• 专利附录
  • 专利说明
  • 权利要求
  • 类似技术
  • 同族专利
  • 引用文献
  • 法律状态
  • 优先权
    • 专利摘要:
    The invention proposes a device and a simple and rapid method of lysis of biological species present in a fluid, implementing a rough surface against which the objects to be lysed are ground by a shearing movement. A device (1) according to the invention for mechanical lysis of biological species (C) comprises a first wall (21) and a second wall (11) movably mounted relative to each other, between an initial position in wherein said walls are spaced from each other, and a lysis position in which the first wall bears against the second wall, the first and second walls being also movably mounted in shear with respect to the other in the lysis position, at least one of the first and second walls having a rough bearing surface against the other wall and having an average surface roughness parameter Ra of between 0.1 μm and 10 μm, and preferably between 0.2 microns and 3 microns.
    公开号:FR3021667A1
    申请号:FR1454866
    申请日:2014-05-28
    公开日:2015-12-04
    发明作者:Anne-Gaelle Bourdat；Antonio Viana；Dulk Remco Den
    申请人:Commissariat a lEnergie Atomique CEA；Commissariat a lEnergie Atomique et aux Energies Alternatives CEA；
    IPC主号:
    专利说明:

    [0001] The invention relates to a device for lysing biological species (microorganisms, bacteria, cells, spores, etc.). etc.) and to a method implemented by this device Biological cell lysis is necessary to recover and study the intracellular material For example, cell lysis is necessary to recover DNA from the cell for a replication and analysis, such as a comparison of DNA., The methods conventionally used for the lysis of biological species are as follows: Chemical lysis: this technique comprises contacting the cells with a lysis solution , usually containing a detergent bursting the cells This technique is effective but can be dangerous for some cellular material.In addition, it is long and requires many manipulations, including a step of purification of the lysis solution and possibly concentration of the lysate, which makes it long, with a risk of losing a large amount of biological material during purification and concentration and requires. the intervention of a specialized person this technique is not effective for the lysis of spores. Thermal shock lysis: this technique comprises, for example, incubating the extract at a temperature below 0 ° C., followed immediately by incubation of the extract at a temperature of at least 95 ° C., preferably about 100 ° C. This technique requires complex equipment, especially if it is desired to lyse spores. Electric field lysis: this technique involves the use of a potential difference leading to rupture of the cell membrane. This technique requires a complex material and is, like the previous methods, adapted to the lysis of spores. 302 16 6 7 2 - Mechanical lysis: this technique is based on the stirring of the extract in the presence of beads so that the shocks blues against the cells burst them. This technique requires complex equipment. In addition, it is necessary to use beads which are completely free of micro-organisms and to recover them after lysis. Examples of devices implementing this method are the biological sample grinders of the Precellys © range of Bertin Technologies. These are expensive and bulky devices, difficult to integrate into a compact instrument. Pressure Lysis: This technique involves the application of hydrostatic pressure to the cells, leading to rupture of their membrane. This technique requires complex equipment. Among the methods of lysis by mechanical grinding, mention may be made of the following documents: FR 2,781,500, which describes a process for mechanical lysis of microorganisms by setting the grinding means in motion by a magnetic field; and US 200310066915, which describes a method of mechanical lysis of microorganisms by setting the grinding means in motion by ultrasound. All of these techniques are complex and require a lot of material. In addition, they require a significant preparation time, and a lysis time of several tens of seconds to several minutes. Moreover, they are difficult to integrate on portable and compact instruments. The object of the present invention is therefore to provide a device and a method for lysis of biological species that is simple, inexpensive, and easily integrable in laboratory equipment or in a portable device, and having a high lysis efficiency, particularly on spores allowing satisfactory lysis (at least 50% of the biological species lysed in less than 10 seconds, or even in 1 second.
    [0002] For this, the invention proposes to provide an apparatus and a mechanical lysis process using a rough surface against which the objects to be lysed are ground by a friction movement (shear) and not by shocks or a simple pressure.
    [0003] To this end, the subject of the invention is a device for mechanical lysis of biological species present in a fluid, characterized in that it comprises a first wall and a second wall movably mounted relative to one another. between an initial position in which the first and second walls are spaced from each other. and a lysis position in which the first wall bears against the second wall, the first and second walls being also movably sheared relative to each other in the lysis region, one at least one of the first and second walls having a rough bearing surface against the other wall and having an average surface roughness parameter Ra of between 0.1 μm and 10 μm, and preferably between 0.2 μm and 3 μm. According to other embodiments of the device according to the invention (i) one of the first wall and second wall may be a microporous wall, that is to say porous to liquids and non-porous to the biological species to be lysed; (ii) the microporous wall may have an average pore diameter of less than 1 μm, or even less than 0.2 μm; (iii) the microporous wall may be rough; (iv) the rough microporous wall may consist of a layer of microporous material having a planar surface on which is positioned a rough layer intended to bear against the other wall in the lysis position; (y) according to a variant of (iv), the rough microporous wall may consist of a layer of microporous material having a rough surface; (Vi) according to another variant of (iv), the rough microporous wall may consist of a layer of macroporous material having a rough surface on which is positioned a microporous layer intended to abut against the other wall; lysis position, the microporous layer being sufficiently flexible and thin to match the roughness of the surface of the layer of macroporous material in the lysis position; (vii) the microporous wall may be non-rough and have a mean Ra surface roughness parameter of less than 0.1 prn, while the non-microporous wall may be rough and have a mean Ra surface roughness parameter of between 0 1 μm and 10 μm, referentially between 0.2 μm and 3 μm; (viii) according to a variant of (vii), the first wall and the second wall may be rough; (ix) the device according to the invention may further comprise a chamber and a piston movably mounted in the chamber between the initial position and the lysis position, the first wall being mounted on the movable piston and the second wall being arranged in look at the first wall; (x) the second wall of the device may then be microporous, and the chamber may further comprise a fluidic outlet arranged opposite the first wall 20 relative to the second microporous wall; (xi) the chamber may then further comprise a fluidic inlet and another fluidic outlet; and (xii) the chamber may further comprise a sealed membrane arranged between the first wall, the fluidic inlet and the fluidic outlet, the membrane being sufficiently thin and flexible to conform to the rune roughness of at least the first and second walls in the lysis position. The invention also relates to a method of mechanical lysis of biological species comprising the following steps: A) Providing a device such as that mentioned above so that the first and second walls are in the initial position; 3021667 5 B) Place the biological species to be lysed between these walls; C) Bring these walls into the desired position; D) Generate shear between these walls in the lysis position to cause the mechanical lysis of these species and obtain a lysate; E) Spread this walls until they are in the initial position; and F) recovering the lysate of biological species. The method according to the invention is effective since it allows a yield at least equivalent to the reference method of lysis of spores by Precellys © (Bertin) mills. The process is fast compared to the reference method (Precellys ™ mill (Bertin)): in less than 10 seconds against 30 seconds for the reference mill.
    [0004] The process according to the invention is economical and straightforward since it does not require the addition of chemical substance, subsequent purification or the addition of foreign matter to the sample (such as beads). In addition, the DNA recovered after lysis by the method according to the invention is directly analyzable (ie without a purification step) using conventional molecular biology techniques, such as "PCR" ( "Polymerase Chain Reaction" in English) or electrophoresis. Finally, the release is carried out without degradation of the nucleic acids of the species. According to other embodiments of the method according to the invention: - step A) may consist in providing a device according to the feature (ix) above, 'step C) slide the piston in the chamber until that the first and second walls are in a lysis position and step D) to rotate the piston on itself so that these walls remain in contact with each other; Step A) may consist in providing a device according to (x) above, step B) in placing a liquid comprising the species to be lysed between the walls, step C) in sliding the piston in the chamber until the liquid has been filtered through the microporous wall, that only these species remain on microporous wall and that these walls are in the lysis position, and step D) to rotate the piston on itself so that these walls remain in contact with each other - step A) may consist in providing a device according to (x above, ['step B) to enter a liquid comprising the species to lyse through the fluidic inlet, between these walls, [step C) to slide the piston into the chamber until these walls are in the lysis position, step D) to rotate the piston on itself so that these walls stay in contact with each other and step F) to circulate an elution liquid between the inlet and the fluidic outlet to recover the lysate through this outlet; and step A) may comprise providing a device according to (xii) above, step B) introducing a liquid comprising the species to be lysed by the fluidic inlet, between the sealed membrane and the second wall, step C) to slide the piston into the chamber until these walls are in the lysis position through the membrane, step D) to rotate the piston on itself such that these walls remain in contact with each other through the membrane, and the step F) to circulate an elution liquid between the fluid inlet and the fluid outlet to recover the lysate through the fluidic outlet.
    [0005] Other features of the invention will be set forth in the detailed description below, with reference to the appended figures which represent, respectively: FIGS. 1 and 2, two schematic sectional views of a first embodiment of FIG. a cell lysis device according to the invention; Figure 3 is a diagrammatic sectional view of a second embodiment of a cell lysing device according to the invention; FIG. 4 is a schematic sectional view of a third embodiment of a cell lysis device according to the invention; FIG. 5 is a diagrammatic sectional view of a fourth embodiment of a device. cell lysing according to the invention; Figure 6 is a diagrammatic sectional view of a fifth embodiment of a cell lysing device according to the invention; Figures 7 and 8, two schematic sectional views of a sixth embodiment of a cell lysis device according to the invention; Figure 9 is a diagrammatic sectional view of a seventh embodiment of a cell lysing device according to the invention; Figure 10 is a schematic sectional view of an eighth embodiment of a cell lysis device according to the invention; and Figures 11 and 12, two schematic sectional views of a ninth embodiment of a cell lysis device according to the invention.
    [0006] In the description, the following four terms are defined as follows: Rough surface: d is a surface having an average surface roughness parameter Ra, corresponding to the arithmetic mean of parameter measurements Ra, greater than 0 , 1 pm, preferably between 0.1 pm and 10 pm and even more preferably between 0.2 pm and 3 pm. (The parameter or coefficient of average roughness Ra surface is established from a measurement with the prefix, as detailed later); Microporous it is a wall or a layer. material comprising open pores large enough to pass liquids, such as water, but narrow enough to prevent passing the biological species to be lysed thus due to biological material from the lysis and to be analyzed. Typically, a microporous wall within the meaning of the invention comprises open pores having a diameter of less than 1 μm, and preferably less than 0.2 μm. By way of example, there may be mentioned sintered filters, porous silica filters, ceramic filters, and porous polymer or porous metal filters; Macroporous: it is a wall, a layer or a material comprising open pores or channels that are wide enough to allow liquids, such as water, to pass, but also wide enough to pass through. the biological species to be lysed or the biological material resulting from the lysis and to be analyzed. Typically, a macroporous wall within the meaning of the invention comprises open pores or channels having a diameter greater than 0.2 μm, and preferably greater than 1 μm. By way of example, there may be mentioned a layer of glass, metal or polymer provided with channels passing through the layer; - Iological species: especially cells, microorganisms, in particular bacteria, spores, viruses, fungi, microalgae. In order to effect lysis of biological species, such as microorganisms, cells, spores, etc., the invention provides a mechanical lysis device comprising a first wall and a second wall mounted movable one by relative to the other: between an initial position in which the first and second walls are spaced from each other, and a lysis position in which the first wall bears against the second wall. The first and second walls are also movable in shear with respect to each other in the lysis position, that is, one in abutment with the other. The shear can be obtained by rotating or translating the first wall relative to the second wall. The invention therefore provides two degrees of freedom between the walls: a degree of freedom to approach the walls one in contact with the other, and a degree of freedom to allow shearing when the walls are in contact with one another. against each other.
    [0007] According to the invention, at least one of the first and second walls has a rough surface which bears against the other wall. In other words, the rough surface has an average surface roughness parameter Ra of between 0.1 μm and 10 μm, preferably between 0.2 μm and 3 μm. As an example of support pressure, tests were carried out using a frosted glass slide and a conventional glass slide held between the thumb and forefinger of one hand. The only force of the fingers applied in pressure and in shear is sufficient to obtain a lysis of the species placed between the two blades.
    [0008] A first embodiment of a device according to the invention is illustrated in FIGS. 1 and 2. In FIG. 1, the device according to the invention is in the initial position in which the first and second walls are spaced apart. The other. The mechanical lysis device 1 comprises a chamber 10 and a piston 20 mounted to be movable in translation in the chamber 10 between the initial position (FIG. 1) and the lysis position (FIG. 2). A seal (not shown) is provided between the piston and the chamber to seal. The first wall 21 is mounted on the movable piston 20 and the second wall 11 is arranged facing the first wall 21. In this embodiment, the second wall 11 is microporous in order to allow the liquid L comprising the C cells to be lysed. to diffuse through the second wall 11 when the piston 20 is lowered 25 to the lysis position in which the two walls 11 and 21 are in contact with each other (Figure 2). The second wall 11 is for example a porous polymer filter. Advantageously, the chamber 10 further comprises a fluidic outlet 12 arranged opposite the first wall 21 relative to the second microporous wall 11, allowing the evacuation of the liquid as well as the iysed species. In an alternative not shown, a reservoir can replace the fluidic outlet, to receive the liquid.
    [0009] The microporous wall 11 has an average pore diameter of between 0.2 μm and 0.5 μm. The pore diameter is adapted to ensure that the liquid diffuses through the wall 11, but that the intact cells (i.e., before lysis) remain on the surface of the wall on the surface from which they are lysed. The biological material to be studied (DNA, proteins, etc.) released by the lysis can pass through the filter or remain on the surface of the filter, to be recovered. In the embodiment illustrated in FIGS. 1 and 2, it is the wall 21 carried by the piston 20 which has a rough bearing surface 22 against the other wall. In other words, the microporous wall 11 has a non-tumbling surface 13, while the non-microporous wall 21 has a rough surface 22, for example an abrasive surface. Alternatively, as illustrated in Figure 3, it is the microporous wall 11 against which is disposed a rough surface 14. For example, the rough microporous wall 11 consists of a layer 11 of microporous material having a flat surface 13 on which is positioned a rough layer 14 intended to bear against the wall 21 of the piston 20 in the lysis position. In this embodiment, the rough layer 14 must be able to pass the liquid in which the cells to be lysed. By way of example, a rough layer 14 of 500 μm, 2 mm thick, and a microporous layer 11 of 500 μm to 5 mm thick can be used. The rough layer 14 is made of a porous abrasive material, for example a metal grid.
    [0010] Figure 4 illustrates an alternative embodiment of rough microporous wall 11. In this embodiment, the rough microporous wall 11 is made of a layer of microporous material having a rough surface, for example microstructured. In other words, the roughness of the surface 15 is achieved by microstructuring a microporous material. This layer 11 of microporous material may also consist of an abrasive filter, such as a sintered filter, a porous silica, a ceramic, a porous polymer. This is for example a stainless steel filter, in the form of a grid. Whatever the embodiment shown in FIGS. 1 to 4, the microporous wall 11 makes it possible to retain the biological species to be lysed, while allowing the biological fluid to flow after the lysis operation, the lysed biological species. flow through the microporous wall 11.
    [0011] Figure 5 illustrates a fourth alternative embodiment of a rough microporous wall 11. In this embodiment, the rough microporous wall 11 consists of a macroporous layer 16 having a rough surface 17, and a microporous layer 18, positioned on the macroporous layer 16 and intended to bear against the first wall 21 carried by the piston 20 in the lysis position. The microporous layer 18 must be sufficiently flexible and thin to conform to the roughness of the surface 17 of the macroporous layer 16 in the lysis position, so that the roughness of the macroporous layer 16 is visible through the microporous layer 18 and the cells can to be lysed. Thanks to the microporous layer 18, the biological species to be lysed are retained and can be lysed. Figure 6 illustrates a fifth embodiment in which the first wall 21 and the second wall 11 are both rough, i.e. they both have a rough surface 22-14. They may be constituted by a combination of the embodiments of FIGS. 1 and 3, a combination of the embodiments of FIGS. 1 and 4 or a combination of the embodiments of FIGS. 1 and 5. FIGS. 7 and 8 illustrate a sixth embodiment embodiment 30 allowing easy implementation of lysis and recovery of the biological material to be analyzed.
    [0012] In this embodiment, the chamber 10 further comprises a fluidic inlet 30 and a fluidic outlet 40 arranged in sectional view between the first and second walls 21-11 when in the initial position.
    [0013] In the initial position, the liquid L comprising the cells C to be lysed is introduced into the chamber 10, between the first wall 21 and the second wall 11, by opening the valve 11 of the fluidic inlet 30, and closing the valves V2 and V3 respectively of the fluidic outlets 12 and 40. This step makes it possible to place the biological cells C to be lysed between the first and second walls 21-11. Once the chamber 10 is filled with liquid, the valve V1 is closed, and the valve V2 is open. The piston 20 is then brought to the lysis position along the arrow F1 (FIG. 8). The liquid can flow through the microporous wall 11 and be discharged along the arrow F3 through the fluidic outlet 12 arranged opposite the first wall 21 relative to the second microporous wall 11. Thus, only the biological cells C to lysate remain on the. When the piston 20 carrying the first wall 21 is in abutment against the second microporous wall 11 in the lysis position, the piston 20 is rotated according to the arrow F2 to generate a shear between the first and second walls. second walls 21 and 11 and cause the mechanical lysis of the biological cells and obtain a lysate. During this step, shown in FIG. 8, the valves V1, V2 and V3 remain closed. Then the first and second walls 21 and they are spaced from each other, until they are in the initial position, by raising the piston 20 in the chamber 10 in the opposite direction of the fl ow. it is then possible to recover the lysate of biological cells.
    [0014] In the embodiment of FIGS. 7 and 8, this recovery is obtained by opening the valves V1 and V3, and by circulating an effluent liquid between the inlet 30 and the fluidic outlet 40 to collect the biological material lysis. Then the cleaning liquid comprising the lysate is recovered by the fluid outlet 40. The fluid outlet 40 can be directly fluidly connected to an analysis apparatus. All these operations can be done in a closed and sealed circuit avoiding any contamination of the lysate. Indeed, it is not necessary to remove beads from the lysate, as in the current processes. In addition, the recovered biological material is not likely to be damaged by chemicals, broken by ultrasound, or destructured by heat. The shearing of biological cells between two walls, of which at least one is rough, allows lysis in a few seconds, with a simple, compact and expensive device.
    [0015] Figure 9 illustrates a seventh embodiment avoiding cleaning the piston after each lysis. In this embodiment, the chamber comprises. in addition, a sealing membrane 50 arranged between the first wall 21, the inlet 30 and the fluidic outlet 40. The membrane 50 must be sufficiently thin and flexible to marry, in the lysis position, the roughness of the rough wall, which can be carried by the piston and / or the chamber. By way of example, it is possible to use a polymer or plastic film with a thickness of between 50 μm and a few hundreds of μm, for example 100 μm, the rough and microporous wall possibly being a stainless steel filter. After lysis, the plunger can be recovered without having to be cleaned, and the chamber can be either cleaned for next lysis or removed. This solution can be envisaged thanks to the possibility of integrating the device according to the invention into an "ab-on-a-chip" system in English, or "lab on a chip". The chamber is then formed in the chip by the space between the fluidic inlet, the fluidic outlet and the second rough wall. In this case, the use of a reported membrane and piston allows the lysis to be performed on a single-use chip, recovering the lysate, and then removing the chip. The membrane can be washed or removed, and the piston is recovered without having to be washed for later use on another consumable. Of course, it is understood that the word "piston" covers, in this case, any room for applying a mechanical shear 10 cells against the wall of the chamber, this piece not necessarily having the conventional shape of a piston . It is thus the membrane which ensures the sealing, and not the piston which can then have any form. It could be, for example, a metal spatula 15 sheared by the user, or an automatic screwdriver. The term "automatic screwdriver" designates a piston 60 having an upper part and a lower part, such that, when the upper part describes a translation, the lower part of the piston describes a rotational movement when a resistance opposes its translation. . The implementation of such a device is illustrated in Figure 10 in which the piston 60 has dimensions (here the width ei, sectional view) less than the same. dimensions (here the width e2, sectional view) of the chamber, so that the shear is not necessarily obtained by rotation of the piston 60, but can also be obtained by translation, as illustrated by the arrow F4.
    [0016] Figures 11 and 12 illustrate another embodiment in which the piston 70 has a microporous wall 71. Thus, when it is lowered in the lysis position against the second wall 11 (smooth or rough) of the chamber 10, the liquid L diffuses through the microporous wall 71 of the piston 70 in the direction of the arrows F5, and can be 30 evacuated, for example by reversing the chamber 10 or by sucking the liquid L.
    [0017] This embodiment may also be combined with a chamber having an inlet and a fluidic outlet (not shown). The device and the method according to the invention make it possible to obtain a very rapid lysis of biological species, without requiring the addition of additional lysis means in the lysis chamber (bilium, chemical reagent, heat input, ultrasound, etc.). The implementation is simple and can be done manually or automatically. In addition to being faster, the process according to the invention is more efficient than the reference method (Precellys © mill (Bertin)). The efficiency of lysis is determined indirectly by the amount of DNA released after lysis. This amount of DNA is evaluated by "q-PCR". The species, in contact with a rough surface, are subjected to shear and pressure forces by either a non-rough surface, such as the surface of a metal spatula or a pipette cone. plastic, or using a second rough surface (case of a frosted glass slide on frosted glass).
    [0018] The species are crushed for a period of 2 seconds in order to release at least one of the cellular constitua.nts in the medium (DNA, RNA, protein, ...). For example, the method was tested in the following configurations: ground glass (Ra = 0.425 μm) / biological cells I ground glass (Ra = 0.425 μm); plastic pipette tip (Ra not measurable / biological cells / etched glass (Ra = 0.425 μm); flat metal spatula (Ra 0.2 μm) I polymer film / biological cells / etched glass (Ra = 0.425 μm) on which applied a porous polymeric filter, for example a "Cyclopore Polycarbonate" filter of 0.4 μm porosity, marketed by Whatmar (reference) and filmed; PrevIA etched by sandblasting (Ra = 0.92 p / biological cells / PMMA) (Ra = 0.92 μm); plastic pipette tip (non measurable Ra) / biological cells / PMEVIA (Ra = 0.92 μm); flat metal spatula (Ra 0.2 μm) plastic film I biological cells / Pfv111, 1A frosted (Ra = 0.92 μm) The use of the plastic film is intended to avoid the contamination of species when using a steel spatula.It should be noted that: - soft types of frosted glass (Ra = 0.425 μm - Ra = 1.98 μm), were tested, the method was validated on e species deemed difficult to lyse, such as Bg spores (Bacillus Qlobigii), Es (Bacillus subtilis), the lyses were made dry or in a volume of 0.5 μl to 5 μl of aqueous solution, the lyses were performed on a spore number of between 100 and 105 spores per lysis. Each of the roughness parameter Ra was determined as follows: measurement of a raw profile using a profiler "Taiysurf", 25 establishment of a primary profile by applying a form filtering (called "form removal ") To the raw profile, establishing a roughness profile by applying a" roughness filter "to the primary profile, determining the parameter Ra from said roughness profile.
    [0019] A first series of tests aimed to compare the efficiency of the process according to the invention (two versions: glass on glass, and glass on plastic) with the reference method by Precellys (Bertin). The amount of DNA released after spore lysis is evaluated by "q-PCR" using a "Stratagene" machine (Agilent) calibrated according to the manufacturer's instructions. The results of the first variation were as follows: 1. With the "PCR" on spores lysed by Precellys (Bertin), a Cl ("Cycle Threshold") was obtained in English) equal to 31. 10 2. The The process according to the invention was applied to the same spore solution in the glass-on-glass variation, that is, the spores were lysed between two frosted glass slides of Ra = 0.425 with PCR. On the spores lysed by the process according to the invention, a Ct of 30 was obtained, indicating that the concentration of lysed spores is two times higher than that of the previous test. 3. Direct PCR was performed on an unlysed control, and a Ct of 35.5 was obtained (most spores are not lysed in the initial solution).
    [0020] The results of the second variation were as follows: 1. With "PCR" on spores lysed by Precellys (Bertin), a Ci was obtained equal to 30. 2. The method according to the invention was applied to the same spore solution in the plastic pipette cone glass declination, i.e. the spores were lysed between a glass slide of Ra = 0.425 μm and a plastic pipette cone. The "FOR" on spores lysed by the process according to the invention resulted in a Ct equal to 29, which indicates that the concentration of lysed spores is twice as high as the previous test. 3. With a direct "FOR" performed on an unlysed control, a Ct of 36 was obtained (most spores are not lysed in the initial solution).
    [0021] These results show: 1. that the method according to the invention leads to lysis of the spores (Ct = 30 or 29 against 35.5 or 36 for the control). There is at least 5% difference between the solution lysed by the process according to the invention and the non-lysed solution, which corresponds to a ratio of 25% .The process is more effective than the lysis process. reference: ACt-e1 (30-31 and 29-30), that is to say that the method according to the invention makes it possible to recover twice as much material.
    [0022] A second series of tests aimed at controlling the non-adsorption and non-degradation of the DNA by the surfaces involved in the process according to the invention. Compared: 1. A direct "PCR" performed on a pure DNA solution + 15 spores, with a Ct equal to 31, at 2. A "FOR" performed on a solution of pure DNA + spores subjected to lysis carried out according to the process according to the invention in the glass declination on a plastic pipette cone, with a Ct equal to 31.
    [0023] These results show that the "PCR" on the pure DNA + spore solution gives the same results as when the solution was subjected to the process according to the invention. Therefore: 1. The process does not destroy the DNA. 2. There is no adsorption of DNA by the surfaces. 3. The recovery of the samples is effective (no losses because even Ct).
    权利要求:
    Claims (14)
    [0001]
    REVENDICATIONS1. Device for mechanical lysis (1) of biological species (C) present in a fluid, characterized in that it comprises a first wall (21, 71) and a second wall (11) movably mounted relative to one another. another, between an initial position in which the first and second walls are spaced apart from each other, and a lysis position in which the first wall bears against the second wall, the first and second walls being also mounted movably in shear with respect to each other in the lysis position, rune at least first and second walls having a rough bearing surface against the other wall, and having a parameter of average surface roughness Ra, between 0.1 pm and 10 pm, and preferably between 0.2 pm and 3 pm.
    [0002]
    The mechanical lysis device (1) according to claim 1, wherein one of the first wall (21, 71) and the second wall (11) is a microporous wall (11, that is to say porous to liquids and non-porous to biological species (C) to be lysed.
    [0003]
    3.3.3. A mechanical lysis device (1) according to claim 2, wherein the microporous wall (11, 71) has an average pore diameter of less than 1 μm, or even less than 0.2 μm.
    [0004]
    4. mechanical lysis device (1) according to any one of claims 2 or 3, wherein the microporous wall (11) is rough.
    [0005]
    Mechanical lysis device (1) according to claim 4, wherein the rough microporous wall (11) consists of a layer of microporous material (11) having a flat surface on which is positioned a rough layer (14) for to bear against the other wall (21) in the lysis position. 30
    [0006]
    The mechanical lysis device (1) according to claim 4, wherein said rough microporous wall (11) is made of a layer of microporous material (11) having a rough surface (15).
    [0007]
    Mechanical lysis device (1) according to claim 4, wherein the rough microporous wall (11) consists of a layer of macroporous material (16) having a rough surface (17) on which is positioned a microporous layer ( 18) intended to bear against the other wall (21) in the lysis position, the microporous layer (18) being sufficiently flexible and thin to conform to the roughness of the surface of the layer of macroporous material (in the lysis position.
    [0008]
    The mechanical lysis device (1) according to any one of claims 2 or 3, wherein the microporous wall (11) is non-rough and has an average surface roughness parameter Ra less than 0.1 μm, while the non-microporous wall (21) is rough and has an average surface roughness parameter Ra of between 0.1 μm and 10 μm, preferably between 0.2 μm and 3 μm.
    [0009]
    9. mechanical lysis device (1) according to any one of claims 4 to 7, wherein the first wall (21) and the second wall (11.) are rough.
    [0010]
    10. mechanical lysis device (1) according to any one of claims 1 to 9, characterized in that it further comprises a chamber (10) and a piston (20) mounted movably in the chamber between the position initial and lysis position, the first wall () being mounted on the movable piston and the second wall (11) being arranged screaming of the first wall.
    [0011]
    11. A mechanical lysis device (1) according to claim 10 when dependent on one of claims 2 to 9, wherein the second wall (11) is microporous, and wherein the chamber (10) further comprises a fluidic outlet (12) arranged opposite the first wall (21) relative to the second microporous wall (11).
    [0012]
    12. mechanical lysis device (1) according to any one of claims 2 to 11, wherein the chamber (10) 5 further comprises a fluidic inlet (30) and another fluid outlet (40).
    [0013]
    13. mechanical lysis device (1) according to claim 12 when dependent on claim 11, wherein the chamber (10) further comprises a sealed membrane (50) 10 arranged between the first wall (21). the fluidic inlet (30) and the fluidic outlet (40), the membrane being sufficiently thin and flexible to conform to the roughness of at least one of the first and second walls (21 and 11) in the lysis position.
    [0014]
    14. Process for mechanical lysis of biological species (C), characterized in that it comprises the following steps: A) Providing a device (1) according to any one of Claims 1 to 13, so that that the first and second walls (21 and 11) are in the initial position; B) Place the biological species to be lysed between the first and second walls; C) Bring the first and second walls to the lysis position; D) Generating shear between the first and second walls in the lysis position to cause mechanical lysis of the biological species and obtain a lysate; E) Spread the first and second walls until they are in the initial position; and. F) Recover the lysate of biological species. 5. A method for mechanical lysis of biological species (C) according to the preceding claim, wherein: step A) comprises providing a device (1) according to claim 10, step C) consists in sliding the piston (20) into the chamber (10) until the first and second walls (21 and 11) are in the lysis position; and step D) is to rotate the piston on itself so that the first and second walls remain in contact with each other. 16. A method for mechanical lysis of biological species (C) according to the preceding claim, wherein: step A) comprises providing a device (1) according to claim 11; step B) comprises placing a liquid comprising the biological species to be lysed between the first and second walls (21 and 11); step C) is to slide the plunger into the chamber until the liquid has been filtered through the microporous wall (11), that only the lysed biological species; remain on the microporous wall (11) and that the first and second walls are in the lysis position; and step D) is to rotate the piston (20) on itself so that the first and second walls remain in contact with each other. 17. A method of mechanical lysis of biological species (C) according to the preceding claim, wherein step A) comprises providing a device (1) according to claim 12; step B) comprises introducing a liquid comprising biological species to be lysed by the fluidic inlet (30) between the first and second walls (21 and 11); step C) is to slide the piston (20) into the chamber (10) until the first and second walls are in the lysis position; step D) is to rotate the piston on itself so that the first and second walls remain in contact with each other; Step F) comprises circulating an elution liquid between the fluid inlet (30) and the fluid outlet (40) to recover the lysate from the fluid outlet. 18. A method of mechanical lysis of biological species (C) according to the preceding claim, wherein: step A) consists of providing a device (1) according to claim 13, step B) comprises entering a liquid comprising the biological species to be lysed by the fluidic inlet (30), between the impervious membrane (50) and the second wall (11), - step C) consists in sliding the piston (20) in the chamber (10) until the first and second walls (21 and 11) are in the lysis position through the sealed membrane; step D) is to rotate the piston on itself so that the first and second walls remain in contact with each other through the sealed membrane; and - step F) is to circulate an elution liquid between the fluid inlet (30) and the fluidic outlet (40) to recover the lysate by the fluidic outlet. 20
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    同族专利:
    公开号 | 公开日
    US10160947B2|2018-12-25|
    WO2015181743A1|2015-12-03|
    US20170218326A1|2017-08-03|
    FR3021667B1|2018-01-26|
    ES2834382T3|2021-06-17|
    EP3149445B1|2020-10-07|
    EP3149445A1|2017-04-05|
    引用文献:
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    FR3098826A1|2019-07-17|2021-01-22|Commissariat à l'Energie Atomique et aux Energies Alternatives|Microfluidic device for preparing and analyzing a biological sample|
    法律状态:
    2015-06-01| PLFP| Fee payment|Year of fee payment: 2 |
    2015-12-04| PLSC| Publication of the preliminary search report|Effective date: 20151204 |
    2016-05-27| PLFP| Fee payment|Year of fee payment: 3 |
    2017-05-30| PLFP| Fee payment|Year of fee payment: 4 |
    2018-05-28| PLFP| Fee payment|Year of fee payment: 5 |
    2019-05-31| PLFP| Fee payment|Year of fee payment: 6 |
    2020-05-30| PLFP| Fee payment|Year of fee payment: 7 |
    2021-05-31| PLFP| Fee payment|Year of fee payment: 8 |
    优先权:
    申请号 | 申请日 | 专利标题
    FR1454866A|FR3021667B1|2014-05-28|2014-05-28|DEVICE FOR LYING BIOLOGICAL SPECIES AND METHOD IMPLEMENTED THEREBY|
    FR1454866|2014-05-28|FR1454866A| FR3021667B1|2014-05-28|2014-05-28|DEVICE FOR LYING BIOLOGICAL SPECIES AND METHOD IMPLEMENTED THEREBY|
    ES15734447T| ES2834382T3|2014-05-28|2015-05-27|Biological species lysis device and procedure implemented by this device|
    US15/313,740| US10160947B2|2014-05-28|2015-05-27|Device for lysing biological species and method implemented by said device|
    EP15734447.4A| EP3149445B1|2014-05-28|2015-05-27|Device for lysing biological species and method implemented by said device|
    PCT/IB2015/053970| WO2015181743A1|2014-05-28|2015-05-27|Device for lysing biological species and method implemented by said device|
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