutter member closes the sample well and a position release in which the closure member at least partially releases the sample well. These provisions make it possible in particular to guarantee the closing of the sample well during the transport of the support strip or the handling of a rack fitted with the support strip. They guarantee the integrity of the sample as well as the safety of the manipulator during any movement during handling (for example from the biological hood to the instrument).
    According to one embodiment of the invention, the shutter member is configured to be moved into the release position when the support bar is positioned on a rack and the rack is loaded into a machine. These provisions ensure that the sample well is completely opened when the rack is loaded into the automated device and that the sample well is completely closed when the rack is removed from the automated device, without operator intervention.
    According to one embodiment of the invention, the support bar is provided with an identification code, such as a bar code. These provisions ensure optimal traceability of the analysis results obtained for each patient.
    According to one embodiment of the invention, the support bar is made of plastic.
    According to one embodiment of the invention, the hollow body of the filtration and transfer device is made of plastic, and for example of injected crystal polystyrene or injected polypropylene.
    According to one embodiment of the invention, the hollow body of the filtration and transfer device has an internal diameter between 2 mm and 10 mm, and a length between 5 cm and 15 cm. The total length of the filtration and transfer device is for example 9 cm in order to have an internal volume sufficient to contain all of the fluids aspirated during the process and thus constitute a retention reservoir for said fluids. The distal part of the filtration and transfer device advantageously has a tapered diameter over a few cm in length, allowing it to i) enter deeply into the different tubes of the extraction system, ii) adapt to the diameter of the locations of a MALDI-TOF MS analysis plate for the transfer operation.
    According to one embodiment of the invention, the hollow body of the filtration and transfer device is rigid or semi-rigid.
    According to one embodiment of the invention, the support strip comprises a plurality of washing wells each intended to contain a washing reagent. The support strip may for example comprise two or three, or even more, washing wells, the volume and composition of the reagents will be adapted if necessary to the application or to the type of sample to be extracted (urine, pus, synovial fluid, etc ... previously cultivated in liquid growth medium).
    According to one embodiment of the invention, the support bar comprises a protective part delimiting the at least one protective and retention housing.
    According to one embodiment of the invention, the support bar comprises a protection and retention housing in which at least partly extend the pipetting element and the filtration and transfer device.
    According to one embodiment of the invention, the support strip comprises a first protection and retention housing and a second protection and retention housing in which the pipetting element and the device extend respectively at least in part. filtration and transfer.
    The present invention further relates to a method of identifying and / or characterizing microorganisms contained in a sample, the method of identifying and / or characterizing comprising the following steps:
    - providing an extraction system according to the invention, in which the reagent well contains a selective lysis buffer and the at least one washing well contains a washing reagent,
    - introduce a predetermined quantity of a sample containing microorganisms into the sample well,
    - withdraw a predetermined quantity of the selective lysis buffer contained in the reagent well using the pipetting element,
    - optionally check the presence and level of filling of the sample in the sample well by an optical method,
    - Introducing into the sample well the selective lysis buffer withdrawn using the pipetting element, and optionally mixing the sample and the selective lysis buffer in said sample well using the pipetting element,
    - lyse non-microorganic cells and / or particles contained in the sample and / or lyse predetermined microorganisms contained in the sample so as to provide a lysed sample, also called a lysate,
    - at least partially immerse the filtration and transfer device in the lysed sample contained in the sample well,
    - filter under suction at least part of the lysed sample through the filter element,
    - capture and accumulate microorganisms in or on the filter element,
    - at least partially immerse the filtration and transfer device in the washing reagent contained in the at least one washing well,
    - washing, in the washing reagent contained in the at least one washing well, the filtration element and the captured and accumulated microorganisms,
    - transfer, using the filtration and transfer device, the washed microorganisms to an analysis support or to a container,
    - analyze the microorganisms transferred so as to identify and / or characterize them.
    According to one embodiment of the extraction process, the volume of sample introduced into the sample well can vary between 0.5 and 1.2 ml, and the volume of selective lysis buffer withdrawn is then modified to respect a 2: 1 ratio (for example if 1.2 ml of sample is introduced into the sample well, then 600 μL of selective lysis buffer is taken).
    According to one embodiment of the extraction process, the step of introducing the sample consists in introducing 1 ml of sample into the sample well, the sample preferably being blood culture positive but the process is applicable to other sterile body fluids previously cultivated in liquid growth media in order to increase the concentration of pathogens potentially present before analysis.
    According to one embodiment of the extraction method, the step of removing the selective lysis buffer consists in taking 500 μl of selective lysis buffer from the reagent well. These 500 μL are mixed and incubated with the ml of sample present in the sample well, in order to lyse the elements represented on blood, in particular the red blood cells.
    According to one embodiment of the extraction process, the step of mixing the sample and the selective lysis buffer consists in carrying out several mixing cycles, each mixing cycle consisting in aspirating and discharging a predetermined quantity of the mixture contained in the sample well, for example 500 pL (if the sample volume is lmL, respect a 1: 2 ratio), using the pipetting element.
    According to one embodiment of the extraction method, the lysis step comprises a step consisting in incubating the mixture for a predetermined period, for example between 1 and 5 minutes, preferably for 2 minutes, at room temperature so as to provide the sample free of non-microorganisms. For example, in the case of a blood culture sample, this step consists in lysing the figured elements of the blood, in particular the red blood cells.
    According to one embodiment of the extraction process, the latter comprises a step prior to the step of immersing the filtration device and transfer to the lysed sample and consisting in applying a vacuum in the hollow body of the device filtration and transfer.
    According to one embodiment of the extraction process, the step of applying a vacuum in the hollow body consists in applying a vacuum between -50 mbar and -980 mbar relative to atmospheric pressure, preferably around -600 mbar compared to atmospheric pressure.
    According to one embodiment of the extraction process, the suction filtration step is carried out for example for 2 minutes.
    According to one embodiment of the extraction method, the analysis support is a MALDI-TOF MS analysis plate or a test slide.
    According to one embodiment of the extraction process, the container is formed by the release well provided on the support bar or inserted directly into the rack.
    According to one embodiment of the extraction process, the latter comprises a step consisting in maintaining the vacuum in the hollow body during ίο the various steps of immersion of the filtration and transfer device, the filtration step and the 'washing step.
    According to one embodiment of the extraction process, the washing step comprises the following step:
    moving for a predetermined period, for example between 30 seconds and 5 minutes, preferably for 2 minutes, the filtration and transfer device in the at least one washing well, for example according to an alternating movement in Z.
    According to one embodiment of the extraction method, the step of moving the filtration and transfer device in the at least one washing well consists in carrying out several cycles of movement of the filtration and transfer device in the '' at least one washing well. Advantageously, the filtration and transfer device is completely extracted from the at least one washing well at each movement cycle, for example at a frequency of approximately 0.5 to 2 Hz, preferably 1 Hz.
    According to one embodiment of the extraction process, the latter comprises a collection step carried out between the washing step and the transfer step and comprising the following steps:
    - remove the filtration and transfer device from the washing reagent contained in the at least one reagent well, and
    - moving the distal end portion of the hollow body transversely to the direction of extension of the hollow body so that the distal end portion of the hollow body comes into contact with an internal wall of the at least one washing well .
    These arrangements make it possible to collect, in the at least one washing well, residual washing drops present at the distal end portion of the filtration and transfer device, and therefore to avoid the subsequent deposition of a too much liquid on the analysis support.
    According to one embodiment of the extraction method, the collecting step comprises a step consisting in holding the distal end portion of the hollow body against the internal wall of the at least one washing well for a predetermined period , for example for 5 to 20 seconds, preferably 10 seconds. Advantageously, the vacuum in the hollow body is maintained during the holding step in order to aspirate all or part of the residual liquid attached to the distal part of the filtration and transfer device.
    According to one embodiment of the extraction process, the latter comprises a step prior to the transfer step, and advantageously subsequent to the step of holding the distal end portion of the hollow body against the internal wall of the at least one washing well, and consisting in putting the hollow body at atmospheric pressure or in slight overpressure.
    According to one embodiment of the extraction process, the step of immersing the filtration device and transfer to the washing reagent contained in the at least one washing well consists in immersing the filtration device and transfer more deeply, for example 1 to 3 mm deeper, than in the lysed sample contained in the sample well.
    According to one embodiment of the extraction method, the transfer step comprises a step consisting in depositing, using the filtration and transfer device, at least a portion of the washed microorganisms on one or more predetermined locations, also called spots, of a MALDI-TOF MS analysis plate.
    According to one embodiment of the extraction method, the latter further comprises the following steps carried out before the analysis step:
    - dry, in a controlled manner, the different locations of the MALDI-TOF MS analysis plate on which microorganisms have been deposited,
    - optionally, take an image of the locations on which microorganisms have been deposited, for example according to two distinct illuminations,
    add a predetermined quantity of matrix, for example from 0.5 to 2 μί, preferably 1 μί, on each location of the MALDITOF MS analysis plate where microorganisms have been deposited,
    - dry, in a controlled manner, the different locations of the MALDI-TOF MS analysis plate on which the matrix has been deposited,
    - optionally, take an image of the locations on which the matrix has been deposited, for example according to two separate illuminations.
    According to one embodiment of the extraction process, the matrix is a DHB matrix, namely a 2,5 Dihydroxybenzoic acid, an HCCA matrix, namely an α-cyano-4-hydroxycinnamic acid, or an SA matrix, namely, anapinic acids (3,5-dimethoxy-4-hydroxycinnamic).
    According to one embodiment of the extraction process, the latter comprises a step carried out prior to the step of adding the matrix and consisting in adding a predetermined amount of formic acid, for example of
    0.5 to 2 pL, preferably 0.5 to 1 pL, on each location of the MALDI-TOF MS analysis plate where microorganisms have been deposited. These provisions make it possible in particular to improve the identification results for Gram positive and yeasts.
    According to one embodiment of the extraction method, the step of depositing the washed microorganisms on one or more predetermined locations on the MALDI-TOF MS analysis plate comprises a step consisting in applying the filtration element against the or each predetermined location of the MALDI-TOF MS analysis plate with a controlled application force of between 100 grams and 1 kg per filtration device, preferably 600 grams.
    According to one embodiment of the extraction method, the step of depositing the washed microorganisms on one or more predetermined locations on the MALDI-TOF MS analysis plate comprises a step consisting in buffering one or more times the filter element on the or each predetermined location of the MALDI-TOF MS analysis plate. These provisions allow a deposit of a sufficient quantity of microorganisms on each predetermined location of the MALDI-TOF MS analysis plate.
    According to an embodiment of the extraction method, a step of taking images of the locations on the MALDI-TOF MS analysis plate can be optionally carried out; depending on the need for control, these image taking steps can be carried out immediately after the transfer of the microorganisms or after deposit of the formic acid and matrix reagents in order to verify the actual presence of a deposit. Preferably, these image taking steps take place after each of the drying steps in order to check the quality and quantity of the deposit of the microorganisms, the good interaction between the formic acid and the deposit of the microorganisms (presence of a halo), the good distribution and crystallization of the matrix with the microorganisms deposited on the MALDI-TOF MS analysis plate.
    According to one embodiment of the extraction method, the buffering step consists of buffering several times, for example between 3 and 7 times, the filtration element on the or each predetermined location of the MALDI analysis plate. -TOF MS according to a buffering frequency between 0.5 and 10 Hz.
    According to one embodiment of the extraction process, the previous step can be combined and combined with a sequence for pressurizing the hollow body of the filtration and transfer device in order to facilitate the transfer of the microorganisms from the filtering membrane on the surface of the MALDITOF MS analysis plate.
    According to one embodiment of the extraction method, the analysis step comprises a step consisting in carrying out a mass spectrometry analysis of the MALDI-TOF type of the microorganisms transferred, and for example of the deposited microorganisms on a MALDI-TOF MS analysis plate.
    According to an embodiment of the extraction method, the analysis step comprises a step consisting in inserting the analysis support in a mass spectrometer.
    According to one embodiment of the extraction process, the washing step comprises a step consisting in separating, isolating or purifying the microorganisms captured and accumulated in the filtration element, for example by facilitating the evacuation of the other components which may be present in the lysed sample, such as cellular debris, non-microorganic elements, and which would be likely to interfere with the subsequent analysis of the microorganisms.
    According to one embodiment of the extraction process, the latter comprises a step, at the start of the process, consisting in piercing sealing films of the at least one washing well and of the reagent well using pipetting element. These provisions allow in particular to create a sufficient opening so that the filtration and transfer device can penetrate into the different washing well (s) without damaging the filtration element.
    According to one embodiment of the extraction process, the drilling step consists in drilling at least one washing well and then the reagent well by a vertical and possibly horizontal movement of the pipetting element.
    According to an embodiment of the extraction method, the latter comprises a step consisting in detecting an identification code provided on the extraction bar in order to associate it with the identification code provided on the container initially containing the sample (for example a positive blood culture flask) and thus ensure optimal traceability of the analysis results.
    According to one embodiment of the extraction process, the latter comprises a step carried out after the step of depositing the microorganisms on the MALDI-TOF MS analysis plate and before the step of adding the matrix and consisting in incubating the MALDI-TOF MS analysis plate between 40 and 80 ° C, for example at 50 ° C, for a predetermined period, for example between 2 and 6 minutes, preferably 4 minutes, and this so as to dry or evaporate any residual washing reagent present on the or each predetermined location.
    According to one embodiment of the extraction process, the latter comprises a step carried out after the step of adding formic acid and consisting in incubating the MALDI-TOF MS analysis plate between 40 and 80 ° C. , for example at 45 ° C or 55 ° C, for a predetermined period, for example between 5 and 7 minutes, preferably 6 minutes, so as to accelerate the evaporation of the drops of formic acid, while ensuring a sufficient contact time of the formic acid with the microorganisms present on the MALDI-TOF MS analysis plate to guarantee effective lysis of the membranes of the microorganisms.
    According to one embodiment of the extraction process, the latter comprises a step carried out after the step of adding the matrix and consisting in incubating the MALDI-TOF MS analysis plate between 40 and 80 ° C. for a period of time. predetermined, for example between 1 and 3 minutes, preferably 2 minutes.
    According to one embodiment of the extraction method, the step of depositing the microorganisms on the predetermined location or locations of the MALDI-TOF MS analysis plate comprises a step consisting in vibrating the filtration device and transfer, and more particularly to vibrate the filtration and transfer device at least during the contact of the filtration element on the or each predetermined location of the MALDI-TOF MS analysis plate. These provisions improve the transfer of microorganisms (quantity and reproducibility) from the filtration element to the MALDI-TOF MS analysis plate.
    According to one embodiment of the extraction method, the step of vibrating the filtration and transfer device consists in applying a vibration of between 0.1 and 200 Hz, for example between 10 and 100 Hz, preferably 50 Hz, for a predetermined duration, and for example between 1 and 5 seconds, preferably for 2 seconds.
    According to one embodiment of the extraction process, the transfer step comprises a step consisting in resuspending the microorganisms washed in a saline solution, for example at 0.45%, contained in the release well.
    According to one embodiment of the extraction method, the resuspension step comprises the following steps:
    - at least partially immerse the filtration and transfer device in the saline solution contained in the release well,
    - contacting repeatedly, for example by a vertical reciprocating movement of the release well and / or the filtration and transfer device, the filtration element with the internal layer of crosslinked silicone covering at least in part the internal surface of the release well for a predetermined duration, and advantageously at a predetermined frequency.
    According to one embodiment of the extraction process, the latter comprises a step consisting in carrying out a measurement of the optical density of the saline solution contained in the release well and containing the washed microorganisms. The optical density measurement can for example be carried out in the release well above the internal layer of crosslinked silicone or directly in the pipetting element.
    According to one embodiment of the extraction process, the latter comprises a step consisting in adjusting, for example to 0.5 McF, the concentration of microorganisms in the saline solution contained in the salting-out well by adding solution saline in the release well. This step can advantageously be carried out automatically by controlling the addition of saline solution calculated from the optical density measurement carried out in the release well.
    According to one embodiment of the extraction process, the latter comprises the following steps:
    - take, from the release well, a predetermined quantity of the saline solution containing the microorganisms previously adjusted to an optical density value of 0.5 McF, for example between 100 and 300 pL and advantageously 145 or 285 pL depending on the type of microorganisms (Gram-positive or Gram-negative),
    - introduce the saline solution taken into an analysis container containing a predetermined amount of saline solution, for example 0.45%, in order to obtain a predetermined concentration of microorganisms, for example 0.05 or 0.025 McF in depending on the type of microorganism (for example gram-negative, gram-positive or yeast), to prepare for the launch of an antibiogram analysis directly from the positive blood culture without going through the culture step on medium.
    According to one embodiment of the extraction process, the analysis container contains between 2 and 4 mL, preferably 3 mL of 0.45% saline solution.
    According to one embodiment of the extraction method, the analysis container is mounted, for example removably, on the support strip. Advantageously, the analysis container is placed near the release well.
    According to one embodiment of the extraction method, the step of analyzing the transferred microorganisms consists in carrying out an antibiogram analysis. Advantageously, the analysis step comprises a step consisting in transferring a predetermined quantity of the consumable, contained in the analysis container and containing the transferred microorganisms, into an antibiogram analyzer, and performing an antibiogram analysis on the consumable transferred.
    According to a variant implementation of the extraction process, the resuspension step comprises a step consisting in rubbing the filter element on a nylon grid previously wetted with 0.45% saline solution.
    According to another variant implementation of the extraction process, the resuspension step comprises the following steps:
    - at least partially introducing the filtration and transfer device into the release well,
    - cut the filtration element, for example using a cutting piece inserted in the release well,
    - release the filter element in the saline solution contained in the release well,
    - set in motion the release well for a predetermined period, for example for 30 seconds to 1 minute, so as to release the microorganisms from the filtration element and to resuspend them in the saline solution.
    According to one embodiment of the extraction method, the cutting step comprises a step consisting in rotating the release well relative to the filtration and transfer device, for example on a half-turn.
    According to one embodiment of the extraction process, the latter further comprises the following steps:
    - provide a rack,
    - position the support bar on the rack.
    According to one embodiment of the extraction method, the step of positioning the support bar comprises a step consisting in automatically moving the shutter member from the shutter position to the release position.
    According to one embodiment of the extraction method, the latter comprises a step consisting in providing an automatic device comprising a manipulation part configured to grip / release and move the pipetting element mounted on the support bar and to grip / release and move the filtration and transfer device mounted on the support bar.
    According to one embodiment of the extraction method, the manipulation part of the planned automaton is configured to move the pipetting element and the filtration and transfer device respectively according to Cartesian displacements.
    According to one mode of implementation of the extraction method, the machine provided comprises a reception area configured to receive a rack equipped with the support bar.
    In any case, the invention will be better understood with the aid of the description which follows with reference to the appended schematic drawings showing, by way of nonlimiting examples, two embodiments of this extraction system.
    Figure 1 is a side view of an extraction system according to a first embodiment of the invention.
    Figure 2 is a sectional view of the extraction system of Figure 1.
    Figure 3 is a partial sectional view of a distal end portion of a filtration and transfer device of the extraction system of Figure 1.
    Figure 4 is a perspective view of a rack equipped with a plurality of extraction systems of Figure 1.
    Figure 5 is a perspective view of an automaton equipped with the rack of Figure 4.
    Figure 6 is a partial perspective view of an extraction system according to a second embodiment of the invention.
    Figure 7 is a perspective view of a rack equipped with a plurality of extraction systems of Figure 6 and further additional containers.
    Figure 8 is a sectional view of the rack of Figure 7, the sealing elements configured to close a reagent well and washing wells of an extraction system fitted to the rack having however been added.
    Figures 9 and 10 are partial perspective views of an extraction system according to a third embodiment of the invention.
    Figure 11 is a perspective view of a MALDI-TOF MS analysis plate.
    Figures 1 to 3 show an extraction system 2, for extracting microorganisms contained in a sample, according to a first embodiment of the invention. The sample can for example be a microbiological culture, such as a positive blood culture. The sample can also be a body fluid, such as a cerebrospinal fluid, a synovial fluid, urine or pus having previously been cultured in a suitable liquid culture medium, in order to increase the concentration of microorganisms per mL.
    The extraction system 2 comprises a support strip 3 made for example of plastic, and advantageously provided with an identification code, such as a bar code.
    As shown more particularly in FIG. 2, the support strip 3 notably comprises a sample well 4 intended to receive a sample containing microorganisms, a reagent well 5 containing a selective lysis buffer L, and one or more washing wells each containing a washing reagent W. According to the embodiment shown in FIGS. 1 to 3, the sample well 4, the reagent well 5 and the washing well (s) are added to the support strip 3. However, according to another embodiment of the extraction system 2, the support strip 3, the sample well 4, the reagent well 5 and the washing well (s) could be produced directly by molding, and including injection molding (see Figure 6).
    According to the embodiment represented in FIGS. 1 to 3, the support strip 3 comprises a washing well 6.1 containing a washing reagent having formulation identical to the formulations described in the document W02013 / 016211, a washing well 6.2 containing a reagent of washing having formulation identical to the formulations described in the document WO2013 / 016211, and a washing well 6.3 containing for example sterile deionized water. Each of the washing wells can for example contain 1400 μL of the respective washing reagent.
    According to one embodiment of the invention, the selective lysis buffer L contained in the reagent well 5 has a formulation identical to the formulations described in document WO2013 / 016211.
    The sample well 4, the reagent well 5 and the washing well (s) 6.1, 6.2, 6.3 are advantageously aligned in the direction of extension of the support strip 3. Advantageously, the reagent well 5 and the washing well (s) 6.1, 6.2, 6.3 are closed by respective sealing lids 7, consisting for example of an aluminum / polyethylene complex heat-sealed on the surface of the reagent and washing wells.
    The support bar 3 also includes a gripping part
    3.1 facilitating its handling by an operator.
    The support strip 3 further comprises a protective part 8 delimiting a protective and retention housing 9 which is open upwards and whose function will be specified below. According to the embodiment shown in Figures 1 to 3, the protective part 8 is arranged between the sample well 4 and the reagent well 5. This position is provided in order to minimize the risk of soiling, during the displacement of the pipetting element above the support strip 3.
    The extraction system 2 further comprises a pipetting element 10, for example of plastic material, removably mounted on the support strip 3, and extending at least partially in the protection and retention housing 9. According to the embodiment shown in Figures 1 to 3, the pipetting element 10 has a tapered distal end portion 10.1 extending in the protection and retention housing 9, and a proximal end portion 10.2 configured to be connected to a vacuum source or a volume displacement member, such as an electronically controlled syringe. Advantageously, the protection and retention housing 9 is configured to retain drops of liquid, such as drops of sample or of reagents, liable to flow by gravity from the tapered distal end portion 10.1 of the pipetting element
    10. Once the pipetting operations have been carried out, the pipetting element 10 is returned to its position in its housing in the extraction bar 3.
    The extraction system 2 also comprises a filtration and transfer device 11 intended to capture and accumulate microorganisms contained in the sample and to transfer the captured and accumulated microorganisms in particular to an analysis support or container. The filtration and transfer device 11 is removably mounted on the support strip 3, and extends at least partially in the protection and retention housing 9.
    The filtration and transfer device 11 more particularly comprises a hollow body 12 having an elongated shape and comprising a proximal end portion 12.1 and a distal end portion 12.2, and a filtration element 13 fixed to the end portion distal 12.2 of the hollow body 12, for example by welding and in particular by thermal welding or ultrasound, or even by solvent. The distal end portion 12.2 of the hollow body 12 and the filter element 13 extend into the protection and retention housing 9, and the protection and retention housing 9 is configured to retain drops of sample or of reagents capable of flowing by gravity from the filtration and transfer device 11. However, according to another embodiment of the extraction system 2 (see FIGS. 6 to 8), the protective part 8 could comprise two protection and retention housings 9.1, 9.2 in which the distal end portion 10.1 of the pipetting element 10 and the distal end portion 12.2 of the hollow body 12 of the filtration and transfer device 11 would respectively be mounted.
    The hollow body 12 of the filtration and transfer device 11 is advantageously rigid or semi-rigid, and can be made of plastic, and for example of injected crystal polystyrene or injected polypropylene.
    According to the embodiment shown in Figures 1 to 3, the hollow body 12 has a tapered intermediate portion 12.3 converging towards the distal end portion 12.2 of the hollow body 12, and the distal end portion 12.2 is cylindrical. However, the hollow body 12 could have a different shape, and the distal end portion 12.2 could in particular be tapered. For example, according to the embodiment shown in Figure 8, the hollow body 12 has a cross section which gradually decreases from the proximal end portion 12.1 to the distal end portion 12.2.
    It should be noted that the proximal end portion 12.1 of the hollow body 12 is configured to be connected to the source of vacuum, and can for example be provided with a retaining flange 14. This retaining flange 14 also serves as a plane of reference in order to precisely position the filtration and transfer device in a Cartesian reference frame during the transfer steps on the MALDI-TOF MS analysis plate.
    As shown more particularly in FIG. 3, the filter element 13 extends at least in part outside the hollow body 12. For example, the filter element 13 projects from the end portion distal 12.2 of the hollow body 12. According to the embodiment shown in Figures 1 to 3, the filter element 13 is of convex shape, and more particularly has a dome shape. However, the filter element 13 could have another shape, and for example be flat and have a disc shape. The filter element 13 could also be cylindrical in shape and have a flat or hemispherical distal part. The filter element 13 could moreover have a diameter adapted to the diameter of a location of a MALDI-TOF MS analysis plate. The filter element 13 could also be produced in situ in the hollow body.
    12.
    The filter element 13 advantageously has a pore size of between 0.1 and 10 μm, and for example around 0.45 μm, and is more particularly configured to retain at least part of the microorganisms contained in the 'sample and to let through other elements contained in the sample, such as non-microorganic elements, for example cellular debris and the figured elements of blood previously lysed with the lysis reagent.
    The filtration and transfer device 11 can further comprise an absorbent material 15 disposed in the hollow body 12 and being at least partly in contact with the filtration element 13. The absorbent material 15 can for example be selected from the group including cotton, cellulose fiber, polyethylene bead aggregate, polyester, absorbent resin, silica gel, hydrogel, molecular sieve, zeolite or other absorbent materials.
    A first method of identifying and / or characterizing microorganisms contained in a sample using an extraction system 2 according to the first embodiment of the invention will now be described.
    The first identification and / or characterization process comprises the following steps:
    - detect the identification code with which the container initially containing the sample is provided (for example a positive blood culture bottle),
    - load the support strip 3 on a rack 16 (see FIG. 4),
    - detect the identification code with which the extraction strip 3 is provided,
    - introduce a predetermined quantity, for example 1 mL, of a sample containing microorganisms, for example a positive blood culture, into the sample well 4 of the extraction system 2,
    load the rack 16 equipped with the support strip 3 into an automaton 17 (see FIG. 5) comprising a handling part 18 configured to grasp / release and move the pipetting element 10 mounted on the support strip 3 according to Cartesian movements and for gripping / releasing and moving the filtration and transfer device 11 mounted on the support bar 3 according to Cartesian movements, the handling part 18 being for example configured to move the pipetting element 10 and the filtration and transfer 11 of the or each support strip 3 loaded on the rack 16, along the Y axis and along the Z axis of a Cartesian coordinate system, and comprising for example several first handling elements 18.1 each configured for grab / release a respective pipetting element 10 and several second handling elements 18.2 each configured to grab / release a filtering device on and transfer 11 respectively, the first manipulation elements 18.1 being for example aligned in a first alignment direction and the second manipulation elements 18.2 being for example aligned in a second alignment direction substantially parallel to the first direction of alignment,
    - optionally, using a camera 19 belonging to the automaton 17, taking images of predetermined locations 21 of a MALDI-TOF MS 22 mass spectrometry analysis plate placed on a support module 23 of the automaton 17, before making deposits on these predetermined locations 21, the support module 23 being for example movable along the axis X of a Cartesian coordinate system,
    - grasp the pipetting element 10 mounted on the support bar 3 using the handling part 18,
    - pierce the sealing films 7 of the washing wells 6.1, 6.2, 6.3 and of the reagent well 5 while progressing from the washing well 6.3 towards the reagent well, for example using the pipetting element 10 ,
    - take a predetermined quantity, for example 500 μL, of the selective lysis buffer L contained in the reagent well 5 using the pipetting element 10,
    - introduce, using the pipetting element 10 and into the sample well 4, the selective lysis buffer removed,
    mix the sample and the selective lysis buffer contained in the sample well 4 using the pipetting element 10,
    - reposition the pipetting element 10 on the support strip 3,
    - incubate the mixture for a predetermined period, for example between 1 and 5 minutes, preferably for 2 minutes, at room temperature so as to lyse cells and / or non-microorganic particles contained in the sample and / or lyse predetermined microorganisms contained in the sample to provide a lysed sample, also called a lysate,
    - grasp the filtration and transfer device 11 mounted on the support bar 3 using the handling part 18,
    - Apply and maintain a vacuum between -50 mbar and -980 mbar relative to atmospheric pressure, preferably about -600 mbar relative to atmospheric pressure, in the hollow body 12 of the filtration and transfer device 11, the vacuum being generated by a pump 20 belonging to the automaton 17 and fluidly connected to the handling part 18, the pump 20 being for example a vacuum and overpressure pump, and being able for example to be connected to a vacuum ballast and a ballast pressure,
    - immerse at least partially the distal end portion 12.2 of the filtration and transfer device 11 in the lysed sample contained in the sample well 4,
    - filter under suction, for example for 2 minutes, at least part of the sample lysed through the filter element 13,
    - capture and accumulate microorganisms in the filtration element 13,
    - immerse at least partially the distal end portion 12.2 of the filtration and transfer device 11 in the washing reagent contained in the washing well 6.1,
    washing, for example by a cycle of vertical reciprocating movement for 30 s to 2 min, preferably 1 min, in the washing reagent contained in washing well 6.1, the filtration element 13 and the microorganisms captured and accumulated,
    - immerse at least partially the distal end portion 12.2 of the filtration and transfer device 11 in the washing reagent contained in the washing well 6.2,
    - wash, for example by a cycle of vertical reciprocating movement for 30 s to 2 min, then statically for 30 s to 5 min, preferably 3 min, in the washing reagent contained in washing well 6.2, the element 13 and the microorganisms captured and accumulated,
    - immerse at least partially the distal end portion 12.2 of the filtration and transfer device 11 in the washing reagent contained in the washing well 6.3,
    - wash, for example by a cycle of vertical reciprocating movement for 30 s to 2 min, then statically for 30 s to 5 min, preferably 3 min, in the washing reagent contained in washing well 6.3, the element 13 and the microorganisms captured and accumulated,
    - remove the distal part of the filtration and transfer device 11 from the washing reagent contained in the washing well 6.3 and move the distal end portion 12.2 of the hollow body 12 transversely to the direction of extension of the hollow body 12 from such that the distal end portion of the hollow body 12 comes into contact with the internal wall of the washing well 6.3 and that the residual washing drops present at the distal end portion 12.2 of the hollow body 12 are collected in the washing well 6.3,
    - maintain the distal end portion 12.2 of the hollow body 12 against the internal wall of the washing well 6.3 for a predetermined period, for example for 5 to 15 seconds, while maintaining the level of depression,
    - return the hollow body 12 of the filtration and transfer device 11 to atmospheric pressure or slightly overpressure (50 to 200 mbar) the time necessary for the next step,
    - deposit, using the filtration and transfer device 11, at least part of the microorganisms washed on one or more predetermined locations 21, also called spots, of the MALDI-TOF MS 22 analysis plate (see Figure 11),
    - reposition the filtration and transfer device 11 on the support strip 3,
    - incubate the MALDI-TOF MS 22 analysis plate between 40 and 80 ° C, for example at 45 ° C or 55 ° C, for a predetermined period, for example between 2 and minutes, preferably 4 minutes, so as to dry or evaporate any residual washing reagent present on the or each predetermined location 21 of the MALDI-TOF MS analysis plate 22, the support module 23 being able to this end be provided with at least one heating element 24,
    optionally, take an image of the predetermined locations 21 using the camera 19,
    - optionally, add a predetermined amount of formic acid, for example from 0.5 to 1 pL, preferably 0.5 pL, on each location 21 of the MALDI-TOF MS analysis plate 22 where microorganisms have been deposited, for example using a pipetting device (not shown in the figures) belonging to the automaton 17 and movable for example along the Y axis and along the Z axis of a Cartesian coordinate system,
    - incubate the MALDI-TOF MS analysis plate between 40 and 80 ° C, for example at 45 ° C or 55 ° C, for a predetermined period, for example between 5 and minutes, preferably 6 minutes so as to dry each predetermined location 21 of the MALDI-TOF MS analysis plate 22,
    -optionnally, take an image of the locations 21 using the camera 19,
    add a predetermined quantity of matrix, for example from 0.5 to 2 μL, preferably 1 μL of HCCA matrix, on each location 21 of the MALDI-TOF MS analysis plate 22 where microorganisms have been deposited, by example using the pipetting device,
    - incubate the MALDI-TOF MS 22 analysis plate between 40 and 80 ° C for a predetermined period, for example between 1 and 3 minutes, preferably 2 minutes,
    -optionnally, take an image of the locations 21 using the camera 19,
    - insert the MALDI-TOF MS 22 analysis plate into a mass spectrometer, and
    - perform a mass spectrometry analysis of the microorganisms transferred to each predetermined location 21 of the MALDITOF MS analysis plate 22 so as to identify and / or characterize the microorganisms.
    According to an embodiment of the first extraction method, each washing step comprises a step consisting in moving for a predetermined period, for example between 30 seconds and 5 minutes, preferably for 2 minutes, the filtration and transfer device 11 in the respective washing well, for example according to a reciprocating movement in Z. Advantageously, each step of displacement of the filtration and transfer device 11 consists in carrying out several cycles of vertical displacement of the filtration and transfer device 11 in the well respective wash. Advantageously, the filtration and transfer device 11 is completely extracted from the respective washing well at each movement cycle, for example at a frequency of approximately 0.5 to 2 Hz, and preferably 1 Hz.
    According to one embodiment of the first extraction method, the step of immersing the filtration and transfer device 11 in the washing reagent contained in the washing well 6.1 consists in immersing the filtration and transfer device 11 deeper, for example 1 to 3 mm deeper than in the lysed sample contained in the sample well 4, the step of immersion of the filtration and transfer device 11 in the washing reagent contained in the washing well 6.2 consists in immersing the filtration and transfer device 11 more deeply, for example from 1 to 3 mm deeper, than in the washing reagent contained in the washing well 6.1, and the immersion step of the filtration and transfer device 11 in the washing reagent contained in the washing well 6.3 consists in immersing the filtration and transfer device 11 deeper, for example from 1 to 3 mm deeper, than in the reagent of washing contained in the washing well 6.2.
    According to an embodiment of the first extraction method, the step of depositing the washed microorganisms on one or more predetermined locations 21 of the MALDI-TOF MS analysis plate 22 comprises a step consisting in buffering several times , for example between 3 and 7 times, the filtration element 13 on the or each predetermined location 21 of the MALDI-TOF MS analysis plate 22 with an application force of between 100 grams and 1 kg, preferably 600 grams , and according to a buffering frequency between 0.5 and 10 Hz. These provisions allow a deposit of a sufficient quantity of microorganisms on each predetermined location of the MALDI-TOF MS 22 analysis plate.
    According to one embodiment of the first extraction method, the step of depositing the microorganisms on the predetermined location or locations 21 of the MALDI-TOF MS analysis plate 22 comprises a step consisting in vibrating the device filtration and transfer 11 at least during the contact of the filtration element 13 on the or each predetermined location of the MALDI-TOF MS analysis plate. These provisions make it possible to improve the transfer of microorganisms (quantity and reproducibility) from the filtration element 13 to the MALDI-TOF MS analysis plate, in particular for microorganisms which are difficult to detach and identify or known to generate low concentrations during of their growth in blood culture flasks.
    According to an embodiment of the first extraction method, the step of vibrating the filtration and transfer device 11 consists in applying to the filtration and transfer device 11 a vibration of between 0.1 and 200 Hz , for example between 10 and 100 Hz, preferably 50 Hz, for a predetermined duration, and for example for 2 seconds.
    According to an embodiment of the first extraction method, the step of depositing the microorganisms on the predetermined location or locations 21 of the MALDI-TOF MS analysis plate 22 comprises a step consisting in applying a cycle of overpressure (adjustment of pressure, number of pulses, duty cycle or duration of the pressure pulse cycle), relative to atmospheric pressure, in the hollow body 12 of the filtration and transfer device 11 during of the contact of the filtration element 13 on the or each predetermined location 21 of the MALDI-TOF MS analysis plate 22 or between two contacts of the filtration element 13 on the MALDI-TOF MS 22 analysis plate. These provisions make it possible to improve the transfer of microorganisms from the filtration element 13 to the MALDI-TOF MS 22 analysis plate.
    According to an embodiment of the first extraction method, the step of mixing the sample and the selective lysis buffer consists in carrying out several mixing cycles, each mixing cycle consisting in aspirating and discharging a predetermined quantity of the mixture contained in the sample well 4, for example 500 μL, using the pipetting element.
    According to an embodiment of the first extraction process, the latter comprises a step carried out prior to the step of adding the matrix and consisting in adding a predetermined amount of formic acid, for example from 0.5 to 2 μL, preferably 0.5 to 1 μL, on each location of the MALDI-TOF MS analysis plate where microorganisms have been deposited. These provisions allow in particular to improve the identification results for Gram positive bacteria and yeasts.
    According to an embodiment of the first extraction method, the latter comprises a step carried out after the step of adding formic acid and consisting in incubating the MALDI-TOF MS 22 analysis plate between 40 and 80 ° C, for example at 45 ° C or 55 ° C, for a predetermined period, for example between 5 and 7 minutes, preferably 6 minutes, and this so as to accelerate the evaporation of the drops of formic acid, while ensuring sufficient contact of the formic acid with the microorganisms present on the MALDI-TOF MS analysis plate to guarantee effective lysis.
    According to an embodiment of the first extraction method, the latter comprises a step of taking an image, for example according to two distinct types of illumination, predetermined locations at different stages of the method and preferably after each drying step (vacuum, microorganisms, formic acid, matrix) in order to control the process at each stage (in particular the following points: correct transfer of the microorganisms, localization and correct mixing of the formic acid and the matrix with the deposited microorganisms, level of satisfactory crystallization of the matrix with microorganisms).
    According to an embodiment of the first extraction method, the latter comprises a step of taking an image of the rack after insertion into the automaton using a low-cost camera (webcam) incorporated in the automaton . This image capture allows, by an adequate image processing, to check the position of the rack, the number and the correct insertion of the bars, the good presence of the pipetting and filtration elements before the launching of a cycle of extraction.
    A second method for identifying and / or characterizing microorganisms contained in a sample using an extraction system 2 according to the first embodiment of the invention will now be described.
    The second identification and / or characterization process comprises the following steps:
    - load the support bar 3 and a release well 26 on a rack 16,
    - detect the identification code with which the extraction bar 3 is provided and detect the identification code with which the container containing the sample is provided (for example the blood culture bottle),
    - introduce a predetermined quantity, for example 1 mL, of a sample containing microorganisms, for example a positive blood culture, into the sample well 4 of the extraction system 2,
    - load the rack 16 fitted with the support strip 3 into an automaton,
    - grasp the pipetting element 10 mounted on the support bar 3 using the handling part 18,
    - take a predetermined quantity, for example 500 μL, of the selective lysis buffer L contained in the reagent well 5 using the pipetting element 10,
    - introduce, using the pipetting element 10 and into the sample well 4, the selective lysis buffer removed,
    mix the sample and the selective lysis buffer contained in the sample well 4 using the pipetting element 10,
    - reposition the pipetting element 10 on the support strip 3,
    - incubate the mixture for a predetermined period, for example between 1 and 5 minutes, preferably for 2 minutes, at room temperature so as to lyse cells and / or non-microorganic particles contained in the sample and / or lyse predetermined microorganisms contained in the sample to provide a lysed sample, also called a lysate,
    - grasp the filtration and transfer device 11 mounted on the support bar 3 using the handling part 18,
    applying and maintaining a vacuum between -50 mbar and -980 mbar relative to atmospheric pressure, preferably at about -600 mbar relative to atmospheric pressure, in the hollow body 12 of the filtration and transfer device 11,
    - immerse at least partially the distal end portion 12.2 of the filtration and transfer device 11 in the lysed sample contained in the sample well 4,
    - filter under suction, for example for 2 minutes, at least part of the sample lysed through the filter element 13,
    - capture and accumulate microorganisms in the filtration element 13,
    - immerse under suction at least partially the distal end portion 12.2 of the filtration and transfer device 11 in the washing reagent contained in the washing well 6.1,
    - wash under suction, in the washing reagent contained in washing well 6.1, the filter element 13 and the microorganisms captured and accumulated,
    immerse under suction at least partially the distal end portion 12.2 of the filtration and transfer device 11 in the washing reagent contained in the washing well 6.2,
    - wash under suction, in the washing reagent contained in the washing well 6.2, the filtration element 13 and the microorganisms captured and accumulated,
    - return the hollow body 12 of the filtration and transfer device 11 to atmospheric pressure or slightly overpressure,
    resuspending the microorganisms in a saline solution SI, for example at 0.45%, contained in the release well 26 for example disposed on the rack 16 near the support bar 3, the resetting step in suspension comprising the following stages:
    - immerse at atmospheric pressure or slightly overpressure (50 to 200 mbar) at least partially the distal end portion 12.2 of the filtration and transfer device 11 in the saline solution SI contained in the release well 26,
    - contacting repeatedly, for example by a vertical reciprocating movement of the release well 26 and / or the filtration and transfer device 11, the filtration element 13 with an internal layer 26.1 of crosslinked silicone, covering at least partly the internal surface of the release well 26, for a predetermined period so as to resuspend the microorganisms accumulated by the filtration element 13 in the saline solution SI contained in the release well 26, the step of setting contact may for example consist in applying to the filtration and transfer device 11 a vibration of between 0.1 and 200 Hz, for example between 10 and 100 Hz, preferably 50 Hz, for a predetermined duration, and for example for 30 seconds,
    - optionally, carry out this contacting repeatedly,
    - measure the optical density (OD), calculate the dilution to be carried out and adjust the OD by pipetting a volume of saline solution present in the washing well 6.3 according to the calculation carried out,
    - take, from the release well 26, a predetermined quantity of the saline solution SI containing the microorganisms, preferably between 100 and 300 pL and for example 145 or 285 pL,
    - Introducing the saline solution taken from an analysis container 27, for example placed on the rack 16 near the release well 26, containing a predetermined amount, for example between 2 and 4 mL, preferably 3 mL, of saline solution S2 , for example 0.45%,
    transfer the consumable contained in the analysis container 27 to an antibiogram analyzer,
    - perform an antibiogram analysis on the transferred consumable.
    According to one embodiment of the second extraction method, the latter comprises a step consisting in carrying out a measurement of the optical density of the saline solution SI contained in the release well 26.
    According to an embodiment of the second extraction process, the latter comprises a step consisting in adjusting the concentration of microorganisms in the saline solution SI contained in the salvage well 26 to 0.5 McF by adding saline solution taken from well 6.3 containing a reserve of 0.45% saline solution.
    According to one embodiment of the second extraction method, the analysis container 27 could be mounted, for example removably, directly on the support strip 3.
    According to a variant implementation of the second extraction process, the step of resuspending the microorganisms could consist in rubbing the filter element 13 on a nylon grid previously wetted with 0.45 saline solution. %.
    According to yet another variant implementation of the second extraction method, the resuspension step could include the following steps:
    at least partially introducing the filtration and transfer device 11 into the release well 26,
    cutting the filtration element 13, for example using a cutting piece inserted in the release well 26,
    release the filter element 13 in the saline solution SI contained in the release well 26,
    - set in motion the release well 26 for a predetermined period, for example for 30 seconds to 1 minute, so as to release the microorganisms accumulated in the filter element 13 and to resuspend them in the saline solution SI contained in the release well 26.
    Figures 9 and 10 show an extraction system 2 according to a third embodiment which differs from the first embodiment essentially in that the extraction system 2 comprises a shutter member 28 movable between a shutter position in which the shutter member 28 closes the sample well 4 and a release position in which the shutter member 28 at least partially releases the sample well 4. The shutter member 28 can by example be pivotally or slidingly mounted on the support strip 3. In addition, the rack 16 could be configured to automatically move the shutter member 28 in the shutter position when positioning the support strip on the rack 16 .
    The support bar 3 could additionally comprise a locking device configured to cooperate with a rack 16 so as to lock the support bar 3 on the rack 16. The locking device could for example include (see FIG. 2) a locking element , such as a locking pin, configured to cooperate with the rack so as to immobilize the support bar 3 in a direction Y of a Cartesian coordinate system, and a retaining element, such as a steel ball, configured to cooperate with a permanent magnet provided on the rack 16 so as to immobilize the support bar 3 in a direction Z of the Cartesian coordinate system during the insertion of the bar into the rack. The steel ball could however be replaced for example by a retaining housing configured to receive a retaining lug provided on the rack.
    It goes without saying that the invention is not limited to the sole embodiment of this extraction system, described above by way of example, on the contrary, it embraces all of the variant embodiments thereof.
    权利要求:
    Claims (15)
    [1" id="c-fr-0001]
    1. Extraction system (2) for extracting microorganisms contained in a sample, the extraction system (2) comprising:
    - a support bar (3) comprising:
    - a sample well (4) intended to receive a sample containing microorganisms, such as a positive blood culture,
    - a reagent well (5) intended to contain a selective lysis buffer,
    at least one washing well (6.1) intended to contain a washing reagent, and
    -at least one protective and retention housing (9, 9.1,
    9.2),
    - a pipetting element (10) removably mounted on the support strip (3) and extending at least partially in the at least one protection and retention housing (9),
    - a filtration and transfer device (11) intended to capture and accumulate microorganisms contained in the sample and to transfer the captured and accumulated microorganisms, the filtration and transfer device (11) being removably mounted on the support bar (3) and extending at least partially in the at least one protection and retention housing (9), the filtration and transfer device (11) comprising:
    a hollow body (12) having an elongated shape and comprising a proximal end portion (12.1) and a distal end portion (12.2), and
    - a filtration element (13) fixed to the distal end portion (12.2) of the hollow body (12).
    [2" id="c-fr-0002]
    2. Extraction system according to claim 1, in which the distal end portion (12.2) of the hollow body (12) and the filter element (13) extend in the at least one protective housing and retention (9).
    [3" id="c-fr-0003]
    3. Extraction system (2) according to claim 1 or 2, wherein the pipetting element (10) comprises a distal end portion (10.1) extending at least partially in the at least one housing protection and retention (9).
    [4" id="c-fr-0004]
    4. Extraction system (2) according to any one of claims 1 to
    3, in which the filtration and transfer device (11) comprises an absorbent material arranged in the hollow body.
    [5" id="c-fr-0005]
    5. Extraction system (2) according to any one of claims 1 to
    4, in which the support bar (3) further comprises a release well (26).
    [6" id="c-fr-0006]
    6. Extraction system (2) according to any one of claims 1 to
    5, in which the at least one washing well (6.1) contains a washing reagent and the reagent well (5) contains a selective lysis buffer (L).
    [7" id="c-fr-0007]
    7. Extraction system (2) according to claim 6, wherein the reagent well (5) and the at least one washing well (6.1) are each closed by a sealing element (7).
    [8" id="c-fr-0008]
    8. Extraction system (2) according to any one of claims 1 to 7, which further comprises a shutter member (28) movable between a shutter position in which the shutter member (28) closes the sample well (4) and a release position in which the shutter member (28) at least partially releases the sample well (4).
    [9" id="c-fr-0009]
    9. A method of identifying and / or characterizing microorganisms contained in a sample, the method of identifying and / or characterizing comprising the following steps:
    - providing an extraction system (2) according to any one of claims 1 to 8, in which the reagent well (5) contains a selective lysis buffer and the at least one washing well (6.1) contains a washing reagent,
    - introduce a predetermined quantity of a sample containing microorganisms into the sample well (4),
    - withdraw a predetermined quantity of the selective lysis buffer contained in the reagent well (5) using the pipetting element (10),
    - introduce, into the sample well (4) and using the pipetting element (10), the selective lysis buffer withdrawn,
    - lyse non-microorganic cells and / or particles contained in the sample and / or lyse predetermined microorganisms contained in the sample so as to provide a lysed sample,
    - at least partially immerse the filtration and transfer device (11) in the lysed sample contained in the sample well (4),
    - filter under suction at least part of the lysed sample through the filter element (13),
    - capture and accumulate microorganisms in the filtration element (13),
    - at least partially immerse the filtration and transfer device (11) in the washing reagent contained in the at least one washing well (6.1),
    - washing, in the washing reagent contained in the at least one washing well (6), the filtration element (13) and the microorganisms captured and accumulated,
    - transfer, using the filtration and transfer device (11), the washed microorganisms to an analysis support or to a container,
    - analyze the microorganisms transferred so as to identify and / or characterize them.
    [10" id="c-fr-0010]
    10. The method of claim 9, wherein the transfer step comprises a step of resuspending the washed microorganisms in a saline solution (SI) contained in a release well (26).
    [11" id="c-fr-0011]
    11. The method according to claim 10, wherein the step of resuspension comprising the following steps:
    - at least partially immerse the filtration and transfer device (11) in the saline solution SI contained in the release well (26),
    - Repeatedly bringing the filter element (13) into contact with an internal layer of crosslinked silicone (26.1) covering at least in part the internal surface of the release well (26) for a predetermined period.
    [12" id="c-fr-0012]
    12. The method according to claim 11, which further comprises the following steps:
    - carry out a measurement of the optical density of the saline solution (SI) contained in the release well (26) and containing the washed microorganisms,
    - adjust, for example to 0.5 McF, the concentration of microorganisms in the saline solution (SI) contained in the release well (26) by adding saline solution in the release well (26),
    - withdraw, from the release well (26), a predetermined amount of the saline solution (SI) containing the microorganisms,
    - introduce the sampled saline solution (SI) into an analysis container (27) containing a predetermined quantity of saline solution (S2).
    [13" id="c-fr-0013]
    13. The method of claim 12, wherein the step of analyzing
    5 includes the following steps:
    transferring a predetermined quantity of the consumable, contained in the analysis container (27) and containing the transferred microorganisms, into an antibiogram analyzer, and
    - perform an antibiogram analysis on the transferred consumable.
    [14" id="c-fr-0014]
    14. The method of claim 9, wherein the transfer step comprises a step of depositing, using the filtration and transfer device (11), at least a portion of the microorganisms washed on one or more predetermined locations (21) of a MALDI-TOF MS analysis plate (22), and
    [15" id="c-fr-0015]
    Wherein the analysis step includes a step of performing MALDI-TOF type mass spectrometry analysis of the microorganisms deposited on said MALDI-TOF MS plate (22).
    1/5
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    引用文献:
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    法律状态:
    2017-11-27| PLFP| Fee payment|Year of fee payment: 2 |
    2018-05-18| PLSC| Publication of the preliminary search report|Effective date: 20180518 |
    2019-11-25| PLFP| Fee payment|Year of fee payment: 4 |
    2020-11-25| PLFP| Fee payment|Year of fee payment: 5 |
    2021-11-24| PLFP| Fee payment|Year of fee payment: 6 |
    优先权:
    申请号 | 申请日 | 专利标题
    FR1661015|2016-11-15|
    FR1661015A|FR3058734B1|2016-11-15|2016-11-15|EXTRACTION SYSTEM AND METHOD FOR EXTRACTING MICRO-ORGANISMS CONTAINED IN A SAMPLE|FR1661015A| FR3058734B1|2016-11-15|2016-11-15|EXTRACTION SYSTEM AND METHOD FOR EXTRACTING MICRO-ORGANISMS CONTAINED IN A SAMPLE|
    PCT/FR2017/053121| WO2018091818A1|2016-11-15|2017-11-15|Extraction system and method for extracting microorganisms contained in a sample|
    EP17811989.7A| EP3541517A1|2016-11-15|2017-11-15|Extraction system and method for extracting microorganisms contained in a sample|
    US16/461,007| US20190270958A1|2016-11-15|2017-11-15|Extraction system and method for extracting microorganisms contained in a sample|
    CN201780070533.9A| CN109963652A|2016-11-15|2017-11-15|For extracting the extraction system and method for the microorganism for including in sample|
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