Handling device, handling assembly and handling method

Abstract

Manipulation apparatus, manipulation assembly and manipulation method. Manipulation apparatus (5) of a sample of biological material by means of capture elements, comprising: - a tube support configured to receive a tube (3);- an aggregation member (15) configured to generate a magnetic attraction force on the capture elements (2), and intended to cooperate with an interface sheath (20), - an interface sheath carriage (30) movable between a high position and a low position, - a excitation system (45) configured to be placed in contact with the interface sheath (20) and to generate in the interface sheath (20) vibrations adapted to separate the capture elements from the interface sheath (20) after the aggregation member (15) and the interface sheath (20) have passed from an active state in which the aggregation member (15) exerts the magnetic attraction force on the capture elements to an inactive state in which the aggregation member (15) does not exert the magnetic attraction force on the capture elements. Abstract figure: Fig. 7;

Description

Title of the invention: Manipulation apparatus, manipulation assembly and manipulation method SCOPE OF DISCLOSURE

[0001] The present disclosure relates to an apparatus for handling a sample of biological material by means of capture elements as well as to a handling assembly and a handling method implementing such a handling apparatus.

[0002] Without being limited thereto, the present disclosure applies in particular to the manipulation of a sample of nucleic acids, proteins, antibodies or other biological material, by means of capture elements, generally in the form of beads, magnetic and configured to be secured to the sample of biological material. STATE OF THE ART

[0003] During its treatment, a sample of biological material is manipulated in particular by being successively immersed in different solutions. This is for example the case of a purification treatment of a sample of nucleic acids as biological material.

[0004] To recover the sample of biological material dispersed in each of the solutions and to be able to release it into one of the other solutions, it is known to use a handling device of the type comprising: - a frame extending along a vertical axis, - a tube holder mounted on the frame and comprising at least one receiving space configured to receive at least one tube adapted to contain a solution comprising the sample of biological material and the capture elements; - an aggregation member configured to generate a magnetic attraction force on the capture elements, the aggregation member being intended to cooperate with an interface sheath in such a way that, in an active state of the aggregation member and the interface sheath, the aggregation member is received in the interface sheath and exerts the magnetic attraction force on the capture elements to aggregate said capture elements on the interface sheath and, in an inactive state of the aggregation member and the interface sheath, the aggregation member does not exert a magnetic attraction force on the capture elements.

[0005] An example of a manipulation apparatus of the aforementioned type is described in document WO 2021 / 056005. The aggregation member comprises a permanent magnet which, in the inactive state of the aggregation member and the interface sheath, is placed at a distance from the interface sheath. Even in the absence of magnetic attraction, capture elements may remain attached to the interface sheath. These capture elements and residues of biological material attached to them may not follow one of the processing steps and may, during a subsequent step, contaminate a solution or the part of the biological material sample that has followed all the processing steps.

[0006] To release the capture elements when the aggregation member and the interface sheath are immersed in the tube solution, the handling apparatus of document WO 2021 / 056005 provides for moving the aggregation member apart and rotating the interface sheath in the tube solution.

[0007] However, capture elements may remain attached to the interface sheath and the turbulence generated in the solution by the rotation of the interface sheath may damage the sample of biological material. SUMMARY OF DISCLOSURE

[0008] The invention aims to solve the problem stated above.

[0009] To do this, according to a first aspect, the invention proposes an apparatus for handling a sample of biological material by means of capture elements, the capture elements being magnetic and configured to be secured to the sample of biological material, the handling apparatus comprising: - a frame extending along a vertical axis, - a tube holder mounted on the frame and comprising at least one receiving space configured to receive at least one tube adapted to contain a solution comprising the sample of biological material and the capture elements; - an aggregation member configured to generate a magnetic attraction force on the capture elements, the aggregation member being intended to cooperate with an interface sheath in such a way that, in an active state of the aggregation member and the interface sheath, the aggregation member is received in the interface sheath and exerts the magnetic attraction force on the capture elements to aggregate said capture elements on the interface sheath and, in an inactive state of the aggregation member and the interface sheath, the aggregation member does not exert a magnetic attraction force on the capture elements, the handling apparatus further comprising: - an interface sheath carriage comprising a holding device configured to hold the interface sheath opposite the receiving space, the interface sheath carriage being movable along the vertical axis relative to the frame between a high position in which said interface sheath carriage places the interface sheath at a distance from the receiving space, and a low position in which said interface sheath carriage places the interface sheath in the receiving space, - a biasing system configured to be placed in contact with the interface sheath and to generate vibrations in the interface sheath suitable for separating the capture elements from the interface sheath after the aggregation member and the interface sheath have passed from the active state to the inactive state.

[0010] Thus, the handling apparatus according to the invention imposes a stress on the interface sheath alone, which makes it possible to make the detachment of the capture elements more reliable while preserving the sample of biological material.

[0011] According to particular provisions, the handling apparatus can make it possible to carry out in a simple and intuitive manner one or more steps of a treatment of a sample of unitary biological material. It can be adapted to different types of treatment of different types of sample of biological material. It can offer a compactness and robustness allowing it to be easily transported.

[0012] The stressing system may be arranged to generate the vibrations in the vicinity of an access end of the interface sheath through which the aggregation member is inserted into the interface sheath, and in which the holding device is configured to allow propagation of the vibrations from the access end to a free end of the interface sheath opposite the access end and in the vicinity of which the capture elements are aggregated in the active state of the aggregation member and the interface sheath.

[0013] The holding device may be configured to hold the interface sheath in the vicinity of the access end.

[0014] The holding device may comprise a ring along a holding axis parallel to the vertical axis and having an inner surface intended to be in peripheral contact with the interface sheath.

[0015] The ring may be configured to receive the interface sheath comprising a side wall along a sheath axis and at least two lugs projecting transversely in an equidistant manner from an outer surface of the side wall, the ring comprising a groove provided on the inner surface around the holding axis and configured to receive the lugs, and at least two grooves extending axially on the inner surface from a passage end to the groove, so as to allow passage of the lugs of the interface sheath in an angular position of the interface sheath relative to the ring and maintenance of the lugs of the interface sheath in the groove in other angular positions of the interface sheath relative to the ring.

[0016] The holding device may further comprise a flange movable relative to the interface sheath carriage between an open position allowing the ring to be placed in position and a closed position ensuring that the ring is held in position relative to the interface sheath carriage.

[0017] The biasing system may be configured to perform at least one shock on the interface sheath.

[0018] The biasing system may comprise a cam driven in rotation about a pivot axis and comprising a radial contact portion arranged to come into contact with the interface sheath in an angular position range of the cam relative to the frame, the cam being at a distance from the interface sheath outside the angular position range.

[0019] The cam may have a biasing state in which said cam is rotated and a stopping state in which said cam is fixed relative to the frame, and the biasing system may include a detection device configured to stop the cam outside the angular position range when the cam transitions from the biasing state to the stopping state.

[0020] The biasing system may be mounted on the interface sheath carriage.

[0021] The handling apparatus may further comprise an aggregation member carriage carrying the aggregation member, the aggregation member carriage being movable along the vertical axis relative to the frame between a disassembled position remote from the interface sheath carriage such that the aggregation member is spaced from the interface sheath, and an assembled position proximate to the interface sheath carriage such that the aggregation member is received in the interface sheath.

[0022] The aggregation member may include a permanent magnet at a distal end for receipt in the interface sheath, the aggregation member and the interface sheath being in the inactive state when the aggregation member carriage is in the disassembled state.

[0023] The tube support may comprise a barrel having a plurality of receiving spaces around a barrel axis, the barrel being rotatable along the barrel axis so as to selectively place one of the receiving spaces opposite the interface sheath.

[0024] According to a second aspect, the invention proposes a handling assembly comprising a handling apparatus as defined previously and at least one interface sheath configured to receive the aggregation member and to be held in the holding device of the interface sheath carriage.

[0025] According to a third aspect, the invention proposes a method for handling a sample of biological material, the handling method implementing a handling assembly as defined above and providing for: - placing in the receiving space of the tube holder a tube containing a solution comprising the sample of biological material and capture elements, the capture elements being magnetic and configured to be secured to the sample of biological matter, - place the interface sheath in the holding device of the interface sheath carriage in the high position, - placing the interface sheath carriage in the low position and the aggregating member and the interface sheath in the active state, the aggregating member being received in the interface sheath and exerting the magnetic attraction force on the capture elements to aggregate said capture elements on the interface sheath, - put the interface sheath carriage in the high position, the aggregation member and the interface sheath remaining in the active state, - put the interface sheath carriage in the low position, the aggregation member and the interface sheath remaining in the active state, - putting the aggregation member and the interface sheath in the inactive state, - activating the stress system to generate vibrations in the interface sheath suitable for separating the capture elements from the interface sheath. BRIEF DESCRIPTION OF THE FIGURES

[0026] Other objects and advantages of the invention will appear on reading the following detailed description of a particular embodiment of the invention made in relation to the appended drawings in which:

[0027] [Fig.l] - [Fig.l] is a perspective representation of a manipulation assembly comprising a manipulation apparatus and an interface sheath for manipulating a sample of biological material by means of capture elements, the capture elements configured to be secured to the sample of biological material, the interface sheath being configured to receive an aggregation member of the manipulation apparatus in such a way that, in an active state of the aggregation member and the interface sheath, the aggregation member is received in the interface sheath and exerts the magnetic attraction force on the capture elements to aggregate them,

[0028] [Fig.2]

[0029] [Fig.3] - Figures 2 and 3 are partial representations respectively in front and side view of the handling assembly of [Fig.l], illustrating the aggregation member and the interface sheath mounted respectively on an aggregation member carriage and an interface sheath carriage movable along a vertical axis on a frame, the interface sheath carriage being in the high position in which it places the interface sheath at a distance from a tube adapted to contain a solution comprising the sample of biological material and the capture elements, the aggregation member carriage being in the disassembled position at a distance from the interface sheath carriage in such a way that the aggregation member is spaced from the interface sheath,

[0030] [Fig.4] - [Fig.4] is an enlarged side view representation of a portion of the handling assembly of [Fig.l], illustrating the interface sheath carriage in the lowered position in which it places the interface sheath in the tube, the aggregation member carriage being in the disassembled position

[0031] [Fig.5] - [Fig.5] is an enlarged side view representation of a portion of the handling assembly of [Fig.l], illustrating the interface sheath carriage in the lowered position and the aggregator member carriage in the assembled position proximate the interface sheath carriage such that the aggregator member is received in the interface sheath,

[0032] [Fig.6] - [Fig.6] is an enlarged side view representation of a portion of the handling assembly of [Fig.l], illustrating the interface sheath carriage in the upper position and the aggregation member carriage in the assembled position,

[0033] [Fig.7] - [Fig.7] is an enlarged perspective view of a portion of the handling assembly of [Fig.l], illustrating the interface sheath carriage comprising a holding device configured to hold the interface sheath opposite the tube, and a biasing system configured to be placed in contact with the interface sheath and generate vibrations in the interface sheath adapted to separate the capture elements from the interface sheath after the aggregation member and the interface sheath have passed from the active state to an inactive state,

[0034] [Fig.8] - [Fig.8] is an exploded perspective view from above of the holding device of [Fig.7] and the interface sheath, the holding device comprising a ring into which the interface sheath is inserted and a flange configured to receive the ring,

[0035] [Fig.9] - [Fig.9] is an exploded perspective view from below of the ring of the holding device of [Fig.8],

[0036] [Fig. 10] - [Fig. 10] is a sectional representation along the orientation referenced XX in [Fig.8] of the ring of the holding device of [Fig.8],

[0037] [Fig. 11]

[0038] [Fig. 12] - Figures 11 and 12 are enlarged representations in front view of the stressing system of [Fig.7], the stressing system comprising a cam driven in rotation to produce at least one shock on the interface sheath generating the vibrations in the vicinity of an access end of the interface sheath by which the aggregation member is inserted into the interface sheath,

[0039] [Fig. 13] - [Fig. 13] is an exploded perspective representation of the biasing system of [Fig.7], illustrating a detection device configured to stop the cam outside an angular position range in which the cam contacts the interface sheath,

[0040] [Fig. 14] - [Fig. 14] is a schematic representation of a particular example, non-limiting, of implementing a manipulation method with the manipulation assembly of [Fig.l] in a binding step of a purification treatment of a nucleic acid sample,

[0041] [FIG.15] - Figures 15a and 15b are schematic representations of an example particular, non-limiting, example of implementing a handling method with the handling assembly of [Fig.l] in a step of washing the nucleic acid sample of the purification treatment of a nucleic acid sample,

[0042] [Fig. 16] - [Fig. 16] is a schematic representation of a particular, non-limiting example of implementing a handling method with the handling assembly of [Fig.l] in a drying step of the purification treatment of a nucleic acid sample,

[0043] [Fig. 17a]

[0044] [Fig. 17b] - Figures 17a and 17b are schematic representations of a particular, non-limiting example of implementation of a handling method with the handling assembly of [Fig.l] in an elution step of the purification treatment of a nucleic acid sample,

[0045] [Fig. 18] - [Fig. 18] is a schematic representation of a particular, non-limiting example of implementing a handling method with the handling assembly of [Fig.l] in a step of washing the capture elements of the purification treatment of a nucleic acid sample. DETAILED DESCRIPTION

[0046] Figures 1 to 3 represent an embodiment of an apparatus 5 for handling a sample of biological material 1. The handling is carried out by means of capture elements 2, generally in the form of magnetic beads and configured to be secured to the sample of biological material 1.

[0047] In a particular example, the manipulation apparatus 5 may be used to manipulate a sample of nucleic acids, proteins, antibodies, or other biological material.

[0048] The handling apparatus 5 comprises a frame 6 extending along a vertical axis A from a base 7 configured to rest on a support surface.

[0049] A tube holder 8 is mounted on the base 7 of the frame 6 and comprises one or more receiving spaces 9 each having a receiving axis R parallel to the vertical axis A. Each receiving space 9 is configured to coaxially receive a tube 3 adapted to contain a solution comprising the sample of biological material 1 and the capture elements 2. Each tube 3 has a side wall around a tube axis T and which extends from a closed bottom 3a to an upper opening 3b.

[0050] In the embodiment shown, the tube support 8 comprises a barrel 10 comprising several receiving spaces 9, for example eight, around a barrel axis B parallel to the vertical axis A. The barrel 10 is driven in rotation along the barrel axis B by a drive system, such as an electric motor placed in the base 7 of the frame 6.

[0051] The manipulation apparatus 5 comprises an aggregation member 15 configured to generate a magnetic attraction force on the capture elements 2. In the embodiment shown, without being limited thereto, the aggregation member 15 comprises a rod 16 which extends along a rod axis I between a proximal end and a distal end. The aggregation member 15 also comprises a permanent magnet 17 extending coaxially with the distal end of the rod 16.

[0052] The aggregation member 15 is configured to cooperate with an interface sheath 20. The interface sheath 20 comprises a side wall around a sheath axis G and which extends between an access end 20a by which the distal end of the aggregation member 15 can be inserted into the interface sheath 20 and a closed free end 20b. The interface sheath 20 constitutes a consumable avoiding soiling and damaging the aggregation member 15.

[0053] The interface sheath 20 is mounted on an interface sheath carriage 30. The interface sheath carriage 30 extends perpendicular to the vertical axis A from a guide end 31 slidably mounted on a vertical mast 35. The interface sheath carriage 30 is movable along the vertical axis A between high and low positions by means of a drive device comprising, in the embodiment shown, a worm screw 36 extending along the vertical axis A and driven in rotation by an electric motor placed in the base 7 of the frame 6.

[0054] In [Fig. 3], the interface sheath carriage 30 is in the high position in which it places the interface sheath 20 at a distance from and opposite one of the receiving spaces 9 with the sheath axis G aligned with the receiving axis R. When a tube 3 is placed in the receiving space 9, the sheath axis G is aligned with the tube axis T and the free end 20b of the interface sheath 20 is opposite the upper opening 3b of the tube 3. The rotation of the barrel 10 makes it possible to selectively place one of the receiving spaces 9 and, where appropriate, the tube 3 which it receives, opposite the interface sheath 20.

[0055] In [Fig.4], the interface sheath carriage 30 is in the low position in which it places the interface sheath 20 in the receiving space 9. The interface sheath 20 can then coaxially penetrate the tube 3 received in the receiving space 9 through the upper opening 3b until the free end 20b of the interface sheath 20 is located in the vicinity of the bottom 3a of the tube 3.

[0056] Similarly, the aggregation member 15 is mounted on a member carriage aggregation member carriage 40. The aggregation member carriage 40 extends perpendicular to the vertical axis A from a guide end 41 slidably mounted on the vertical mast 35, above the interface sheath carriage 30. The aggregation member carriage 40 is movable along the vertical axis A between disassembled and assembled positions by means of a drive device comprising, as for the interface sheath carriage 30, another worm screw 36 extending along the vertical axis A and driven in rotation by an electric motor placed in the base 7 of the frame 6.

[0057] In Figures 3 and 4, the aggregation member carriage 40 is in the disassembled position away from the interface sheath carriage 30 such that the aggregation member 15 is spaced from the interface sheath 20, with the rod axis T aligned with the sheath axis G and the distal end of the aggregation member 15 facing the access end 20a of the interface sheath 20.

[0058] In Figures 5 and 6, the aggregation member carriage 40 is in the assembled position near the interface sheath carriage 20 such that the aggregation member 15 is received coaxially in the interface sheath 20, with the permanent magnet 17 in the vicinity of the free end 20b of the interface sheath 20.

[0059] In [Fig. 5], the interface sheath carriage 30 in the low position and the aggregating member carriage 40 in the assembled position place the aggregating member 15 in the interface sheath 20 itself placed in the tube 3 in the vicinity of the bottom 3a of the tube 3. This arrangement constitutes an active state of the aggregating member 15 and the interface sheath 20 in which the aggregating member 15 exerts the magnetic attraction force on the capture elements 2 to aggregate them on the interface sheath 20.

[0060] In Figures 3 and 4, the disassembled position of the aggregation member carriage 40 places the aggregation member 15 at a distance from the interface sheath 20 where it does not exert a magnetic attraction force on the capture elements 2. The aggregation member 15 and the interface sheath 20 are then in an inactive state.

[0061] Figures 7 to 10 illustrate a holding device 24 arranged on the interface sheath carriage 30, opposite the guide end 31, and configured to hold the interface sheath 20.

[0062] The holding device 24 comprises a ring 25 along a holding axis M parallel to the vertical axis A and having an inner surface intended to be in peripheral contact with the interface sheath 20, in the vicinity of the access end 20a.

[0063] In the embodiment shown, the side wall of the interface sheath 20 comprises lugs 22 projecting transversely relative to the sheath axis G from an outer surface. In [Fig. 8], the interface sheath 20 comprises two diametrically opposed lugs 22. Any other arrangement of equally distributed lugs 22 could however be provided. The ring 25 comprises a groove 26 formed on the inner surface around the holding axis M and configured to receive the lugs 22. Two grooves 27 extend along the holding axis M on the inner surface from a passage end to the groove 26, so as to allow passage of the lugs 22 of the interface sheath 20 in an angular position of the interface sheath 20 relative to the ring 25 and maintenance of the lugs 22 of the interface sheath 20 in the groove 26 in other angular positions of the interface sheath 20 relative to the ring 25.

[0064] The holding device 24 further comprises a flange 28 provided with a housing along a housing axis L parallel to the vertical axis A and configured to receive the ring 25 with the holding axis M aligned with the housing axis L. The flange 28 is movable relative to the interface sheath carriage 30 between an open position allowing the ring 25 to be placed in position and a closed position ensuring that the ring 25 is held in position relative to the interface sheath carriage 30.

[0065] Figures 11 and 12 show a stressing system 45 configured to be placed in contact with the interface sheath 20 and generate in the interface sheath 20 vibrations adapted to separate the capture elements 2 from the interface sheath 20 after the aggregation member 15 and the interface sheath 20 have passed from the active state to the inactive state.

[0066] In the embodiment shown, the biasing system 45 is mounted on the interface sheath carriage 30 and comprises a cam 46 pivotally mounted about a pivot axis P perpendicular to the vertical axis A on the interface sheath carriage 30. The cam 46 comprises a radial contact portion 47 arranged to come into contact with the interface sheath 20, in the vicinity of the access end 20a, in an angular position range of the cam 46 relative to the interface sheath carriage 30. The cam 46 is at a distance from the interface sheath 20 outside the angular position range.

[0067] The cam 46 has a stop state in which it is fixed relative to the frame 6 and to the interface sheath carriage 30. It also has a stress state in which it is rotated about the pivot axis P so as to intermittently stress, in the form of shocks, the interface sheath 20 and generate vibrations in the vicinity of the access end 20a. The holding device 24 is then configured to allow propagation of the vibrations from the access end 20a to the free end 20b of the interface sheath 20.

[0068] In [Fig. 13], in order to stop the cam 46 outside the angular position range when the cam 46 passes from the biasing state to the stopping state, the biasing system 45 comprises a detection device 50. In the embodiment shown, the detection device 50 implements a REED type sensor 51 mounted on the interface sheath carriage 30 outside the angular position range of the cam 46. The cam 46 comprises a flange 48 which extends over an angular sector perpendicular to the pivot axis P and parallel to the contact portion 47 of the cam 46, being centered on this contact portion 47. On a surface facing the REED type sensor 51, the flange 48 comprises a magnetic member 52, in the form of two magnets in the embodiment shown. Thus, when the cam 46 passes from the stress state to the stop state, it is driven by pulses until the magnetic member 52 is detected by the REED type sensor 51 and stops the cam 46 outside the angular position range of the cam 46.

[0069] The handling apparatus 5 can be implemented in a handling method providing for placing in the receiving space 9 of the tube support 8 a tube 3 containing a solution comprising the sample of biological material 1 and capture elements 2.

[0070] The interface sheath carriage 30 is in the high position and the flange 28 of the holding device 24 is placed in the open position. The ring 25 is placed in the housing of the flange 28. The interface sheath 20 is inserted into the ring 25 of the holding device 24 in the angular position putting the lugs 22 of the interface sheath 20 in correspondence with the grooves 27. When the lugs 22 reach the groove 26, the interface sheath 20 is pivoted about its sheath axis G to lock the interface sheath 20 in the ring 25. The flange 28 is placed in the closed position.

[0071] The interface sheath carriage 30 is moved to the lower position and then the aggregation member carriage 40 is placed in the assembled position. The aggregation member 15 and the interface sheath 20 are then in the active state with the aggregation member 15 received in the interface sheath 20 and exerting the magnetic attraction force on the capture elements 2 to aggregate them on the interface sheath 20. The biological material sample 1 can thus be collected on the interface sheath 20.

[0072] The interface sheath carriage 30 is moved into the high position, the aggregation member 15 and the interface sheath 20 remaining in the active state to extract the sample of biological material 1 from the solution contained in the tube 3.

[0073] The barrel 10 can be pivoted to place another tube 3 with another solution opposite the interface sheath 20. The interface sheath carriage 30 can then be placed in the low position, the aggregation member 15 and the interface sheath 20 remaining in the active state, then the aggregation member carriage 40 is moved to the disassembled position to place the aggregation member 15 and the interface sheath 20 in the inactive state.

[0074] The stress system 45 can then be activated to generate in the sheath interface 20 vibrations adapted to separate the capture elements 2 from the interface sheath 20.

[0075] In relation to figures 14 to 18, a particular, non-limiting example of implementation of the manipulation method during different stages of a purification treatment of a DNA sample is now described.

[0076] The experiment proposes a comparison of results of a “manual” extraction and an “automated” extraction, i.e. using the manipulation apparatus, of Listeria monocytogenes DNA from a culture in the presence or absence of an agri-food matrix. The two types of extraction are carried out using the same extraction kit and on the same sample. Protocol followed Culture of Listeria monocytogenes

[0077] Strain used: Listeria monocytogenes American Type Culture Collection (ATCC) 13932

[0078] Off-matrix culture: Deposition of a cryobead in 10ml of Buffered Peptone Water (BPE)

[0079] Culture with matrix: Deposit of a cryoball in 9ml of EPT + 1ml of semi-skimmed milk

[0080] Incubation at 37°C (±2°C) for 18 hours.

[0081] Lysis of samples (release of genetic material)

[0082] 1- Taking 200pl of sample with a single-channel pipette

[0083] 2- Deposit in 200pl of lysis buffer. Vortex

[0084] 3- Incubate for 5 minutes at 95°C. Leave to cool to room temperature. Manual extraction:

[0085] 1- Deposit of 400pl of lysed sample in the binding buffer mixture + elements magnetic capture

[0086] 2- Homogenize the capture elements in the sample by suction-re crowding (lOx)

[0087] 3- Incubate for 1 minute at room temperature.

[0088] 4- Place the aggregation organ + interface sheath in the capture elements. Leave to incubate until the capture elements are captured (approximately 1 min)

[0089] 5- Transfer the capture elements into the first wash tube (wash buffer) wash no. 1).

[0090] 6- Remove the aggregation member from the interface sheath and homogenize the elements capture in wash buffer 1.

[0091] 7- Replace the aggregation organ in the interface sheath until all the the capture elements (approximately 1 min).

[0092] 8- Let the capture elements dry at room temperature for 5 min.

[0093] 9- Transfer the capture elements into the elution tube.

[0094] 10- Remove the aggregation organ from the interface sheath and homogenize the capture elements in the elution buffer.

[0095] 11- Incubate for 1 min at room temperature.

[0096] 12- Replace the aggregation organ in the interface sheath until all the the capture elements (approximately 1 min).

[0097] 13- Remove the aggregation organ from the interface sheath of the elution tube. The DNA are in the elution tube.

[0098] Duration of manipulation = 15 min

[0099] Operator handling time = 10 min Automated extraction:

[0100] See description below.

[0101] Duration of manipulation = 8 min

[0102] Operator handling time = 2 min Amplification of extracted DNA samples:

[0103] The extracted DNA samples are amplified by a qPCR method with DNA intercalators and specific for Listeria monocytogenes. The qPCR analysis is carried out on a CFX 96 type thermocycler.

[0104] In order to be able to perform a quantification of the number of genome units present in each reaction, a Listeria monocytogenes quantification range specific to the amplification is launched at the same time as the samples.

[0105] A negative control (sample free from any trace of Listeria monocytogenes DNA - here Molecular Biology quality water) is also analyzed.

[0106] The amplification conditions are as follows:

[0107] - Initial denaturation: 95°C 2 min

[0108] - Cycling: 40x [95°C-5s / 60°C-10s / 72°C-20] + FAM reading

[0109] - Melt: ramping of 0.3°C ranging from 75°C to 95°C

[0110] A sample is considered positive if, following amplification, it shows a sigmoidal curve crossing the positivity threshold (threhsold) defined for this analysis. Positive curves are confirmed by the melting value of the samples. Compliance criteria:

[0111] The qPCR is considered compliant if:

[0112] - The first range point has a CT (Cycle Threshold = Value of the intersection of the amplification curve and the positivity threshold) equal to the reference value ±1.

[0113] - The amplification range used presents:

[0114] o A slope between -2.8 and -4.1

[0115] oUnr2>0.95

[0116] o Each point in the range has a melt value equal to the reference value ±2.

[0117] - The negative control shows a negative result:

[0118] o No amplification curve

[0119] o Presence of an amplification curve but melt value outside the acceptable range.

[0120] The measured accuracy of this qPCR analysis is 30%. The two methods will therefore be judged as giving equivalent results if the genomic unit values ​​calculated following amplification of one compared to the other are located within a gap ranging from 70% to 130%.

[0121] Description of the automated extraction method:

[0122] This analysis is a 4-step DNA sample purification analysis:

[0123] - binding in English,

[0124] - washing or washing in English,

[0125] - drying or drying in English

[0126] - elution or elution in English.

[0127] For this analysis, the barrel contains 8 tubes distributed as follows:

[0128] Tube No. 1: Binding buffer + magnetic beads (600pl)

[0129] Tube No. 2: Wash buffer 1 (1000pl)

[0130] Tube No. 3: empty

[0131] Tube No. 4: empty

[0132] Tube No. 5: empty

[0133] Tube No. 6: empty

[0134] Tube No. 7: Wash buffer 4 (1000pl)

[0135] Tube No. 8: Elution buffer (400pl) Sample preparation:

[0136] - Taking 200pl of sample with a single-channel pipette

[0137] - Deposit in 200pl of lysis buffer. Vortex

[0138] - Incubate for 5 minutes at 95°C. Allow to cool to room temperature. Preparation of the handling device:

[0139] - Installation of the interface sheath in the ring of the holding device

[0140] - Deposit of 400pl of extracted sample in the reception space n°1 of the barrel containing the binding buffer

[0141] - Installing the barrel on the frame

[0142] - Turning on the manipulation device

[0143] - Launch of the purification sequence DNA purification:

[0144] Initialization of the sequence:

[0145] The initial position of the barrel 10 presents the tube No. 3 under the aggregation member 15.

[0146] During initialization, the barrel 10 rotates to position the tube No. 1 under the aggregation member 15.

[0147] Binding step (duration 90s)

[0148] In a preliminary phase, the interface sheath 20 can be outside the tube No. 1 (interface sheath carriage 30 in the high position) and the aggregation member 15 can be outside the interface sheath 20 (aggregation member carriage 40 in the disassembled position). The interface sheath 20 can descend alone into the tube No. 1 (interface sheath carriage 30 in the low position and aggregation member carriage 40 in the disassembled position) then make back-and-forth movements along the tube axis T to homogenize the solution.

[0149] In [Fig.14], the interface sheath 20 is outside the tube No. 1 (interface sheath carriage 30 in the high position) and the aggregation member 15 is outside the interface sheath 20 (aggregation member carriage 40 in the disassembled position) (a). The aggregation member 15 enters the interface sheath 20 (aggregation member carriage 40 in the assembled position) (b). The interface sheath 20 descends into the tube No. 1 with the aggregation member 15 inside (interface sheath carriage 30 in the lower position and aggregation member carriage 40 in the assembled position) until the interface sheath 20 is close to the bottom 3a of the tube 1 with the aggregation member 15 in the vicinity of its free end 20b (c). In this position, the aggregation member 15 can exert the magnetic attraction force to aggregate the capture elements 2 to which the DNA sample 1 is bound on the interface sheath 20.The interface sheath 20 rises to exit the tube 1 with the aggregation member 15 in the interface sheath 20 (interface sheath carriage 30 in the high position and aggregation member carriage 40 in the assembled position) so that the capture elements 2 and the DNA sample 1 are extracted from the solution of tube No. 1 (d).

[0150] Transfer of capture elements

[0151] The barrel 10 rotates to position the tube No. 2 under the interface sheath 20 and the aggregation member 15.

[0152] Washing step (duration 90s)

[0153] In Figures 15a and 15b, the aggregation member 15 is in the interface sheath 20 (aggregation member carriage 40 in the assembled position) with the interface sheath 20 outside the tube No. 2 (interface sheath carriage 30 in the high position) (a). The capture elements 2 and the DNA sample 1 are aggregated on the interface sheath 20. The interface sheath 20 descends into tube No. 2 (interface sheath carriage 30 in the low position) with the aggregation member 15 inside (aggregation member carriage 40 in the assembled position) then the aggregation member 15 comes out of the interface sheath 20 (aggregation member carriage 40 in the disassembled position) (b). The biasing device 45 is activated so that the cam 46 periodically comes into contact with the access end 20a of the interface sheath 20 (biased state) and thus produces shocks generating vibrations which propagate to the free end 20b of the interface sheath 20 (c and d). The capture elements 2 detach from the interface sheath 20 which can make back and forth movements along the tube axis T. The interface sheath 20 ends its movement outside the tube No. 2 (interface sheath carriage 30 in the high position) (e).The aggregation member 15 is inserted into the interface sheath 20 (aggregation member carriage 40 in the assembled position) (f) then the interface sheath 20 and the aggregation member 15 descend into the tube No. 2 (interface sheath carriage 30 in the lower position and aggregation member carriage 40 in the assembled position) (g). The interface sheath 20 and the aggregation member 15 are in the active state. The interface sheath 20 and the aggregation member 15 exit the tube 2 with the capture elements 2 and the DNA sample 1.

[0154] Transfer of capture elements

[0155] The barrel 10 rotates to position the tube No. 5 under the interface sheath and the aggregation member.

[0156] Drying step (duration 120s)

[0157] In [Fig. 16], the aggregation member 15 is in the interface sheath 20 (aggregation member carriage 40 in the assembled position) with the interface sheath 20 outside the tube No. 5 (interface sheath carriage 30 in the upper position) (a). The capture elements 2 and the DNA sample 1 are aggregated on the interface sheath 20. The interface sheath 20 descends into the empty tube No. 5 (interface sheath carriage 30 in the lower position) with the aggregation member 15 inside (aggregation member carriage 40 in the assembled position) (b). The cam 46 of the biasing system 45 moves from the stopped state to the biased state to move the interface sheath 20 and the aggregating member 15 into the empty tube No. 5 (c). The interface sheath 20 rises to exit the tube No. 5 with the aggregating member 15 in the interface sheath 20 (interface sheath carriage 30 in the high position and aggregating member carriage 40 in the assembled position).

[0158] Transfer of capture elements

[0159] The barrel rotates to position tube No. 8 under the interface sheath 20 and the aggregation member 15.

[0160] Elution step (duration 90s)

[0161] In Figures 17a and 17b, the aggregation member 15 is in the interface sheath 20 (aggregation member carriage 40 in assembled position) with interface sheath 20 out of tube No. 8 (interface sheath carriage 30 in upper position) (a). Capture elements 2 and DNA sample 1 are aggregated on interface sheath 20. Interface sheath 20 descends into tube No. 8 (interface sheath carriage 30 in lower position) with aggregation member 15 inside (aggregation member carriage 40 in assembled position) then aggregation member 15 comes out of interface sheath 20 (aggregation member carriage 40 in disassembled position) (b). The biasing device 45 is activated so that the cam 46 periodically comes into contact with the access end 20a of the interface sheath 20 (biased state) and thus produces shocks generating vibrations which propagate to the free end 20b) of the interface sheath 20 (c and d).The capture elements 2 are detached from the interface sheath 20 which can make back and forth movements along the tube axis T. The interface sheath 20 ends its movement at the bottom 3a of the tube No. 8 (interface sheath carriage 30 in the low position) and remains in position for a determined duration (e). The aggregation member 15 is inserted into the interface sheath 20 (aggregation member carriage 40 in the assembled position) and held in this position for a determined duration (f). The DNA sample 1 is detached from the capture elements 2. The interface sheath 20 is extracted from the tube No. 8 with the aggregation member 15 inside (interface sheath carriage 30 in the high position and aggregation member carriage 40 in the assembled position), so as to extract the capture elements 2 while leaving the DNA sample 1 in the tube No. 8 (g).

[0162] Transfer of capture elements

[0163] The barrel rotates to position tube No. 7 under the interface sheath 20 and the aggregation member 15.

[0164] System cleaning step (duration 60s)

[0165] In [Fig. 18], the aggregating member 15 is in the interface sheath 20 (aggregating member carriage 40 in the assembled position) with the interface sheath 20 outside the tube No. 7 (interface sheath carriage 30 in the upper position) (a). The capture elements 2 are aggregated on the interface sheath 20. The interface sheath 20 descends into the tube No. 7 (interface sheath carriage 30 in the lower position) with the aggregating member 15 inside (aggregating member carriage 40 in the assembled position) then the aggregating member 15 comes out of the interface sheath 20 (aggregating member carriage 40 in the disassembled position) (b). The biasing device 45 is activated so that the cam 46 periodically comes into contact with the access end 20a of the interface sheath 20 (biased state) and thus produces shocks generating vibrations which propagate to the free end 20b of the interface sheath 20 (c and d).The capture elements 2 detach from the interface sheath 20. The interface sheath 20 rises to exit the tube No. 7 (interface sheath carriage 30 in . high position) (e)

[0166] Return to initial position:

[0167] The barrel 10 rotates to position the tube No. 3 under the interface sheath 20 and the aggregation member 15. Following the end of the purification:

[0168] The operator takes the volume necessary for the analysis (here 5pl) directly from tube 8. He can then take the entire elution volume and place it in a suitable tube to preserve this extracted sample. Results obtained: Validation of qPCR compliance

[0169] Results obtained with the DNA quantification range: Log load (UG / reaction) CT melt Confirmation amplification 6 13.78 81.9 Listeria monocytogenes 5 16.36 82.2 Listeria monocytogenes 4 19.36 82.2 Listeria monocytogenes 3 22.88 82.2 Listeria monocytogenes 2 26.58 82.2 Listeria monocytogenes Negative no and none negative Criteria Expected result Observed result Compliance Slope Between -2.8 and -4.1 -3.07 OK r2 >0.95 0.995 OK Ct value of the first point Between 12.6 and 14.6 13.78 OK Melt reference value Between 81.2 and 83.2 See table above OK Value of the negative no ct or non-compliant melt no ct OK

[0170] Conclusion: The qPCR range showing consistent results, the samples can be analyzed. Sample results:

[0171] Sample Culture medium Extraction method CT observed nb UG Ratio automated method / manual method Compliance Listeria monocytogenes EPT Manual 19.86 9524 83% OK Listeria monocytogenes EPT Automated 20.12 7905 Listeria monocytogenes EPT + milk Manual 20.96 4329 86% OK Listeria monocytogenes EPT + milk Automated 21.17 3724

[0172]

[0173]

[0174]

[0175] Conclusion: The presence of Listeria monocytogenes was detected in all samples. The quantification results obtained show a deviation within the tolerable range of accuracy. General conclusion: Automated extraction using the sample preparer achieves results similar to those obtained by manual extraction. However, automated extraction has two advantages: - significantly reduced operator handling time and difficulty, - a greatly reduced overall analysis time.

Claims

Claims

1. Apparatus (5) for handling a sample of biological material (1) by means of capture elements (2), the capture elements (2) being magnetic and configured to be secured to the sample of biological material, the handling apparatus (5) comprising: - a frame (6) extending along a vertical axis (A), - a tube support (8) mounted on the frame (6) and comprising at least one receiving space (9) configured to receive at least one tube (3) adapted to contain a solution comprising the sample of biological material (1) and the capture elements (2); - an aggregation member (15) configured to generate a magnetic attraction force on the capture elements (2), the aggregation member (15) being intended to cooperate with an interface sheath (20) in such a way that, in an active state of the aggregation member (15) and the interface sheath (20), the aggregation member (15) is received in the interface sheath (20) and exerts the magnetic attraction force on the capture elements (2) to aggregate said capture elements (2) on the interface sheath (20) and, in an inactive state of the aggregation member (15) and the interface sheath (20), the aggregation member (15) does not exert a magnetic attraction force on the capture elements (2), the handling apparatus (5) being characterized in that it further comprises: - an interface sheath carriage (30) comprising a holding device (24) configured to hold the interface sheath (20) opposite the receiving space (9), the interface sheath carriage (30) being movable along the vertical axis (A) relative to the frame (6) between a high position in which said interface sheath carriage (30) places the interface sheath (20) at a distance from the receiving space (9), and a low position in which said interface sheath carriage (30) places the interface sheath (20) in the receiving space (9), - a biasing system (45) configured to be placed in contact with the interface sheath (20) and generate in the interface sheath (20) vibrations adapted to separate the capture elements (2) from the interface sheath (20) after the aggregation member (15) and the interface sheath (20) have changed from the active state to the inactive state.

2. A handling apparatus (5) according to claim 1, wherein the biasing system (45) is arranged to generate the vibrations in the vicinity of an access end (20a) of the interface sheath (20) by which the aggregation member (15) is inserted into the interface sheath (20), and in which the holding device (24) is configured to allow propagation of the vibrations from the access end (20a) towards a free end (20b) of the interface sheath (20) opposite the access end (20a) and in the vicinity of which the capture elements (2) are aggregated in the active state of the aggregation member (15) and of the interface sheath (20).

3. A handling apparatus (5) according to claim 2, wherein the holding device (24) is configured to hold the interface sheath (20) in the vicinity of the access end (20a).

4. Handling apparatus (5) according to any one of claims 1 to 3, in which the holding device (24) comprises a ring (25) along a holding axis (M) parallel to the vertical axis (A) and having an inner surface intended to be in peripheral contact with the interface sheath (20).

5. Handling apparatus (5) according to claim 4, wherein the ring (25) is configured to receive the interface sheath (20) comprising a side wall along a sheath axis (G) and at least two lugs (22) projecting transversely in an equally distributed manner from an outer surface of the side wall, the ring (25) comprising a groove (26) formed on the inner surface around the holding axis (M) and configured to receive the lugs (22), and at least two grooves (27) extending axially on the inner surface from a passage end to the groove (26), so as to allow passage of the lugs (22) of the interface sheath (20) in an angular position of the interface sheath (20) relative to the ring (25) and holding of the lugs (22) of the interface sheath (20) in the groove (26) in other angular positions of the interface sheath (20) relative to the ring (25).

6. Handling apparatus (5) according to any one of claims 3 and 4, wherein the holding device (24) further comprises a flange (28) movable relative to the interface sheath carriage (30) between an open position allowing the ring (25) to be placed in position and a closed position ensuring that the ring (25) is held in position relative to the interface sheath carriage (30).

7. Manipulation apparatus (5) according to any one of the claims 1 to 6, wherein the biasing system (45) is configured to perform at least one impact on the interface sheath (20).

8. Handling apparatus (5) according to any one of claims 1 to 7, in which the biasing system (45) comprises a cam (46) driven in rotation about a pivot axis (P) and comprising a radial contact portion (47) arranged to come into contact with the interface sheath (20) in a range of angular position of the cam (46) relative to the frame (6), the cam (46) being at a distance from the interface sheath (20) outside the range of angular position.

9. A handling apparatus (5) according to claim 5, wherein the cam (46) has a biasing state in which said cam (46) is rotated and a stopping state in which said cam (46) is fixed relative to the frame (6), and wherein the biasing system (45) comprises a detection device (50) configured to stop the cam (46) outside the angular position range when the cam (46) transitions from the biasing state to the stopping state.

10. A handling apparatus (5) according to any one of claims 1 to 9, wherein the biasing system (45) is mounted on the interface sheath carriage (30).

11. A handling apparatus (5) according to any one of claims 1 to 10, further comprising an aggregation member carriage (40) carrying the aggregation member (15), the aggregation member carriage (40) being movable along the vertical axis (A) relative to the frame (6) between a disassembled position remote from the interface sheath carriage (30) such that the aggregation member (15) is spaced from the interface sheath (20), and an assembled position proximate to the interface sheath carriage (30) such that the aggregation member (15) is received in the interface sheath (20).

12. A handling apparatus (5) according to claim 11, wherein the aggregating member (15) comprises a permanent magnet (17) at a distal end for receipt in the interface sheath (20), the aggregating member (15) and the interface sheath (20) being in the inactive state when the aggregating member carriage (40) is in the disassembled state.

13. A handling apparatus (5) according to any one of claims 1 to 12, wherein the tube holder (8) comprises a barrel (10) having a plurality of receiving spaces (9) around a barrel axis (B), the barrel (10) being rotatable along the barrel axis. barrel (B) so as to selectively place one of the receiving spaces (9) opposite the interface sheath (20).

14. Handling assembly comprising a handling apparatus (5) according to any one of claims 1 to 13 and at least one interface sheath (20) configured to receive the aggregation member (15) and to be held in the holding device (24) of the interface sheath carriage (30).

15. A method of handling a sample of biological material (1), the handling method implementing a handling assembly according to claim 14 and providing for: - placing in the receiving space (9) of the tube holder (8) a tube (3) containing a solution comprising the sample of biological material (1) and capture elements (2), the capture elements (2) being magnetic and configured to be secured to the sample of biological material (1), - placing the interface sheath (20) in the holding device (24) of the interface sheath carriage (30) in the high position, - putting the interface sheath carriage (30) in the low position and the aggregation member (15) and the interface sheath (20 in the active state,the aggregating member (15) being received in the interface sheath (20) and exerting the magnetic attraction force on the capture elements (2) to aggregate said capture elements (2) on the interface sheath (20), - putting the interface sheath carriage (30) in the high position, the aggregating member (15) and the interface sheath (20) remaining in the active state, - putting the interface sheath carriage (30) in the low position, the aggregating member (15) and the interface sheath (20) remaining in the active state, - putting the aggregating member (15) and the interface sheath (20) in the inactive state, - activating the stressing system (45) to generate in the interface sheath (20) vibrations adapted to separate the capture elements (2) from the interface sheath (20).,

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