DEVICE FOR OBTAINING BIOLOGICAL MATERIAL AND / OR BIOLOGICAL INFORMATION FROM A HETEROGENEOUS MATRIX

专利摘要:
Device for obtaining biological material and / or biological information from a heterogeneous matrix sample, uses of said device, methods implementing said device and kits for obtaining biological material and / or biological information to from a heterogeneous matrix sample comprising the various constituents of said device.
公开号:FR3025315A1
申请号:FR1458143
申请日:2014-08-29
公开日:2016-03-04
发明作者:Herve Rostaing；Filliard Agnes Dupont；Patrick Broyer；Sandrine Gicquel；Carole Vachon；Jerome Blaze
申请人:Biomerieux SA；
IPC主号:

专利说明:

[0001] Device for obtaining biological material and / or biological information from a heterogeneous matrix sample Technical field The present invention relates to the field of obtaining biological material and / or biological information from a heterogeneous matrix sample for further analysis, in particular for diagnostic purposes. STATE OF THE ART The control of biological material and / or biological information from samples of various origins (for example of industrial, agro-alimentary or clinical origin) requires the implementation of techniques which make it possible to detect - for example for the purpose of identification and / or enumeration of microorganisms - and whose performance in terms of results must be as efficient as possible. In general, if one considers said biological material, the latter may be non-pathogenic in nature. However, in the area of diagnosis, the biological material being checked is the most frequently pathogenic, therefore requiring rapid and precise detection of it in order to initiate ad hoc corrective actions as soon as possible . Of course, the toxins or other metabolites produced by this pathogenic biological material can also be sought. As a standard, the detection of biological material and / or biological information can be carried out by immunological tests, enzymatic tests, molecular biology techniques, or, with respect to said biological material, by enumeration and / or identification on culture box. In any case, whether it is biological material and / or biological information, detection sensitivity and detection specificity are two important factors in evaluating the effectiveness of the test performed. For example, the detection of microorganisms is conventionally carried out on selective culture media plated on a Petri dish, as recommended by the International Organization for Standardization (ISO) standards. English) or the BAM-FDA methods ("Bacteriological Analytical Manual of the Food and Drug Administration"). These standards recommend, in particular, the use of media such as "polymyxin egg yolk mannitol bromothymol blue agar" (whose acronym is PEMBA) or "mannitol-egg yolk-polymyxin agar" (whose acronym is MYP), according to their denominations in the English language. The identification of microorganisms is carried out, conventionally, according to morphological, cultural and / or metabolic criteria. Nevertheless, these Petri dish detection methods can be negatively impacted by the presence of non-specific elements from the sample to be analyzed. This is particularly true with regard to heterogeneous matrix samples, ie having differences in consistency (solid parts, more or less liquid parts, semi-solid parts etc.), such as soil samples, human stool or animal, 20 mucus, sputum or tissue samples (forensic samples), which are generally rich in polysaccharides, cell debris, and inhibitors of all kinds (bile salt, polyphenols, polysaccharide, fiber, hemoglobin, ... ), which may disturb the growth of the microorganisms sought and thus give rise to false negative results (problem of detection sensitivity). Alternatively, the detection of biological information from the biological material can be achieved, for example, by conventional nucleic acid amplification methods, specific for one or more types of microorganisms and viruses of interest. However, these techniques are sensitive and likely to be significantly inhibited by compounds such as polysaccharides, cell debris and inhibitors of all kinds. This may lead to false-negative results (detection sensitivity problems) or inaccurate diagnosis and result in serious consequences for the human or animal patient. Therefore, it is essential to isolate microorganisms and / or viruses so that a minimum of inhibitors are present in the isolate. This is particularly important for heterogeneous matrix samples as mentioned above. Indeed, these include a large number of non-specific constituents (polysaccharides, cell debris, inhibitors of all kinds), capable of generating false negatives during the implementation of the abovementioned nucleic acid amplification methods. .
[0002] In addition, and whatever the type of detection method used, these heterogeneous matrix samples have, in addition to conventional problems of contamination of the sample and / or the environment, more specific dosing / calibration problems. (Difficult correlation between quantity of sample taken and mass thereof, means of constant calibration from one sample to another) and suspension in a liquid medium, inherent to the nature of the sample. Currently, there are different methods for analyzing the genetic material contained in a heterogeneous matrix sample. By way of example, the "QIAamp® DNA" kit from QiaGen makes it possible to isolate and purify the total genomic DNA of a solid or liquid sample (such as blood, serum, plasma, etc.). . This kit utilizes a device consisting of tubes and filtration columns adapted for use with standard micro-centrifuges. According to the supplier's recommendations, the method for obtaining the total genomic DNA comprises the following steps: taking a determined quantity of the sample of interest (by weighing), depending on its nature, introducing it into a tube containing a solution for its suspension, put the sample in suspension by vigorous stirring vortex type. Once the sample is vigorously mixed, a lysis solution is added and an incubation step is performed for several minutes to break the cell membranes and release the genetic information into the solution.
[0003] Then, in order to remove debris, the solution is centrifuged and the supernatant removed. It follows several laborious steps of additions of solutions and centrifugations to purify the DNA sample, before eluting on a column by centrifugation.
[0004] However, this method, using the aforementioned "QIAamp® DNA" kit, has several drawbacks, especially when the sample tested is a heterogeneous matrix sample. Indeed, the first steps of weighing and transferring the sample into the microcentrifuge tubes are critical because of the narrowness of the opening of said tubes. This transfer can therefore be difficult and require additional manipulations and equipment, and elongated implementation times. In addition, during these weighing and / or transfer operations, there is a significant risk of: loss of material, contamination of the sample and / or the environment and / or the laboratory technician; Laboratory technician's discomfort with repeated handling of stool, sputum or mucus specimens, the difficulty of calibrating the sample by weighing.
[0005] Moreover, the numerous steps constituting the protocol of the "QIAamp® DNA" kit, which involve the manipulation of a large quantity of different tubes, are liable to lead to handling errors. In addition, the handling of small tubes, such as microcentrifuge-adapted tubes, is inconvenient for the laboratory technician, which is also likely to lead to loss of time and efficiency. . On the other hand, the kit "QIAamp® DNA" only allows nucleic acid isolation of a sample, but does not detect the presence of viable microorganisms, such as a detection on culture medium. In addition, the "QIAamp® DNA" kit does not allow the detection of yeast from a stool sample.
[0006] In another example, US Patent 4,081,356 discloses a device and method for recovering small sediments containing parasite eggs and larvae from a faecal sample. The disclosed device consists of two compartments separated by a summary filtration element (i.e., very non-selective), such as a filter and a steel mesh; the upper compartment being obstructed at its outer end by a spatula for taking the sample. The fecal sample is taken using the spatula, which is adapted to be connected to the upper part of the compartment. Then, the sample is emulsified using a solution of formaldehyde, optionally supplemented with a surfactant, mechanically with the aid of a stick. The emulsification is finalized by a horizontal stirring step in the presence of glass beads. Then a vertical stirring step makes it possible to pass all of the emulsified sample in the lower compartment of the device, through the summary filtration element. Ether is then used to rinse this summary filter element and allow a mixture to form at the bottom compartment. Finally, the lower compartment is agitated and centrifuged to separate the mixture thus obtained into different layers of materials of different densities, in order to isolate the sediment layer containing the parasite eggs and the larvae.
[0007] However, the device used in the aforementioned method has several disadvantages, particularly related to the lack of blatant selectivity of the summary filtration element, which allows the passage, in addition to eggs and larvae referred to above, non-specific elements that will then sediment in different layers. Moreover, the first sampling steps, in particular the step of connecting the spatula with the upper compartment, do not allow a clean and easy sample collection, and therefore require additional manipulations and elongated realization times. Moreover, this inevitably affects the reproducibility of the method implementing this device, in that it proves extremely difficult - if not impossible - to take a precise and calibrated volume of said sample, in particular on hard Bristol-type stools. 1 and 2. The human stool is commonly characterized and classified according to the Bristol scale consisting of a visual classification distributing human stool into seven types. Types 1-2 correspond to hard stools, types 3-4 are considered normal, and types 5-6-7 correspond to stools of liquid consistency.
[0008] In view of the problems presented above, an object of the present invention is to develop a device and its associated method (s) for: dosing / calibrating the quantity of heterogeneous matrix sample taken 10 without the need to weigh the latter, to effectively suspend said sample in a liquid medium, - to limit the problems related to clogging (bulk) filters to facilitate filtration and improve the recovery of the biological material 15 and / or biological information, - to limit the obtaining of non-specific elements likely to distort the result of the analysis in fine, to avoid the loss of a part of the sample during the transfer of sample in the container (eg in the tube), to improve efficiency and practicality, to minimize the risk of contamination of the sample and / or the environment and / or laborato technician To simplify the heterogeneous matrix sample preparation protocol in order to reduce the operator time, the number of materials and consumables used, the risks of errors and to improve the reproducibility, to propose a device compatible with the whole system. known methods of analysis (microbiology, culture, virology, bacteriology, immunoassay, metasequencing, PCR, etc.), to isolate yeasts.
[0009] Other objectives will become apparent, if any, from reading the present patent application.
[0010] SUMMARY OF THE INVENTION Accordingly, the object of the present invention relates to a device for obtaining biological material and / or biological information from a heterogeneous matrix sample, for example a stool sample. (Feces) human or animal, said device comprising: a container adapted to receive a content comprising said sample and at least one suspension solution, intended to allow the suspension of said sample, a stopper for closing said sample; preferably containing hermetically, said stopper comprising: at least one calibrated sampling means, for taking a predetermined volume of said sample corresponding to a given mass, said sampling means comprising at least one calibrated hollow portion connected to the inner portion of the plug and extending from said inner portion of the plug to the interior of the container when said plug and said container are assembled, - at least one opening, preferably closable by a removable closure means, allowing the communication of fluid between the inside of said container and the outside of said device when said plug and said container at least one filtering means is arranged, positioned with respect to said opening so as to filter the contents during the passage of said contents from the inside of said container to the outside of said device via said opening, said filtering means being adapted to allow the selective passage of said biological material and / or biological information outwardly of said device. Thus, the device according to the invention makes it possible to simplify and facilitate the sampling of a given mass of a heterogeneous matrix sample (in particular by avoiding a delicate weighing step), its suspension and the isolation of the biological material and / or the biological information it contains, for later analysis. In particular, the device according to the invention has the advantage of being adapted to process heterogeneous matrix samples of very varied consistency and / or texture. The simplification of the isolation of the biological material and / or the biological information obtained by means of the device according to the invention results notably in a reduction of the "open tube" stages, which require consumables and technical manipulations (pipetting, removal of supernatant, centrifugation, etc.), which can lead to errors and contamination of the sample and / or the external medium. The device according to the invention also makes it possible to dispense with the use of a multitude of equipment, such as scales, ball mills ("bead beaters" in English) or centrifuges.
[0011] By "heterogeneous matrix sample" is meant a small portion or a small amount of isolated heterogeneous matrix material for analysis. The heterogeneous matrix material from which the sample is taken is characterized by intrinsic differences in consistency and / or texture (solid parts, more or less liquid parts, semi-solid parts, viscous / viscoelastic parts). . This heterogeneous matrix material is likely to contain the biological material and / or the biological information sought (s). By way of example, the heterogeneous matrix sample may be a soil sample, human or animal stool, mucus, sputum, an industrial sample or a tissue sample (forensic samples). Preferably, the heterogeneous matrix sample is a sample of human or animal stool. According to a particularly preferred embodiment, this heterogeneous matrix sample is a human stool sample. The device according to the present invention makes it possible to obtain biological material and / or biological information from all types of stool defined by the Bristol scale, namely types 1-7.
[0012] For the purposes of the present invention, the term "biological material" is intended to mean any material containing genetic information that is self-reproducible or reproducible in a biological system. As an example of self-reproducing biological material, there may be mentioned a microscopically self-reproducing "biological material" such as one or more human, animal or vegetable cell (s) or a / several bacteria / yeasts. According to a preferred embodiment of the invention, said biological material is a self-replicating microbiological material (such as one or more bacterium (s), yeast (s), fungus (s)) or reproducible (such as one or several viruses) in a biological system. The device according to the present invention is particularly well suited for detecting and / or identifying and / or counting pathogenic microorganisms for humans and / or animals.
[0013] For the purposes of the present invention, the term "biological information" is intended to mean any element constituting said biological material or produced by the latter, such as nucleic acids (DNA, RNA), proteins, peptides or metabolites. . The biological information may in particular be contained within said biological material or excreted / secreted by the latter.
[0014] Said "calibrated hollow portion", preferably of concave shape, makes it possible to delimit a predetermined volume, adapted to take a predefined mass of the heterogeneous matrix material. More specifically, the volume of this hollow portion is determined to contain a quantity of biological material and / or biological information sufficient to enable it to be analyzed.
[0015] According to a preferred embodiment, the calibrated sampling means comprises a rod and a calibrated hollow portion. According to this preferred embodiment, said calibrated hollow portion is connected to the inner portion of the plug via said rod. Advantageously, in order to avoid collecting a mass / excess quantity of biological material and / or biological information, said rod is adapted so that only said at least one calibrated hollow portion is capable of containing the heterogeneous matrix sample. . For this purpose, said rod is preferably devoid of any recess or cavity, obviously with the exception of said calibrated hollow portion. According to a particular embodiment, said rod is made of solid material.
[0016] According to a preferred embodiment, said calibrated hollow portion is a recessed part of said rod, advantageously situated at the end of the rod opposite to the internal part of the plug. According to a preferred embodiment, said hollow portion has at least one wall adapted to remove the surplus of heterogeneous matrix sample by "scraping" said calibrated hollow portion against a suitable surface of sufficiently rigid constitution. Thus, by removing the excess heterogeneous matrix sample protruding outside the volume defined by the calibrated hollow portion, the operator ensures that the sampled heterogeneous matrix sample mass does not exceed, significantly, the desired mass, corresponding to the volume of the calibrated hollow portion. Advantageously, said hollow portion is calibrated to contain a mass of between 10 mg and 2000 mg, preferably between 100 mg and 1000 mg, advantageously between 200 mg and 300 mg, of said heterogeneous matrix material.
[0017] According to a particular embodiment of the invention, the sampling means is adapted to contain a plurality of hollow portions, preferably from 2 to 10, preferably from 2 to 6, advantageously 3 or 6, each of which may contain between 10 mg and 2000 mg of said heterogeneous matrix material, preferably between 100 mg and 1000 mg, preferably between 200 mg and 300 mg, of said heterogeneous matrix material. Consequently, according to this particular embodiment, the sampling means according to the invention can be adapted to take a quantity of heterogeneous matrix sample of between 20 mg and 20000 mg, preferably between 200 mg and 6000 mg. Obviously, those skilled in the art will be able to adapt the sampling means according to the invention, in particular as regards the number and / or the volume of the calibrated hollow part (s), in particular in order to ultimately obtain a concentration of biological material and / or biological information compatible with the minimum sensitivity level of the analysis techniques. By way of example, it is well known to those skilled in the art that the microorganisms responsible for bovine tuberculosis (mycobacteria of the tuberculosis complex) are difficult to detect and therefore require a larger quantity of sample to to obtain a concentration of analyzable microorganisms.
[0018] In a particular embodiment, said sampling means has, on each side of the stem, 3 calibrated hollow portions which may each contain a volume corresponding to 1000 mg, ie a total volume corresponding to 6000 mg, of a heterogeneous matrix sample. , such as cattle, sheep, goats or porcine feces, advantageously cattle.
[0019] Typically, stool analysis of human origin is performed from 200 to 300 mg of sample. However, in special cases, this can be up to 1000 mg. On the other hand, in the case of veterinary applications, the sample mass required for the analysis may be several grams or even several tens of grams.
[0020] According to a preferred embodiment of the invention, the assembly of the cap and the container is effected by close cooperation of the two entities via a closure system, such as a screwing or clipping system, to close said container so that the contents can only exit as filtrate through said at least one opening of said stopper when said container is closed by said stopper. For the purposes of the present invention, the term "removable closure means" means any means making it possible to prevent any uncontrolled release of filtrate. By way of example, said closure means may be a cap or a breakable portion of protection. In the latter case, said breakable protective portion is integral with said opening, preferably is sealed thereto (for example by heat sealing) and proves to be breakable, for example, by twisting or pulling. Alternatively, this breakable portion of protection may be cut under the action of a cutting means, such as a knife or a pair of scissors. It is interesting to note that said breakable part also fulfills the function of inviolability witness, insofar as the fact that this breakable part is disassociated from the device means, for the operator, that the device in question has already been used. . In other words, this breakable part makes it possible to ensure the integrity of the device according to the invention. Advantageously, when the removable closure means according to the invention is a breakable portion of protection, the latter has on the end opposite to said opening, a shape complementary to said opening. Thus, when said breakable protective portion is disengaged from said opening, for example by applying a traction or torsion force on said breakable part, the end of this breakable part having a shape complementary to said opening can be positioned on the latter in order to seal it. In this preferred embodiment, said breakable protection part, once disconnected from the opening, acts as a cap; said breakable portion of protection may therefore be described as "breakable protective cap".
[0021] As indicated above, the calibrated sampling means according to the invention is connected to the internal part of the cap by any appropriate means. Thus, said calibrated sampling means can be screwed, glued, assembled by elastic casing (for example by clipping) or else plugged into force in an opening 5 made in said plug. According to a particular embodiment, this calibrated sampling means can be heat sealed to the inner part of the plug. According to a preferred embodiment of the invention, said container further comprises at least one mechanical suspension means, preferably of spherical shape such as a ball, adapted to facilitate the suspension of said sample in said solution. suspending. By "mechanical suspending means" is meant an element adapted to be disposed within the container according to the invention and to be moved by one or more external force system (s) ( s) to the device according to the invention, for example by a manual stirring or induced by the vortex type mixing device, sonifier or magnetic stirrer, ball mill ("bead beater" in English) to allow / facilitate the mechanical suspension of the sample in the said suspension solution. Advantageously, the device comprises a number of mechanical suspending means, such as balls, selected between 1 and 200, preferably between 5 and 50, advantageously between 10 and 40, particularly preferably between 25 and 35; said mechanical suspending means having a size of between 1 mm and 10 mm, preferably between 2.5 mm and 3.5 mm, advantageously of approximately 3 mm; preferably, said mechanical suspension means being made of glass, iron, plastic (s), ceramic (s), particularly preferably glass.
[0022] The size of the means (s) of mechanical suspension means the largest dimension of this (s) last (s). For example, the size of a mechanical suspension means in the form of a rectangular parallelepiped (right-hand block) is the length (largest dimension) of this rectangular parallelepiped. As previously indicated, said at least one mechanical suspending means is spherical in shape and advantageously consists of a ball. In the case of a mechanical suspension means of spherical shape, the size refers to the diameter thereof. Thus, when a plurality of balls having a size of between 2 mm and 10 mm, preferably between 2.5 mm and 3.5 mm, advantageously 10 mm, is used as mechanical suspending means. about 3 mm, it should be understood that the diameter of the balls is between 2 mm 10 mm, preferably between 2.5 mm and 3.5 mm, preferably said beads have a diameter of about 3 mm. Preferably, said size of said beads is in agreement with the shape and dimensions of said calibrated hollow portion.
[0023] Preferably, the shape and / or size of said at least one mechanical suspending means is / are adapted to not seal said at least one opening for fluid communication between the interior of said container and the outside said device when said cap and said container are assembled. Conversely, when it is known, in advance, the size and / or the shape of said at least one mechanical suspending means, the shape and / or the size of said opening can (wind) be adapted (s). ) accordingly, in order to achieve the same goal.
[0024] According to a particular embodiment, the mechanical suspension means (s) has a size (a diameter in the context of means (s) of mechanical suspension of spherical shape) adapted to cooperate with said at least one hollow portion of the calibrated sampling means, in particular to facilitate the suspension of the heterogeneously sampled sample taken beforehand.
[0025] Preferably, the means (s) for mechanical suspension is / are constituted (s) of a material adapted to interact with an external suspension system / device, said system / device allowing to put in place movement or vibration means (s) mechanical suspension (preferably the balls). Such a system / device may consist, for example, of a sonifier (also called "sonicator") or a magnetic stirrer. Optimally, the material of which is / are constituted (s) means (s) mechanical suspension interacts little or not with said biological material and / or biological information, especially in order not to alter / degrade said biological material and / or said biological information. Advantageously, in order to facilitate handling, said calibrated sampling means has a rigidity sufficient to prevent said calibrated sampling means from bending during sample collection and to guarantee a low coefficient of variation on the mass of biological matrix removed. . By "sufficient rigidity" is meant a rigidity adapted to take samples of all types of heterogeneous matrix sample, and in particular those having a relatively solid consistency. The sufficient rigidity - and, consequently, the low elasticity - of the calibrated sampling means makes it possible to facilitate sampling and to avoid heterogeneous matrix sample projections during the sampling operation. Advantageously, the sufficient rigidity of the sampling means is obtained by the use of materials making it possible to achieve the required degree of rigidity, such as polypropylene (PP), Delrin resin, thermoplastic polyamide materials (PA), polycarbonate (PC) , polymethylmethacrylate (PMMA), low density polyethylene (LDPE), acrylonitrile-butadiene-styrene (ABS) and / or a form that promotes the rigidity of the sampling means. In addition, in order to prevent the calibrated sampling means according to the invention from bending during sampling, it is important that the connection between said sampling means and the internal part of the cap is sufficiently rigid. in order to prevent it from bending / bending during the sample taking operation. Indeed, during this operation, the operator usually holds between at least two of his fingers the outer portion of the cap then performs the desired sample. It is therefore important that there is no weak point in the connection between the inner part of the plug, on the one hand, and calibrated sampling means, on the other hand. According to a particular embodiment, said calibrated hollow portion comprises at least one opening to facilitate the suspension of said sample in said suspension solution.
[0026] Preferably, said at least one aperture has a circular, ovoid or elongate (preferably oblong) shape. Preferably, said opening is elongate, advantageously oblong, having, for example, a rod shape. This elongate - and preferably elongated - shape ensures a calibrated and reproducible sample of the heterogeneous matrix sample while promoting suspension thereof in the suspending solution. According to a particularly preferred embodiment, said at least one opening has a shape and / or a size adapted to cooperate with at least one mechanical suspension means as defined above, in order to promote the suspension in suspension. of the heterogeneous matrix sample taken by the operator. According to a particular embodiment, in order to further facilitate this suspension, said at least one hollow part is coated on all or part of its surface (preferably on its entire surface) with a non-stick coating, such as one or more layer (s) of water-soluble material (s) or a coating of hydrophobic type. Preferably, and in order to facilitate the manufacture of the device, the material chosen for producing the calibrated hollow portion is naturally hydrophobic and / or has a low roughness. By way of example, this low roughness can in particular be obtained by polishing to give a mirror polish finish.
[0027] According to a preferred embodiment, said container comprises said suspension solution, for example a buffer solution, in a volume sufficient to allow the suspension of the sample.
[0028] This suspension can be carried out: directly, namely by immersing partially or completely, preferably completely, said at least one calibrated hollow portion containing the sample in the suspension solution, or indirectly by inducing a level variation of the suspending solution so that the latter comes into intermittent contact (at regular or irregular intervals) with all or part of said at least one calibrated hollow portion containing the sample. The "indirect" suspension of the heterogeneous matrix sample in the suspension solution can in particular occur during a mechanical suspending step, namely, for example, during a step of stirring / mixing. The latter can be obtained, for example, using a stirrer or a vortex mixing apparatus.
[0029] By way of example of "suspension solution", any solution suitable for: i) allowing the said "heterogeneous matrix sample" to be suspended, and / or ii) preserving the biological material and / or the biological information of interest (ie to avoid any degradation of the latter). Thus, said suspending solution can be a phosphate buffer (mono / dibasic, 10 mM EDTA, pH 8), a PBS buffer or a TE buffer ("TrisEDTA", 10 mM Tris, 10 mM EDTA (Ethylene Diamine Tetra Acid). acetic acid), pH 8). Interestingly, said suspending solution may also comprise a preservative element preserving the microbial fauna of the sample taken, ie avoiding any modification thereof, in order to allow the sample to be transported at room temperature from the sample. Instead of sampling at the place of analysis, or to postpone the analysis step. A preservative of this type may, for example, consist of a preservative of "Cary-Blair" type, "modified Cary-Blair", as described in the publication Srijan et al. Pl, or a tryptic soy broth optionally supplemented with glycerol and / or 5% sheep blood (in accordance with the teachings of Wasfy et al [2]). Of course, those skilled in the art will be able to adapt both the nature and the volume of the suspending solution depending on the nature and volume of sample taken. Advantageously, said suspension solution comprises a calibrator of the plant DNA type, bacteria derived from heavy plants or peptides, etc., which make it possible to monitor and control the effectiveness of the suspension, filtration and filtration stages. lysis (out of the device). In addition, the use of said calibrator is particularly interesting for validating and interpreting the results. In addition, the use of said calibrator directly with the suspension solution makes it possible to dispense with an additional pipetting step in the protocol.
[0030] The suspending solution may also include means for capturing non-targeted elements (such as inhibitors, which may interfere with the subsequent analysis steps). For example, activated charcoal or polyvinylpolypyrrolidone (PVPP) can be used as a means of capturing non-targeted elements to improve detection sensitivity, retaining elements that can disrupt / inhibit steps. In addition, when it is desired to analyze biological information contained in biological material, preferably in self-reproducing biological material, the suspension solution as defined above may be in a In particular embodiment of the invention, further contain a lysis solution, to induce the lysis of said biological material and the release of the desired biological information within said sample suspension solution. According to a preferred embodiment, said filtration means is selected from: a gradient filter, and a superposition of at least two filters, preferably two or three filters, whose pore size decreases from inside to the outside of the device.
[0031] According to a preferred embodiment, the device is adapted to contain one or more filters each having an area greater than 50 mm 2, preferably between 100 and 350 mm 2, advantageously between 290 and 330 mm 2, in order to limit the clogging of the filters. while ensuring maximum filtered volume.
[0032] By "gradient filter" (also called "complex filter") is meant a filter having a plurality of different pore sizes. Specifically, when a gradient filter is employed for purposes of the present invention, the pore size of this filter decreases from the inside to the outside of the device to allow for progressive and differential filtration of the template sample. Heterogeneous of interest. In any case, whether using a gradient filter or a superposition of at least two filters, as described above, the pore size decreases from the inside to the outside of the device, preferably from 500 0.1 μm, preferably 200 μm to 10 μm, particularly preferably 200 μm to 20 μm. Of course, the pore size of the filter (s) used (s) is adapted depending on the size of the biological material and / or biological information that is desired to recover in the filtrate.
[0033] Thus, when it is desired to isolate, from the sample taken, viruses, toxins, metabolites or other biological information present within said sample, a pore size of between 0.1 μm and 20 μm, preferentially 0.1 μm and 5 μm, can be used. In the case of a superposition of at least two filters, a superposition of two to four filters is preferably used. Advantageously, a superposition of two or three filters is used. In a particular embodiment, said filtering means comprises at least one means for capturing non-targeted elements (such as inhibitors, which may interfere with subsequent analysis steps). By way of illustration, said means for capturing non-targeted elements may consist of active charcoal, or of a material having a particular affinity for inhibitors that can be contained in biological material (proteins, polysaccharides, etc.), for example the PVPP.
[0034] According to a preferred embodiment, the container comprises at least one wall comprising at least one zone of flexible material, adapted to undergo compression and generate in response an overpressure inside said container to allow or facilitate the filtration of the content. through said filtering means. Said at least one zone of flexible material proves particularly advantageous, insofar as the operator can generate on said at least one zone a manual pressure without excessive force, in order to allow / facilitate the filtration of the content through said means. filtration, as indicated above. The device according to the invention thus enables any (even) less experienced laboratory technician to obtain, by using the device according to the invention, a filtrate containing the biological material. and / or the biological information to be analyzed. In general, the device according to the invention can be adapted to any system / device making it possible to facilitate the filtration operation. For example, said device may be adapted to a vacuum filtration system or a centrifugal filtration system. It follows from the foregoing that the device according to the invention can, contrary to the protocol for obtaining biological material and / or biological information of the prior art, be easily automated. In a preferred embodiment, said plug comprises at least one rigid zone, said rigid zone having a shape adapted to cooperate with at least one holding member, such as a clamp, connected to a mixing device, such as a vortex, so as to allow the maintenance of said device on the mixing apparatus during the mixing of said device to facilitate the suspension of said sample in said suspension solution.
[0035] According to a preferred embodiment, said rigid zone having a shape adapted to cooperate with a clamp connected to said mixing apparatus, said rigid zone comprising: at least one anti-rotation means, comprising two distinct bearing surfaces, example constituted by two shoulders or by two tongues, each of the two separate bearing surfaces being adapted to be or abut against one of the two ends of the clamp when the cap undergoes a rotational movement, to prevent or stop said rotation, and / or - at least one anti-translational means, comprising at least one bearing surface, such as a flange, a flange or a shoulder, said at least one bearing surface being adapted to be or come in abutment against said clamp when the plug undergoes a translation movement, in order to prevent or stop said translational movement.
[0036] Said at least one rigid zone allows the device of the invention to easily adapt to commercially available mixing / homogenization devices, such as vortex-type devices, and to obtain results at the very least equivalent to those observed with devices specifically designed for this purpose, of the type of ball mill ("bead beater" in English).
[0037] The anti-rotation means and / or the aforesaid anti-translation means allow / allow to improve the maintenance of the device according to the invention by the holding member (for example a clamp of the apparatus of vortex mixing). The anti-rotation means makes it possible to limit, and preferably to suppress, any rotation of said plug (and therefore, by extension, of the device according to the invention) with respect to the aforementioned holding member. In addition to the fact that the anti-rotation means makes it possible to ensure a completely satisfactory maintenance between the plug - and therefore the device according to the invention - and the holding member of the mixing device, this means anti-rotation also serves to orient the plug, and therefore the calibrated sampling means, integral with said plug, so that the calibrated hollow portion of said sampling means is oriented downwards or upwards, preferably downwards, when the device according to the invention is positioned on the holding member of the kneading apparatus.
[0038] With regard to said anti-translational means, it makes it possible to limit, and preferably to suppress, any translational movement of the plug (and therefore, by extension, the device according to the invention) along a vector oriented in the plug-and-socket direction. containing during the kneading operation. This anti-translation means thus makes it possible to ensure efficient mixing and to prevent the device according to the invention from escaping from the holding member during said kneading operation. Advantageously, this anti-translational means comprises at least one flange (projecting portion) positioned on all or part of the periphery of the upper part of the cap.
[0039] According to a particular embodiment, this anti-translational means consists of a flange (circular part) positioned on the upper part of the plug and connected thereto, of diameter greater than the diameter of the plug body. The flange formed on all or part of the periphery of the upper part of the plug 30 constitutes a bearing face, which will cooperate with the aforementioned holding member (such as a clamp) to ensure the maintenance of the plug - and therefore of the device according to the invention - during the kneading operation. According to a preferred embodiment, when the holding member is a clamp, the bearing face formed by the flange or collar, as mentioned above, will abut against said clamp to prevent or stop any translation movement, as explained below, under the heading "detailed description of the invention".
[0040] According to a preferred embodiment of the invention, the upper part of the container - advantageously the shoulder of the bottle in the event that said container is a bottle - performs the function of anti-translational means by limiting or even eliminating any translation movement of the plug 10 (and therefore, by extension, the device according to the invention) along a vector oriented in the container-cap direction during the kneading operation. Indeed, the bearing surface constituted by the upper part of said container (for example the shoulder of the bottle) is / abuts against said holding member to prevent or stop any translational movement of the device according to the invention. invention according to a vector oriented in the container-cap direction. According to a variant of the invention, a second anti-translation means can be positioned on all or part of the periphery of the lower part of the plug, in order to limit or even eliminate any translational movement of the plug (and therefore, by extension of the device according to the invention) along a vector oriented in the container-cap direction during the kneading operation. In a general manner, said device is a device for obtaining biological material, preferably microbiological material, and said filtering means is adapted to allow the selective passage of said biological material, preferably of said microbiological material, to outside said device. The invention also relates to the cap per se as defined above.
[0041] In addition, the invention also relates to the use of the aforementioned device for obtaining biological material and / or biological information from a heterogeneous matrix sample, preferably for obtaining biological material 3025315 24 , preferably for obtaining microscopic biological material, advantageously for obtaining microbiological material. Said filtering means is adapted accordingly, in order to allow the selective passage of said biological material, preferably said microscopic biological material, advantageously of said microbiological material, to the outside of said device. According to a preferred embodiment, and as indicated above, the heterogeneous matrix sample is selected from a soil sample, a stool sample, a necrotic tissue sample, a food sample, an industrial sample, preferably said sample being a sample of human or animal stool. Advantageously, said heterogeneous matrix sample is a stool sample. Another subject of the invention relates to a process for obtaining biological material and / or biological information from a heterogeneous matrix sample, said method implementing the device according to said method comprising the following steps: ) taking a predetermined sample volume corresponding to a given mass using said calibrated sampling means, b) suspending said sample in said suspension solution, optionally by mechanical suspension, c) filtering the sample. suspension obtained in step b) through said filtration means to obtain a filtrate containing said biological material and / or said biological information. According to one particular embodiment, the operator can store the suspension 30 obtained at the end of the above-mentioned step b) and, if necessary, defer the filtration step c) in time. This makes it possible, in particular, to preserve the biological material and / or the biological information of interest for the purpose of subsequent analysis (s) or, in the case of samples taken "in the field" (ie out of the laboratory), to send to a laboratory the device according to the invention comprising the suspension obtained in step b), namely comprising the biological material and / or the biological information of interest, obtained (s) from the sample collection with heterogeneous matrix. This proves, in fact, particularly advantageous when samples of heterogeneous matrix samples are taken in remote areas (for example in bush areas), distant from any analytical laboratory. In this embodiment, preferably at least one preservative adapted to the nature of the biological material and / or the biological information to be analyzed is used. Thus, said suspension solution may, for example, contain one or more chemical preservative (s) (for example of the Cary-Blair type) and / or one or more means of freezing (allowing, at illustrative title, freezing at a temperature of the order of -80 ° C). Of course, the sine qua non condition is that the means (s) of preservation used (s) does not (or, where appropriate, not significantly) alter the biological material and / or the biological information to be analyzed. According to a particular embodiment, said suspension solution comprises at least one culture means. This culture means can be at least one nutrient or a plurality of nutritive elements (for example a culture medium) making it possible to promote the growth of the set of microorganisms present in the sample taken or to guide , more or less selectively, the growth of one or more type (s) of microorganisms.
[0042] Said at least one culture means may also be at least one selective agent having inhibitory properties as regards the growth of at least one type of microorganism, such as an antibiotic (bactericidal or bacteriostatic, preferably bactericidal), antifungal. The use of at least one selective agent can guide the growth of at least one type of microorganism.
[0043] Of course, those skilled in the art will adapt the culture medium (s) present in the suspension solution according to the type (s) of microorganisms of interest.
[0044] Another object of the invention is a method of extracting biological information (e.g., nucleic acids, such as DNA and / or RNA) from a heterogeneous template sample, said method comprising the steps of: a) carrying out the method of obtaining said material and / or biological information to obtain a filtrate containing said biological material and / or said biological information, b) when the biological information to be extracted is contained within said biological material, such as a cell, a bacterium, a fungus or a yeast, performing a lysis step of said biological material, preferably a mechanical lysis step, in order to obtaining a lysate comprising the biological information to be extracted, c) extracting the biological information from the filtrate obtained in step a) or the lysate obtained in step b) using an extraction process of in adapted biological training.
[0045] According to a preferred embodiment, this extraction method is a nucleic acid (s) extraction process (genetic information). In this embodiment, the lysis step b) of the abovementioned extraction process can be integrated in the extraction step c). For example, some automated easyMAG® gene information extraction systems (bioMérieux) incorporate a lysis step into the extraction protocol. Said lysis can in particular be of chemical, mechanical or thermal type, depending on the nature of heterogeneous matrix sample, the biological material and / or biological information sought (s) or further analysis techniques that will be used later.
[0046] According to a preferred embodiment, said process for extracting biological information comprises, before step c), and before step b) when this has to be carried out, a step of concentration of said biological material. and / or of said biological information, preferably by centrifugation (with or without cesium chloride type density pad for example) or by flocculation. When said concentration step is carried out by centrifugation, this is carried out at a speed of between 6000g and 12000g, advantageously about 12000g. Another subject of the invention relates to a biological information analysis method, said method comprising the following steps: a) extracting said biological information to be analyzed by using the above-mentioned method of extracting biological information, b) identifying and / or quantifying said biological information by any appropriate method of analysis, for example a genetic analysis method such as a PCR, an immunoassay or enzymatic test method.
[0047] According to a preferred embodiment, this method of analysis is a genetic analysis method (nucleic acid (s) analysis). In this case, said biological information is genetic information and the identification and / or quantification of this genetic information is / are performed by any appropriate genetic analysis method, for example by PCR.
[0048] In addition, a purification step may, if necessary, be carried out before step d), in order to eliminate any non-targeted element (such as one or more inhibitors that may interfere with the or the method (s) of analysis used in step (d)).
[0049] Another object of the invention is a method of analyzing biological material from a heterogeneous matrix sample, said method comprising the steps of: a) obtaining a filtrate containing the biological material to be analyzed using the aforesaid process for obtaining biological material, b) where appropriate, inoculating a reaction medium with the filtrate obtained in step a), said reaction medium being adapted to allow the growth and / or expression of at least a metabolism of said biological material, c) if necessary, incubate the filtrate obtained in step a), or the inoculated reaction medium obtained in step b), for a suitable period of time and at an appropriate temperature, and) analyze the biological material at the end of step (a), (b) or (c) by any appropriate means of biological analysis. According to a preferred embodiment, said biological material is a microbiological material. In a particular embodiment, step a) of the biological material analysis method can be adapted for the analysis of so-called fragile biological materials, such as Gram-based bacteria, in particular by using setting means. suspension resulting in less mechanical stress on the bacterial walls. In order to allow a less degrading suspension for these bacteria, the operator can reduce the mixing frequency, use mechanical suspension means having a larger size, or eliminate the use of mechanical suspension means. In the case where a mechanical suspension means would not be used, and where the sample would be difficult to suspend, a calibrated hollow portion having at least one opening - preferably a plurality of openings - can be used to facilitate suspension. Obviously, those skilled in the art will be able to adapt these parameters as a function of the biological material of interest called "sensitive" and of the nature of the sample. Preferably, the abovementioned microbiological material analysis method comprises step b) and optionally step c), advantageously steps b) and c), in which: step c), when: is present, incubates the inoculated reaction medium obtained in step b) for a suitable period of time and at an appropriate temperature, and - the biological material analysis step d) consists of the detection and / or identifying and / or counting said biological material on / in said reaction medium, preferably by visual or optical reading. Another subject of the invention relates to a kit for obtaining biological material 10 and / or biological information from a heterogeneous matrix sample, said kit comprising: said container and said stopper, in assembled or disassembled form at least one suspension solution adapted to allow said sample to be suspended, preferably at least one mechanical suspending means, such as at least one bead. Advantageously, said container and said stopper are in disassembled form and are packaged, preferably, sterile. Another subject of the invention relates to the use of the aforementioned kit for carrying out said process for obtaining biological material and / or biological information.
[0050] According to a preferred embodiment of the present invention, the device according to the invention is suitable for taking a sample of faecal material for microbiological analysis.
[0051] The biological material and / or biological information present in the filtrate obtained by using the device according to the invention can be analyzed by various biological analysis methods, such as enzymatic tests. or immunological, nucleic sequence amplifications or Petri dishes or by sequencing. In addition, the biological material and / or biological information present in the above-mentioned filtrate may, if necessary, be concentrated, purified, lysed or cultured, enriched. (s) or be directly analyzed. By way of example, the biological material and / or biological information present in the above filtrate can be directly analyzed by MALDI-TOF mass spectrometry (deposition of at least one drop of MALDI-TOF plate filtrate), by Raman spectroscopy, by membrane or strip immuno-chromatography (lateral flow test or "lateral flow test" in English), or by API-type bacterial identification systems. Brief description of the drawings The invention, its functionality, its applications as well as its advantages will be better understood on reading the present description, with reference to the figures, in which: FIGS. 1A and 1B respectively show a view of FIG. 2 is a front view of the cap, disengaged from the container as shown in FIGS. 1A and 1B, FIG. 3, and FIG. 3 shows a view of the first embodiment of the device according to the present invention. FIG. 4 is a front view of a cap according to a second embodiment, separated from the container according to the invention, FIG. 5 represents a view from below of FIG. FIG. 6 shows a side view of a device according to the invention connected to a kneading apparatus by means of a clamp, FIG. In the face of the device assembly according to the invention, the mixing device-blade as shown in FIG. 6, FIGS. 8A and 8B are perspective views of a plug according to a third embodiment, FIG. 9 shows a front view of the spout of the plug shown in Figures 8A and 8B, - Figure 10 is a perspective view of the upper plane of the central portion of the plug shown in Figures 8A and 8B, - Figure 11 shows a perspective view of the lower plane of the central portion of the plug shown in FIGS. 8A and 8B; FIG. 12 is a front view of a second embodiment of the spout of the plug shown in FIGS. 8A and 8B; FIGS. 13A and 13B show two perspective views of the sampling means adapted to be connected to the plug shown in FIGS. 8A and 8B; FIG. 14 is a perspective view of a second embodiment; of the calibrated sampling means according to the invention, - Figure 15 shows a perspective view of a third embodiment of the calibrated sampling means according to the invention, - Figure 16 is a perspective view of a plug. comprising a fourth embodiment of the calibrated sampling means according to the invention; FIG. 17 is a diagram schematizing the various steps of the biological information and biological material analysis methods according to the present invention; is a graphical representation of the results obtained for a linearity test, quantifying the DNA (by PCR, Number of Cq) of Gram-negative bacteria (KPC) by the biological information analysis method according to the invention, according to the log of the inoculated KPC concentration. A linear regression (R2) is shown and calculated (Linear). FIG. 19 is a graphical representation of the results obtained for a linearity test, quantifying the DNA (by PCR, Number of Cq) of Gram-positive bacteria (MRSA) by the method of analysis of biological information according to the invention, as a function of the log of the inoculated MRSA concentration. A linear regression (R2) is represented and calculated (Linear). FIG. 20 is a graphical representation of the results obtained for a linearity test, quantifying the DNA (by PCR, Number of Cq) of S. pombe yeasts by the biological information analysis method according to the invention, according to the log of the concentration of S. pombe inoculated. A linear regression (R2) is represented and calculated (Linear). FIG. 21 compares the PCR quantitation of adenovirus DNA using the Adenovirus R-gene kit (Reference: 69-010B) with the QIAamp® DNA stool extraction kit (QiaGen) and the method of extraction of biological information according to the invention. FIG. 22 (left scale ngAIL, right ratio) shows the impact of the Bristol stool type on the amount and purity of the extracted microorganism DNA. DETAILED DESCRIPTION OF THE INVENTION The purpose of the following detailed description is to explain the invention in a sufficiently clear and complete manner, in particular with reference to the figures mentioned above, but should in no way be regarded as limiting the scope of the invention. protection to the particular embodiments object of said figures.
[0052] As shown in FIG. 1A, the device according to the invention comprises a container 10, in the form of a flexible plastic bottle, which can be deformed in particular by a pressure exerted on said container by the fingers of the operator. .
[0053] The flexible wall 101 of the above-mentioned container 10 is made of flexible material. This flexible wall 101 contains, for example, rigid paper, cardboard, polyethylene, polyvinyl chloride, polypropylene, low density polyethylene, polyethylene terephthalate, as well as any suitable combination of these materials and / or any other biobased material so that the flexible wall 101 of the container 10 has satisfactory properties especially in terms of rigidity (in order to be able to place the container on a flat surface), sealing and compressive deformation when pressure is exerted (For example by the fingers of the operator) on the flexible wall 101 of the container 10, during the filtration step. As shown in this FIG. 1A, the container 10 is closed by means of a stopper 11 comprising, from its upper part to its lower part: a tubular-shaped orifice 111, which makes it possible to harvest the filtrate (closable by a cap 12), a plug body 112 (central part), a calibrated sampling means 113 connected to the lower part of the plug 11 (not numbered in FIG. 1A).
[0054] More specifically, this calibrated sampling means 113 comprises a calibrated hollow portion 1132 connected to the lower part of the plug (not numbered in FIG. 1A) via a rod 1131. The volume of the calibrated hollow portion 1132 a has been previously calculated to correspond to a predetermined mass of heterogeneous matrix sample, which may contain the biological material and / or the biological information of interest. The device 1 is constituted by assembling the plug 11 and the container 10.
[0055] The container 10 is filled with a sufficient volume of suspension solution 13 as previously defined. The level of suspension solution 13 is shown in FIGS. 1A and 1B for illustrative purposes only. The internal structure of the plug 11 - and in particular of the plug body 112 - is clearly visible in FIG. 1B, which shows a sectional view of the device 1. The body of the plug 112 comprises an inner portion 117 and an outer portion 119 these two parts 117 and 119 delimiting a cavity in which a filtering means 116 is arranged. This filtering means 116 consists of a superposition of three filters 1161, 1162 and 1163, each having a surface area of 314. mm2; each of the three filters 1161, 1162 and 1163 further having degressive size pores of the inner portion 117 to the outer portion 119 of the plug body 112. More specifically, the first filter 1161 has pores whose size is between 100 iim and 200 iim while the two "upper" filters 1162 and 1163 each have pores less than 50 iim. The first filter 1161 makes it possible to eliminate the larger entities (for example cellular debris, etc.) while the two "upper" filters 1162 and 1163 make it possible to ensure the selectivity of the filtration operation. As illustrated in FIG. 1B, the filter 1163 having the smallest pore sizes (equal to those of the filter 1162) comes into contact with the outer portion 119 of the plug body 112. The rod 1131 of the calibrated sampling means 113 is connected to the inner portion 115 of the plug 11 via a connection (or attachment) means 115, positioned on said inner portion 117. Apertures 118 are formed within the portion 117 of the plug 11 to allow the passage of the suspending solution 13 comprising the heterogeneous matrix sample (not shown) to the tubular shaped orifice 111 via the filtering means 116, comprising the superimposition of the three filters 1161, 1162 and 1163. For purposes of For clarity, the use of the device 1 according to the invention is briefly described hereinafter. The operator takes, using the hollow portion 1132 of the calibrated sampling means 113, a volume corresponding to the volume defined by the calibrated hollow portion 1132 and corresponding to a desired heterogeneous matrix sample mass.
[0056] The soft-walled container 10 is filled with a sufficient volume of a suspending solution 13. As illustrated in FIGS. 1A and 1B, the container 10 is closed / closed by the stopper 11 (for example the stopper 11). 3025315 is screwed on a screw thread positioned on the neck of said container) and, optimally, the tubular-shaped orifice 111 is closed off by the cap 12. The device 1 is then agitated to allow the suspension to be suspended. the heterogeneous matrix sample contained in the calibrated hollow portion 1132 of the sampling means 113. Once the suspension is carried out, the operator removes, if necessary, the cap 12 and "returns" the device 1. Because of this reversal operation, the suspension solution 13, charged with heterogeneous matrix sample, passes through the openings 118 made within the inner part 117 of the plug 11, passes successively through the three s filters 1161, 1162 and 1163 of the filter means and flows outward, in the form of filtrate, via the tubular-shaped orifice 111. The filtrate is then collected in any type of suitable container, for example in a Eppendorf tube.
[0057] Advantageously, and as indicated above, the passage of the heterogeneous matrix sample loading solution 13 through the filtration means 116 is facilitated / accelerated when the operator exerts pressure on the flexible wall 101 of the container 10, resulting in an overpressure within said container 10 and in particular at the level of the filtration means 116. This operation 20 of compressing the flexible wall 101 by the operator is particularly advantageous for facilitating filtration, given the smallness / narrowness of the pores of the two upper filters 1162 and 1163. The view represented in FIG. 2 makes it possible to visualize the plug 11, when it is disassembled from the container 10, for example by unscrewing said plug 11. FIG. 2 clearly shows the tubular orifice 111 which can be closed by means of the cap 12, the plug body 112, the calibrating means 113. As shown in FIGS. 1A and 1B, the latter comprises a rod 1131 connected, at one of its ends, to the internal part of the cap body 112 and comprising, at its opposite end, a portion hollow calibrated 1132.
[0058] As indicated above, FIG. 3 is a bottom view of the stopper 11. In this FIG. 3, it is clearly seen the radial arrangement of the openings 118 formed within the internal portion 117 of the stopper 11. around the rod 1131 of the sampling means 113. Although the number and arrangement of the openings 118 may vary according to the wishes of those skilled in the art, the radial arrangement of the openings 118 as shown in FIG. distributing the volume of the suspension solution 13 charged in heterogeneous matrix sample to the filtration means (not shown in FIG. 3) via said openings 118.
[0059] FIG. 4 illustrates a second embodiment of a plug 21 according to the invention. The plug body 212 is provided with: an anti-rotation means 220, and an anti-translational means 221, both adapted to cooperate with a holding member such as a clamp (not shown), a mixing apparatus, such as a vortex (also not shown in Figure 4). The anti-rotation means 220 is positioned on the outer surface of the plug body 21. As shown in FIG. 4, the anti-rotation means 220 is constituted by an extra thickness on the surface of the plug body 21. This extra thickness defines two shoulders 2201 and 2202 on the plug body 212. When the holding member of the mixing device (not shown in Figure 4) is a clamp, each of the two branches of this clamp will come to position 25 from and other anti-rotation means 220. When a rotational movement is impelled to the plug 21 during the mixing step, one of the two shoulders 2201 or 2202 (depending on the direction of rotation) will abut on the end of one of the two branches of the clamp, preventing or stopping de facto the rotation of said plug 21. Preferably, the plug is positioned on the clamp so that the shoulders 2201 and 2202 abut against the ends of each of the two branches of the clamp.
[0060] This anti-rotation means proves particularly advantageous insofar as it makes it possible to impose a particular orientation on the calibrated sampling means 213 connected to the internal part (not shown in FIG. 4) of the stopper 21. anti-rotation element 220 is positioned on the outer surface of the plug body 212 so that the calibrated hollow portion 2132 of the sampling means 213 is oriented downwardly when the device comprising the plug 21 is disposed on the holding member (for example a clamp) of the vortex mixing apparatus. This orientation of the hollow part "downwards" makes it possible to improve the suspension of the heterogeneous matrix sample that it contains. The anti-translational means 221, as represented in FIG. 4, consists of a flange with a diameter greater than that of the plug body 212. The function of this anti-translation means 221 will be described below, in connection with the figure 5.
[0061] The arrangement of anti-rotation and non-translational means 221 relative to plug body 212, and in particular to inner portion 217 of plug body 212, is clearly illustrated in FIG. 5, which shows a view from below. the plug 21 according to the second embodiment of the invention, said cap 21 being detached from the container 10. As is the case in Figure 3, there is, here again, the radial arrangement of the openings 218 around the rod 2131 of the sampling means 213. FIG. 6 shows the holding of the device 2 (comprising the cap 25 according to a second embodiment and the container 10) on a mixing device 71 via the cooperation of the plug body 212. and a clamp 40, connected to the kneading apparatus 71. In this regard, it should be noted that the clamp 40 may be an integral part of the kneading apparatus 71 or may be connected thereto by any means appropriate. More precisely, it is observed in this FIG. 6 that the shoulder 2201 of the anti-rotation means 220 abuts against the end 402 of the corresponding branch of the clamp 40. Of course, the shoulder 2202 it also abuts against the end 403 of the corresponding branch of the clamp 40, even if this can not be observed in FIG. 6. In other words, the anti-rotation means 220 "abuts" on each of the two ends 402 and 403 of the clamp 40, so that the plug 21, and therefore the device 2, can not undergo any rotational movement in one direction or the other. In a very interesting way, it can be seen in this FIG. 6 that the anti-rotation means 220 has been positioned on the outer surface of the plug body 212 so as to orient the calibrated hollow portion 2132 "downwards". when the stopper 21 is held by the gripper 40. As indicated above, the orientation of the calibrated hollow portion 2132 downwards ensures optimum suspension of the heterogeneous matrix sample. In addition, the anti-translational means 221 is also clearly visible in FIG. 6. As indicated above, in this embodiment, it is a collar of diameter greater than that of the plug body 212. When a translational movement occurs in a vector oriented in the plug-21-containing direction 10, the flange acts as a bearing face, which abuts against the portion 401 of the gripper 40, thereby preventing or stopping this translational movement. As shown in FIG. 6, the container 10 is filled with a sufficient volume of suspending solution 13 as previously defined. The level of this suspension solution 13 is shown for information only in FIG. 6. The tubular orifice 221 of the stopper 21 is furthermore closed off by the cap 12, in order to avoid any untimely exit of the solution. suspending agent 13 during the mixing step.
[0062] FIG. 7 shows a front view of the stopper 21, held by the clamp 40, itself connected to the kneading apparatus 71. In this FIG. 6, the positioning of the clamp 40 from else of the anti-rotation means 220, so that each of the two shoulders 2201 and 2202 abut against the corresponding ends 402 and 403 of each of the two branches of the clamp 40.
[0063] This clamp 40 is furthermore positioned under the anti-translational means 221. In the event of a translation-type movement (following a vector oriented in the plug-21-containing direction 10), the collar constituting the antitranslation means 221 will abut against the portion 401 (not shown in FIG. 7) of the clamp 40 to prevent or stop any translational movement. A third embodiment of the plug 31 is illustrated in FIGS. 8A and 8B. The latter clearly represent this plug 31, which comprises, from the upper part to the lower part: a tubular-shaped orifice 311, an upper part 319, an anti-translational means 321, a plug body 312, an anti-translational means rotation 3201, 3202 a calibrated sampling means 313 comprising a rod 3131 and a hollow portion 3132. In addition, the tubular-shaped orifice 311 is closable by means of the cap 326. As can be seen in the light of 8A and 8B, this hollow portion 3132 is oppositely oriented against the anti-rotation means 3201, 3202. In this manner, when the device comprising the plug 31 and the container 10 is held by a clamp connected to a mixing apparatus , the anti-rotation means 3201, 3202 is oriented upwards, as illustrated in FIG. 6 while said calibrated hollow portion 3132 is deliberately oriented downwards, in order to promote the setting in spension of the heterogeneous matrix sample present in the calibrated hollow portion 3132. In addition, unlike the cap according to a second embodiment of the invention, shown in FIGS. 4-7, in which the anti-rotation means 3201, 30 3202 is constituted by a thickness of material, (for example a thickness of plastic material), positioned over the width of a portion of the body of the cap 212, the anti-translational means 3201, 3202 of the cap 31 3025315 comprises, over the entire length of the plug body 312, two parallel tongues 3201, 3202, present on the surface of the plug body 312. As for the shoulders 2201 and 2202 shown in FIG. 5, when a rotational movement is impulsed to the plug 21 during the kneading step, one of the two tongues 3201, 3202 (depending on the direction of rotation) abuts on the end of one of the two branches of the clamp, preventing or stopping de facto the rotation of said bou chon 31.
[0064] Preferably, the plug 31 is positioned on the clamp so that the tabs 3201, 3202 abut the ends of each of the two legs of the clip. The various parts constituting the plug 31, previously shown with reference to FIGS. 8A and 8B, with the exception of the calibrated sampling means, are described below. The pouring spout 31 'of this plug 31 is shown in FIG. 9. On the latter, a removable cap 326 of shape complementary to that of the tubular orifice is observed from the upper part to the lower part. 311. This cap 326 may in particular be of cylindrical shape or truncated conical shape. The spout 31 'also comprises the upper part of the body of the cap 319 and two rings 327 and 328, arranged parallel to each other around the upper part of the cap 319. The assembly constituted by the two rings 327 and 328 represents a male elastic casing element (commonly referred to as a "male clipping element") adapted to cooperate with a female elastic casing element (commonly referred to as a "female clipping element"). This latter is obtained by boring the central element 31 ", shown in FIG. 10 and described hereinafter.
[0065] FIGS. 8A and 8B also show a positioning means consisting of a flattened zone 323 of the anti-translational means 321, making it possible to position the plug with respect to the ends 402 and 403 of each of the two branches of the clamp 40 and thus to avoid that the anti-translational means 321 comes into contact with the mixing apparatus 71. FIG. 10 makes it possible to precisely observe the upper part of the central element 31 ", comprising various filter supports ( s) 325, 330 adapted to receive the filtering means represented by numeral 116 in Figure 1B, said filter supports (s) comprise a plurality of lugs 325 positioned near openings 318, said lugs 325 extending to at the level of the peripheral filter support 330, consisting of a flange As previously indicated, the central element 31 "also has a female clipping element (not shown in FIG. Figure 10), including two grooves bored in the inner side wall 329 of the central member 31 "; these two grooves (not shown) being adapted to respectively receive the rings 327 and 328 of the male clipping element of the spout 31 ', and thus allow the elastic casing of the spout 31' in the central element 31 ".
[0066] In addition, an opening 315 passes right through the central part of the central element 31 ". This opening 315 has a shape complementary to that of the male clipping means 3133 of the sampling means 313, as shown in FIG. FIGS. 13A and 13B More precisely, said opening 315 is of trapezoidal shape and is dimensioned so that, when the male clipping means 3133 of the sampling means 313 (see FIGS. 13A and 13B infra) is plugged into the opening 315, the sampling means 313 is: (i) immobilized, and also (ii) oriented relative to the anti-rotation means 3201, 3202, as previously explained, in order to impose the desired orientation on the calibrated hollow portion 3132 (see above).
[0067] In addition, the complementarity between the shape of the opening 315, on the one hand, and that of the male clipping means 3133, on the other hand, imposes and guarantees the desired orientation of the sampling means 313 - and in that particular of its hollow part 3132 during the assembly operation of the sampling means 313 with the central element 31 ". In addition, the elastic casing of the part 3133 of the sampling means 313 in the opening 315 makes it possible to to give the assembly "central element 31" -means of sampling 313 ", sufficient rigidity so that the rod 3131 of the sampling means 313 does not bend during the sampling of heterogeneous matrix sample, thereby avoiding same, the risks of projection of heterogeneous matrix sample. The openings 318 as shown in FIG. 10 allow the flow of the heterogeneous matrix sample-loading solution 13 from the container 10 to the intermediate portion 340 of the central member 31 "containing the filter means 116. Although the assembly of the spout 31 'with the central element 31 "and the latter with the sampling means is obtained by elastically fitting male clipping systems (327, 328 and 3133) and females (grooves dug in the inner side wall 329 and opening 315), the assembly of these various elements can, of course, be obtained, alternatively, by any connection / fixing system known to those skilled in the art, such as a system screwing, gluing or welding (eg heat sealing).
[0068] FIG. 11 shows a bottom view, in slight perspective, of the central portion 31 "of the plug 31, on which are observed the openings 318, arranged radially around the opening 315. Playing on the arrangement relative to the trapezoidal opening 315 relative to that of the anti-rotation means 3201, 3202, it is possible to orient the hollow portion 3132 of the sampling means 313 30 so that the latter is oriented downwards, in order to to promote the suspension of the heterogeneous matrix sample (as explained above) In this FIG. 11, it is also noted that the inner portion 317 has a truncated cone-shaped shape, making it possible to limit the risks of leakage. the suspension comprising the heterogeneous matrix sample. When screwing the cap onto the container, said truncated cone-shaped portion comes into contact with the edge of the container and crushes the edge angle of the container on a weakened e surface, thus ensuring a good seal. In this FIG. 11, an internal thread 324 obtained by tapping is also observed, designed to cooperate with an external thread made on the upper part of the container 10, preferably on the neck of the container 10, in order to allow the assembly cap 31 and the container 10 by screwing. Of course, other closure systems, preferably hermetic, may be used to allow the assembly of the cap and the container, for example, a system of elastic casing (clipping).
[0069] Figure 12 shows a second embodiment of the spout 41 'of the plug according to the invention. This spout 41 'is adapted to be fitted by elastic casing inside the central element 31 "of the cap 31. This is achieved by introducing the rings 427 and 428 into the corresponding grooves formed in the internal lateral wall 329 of the the central element 31 "(see Figure 10).
[0070] As shown in FIG. 12, the tubular orifice 411 of this spout 41 'is integral with the breakable and repositionable cap 426 (for example heat-sealed to said cap or simply molded together with the cap, in the manner of the pipettes. physiological saline solution), so that said breakable and repositionable cap 426 prevents, in this configuration, any uncontrolled liquid outlet via the tubular-shaped orifice 411, outwardly. In other words, when this breakable and repositionable cap 426 is integral with the tubular-shaped orifice 411, this makes it possible to avoid any uncontrolled leakage of liquid towards the outside of the device according to the invention. In addition, the fact that this breakable and repositionable cap 426 is integral with and obstructs the tubular orifice 411 indicates to the operator that no liquid has been previously poured through the spout 41 '. The breakable and repositionable cap 427 thus plays, indirectly, the role of inviolability witness.
[0071] The breakable and repositionable cap 426 can be disengaged from the end of the tubular-shaped opening 411 by the action of a pull-type force or, preferably, a twist on said breakable and repositionable cap 426, particularly in exerting a rotational force on one, or preferably two, fins 4261 and 4262 of said cap 426.
[0072] The end of the breakable and repositionable cap 426, opposite the end in contact with the tubular-shaped orifice 411, comprises a hollow portion 4263 of substantially cylindrical or truncated conical shape. This hollow portion 4263 is of complementary shape to that of the end of the tubular-shaped orifice 411, so that when the breakable and repositionable cap 426 is disengaged from the tubular-shaped orifice 411, for example by applying a pull on one or two of the fins 4261 and 4262 of said cap 426, the latter may be used, later, to seal again the tubular-shaped orifice 411 spout 41 '. This obturation is obtained by interlocking the tubular-shaped opening 411 in the hollow part 4263 of the breakable and repositionable cap 426. It should be noted that according to a simpler embodiment, the breakable and repositionable cap 426 can be replaced by a simple breakable portion of protection, which does not allow to re-seal the tubular-shaped opening 411 after separation of the breakable portion of protection thereof, unlike the breakable cap and repositionable 426, shown in Figure 12. FIGS. 13A and 13B show, respectively, a three-quarter perspective view of the calibrated sampling means 313 and a back view of the same calibrated sampling means 313. These two FIGS. 13A and 13B allow us to observe the average male clipping 3133, the solid material rod 3131 and the calibrated hollow portion 3132 3132, of oblong shape (more precisely having a shape called "Rod"), said calibrated hollow portion 3132 having been obtained by recess of a region located at the end of the rod 3131 opposite to the inner part of the plug (not shown in Figures 13A and 13B).
[0073] A second embodiment of the hollow portion 4132 is shown diagrammatically in FIG. 14. In this front view, it can be seen that said calibrated hollow portion 4132 comprises openings 41321 intended to facilitate the suspension of the sample at heterogeneous matrix present at the calibrated hollow portion 4132. This advantageous technical effect is obtained by allowing the suspension solution 13 to pass through the calibrated hollow portion 4132 during the step of suspending the suspension. 'sample. A calibrated hollow portion 4132 according to this second embodiment is particularly suitable for facilitating the suspension of heterogeneous matrix samples having significant adhesive properties, such as saddles having a Bristol type of 1 or 2. The FIG. 15 illustrates a third embodiment of the calibrated hollow portion 5132, having openings 51321 of ovoid shape and dimensions greater than those of the openings 41321 schematized in FIG. 14. These openings 51321 make it possible to further facilitate the suspension of a heterogeneous matrix sample contained within the calibrated hollow portion 5132. The hollow portion 5132 according to this third embodiment proves particularly suitable for allowing the suspension of sample having adhesive properties more marked than those of the heterogeneous template sample to be collected using or via the calibrated hollow portion 4132 of Fig. 14, such as saddles having a Bristol type of 1-2. FIG. 16 shows a particular embodiment, in which the sampling means 613 comprises, in a large part of its stem 6131, three calibrated hollow portions 6132, 6134 and 6135 of square or rectangular shape, each having a predetermined volume. to allow each of these three calibrated hollow parts 6132, 6134 and 6135 to take a mass of 1000 mg. The calibrated sampling means 613, shown in FIG. 16, thus makes it possible to take a total mass of 3000 mg of heterogeneous matrix sample. A calibrated sampling means 613 of this type may be particularly important for recovering in fine a filtrate containing a concentration of biological material and / or sufficient biological information to allow their analysis, especially in the case where the biological material and / or the biological information sought is (are) known to be present in a very small amount in the heterogeneous matrix material from which the sample is taken. This sampling means 613 may furthermore advantageously be used in veterinary applications, increasing the volume defined by each of the hollow portions 6132, 6134 and 6135 as necessary. According to a particular embodiment, the rod 6131 of the On the opposite side to that comprising the three calibrated hollow portions 6132, 6134 and 6135, the sampling means 613 has three additional calibrated hollow portions. Preferably, these three additional calibrated hollow portions each make it possible to take a mass of heterogeneous matrix sample of approximately 1000 mg. Thus, this adapted sampling means comprising 6 calibrated hollow portions makes it possible to take a total mass of heterogeneous matrix sample of about 6000 mg of heterogeneous matrix sample. FIG. 17 is a diagram showing: (i) the various essential (solid line) and optional (dashed) steps of the process for obtaining biological material and / or biological information according to the invention (ii) the various necessary steps (solid line) and optional (dashed) different subsequent analysis pathways, allowing the identification and / or detection and / or quantification of said biological material and / or said biological information.
[0074] Steps 171, 172 and 173, shown in Figure 17, are described in connection with the device 1, shown in Figures 1A and 1B.
[0075] In order to carry out the sampling step 171, the operator manually grasps the plug, preferably by the plug body, and fills the volume defined by the hollow portion calibrated with the heterogeneous matrix sample (for example a 5 stool sample). The stopper and the container, previously filled with a suspension solution, are assembled, for example by screwing the stopper onto the neck of the container, so that the heterogeneous matrix sample contained in the hollow portion is in contact with the suspending solution.
[0076] It should be noted that the container may contain, in addition to the suspending solution, one or more suspending means, for example 30 glass beads each 3 mm in diameter. The suspending step 172 may be performed by shaking the device to allow the suspension of the heterogeneous matrix sample contained in the calibrated hollow portion in the suspending solution. In order to facilitate and / or accelerate this mechanical suspending step 172, the device may be connected to a mixing means, such as a vortex, as shown in FIG. 6. Advantageously, the device is connected to said means of mixing by a holding member cooperating closely with the rigid portion of the plug, to ensure effective maintenance of the device during the mechanical suspending step 172, consisting, in this case, a kneading step. Once the operator estimates that the heterogeneous matrix sample has been properly suspended within the suspending solution, the suspending step 172 is complete. At this time, the device containing the heterogeneous matrix sample suspension may optionally be stored, in step 1721, preferably after adding at least one preservation means and / or by freezing. Alternatively, the device may also be incubated at a temperature, for example at 37 ° C., for a duration of a few hours to several days in order to enrich the suspension of certain microorganisms present in the sample. This incubation step may in particular be carried out in the presence of a selective culture medium contained in the device according to the invention. The maximum storage time obviously depends on the means of preservation used, if any, and the nature of the biological material and / or biological information to be analyzed. As indicated above, this storage step may be particularly useful in the context of "non-laboratory" heterogeneous matrix sampling operations. Indeed, the device optionally comprising a means of preservation may, after the tubular orifice has been closed by a cap, be sent to an analysis laboratory in order to perform all the desired analyzes of the biological material. and / or biological information capable of being contained in the heterogeneous matrix sample taken in step 171. Alternatively, the operator can directly proceed to the filtration step 173. In order to to perform this filtration step 173, the removable closure means is removed from the tubular-shaped orifice, then the device is then inverted and at least one pressure is applied to at least one zone of the flexible wall of the container, in order to create an overpressure in this container and thus force the passage of the suspension solution charged in heterogeneous material sample through the plug filtration means through the openings. Thus, the heterogeneous material sample-loaded slurry solution which flows along the tubular-shaped orifice has necessarily been filtered by the filtration means 116 positioned in the plug body. At the end of this filtration step 173, the filtrate containing said biological material and / or biological information is poured via the tubular-shaped orifice into a collection tube, for example an Eppendorf tube. Once said biological material and / or biological information isolated (s) in the collector tube, the operator can perform various biological analyzes, depending on the nature of the biological material and / or biological information sought (s).
[0077] When it is desired to analyze the viable biological material, conventional microbiology techniques can be performed for these purposes, such as seeding and culturing said viable biological material on solid, liquid or aqueous reaction media. semi-solid, selective or non-selective, preferably comprising a culture medium. Metabolic expression of this viable biological material can also be assessed through the use of enzyme assays (eg API galleries, MALDI-TOF plate, lateral flow test).
[0078] These microbiology techniques make it possible to detect and / or identify and / or enumerate, at step 1752, the desired viable biological material. However, depending on the analytical techniques used, the type of viable biological material of interest, an additional enrichment step may be necessary to promote the growth of said biological material and thereby increase its concentration in the medium. reaction. Of course, said viable biological material, as well as biological information in the broad sense, can also be the subject of genetic, protein and / or metabolic analyzes. For this purpose, the reader will refer to the biological information analysis, which is described hereinafter. After the filtration step 173, when the operator wishes to analyze the biological information contained in the filtrate, and in particular comprising metabolites, proteins or nucleic acids, specific detection and / or identification techniques. and / or quantization are performed. In a particularly advantageous manner, a step of concentration of said biological information 1741 contained in the filtrate is carried out by centrifugation at 6000-12000 g or by flocculation, in order to concentrate the biological information of interest and to reduce the quantity of non-targeted elements. (such as inhibitors) present in the suspension. If appropriate, a lysing step 1742 is carried out, when the biological information of interest is contained in said biological material, in particular when said biological material is a self-reproducing biological material, in order to make the biological information accessible to (x ) average (s) of analysis (s).
[0079] According to one variant, the lysis step 1742 can be carried out before the concentration step 1741.
[0080] Subsequent steps depend on the nature of the biological information sought and the analytical techniques employed, genetic, protein or metabolic analysis techniques. When the operator wishes to analyze the biological information of interest by means of a genetic analysis technique (nucleic acid (s) analysis), a nucleic acid extraction step 17431 is carried out by implementing any suitable nucleic acid extraction protocol. The nucleic acids obtained at the end of the extraction step 17431 are detected and / or identified and / or quantified by any appropriate genetic analysis method 17432, for example by PCR. Protein and / or metabolic analysis 17441 involves, for its part, adapted analysis techniques, such as immunoassays (immunoassays) or enzymatic tests (non-limiting list), which allow, in step 17450, to detect and / or identify and / or quantify the proteins and / or metabolites of interest initially present in the heterogeneous matrix sample. The examples below will make it possible to better understand the present invention.
[0081] However, these examples are for illustrative purposes only and should in no way be construed as limiting the scope of said invention in any way.
[0082] Examples Example 1 - Assembly of a device according to the invention For the purposes of Example 1, the device for obtaining biological material and / or biological information from a heterogeneous matrix sample is assembled from the following way: 3025315 51 - insertion of the first filter 1161 (Filtrona reference: BNW440148) in the central part of the central element 31 "of the plug 31, insertion of a second filter 1162 (Pall Pad reference: 66025) in superposition of first filter 1161, 5 installation of the spout 41 'by elastic fitting male clipping systems 427 and 428 and females (grooves cut in the inner side wall 329 of the central element 31 "), - clipping of the sampling means calibrated 313, having a calibrated hollow portion 3132 (volume: 300 μl), in the opening 315 of the central element 31 ", addition of 30 glass beads with a diameter of 3 mm in the container 10, addition of 5 mL of application solution suspension (in this case TE buffer suspension buffer) in the container 10, then screwing the stopper comprising the spout 41 'and the calibrated withdrawal means 313 on the neck of the container 10. EXAMPLE 2 - PROCESS for obtaining biological material and / or biological information using the device of Example 1 The method for obtaining biological material and / or biological information from a heterogeneous matrix sample according to the invention The present invention, implementing the device described in Example 1 comprises the following steps: 1) Sampling of the sample More precisely, the user opens, first, the pot containing the stool to be analyzed, and taking a sample using the calibrated sampling means 313, so that the calibrated hollow portion 3132 of the calibrated sampling means 313 is filled with the sample of interest. In order to allow optimal calibration, the calibrated sampling means 313 is then "scraped" on the rim of the aforementioned pot in order to eliminate the possible excess of stool present on the calibrated sampling means 313 and / or overflowing the calibrated hollow portion 3132. 2) Suspending the sample 5 In a second step, the user introduces the calibrated sampling means 313 into the container 10, and firmly screws the stopper 31 before fixing the device according to the invention on a kneading apparatus 71 (Genie II vortex, Scientific Industries, Inc.). More specifically, as shown in FIG. 6, the plug of the device according to the invention is held by means of a clamp 40 connected to a "vortex tray", itself disposed on the mixing apparatus 71 (" vortex "). The device thus fixed is kneaded ("vortexed") to obtain the complete suspension of the sample, namely for a period of time generally between about 30 seconds and about 2 minutes depending on the Bristol type. Of course, the optimal mixing time ("vortexing") of the sample can easily be determined by the user on the basis of his general knowledge and, where appropriate, routine tests. 3) Filtration of the sample suspended in step 2) In a third step, once the suspending step is completed, the user removes the device according to the invention from the "vortex tray", then removes the cap 426 by pulling on the fins 4261 and 4262, returns the device over a collection tube (Eppendorf 2 mL), then exerts pressure with its fingers on the flexible walls 101 of the container 10 to collect the desired volume of filtrate, namely about 2 mL. The filtrate thus obtained is ready for analysis. Example 3 - Effectiveness of the calibration of the sampling means 313 on stool samples of different types Bristol.
[0083] The sample is taken from stool samples from healthy donors stored at -80 ° C. Stool is previously thawed.
[0084] The samples are taken by carrying out step 1 of the method of Example 2. In order to precisely determine the mass of stool taken from the calibrated hollow portion 3132 of the sampling means 313, the stopper is weighed before ( tare) and after the sampling operation. The results obtained are shown in Table 1 below: Bristol Bristol 2 Bristol 3 Bristol 3 Bristol 4 Bristol 5 Bristol 6 Weight of stool by levy 189 201 214 217 217 191 (mg) 199 200 215 271 209 191 143 259 223 211 206 242 216 236 207 187 220 216 258 234 220 236 254 224 209 204 192 244 192 191 198 200 Average 184 201 218 223 213 214 CV (%) 15 0.4 10 12 3 9 10 Table 1 Example 3 calculates the variability sampling (CV), corresponding to all the steps used to perform the sampling.
[0085] The CV is a percentage calculated by the ratio of the standard deviation to the mean, applying the following formula: where Y: is the mean (number 1, number 2 ...) of the sample and n is the size of the sample. The calibrated hollow portion 3132 of the sampling means 313 makes it possible to collect a given / predetermined mass of stools in an easy, efficient and reproducible manner (robustness of the process for obtaining biological material and / or biological information using the device according to the invention) and this for different Bristol types. Minor variations in mass observed in repeatability are considered acceptable in that they do not significantly affect the amount of biological material and / or biological information obtained by the use of the device. Example 4 - Detection linearity test for nucleic acids of different types of microorganisms using the biological information analysis method according to the invention.
[0086] In order to control the effectiveness of the biological information analysis method (in this case DNA) from a heterogeneous matrix sample, object of the present invention, the following three types of microorganisms have were inoculated into the suspension solution contained within the device according to the invention: - carbapenem-resistant Klebsiella pneumoniae (KPC), - methicillin-resistant Staphylococcus aureus (MRSA), - Schizosaccharomyces pombe (S. pombe). These three types of microorganisms were selected based on their size and characteristics, KPC being a Gram-negative bacillary of 5 μm long carbapenem-resistant, MRSA being a methicillin-resistant Gram-positive shell of 1 μm diameter. and S. pombe being a yeast 10 μm long and cylindrical in shape. Inoculation of the suspension solution is prepared in the following manner: 3025315 - MRSA and KPC: MRSA and KPC are grown on TSA (Trypticase Soy Agar) agar for 1 night at 37 ° C. A solution of 7 McFarland corresponding to 2.109 CFU / mL (using a densitometer) is prepared and diluted in a cascade (at 1 / 10th) until the dilution is 103 CFU / mL. 100 g of the dilutions concerned are used to inoculate the suspension solution before adding the stool sample. 100 g of the 103 CFU / mL dilution are then loaded onto COS plates (Reference: 43041, bioMérieux) (Columbia Agar + 5% sheep blood) in triplicate to check the concentration of the inoculum. Colonies are counted after 24 hours of incubation (37 ° C). - S. pombe: S. pombe is cultured for 2 to 3 days on agar SDC (Sabouraud agar glucose, reference: 43555, bioMérieux) at 30 ° C, then 1 night in a Sabouraud broth 15 (30 ° C) (Reference: 42108, bioMérieux), the optical density is measured spectrophotometrically and the broth is diluted in a cascade up to the dilution of 105 CFU / mL. 100 g of the dilutions concerned are used to inoculate the suspension solution before adding the stool sample. Direct counting of the yeasts under a microscope using a Kova cell is then performed from the 106 CFU / mL dilution to verify the inocula concentration. Each suspension solution of each device is respectively inoculated with the following quantities of MRSA and KPC: 0, 105, 107, 108 and 2.109 CFU. For S. pombe, the quantities inoculated are: 0, 105, 106 and 107 CFU.
[0087] Three checks are systematically performed during the tests: Negative Control No. 1: Device according to the invention, comprising a suspension solution (Tampon TE) not inoculated and a stool sample analyzed beforehand to ensure the absence of MRSA, KPC and S. pombe in these stools, Negative Control No. 2: Device according to the invention comprising uninoculated suspension solution (Tampon TE), without stool sample, in order to verify if there has been no contamination during handling, positive control: Device according to the invention comprising a suspension solution (TE buffer) inoculated with all three microorganisms tested at a known concentration, without a stool sample, to determine the impact of the stool on the quantitative PCR detection of microorganisms. In the container 10 were added: 5 mL of TE buffer and 30 glass beads with a diameter of 3 mm. Inoculation of the suspension solution is then carried out by adding 100 g of the previously prepared suspensions. After inoculation, the device is used to perform the sampling, suspension and filtration steps as described in Example 2. The sampling step 1) is performed from a stool sample of healthy donors, stool stored in pots and frozen at -80 ° C. Stools known to naturally contain KPC, MRSA or S. pombe are discarded. The filtrate collected at the end of the filtration step 3) (see Example 2) is centrifuged for 3 minutes at 12000 g in order to concentrate the microorganisms. The supernatant is discarded and the pellet is resuspended in 600 g of TE buffer by vigorous vortexing.
[0088] The 600 g thus obtained are then poured into a lysis tube (Eppendorf 1.5 ml) pre-filled with a mixture of beads consisting of 150 μl of zirconium beads 0.1 mm in diameter and 600 μl of beads. in glass 1 mm in diameter. Subsequently, the Eppendorf tube is placed on a "vortex tray" (25-position horizontal tray), itself placed on a Genie 2 vortex (Scientific Industries, Inc.), for 20 minutes and at maximum power, in order to lyse the microorganisms contained in the Eppendorf tube. The solution thus lysed is recovered by pipetting and the beads are washed with 200 g of TE buffer. The TE buffer used for washing the beads is recovered and added to the solution previously recovered by pipetting.
[0089] The entire volume recovered is then separated into two aliquots, each aliquot being introduced into a tube containing 2 mL of easyMAG® lysis buffer (Reference: 280134, bioMérieux) and 140 g of EasyMAG® silica (Reference: 280133, bioMérieux). Each mixture is then placed in a well of an EasyMAG® shuttle (bioMérieux). The specific protocol "B" is launched, including an "off-board" lysis and an elution in a volume of 50 g. The 2x50 g of eluate coming from the same device are mixed, after extraction, in the same tube.
[0090] The reagents and consumables of easyMAG® are listed below: Part Number: 280134 NucliSENS® easyMAG® Lysis Buffer (4 x 1000 mL / bottle) Part Number: 280130 NucliSENS® easyMAG® Extraction Buffer 1 (4 x 1000 mL / bottle) Reference: 280131 NucliSENS® easyMAG® extraction buffer 2 (4 x 1000 15 mL / bottle) Reference: 280132 NucliSENS® easyMAG® extraction buffer 3 (4 x 1000 mL / bottle) Reference: 280133 Magnetic silica NucliSENS® easyMAG® (48 x 0.6 mL / vial) Reference: 280135 NucliSENS® easyMAG® Consumables 20 Item No .: 280146 Multipette tips After extraction, 5 g of eluates are used to perform a PCR analysis targeting each of the three micro -organisms inoculated. The results are expressed in Cq (quantization cycle), which is directly related to the concentration of DNA present in the amplification tube as a function of the log of the concentration of inoculated microorganisms. The primers and probes used for the molecular analysis are specific for KPC genes (gene encoding carbapenemases) in K pneumoniae, SCCmec genes (meticillin resistance gene) in MRSA and genes SPBPJ4664.02 (glycoprotein). at S. pombe.
[0091] For each of the sequences of interest, the PCR mixes are prepared according to the indications given in Tables 2-4 below: Reagents Final concentration Acros Water Buffer pH 8.6 (X) 1 Solution MgC12 (mM) 3.5 dNTP ( mM) 0.2 ASB (μg / μL) 0.5 Anti-sense primer (i_tM) 0.2 Sense primer (i_tM) 0.2 Probe (i_tM) 0.1 DNA Polymerase (Fast Start) (U / g) 0.08 Table 2 - Mix of PCR reagents for l Assay KPC Reagents Final concentration Water Acros Buffer pH 8.6 (X) 1 Solution MgC12 (mM) 5 dNTP (mM) 0.2 BSA ASB (μg / μL) 0.5 Anti-sense primer (i_IM) 0.65 Sense primer (i_tM) 0.65 Probe (i_tM) 0.17 DNA Polymerase (Fast Start) (U / g) 0.08 Table 3 - Mix of PCR reagents for the analysis of S. pombe Reagents Final concentration Acros Water Buffer pH 8.6 (X) 1 Solution MgC12 ( mM) 5 dNTP (mM) 0.2 ASB (μg / μL) 0.5 Anti-sense primer (i_tM) 0.2 Sense primer (i_tM) 0.2 Probe (i_tM) 0.1 DNA polymerase (Fast Start) (U / g) 0.144 5 Table 4 - Mix of PCR reagents for analysis For each PCR reaction, the PCR reagent mixes are prepared in a final volume of 20 g, to which are added 5 g of the eluates obtained for each sample treated. The mixes are then amplified using a thermocycler, according to the amplification cycles shown in Table 5 below: Activation Denaturation Hybridization / Enzymatic Elongation (Fast Start) Temperature (° C) 95 95 65 ° C Time 5 min 15 sec 45 sec Cycle (s) 1 50 Table 5 The results obtained (shown in Figures 18, 19 and 20) show a linear relationship between the amount of microorganisms previously inoculated in the suspension solutions and the amount of PCR amplified DNA found in the eluate after extraction. Therefore, it demonstrates that the method of biological information analysis according to the invention allows to detect in a "LOG-linear" manner the microorganisms present in the stool samples, and this over the entire range of quantities tested. . In conclusion, in view of the results thus obtained, it appears that the biological information analysis method according to the invention is effective for isolating and identifying the microorganisms present in stool samples (including yeasts), 20 both quantitatively and qualitatively. EXAMPLE 5 Comparison of the Biological Information Analysis Method According to the Invention and the Protocol Using the QIAamp® DNA Stool Kit (QiaGen, Reference: 51504) The preparation of the inocula was carried out according to the methodology set forth in US Pat. Example 4, from a 0.5 McF solution corresponding to 108 CFU / mL. Inoculation of the suspension solution is carried out with 108 CFU of MRSA, S. pombe and KPC. Inocula control is performed as in Example 4. The same goes for negative controls # 1 and # 2 and for the positive control.
[0092] The obtaining and the quantification of the biological information is carried out by implementing the method of analysis of biological information (in this case of DNA) according to the invention, described in Example 4.
[0093] Obtaining biological information with the QIAamp® DNA stool kit is performed following the experimental protocol provided by the supplier (QiaGen). Nevertheless, considering the present metagenomic application, a mechanical lysis step was added after addition of the ASL buffer and before incubation at 95 ° C. for 10 minutes, in order to obtain a better yield of lysis of the microorganisms. This is a general knowledge of the skilled person. The biological information obtained by using the QIAamp® DNA stool kit is quantified under the same conditions as those making it possible to quantify the biological information obtained by implementing the method according to the present invention. The results obtained for each of the two protocols are presented in Table 6 below. Protocol Amount of DNA Result qPCR (Cq) extract (lig) MRSA KPC S. pombe Invention 2.5-3 26.6 28.1 35.2 QIAamp® DNA 7-10 28.4 29.2 Not detected (> 40) stool Table 6 - Comparison of the results obtained by PCR quantitatively from the DNA extracted by the method according to the invention and by the method according to the kit QIAamp® DNA stool (QiaGen). This table 6 shows the amount of extracted DNA for each of the two protocols, as well as the value of the Cq obtained by the PCR analysis (Cq corresponds to the Ct or threshold cycle, the cycle at which the emitted fluorescence value reaches the predefined threshold) .
[0094] The results thus obtained clearly demonstrate that the molecular analysis of the biological information (DNA) by PCR, carried out from the eluates obtained by the implementation of the method according to the invention, is effective and has improved sensitivity. compared to the process using the QIAamp® DNA stool kit. In addition, it is important to note that only the method according to the invention allows the detection of yeasts (here S. pombe yeasts). In conclusion, the method of analysis of biological information according to the invention, using the device according to the invention, allows efficient detection of the biological information contained in the microorganisms present in a stool sample ( including yeasts), while simplifying sampling steps 1), suspending 2) and filtering 3) samples, as indicated in Example 2.
[0095] EXAMPLE 6 Comparison of the biological information analysis method according to the invention and of the protocol implementing the QIAamp® DNA stool kit for an application of sequencing of the microbiota of the human organism. The QIAamp® DNA stool kit (QiaGen; Reference: 51504) is implemented according to the experimental protocol provided by the supplier (QiaGen) and modified as mentioned in Example 5 supra. The devices of the present invention are assembled according to the indications given in Example 1. The filtrate is collected according to Example 2 and the DNA is extracted according to the extraction method according to the invention. The filtrate treatment protocol is identical to that mentioned in Example 3 for genetic material. However, the eluate thus obtained is then sequenced by a new generation PGM sequencer (IonTorrent) with a chip 318 (Reference: see Table 7 below). Reagents / kits Storage Supplier Ref. Lot 3025315 62 Ion PGM Sequencing RT Life Technologies 4482003 057A03-13 Supplies 400 Ion PGM Sequencing -20 ° C Life Technologies 4482004 057A03-13 Reagent 400 Ion PGM Sequencing + 4 ° C Life Technologies 4482005 057A03-13 Solutions 400 Ion 318 Chip v2 kit TABLE 7 Reagents / Kits Used for the Sequencing of the Microbiota In order to test the reproducibility of the protocol embodying the present invention, three repetitions per experimental condition were performed to allow the calculation of the variability of the microbiota. sampling (CV), corresponding to all the steps implemented by the protocol. CV calculation was performed on the 10 phyla predominantly present in the stools. The results, presented in Table 8 below, show that the CVs are equivalent between the two protocols (10-14%), with however a strong variation in value between phylum for the same protocol (6-23% for the process). according to the invention and 2-35% for QiaGen). Consequently, this experiment shows that the DNA analysis method according to the invention makes it possible to obtain equivalent results in terms of reproducibility, while being simpler to achieve than the method according to the QIAamp® DNA stool kit. . CV of the 10 phyla predominantly present in the stool QiaGen Method according to the invention No. Taxon Rank Taxon CV (%) CV (%) 1239 phylum Firmicutes 2% 1% 976 phylum Bacteroidetes 15% 6% 201174 phylum Actinobacteria 2% 21% 1224 Phylum Proteobacteria 12% 13% 508458 Phylum Synergistetes 6% 15% 544448 Phylum Tenericutes 14% 23% 203691 Phylum Spirochaetes 2% 17% 3025315 63 1117 Phylum Cyanobacteria 35% 5% 1090 Phylum Chlorobi 17% 23% 3201, 3202 66 Phylum Fusobacteria 22 % 17% Mean 13% 14% CV (CV variation) 82% 55% Table 8 Example 7 - Comparison in terms of quantification of adenovirus DNA by PCR using the Adenovirus R-gene® kit (Reference: 69 -010B) after DNA extraction with a) the QIAamp® DNA stool extraction kit and with b) the biological information extraction method according to the invention.
[0096] In order to control the concentration of adenovirus contained in the tested samples, stools containing a known concentration of adenovirus ("positive stool") are diluted in a stool mixture not containing adenovirus. Dilution of positive stool is performed in cascade at the following concentrations: 104, 105, 106 and 108 copies of virus per gram of stool. In the case of stools containing adenoviruses, Bristol types are generally between 4 and 7. Controls are routinely performed: - 2 Negative controls: Noninoculated stools detected negative and uninoculated EDTA phosphate buffer. For the extraction of adenovirus DNA according to the QIAamp® DNA stool kit (Ref 51504, QiaGen), the experimental protocol of the supplier is followed. The process of extracting biological information (adenovirus DNA) is described below. The steps of 1) stool collection, 2) suspension and 3) filtration are performed according to the teaching of Example 2, using as a suspension solution a phosphate solution. (0.2M), EDTA (50mM), pH8. The filtrate thus obtained is vortexed a few seconds for homogenization purposes, then 400 g of homogenized filtrate are then poured into the well of an easyMAG® shuttle (bioMérieux) and the "Dispense Lysis" program is launched in order to distribute 2 mL of lysis buffer (bioMérieux, reference: 280134). Once the "Dispense Lysis" program is completed, 10 g of IC2 (internal control of the Adenovirus R-gene® kit), then 740 g of a mixture, containing 600 g of lysis buffer (bioMérieux, reference: 280134) and 140 g of silica are added to each easyMAG® shuttle well. Once the mixture has been completed, the easyMAG® program specific B, "off-board" lysis, elution in 50 III is launched. The eluate thus recovered is transferred to a new tube within 30 minutes, following the end of the elution.
[0097] Quantitative PCR analysis is carried out in order to quantify the adenovirus DNA extracted according to the method according to the invention on the one hand, and the method according to the QIAamp® DNA stool method on the other hand, depending on the quantity theoretically inoculated into the suspending buffer. Thus 10 IU of eluates were removed for quantitative PCR analysis using the Adenovirus R-gene kit. The results are expressed in log of the concentration of inoculated microorganisms. As shown by the results presented in FIG. 21, there is a linear relationship between the number of viruses previously inoculated in the suspending buffer and the amount of DNA detected in quantitative PCR from the eluate obtained in implementing the biological information extraction method according to the invention. Thus, the process for extracting biological information according to the invention (viral DNA) makes it possible to detect and quantify the adenoviruses initially present in a stool sample, and to obtain a result equivalent to the extraction method implementing The kit QIAamp® DNA stool, and this on a wide range of linearity (from 104 to 108 copies of virus per gram of saddle).
[0098] Example 8 - Test showing the amount of DNA extracted from different Bristol-type stool samples, using the biological information extraction method according to the invention.
[0099] In order to evaluate cross-stool reproducibility in the amount of DNA extracted from stool samples of different Bristol types, several nucleic acid extractions were performed on six different stool samples, the type of which was Bristol is between 1 and 6, using the process of extracting biological information (in this case DNA) according to the invention.
[0100] The aforesaid method of extracting biological information according to the invention is identical to the method described in Example 4 up to the step involving easyMAG®. The eluates obtained are analyzed by Nanodrop (ThermoScientific), in order to quantify and control the purity (ratio 260/280 and ratio 260/230) of the extracted DNA. As illustrated in FIG. 22, a significant variation in the amount of DNA extracted is observed as a function of the Bristol type of the stool sample. In fact, for Bristol-type stools 1 to 3, about 500 ng / lIL of nucleic acids (5 lig of DNA) are extracted, whereas for Bristol-type stools 4 to 6, the concentration of nucleic acids is approaching 300 ng / ml nucleic acid (3 μg DNA). The mass yield of DNA extracted is, therefore, about half as important for Bristol-type stools 4 to 6. However, the amount extracted remains sufficient to allow analysis by PCR or sequencing.
[0101] This variation in terms of amount of DNA extracted is not due to the nature of the extraction process used but to the Bristol stool type. Indeed, a DNA extraction carried out using the Macherey Nagel kit (NucleoSpin Blood L), adapted for DNA extractions from liquid samples (for example from blood) is performed on the stool Bristol type 5 and 6, and gives similar results (results not shown). This phenomenon is explained by the fact that Bristol 5 and 6 type stools are more liquid - therefore more diluted by definition - which results in a decrease in the concentration of microorganisms and therefore de facto their DNAs. . In conclusion, the method of extracting biological information (in this case DNA) according to the invention, using the device according to the invention, makes it possible to extract a sufficient amount of microorganism DNA. organisms in Bristol-type stool 1 to 6 to allow subsequent analysis by PCR or sequencing, despite variations observed in Figure 22.
[0102] Note that the method of extracting biological information (in this case DNA) according to the invention, implementing the device according to the invention, also works for Bristol 7 type saddles; the lack of results for this Bristol type is simply due to the non-availability of Bristol 7 type of stool at the time this example was made.
[0103] Example 9 - Test demonstrating the effectiveness of the device according to the invention for taking and analyzing bovine stool samples.
[0104] Sampling and analysis of a sample of bovine faeces is carried out using the device whose assembly is described in Example 1, except that: the calibrated sampling means 613 (comprising three hollow portions) calibrated 6132, 6134 and 6135) of the device shown in FIG. 16 is used to collect 3000 mg of bovine stool, and the volume of the TE buffer is increased to 10 ml. The device according to the invention was used as indicated in Example 2 until the filtration step included.
[0105] The device according to the invention thus made it possible to obtain 2 ml of a bovine stool filtrate, and this without clogging (bulkiness) of the filters of said device 3025315 67 In conclusion, with some adjustments, the device according to the invention is quite suitable for veterinary application (s), for example in order to obtain all or part of the microorganisms present in a stool sample of animal origin, for example bovine stool.
[0106] EXAMPLE 10 Comparison of Implementation Time and Convenience of Use Between the Biological Information Extraction Process of the Invention and the QIAamp® DNA Stool Kit (QiaGen)
[0107] In order to compare the time of implementation and the usability of use between the biological information extraction method (in this case of DNA) according to the invention and the protocol implementing the QIAamp® DNA kit stool, different DNA extractions were performed and the completion time of each step was reported in Tables 9, 10 and 11 below, depending on the type of process used.
[0108] The process using the QIAamp® DNA stool kit was used to process 5 stool samples. A total of 25 manual steps (including 15 sample preparation) and a total duration of 2h20 (of which 1h57 for sample preparation) were required. The various steps of the process using the QIAamp® DNA stool kit and their completion times are presented in Table 9 below: For 5 devices Item No. Step of the "QIAamp® DNA Stool" Method Time (hr: min) Duration cumulative (hr: min) 1 Reactive and consumable preparation 00:09 2 Sampling 200-250 mg 00:10 00:10 3 Preparation of lysozyme 00:10 00:20 4 Addition of lysozyme 00:06 00:26 5 Incubation 37 ° C 00:30 00:56 6 Adding ASL + vortex 15s / sample 00:05 01:01 7 Adding balls + vibroBrushing 00:08 01:09 8 Incubation 95 ° C + Preparation tubes 2mL 00:10 01:19 9 Vortex 15s / sample. + Centrifugation + Transfer supernatant 00:06 01:25 10 Add Inhibitex + Vortex + Incubation TA 00:04 01:29 3025315 68 11 Centrifugation 00:06 01:35 12 Transfer supernatant + Centrifugation 00:05 01:40 13 Preparation tubes + PK 00:01 01:41 14 Transfer supernatant + Add AL + Homogenization 00:04 01:45 15 Incubation at 70 ° C 00:10 01:55 16 Add Et0H + vortex 00:02 01:57 17 Deposit on column 00 : 02 01:59 18 Centrifugation 00:02 02:01 19 Addition AW1 + Centrifugation 00:02 02:03 20 Addition AW2 + Centrifugation 00:04 02:07 21 Addition AW2 + Centrifugation 00:05 02:12 22 Add AE 00 : 01 02:13 23 Incubation 00:05 02:18 24 Centrifugation 00:01 02:19 25 Conservation of the Eluat 00:01 02:20 Table 9 In addition, the process of extracting biological information (in viral DNA species and bacterial DNA) according to the invention has also been used to also treat 5 stool samples. The method of extracting viral DNA according to the invention - for 5 samples comprises 8 steps (including 3 sample preparation) and is carried out in 1:30 to 30 minutes (including 30 minutes of sample preparation). The various steps of the viral DNA extraction process and their completion time are reported in Table 10 below: For 5 devices Item No. Step of the "Viral DNA" method Time (h: min) Cumulative duration (h: min ) 1 Preparation of reagents and consumables 00:14 NA 2 Start of easyMAG® 00:13 NA 3 Sampling of stools 200-250 mg 00:04 00:04 4 Suspended 00:05 00:09 5 Filtration 00:04 00:15 6 easyMAG® shuttle planning 00:10 00:25 7 easyMAG® protocol start 00:05 00:30 3025315 69 1 8 Extraction in easyMAG® 01:00 01:30 Table 10 The extraction process of Bacterial DNA - for 5 samples - comprises 12 steps (7 of which are sample preparation) and is performed in 2:13 (including 58 minutes of sample preparation). The various steps of the bacterial DNA analysis method and their time of completion are reported in Table 11 below: For 5 devices Item No. Step of the "Bacterial DNA" method Time (h: min) Cumulative duration (h: min ) 1 Preparation of reagents and consumables 00:14 NA 2 Start of easyMAG® 00:13 NA 3 Sampling of stools 200-250 mg 00:04 00:04 4 Suspended 00:05 00:09 5 Filtration 00:04 00:15 6 Centrifugation 00:06 00:21 7 Resume and resuspension of the butt 00:12 00:33 8 Mechanical lysis 00:21 00:54 9 Transfer supernatant 00:04 00:58 10 easyMAG® shuttle preparation 00:10 01 : 08 11 EasyMAG® protocol start 00:05 01:13 12 Extraction in easyMAG® 01:00 02:13 10 Table 11 In the light of the data presented in Tables 9, 10 and 11 above, it appears that the processes extraction of bacterial DNA and viral DNA according to the invention, using the device of the invention, allow to limit the number of steps and re significantly reduce the sample preparation time. In contrast, the method employing the QIAamp® DNA stool kit requires a high number of manual and technically delicate steps, requiring the presence and continued attention of a qualified laboratory technician.
[0109] In conclusion, the device according to the invention makes it possible to simplify the experimental protocols ("viral DNA" and "bacterial DNA") with respect to the protocols of the prior art (fflamp® DNA stool, QiaGen (Ref 51504)), by grouping together in the same device the elements necessary to carry out the sampling, suspension and filtration steps. This simplification significantly reduces the number and complexity of handling, and reduces the risk of errors and cross contamination, while providing increased working comfort for the normally qualified laboratory technician.
[0110] Bibliography [1] Srijan A, Bodhidatta L, Mason C, Bunyarakyothin G, Jiarakul W, Vithayasai N. Field Evaluation of a Transport Medium and Enrichment Broth for Isolation of Campylobacter Species from Human Diarrheal Stool Samples. J Med Microbiol, 2013; 3, 48-52. [2] Wasfy M, Oyofo B, Elgindy A, Churilla J Clin Microbiol 1995; 33: 2176.

权利要求:
Claims (21)
[0001]
REVENDICATIONS1. Device for obtaining biological material and / or biological information from a heterogeneous matrix sample, for example a human or animal stool sample, said device comprising: a container adapted to receive a content comprising said sample and at least one suspension solution, intended to allow the suspension of said sample, - a stopper for closing said container, preferably hermetically, said stopper comprising: - at least one calibrated sampling means, for sampling a predetermined volume of said sample corresponding to a given mass, said sampling means comprising at least one calibrated hollow portion connected to the inner part of the plug and extending from said inner part of the plug towards the inside of the container when said plug and the said container are assembled, - at least one opening, preferably closable by detachable sealing means, allowing the communication of fluid between the inside of said container and the outside of said device when said cap and said container are assembled, at least one filtering means, positioned relative to said opening so as to filtering the content during the passage of said content from the inside of said container to the outside of said device via said opening, said filtering means being adapted to allow the selective passage of said biological material and / or biological information to the outside of said device 3025315 73
[0002]
2. Device according to claim 1, wherein said container further comprises at least one mechanical suspension means, preferably of spherical shape such as a ball, adapted to facilitate the suspension of said sample in said setting solution. in suspension. 5
[0003]
3. Device according to claim 2, said device comprising a number of mechanical suspending means, such as balls, selected between 1 and 200, preferably between 5 and 50, advantageously between 10 and 40, particularly preferably between 25 and 35; said mechanical suspending means having a size of between 2 mm and 10 mm, preferably between 2.5 mm and 3.5 mm, advantageously about 3 mm; preferably, said mechanical suspension means being made of glass, iron, plastic (s), ceramic (s), particularly preferably glass. 15
[0004]
4. Device according to one of the preceding claims, wherein said calibrated sampling means has a sufficient rigidity to prevent said calibrated sample means bends during sampling. 20
[0005]
5. Device according to one of the preceding claims, wherein said container comprises said suspension solution, for example a buffer solution, in a volume sufficient to allow the suspension of the sample. 25
[0006]
6. Device according to one of the preceding claims, wherein said filtering means is selected from: - a gradient filter, and - a superposition of at least two filters, preferably two or three filters, whose size of pores decreases from the inside to the outside of the device 30
[0007]
7. Device according to one of the preceding claims, wherein said container comprises at least one wall comprising at least one flexible material area adapted to undergo compression and generate in response an overpressure inside said container in order to allow or facilitate filtration of the content through said filtering means. 5
[0008]
8. Device according to one of the preceding claims, wherein said calibrated hollow portion comprises at least one opening to facilitate the suspension of said sample in said suspension solution.
[0009]
9. Device according to one of the preceding claims, wherein said plug 10 comprises at least one rigid zone, said rigid zone having a shape adapted to cooperate with at least one holding member, such as a clamp, connected to a device mixing apparatus, such as a vortex, so as to allow said device to be held on the mixing apparatus during mixing of said device in order to facilitate the suspension of said sample in said suspension solution.
[0010]
10. Device according to claim 9, said rigid zone having a shape adapted to cooperate with a clamp connected to said mixing apparatus, said rigid zone comprising: at least one anti-rotation means, comprising two distinct bearing surfaces, for example example constituted by two shoulders or two tongues, each of the two separate bearing surfaces being adapted to be or abut against one of the two ends of the clamp when the cap undergoes a rotational movement, to prevent or stop said rotation, and / or at least one anti-translational means, comprising at least one bearing surface, such as a flange, a flange or a shoulder, said at least one bearing surface being adapted to be or come in abutment against said clamp when the plug undergoes a translation movement, in order to prevent or stop said translational movement. 30
[0011]
11. Stopper as defined in one of the preceding claims. 3025315 75
[0012]
12. Use of the device according to one of claims 1 to 10 for obtaining biological material and / or biological information from a heterogeneous matrix sample, preferably for obtaining biological material, preferably for obtaining microscopic biological material, advantageously for obtaining microbiological material.
[0013]
13. Use according to the preceding claim, wherein said heterogeneous matrix sample is selected from a soil sample, a stool sample, a forensic sample such as a sample of necrotic tissue, a food sample, an industrial sample, preferably said sample being a sample of human or animal stool.
[0014]
14. A method for obtaining biological material and / or biological information from a heterogeneous matrix sample, said method implementing the device according to one of claims 1 to 10, said method comprising the following steps a) withdrawing a predetermined sample volume corresponding to a given mass using said calibrated sample means, b) suspending said sample in said suspension solution, optionally by mechanical suspension, c) filtering the suspension obtained in step b) through said filtration means to obtain a filtrate containing said biological material and / or said biological information. 25
[0015]
A method of extracting biological information from a heterogeneous matrix sample, said method comprising the steps of: a) performing the method of claim 14 to obtain a filtrate containing said biological material and / or said biological information, b) when the biological information to be extracted is contained in said biological material, such as a cell, bacteria, fungus or yeast, performing a lysis step of said biological material, preferably a mechanical lysis step, in order to obtain a lysate comprising the biological information to be extracted, c) extracting the biological information from the filtrate obtained in step a) or the lysate obtained in step b ) by implementing a suitable biological information extraction method.
[0016]
16. Process for extracting biological information according to the preceding claim, said process comprising, before step c), and before step b) when this is to be performed, a step of concentration of said biological material and or of said biological information, preferably by centrifugation or by flocculation.
[0017]
17. A method for analyzing biological information, said method comprising the following steps: a) extracting the biological information to be analyzed by implementing the method according to claim 15 or 16, b) identifying and / or quantifying said information biological method by any suitable method of analysis, for example a genetic analysis method such as a PCR, an immunoassay method or an enzymatic assay.
[0018]
18. A method of analyzing biological material from a heterogeneous matrix sample, said method comprising the steps of: a) obtaining a filtrate containing the biological material to be analyzed using the method of claim 14, b) the where appropriate, inoculate a reaction medium with the filtrate obtained in step a), said reaction medium being adapted to allow the growth and / or the expression of at least one metabolism of said biological material, c) if necessary incubating the filtrate obtained in step a), or the inoculated reaction medium obtained in step b), for a suitable period of time and at an appropriate temperature, d) analyzing the biological material at the end of the step a), b) or c) by any means of appropriate biological analysis.
[0019]
19. An analysis method according to claim 18, said method comprising step b) and optionally step c), advantageously steps b) and c), wherein: step c), when is present, incubates the inoculated reaction medium obtained in step b) for a suitable time and temperature, and - the biological material analysis step d) consists of the detection and / or the identifying and / or counting said biological material on / in said reaction medium, preferably by visual or optical reading. 15
[0020]
20. Kit for obtaining biological material and / or biological information from a heterogeneous matrix sample, said kit comprising: the container as defined in one of claims 1 to 10 and the stopper according to claim 11, in assembled or disassembled form, at least one suspending solution, adapted to allow the suspension of said sample, preferably at least one mechanical suspending means, such as at least one ball. 25
[0021]
21. Use of the kit according to the preceding claim for carrying out the method according to claim 14.

类似技术:

---

公开号 | 公开日 | 专利标题

---

EP3185782B1|2021-06-02|Device for obtaining biological material and/or biological information from a sample with a heterogeneous matrix

---

EP2152850B1|2020-04-01|Device for the lysis of microorganisms present in an environmental or clinical sample and the extraction of nucleic acids from said microorganisms for analysis

---

EP3434759A1|2019-01-30|Method and kit isolating microorganisms from culture

---

US20150307869A1|2015-10-29|Device for capture and lysis of microorganisms from liquids and methods of use thereof

---

WO2010067019A2|2010-06-17|Automated system for the lysis of microorganisms in a sample, and for the extraction and purification of the nucleic acids of said microorganisms for analysis

---

WO2014114896A1|2014-07-31|Method for the specific isolation of nucleic acids of interest

---

EP3209793A1|2017-08-30|Method and devices for treating biological samples

---

US9932620B2|2018-04-03|Methods, devices, and systems of detecting microorganisms

---

EP0815258A1|1998-01-07|Method for detecting microorganisms by separation and culture on a gelled system, gelled system and assay kit therefor, and use thereof in microbiology

---

US20130309700A1|2013-11-21|Methods, devices, and systems of detecting microorganisms

---

EP2802875A1|2014-11-19|Method for directly detecting and identifying a microorganism in a biological sample via optical means

---

Schober et al.2018|Isolation of plastid fractions from the diatoms Thalassiosira pseudonana and Phaeodactylum tricornutum

---

US9790462B2|2017-10-17|Device and method for treating a filtration medium

---

EP2872867B1|2020-04-01|Automated system for the lysis of microorganisms present in a sample, for extraction and for purification of the nucleic acids of said microorganisms for purposes of analysis

---

CN108603221A|2018-09-28|Comprehensive sample processing system

---

FR3057276A1|2018-04-13|METHOD AND DEVICE FOR CAPTURING AND / OR CONCENTRATING MICROORGANISM.

---

FR3057275A1|2018-04-13|METHOD AND DEVICE FOR PREPARING SAMPLES

---

US20150140583A1|2015-05-21|Methods, devices, and systems of detecting microorganisms

---

Møller et al.2022|Isolation of Highly Purified, Intact, and Functional Mitochondria from Potato Tubers Using a Two-in-One Percoll Density Gradient

---

EP3523624A1|2019-08-14|Method and device for preparing samples

---

FR3086952A1|2020-04-10|METHOD FOR COLLECTING MICROORGANISMS FROM A LIQUID OR VISCOUS BIOLOGICAL SAMPLE OR A SUSPENSION OF HIGHLY CONTAMINATED MICROORGANISMS

---

EP2255197A2|2010-12-01|METHOD FOR IN VITRO DETECTION AND/OR QUANTIFICATION AND/OR IDENTIFICATION OF BACTERIA IN A BIOLOGICAL MATERIAL& xA;

---

WO2016187607A1|2016-11-24|Method for dedifferentiating and culturing microbial species

---

FR2674632A1|1992-10-02|Process and device for the rapid detection of undesirable microorganisms in a product of the agrifood or medicoveterinary sector

同族专利:

---

公开号 | 公开日

---

EP3185782B1|2021-06-02|

---

WO2016030642A1|2016-03-03|

---

CN107072642A|2017-08-18|

---

FR3025315B1|2018-08-24|

---

JP2017526355A|2017-09-14|

---

CN107072642B|2021-06-15|

---

JP6817932B2|2021-01-20|

---

EP3185782A1|2017-07-05|

---

US10856854B2|2020-12-08|

---

US20170273670A1|2017-09-28|

引用文献:

---

公开号 | 申请日 | 公开日 | 申请人 | 专利标题

---

WO2003014705A1|2001-08-06|2003-02-20|Immunoassay Indústria E Comércio Ltda|Multiple collection kit for stool laboratory examination|

---

EP1656887A1|2004-11-15|2006-05-17|JAPAN as represented by PRESIDENT OF NATIONAL CANCER CENTER|Container for suspension and filtration of stool|

---

US20060115385A1|2004-12-01|2006-06-01|Meridian Bioscience, Inc.|Specimen collection system|

---

US20110048981A1|2009-08-27|2011-03-03|Arkray, Inc.|Sample collection implement|

---

US4018356A|1976-05-28|1977-04-19|Szasz I Emery|Floating deck for liquid storage tank|

---

US4081356A|1976-09-24|1978-03-28|The United States Of America As Represented By The Department Of Health, Education And Welfare|Fecalator, an apparatus and method for concentration of parasite eggs and larvae|

---

JPS6173054A|1984-09-17|1986-04-15|Medical Technol Corp|Small bottle for adjusting sample|

---

IT206959Z2|1986-04-16|1987-10-26|Sta Te Srl|DISPOSABLE DEVICE FOR THE COLLECTION OF PHYSIOLOGICAL SAMPLES, IN PARTICULAR BODY SAMPLES.|

---

US4735905A|1986-08-15|1988-04-05|V-Tech, Inc.|Specimen-gathering apparatus and method|

---

US4895453A|1988-08-24|1990-01-23|E. I. Du Pont De Nemours And Company|Vortex mixer drive|

---

JPH08271511A|1995-03-28|1996-10-18|Dainippon Printing Co Ltd|Stool smapling stick|

---

US5543115A|1995-07-17|1996-08-06|Mizuho Usa, Inc.|Specimen handling device|

---

US5730147A|1996-01-11|1998-03-24|Craig; Robert J.|Combined thermometer and fecal sampling apparatus|

---

US6090572A|1998-06-26|2000-07-18|Biostar, Incorporated|Filtration and extraction device and method of using the same|

---

CN100538357C|2003-11-14|2009-09-09|香港澳维有限公司|Sample collection cup with sample analysis system of integrated activatable|

---

WO2005072620A1|2004-01-16|2005-08-11|Andx, Inc.|Sample collection device and method|

---

AT500459B1|2004-01-23|2010-08-15|Greiner Bio One Gmbh|METHOD FOR ASSEMBLING A CAP WITH A RECEIVING CONTAINER|

---

US20060147348A1|2004-11-13|2006-07-06|Greg Liang|Sampling device and methods|

---

JP4339906B2|2006-10-19|2009-10-07|デンカ生研株式会社|Simple membrane assay method and kit using sample filtration filter|

---

JP2013217707A|2012-04-05|2013-10-24|Bl:Kk|Specimen sampling tool and specimen sampling kit|

---

US20140161688A1|2012-11-30|2014-06-12|Rarecyte, Inc.|Apparatus, system, and method for collecting a target material|US10054522B1|2015-05-15|2018-08-21|Colleen Kraft|Systems, devices, and methods for specimen preparation|

---

RU2663629C1|2017-09-28|2018-08-07|Федеральное государственное бюджетное образовательное учреждение высшего образования Санкт-Петербургская государственная академия ветеринарной медицины ВПО СПбГ АВМ)|Cap for collecting larvae and small nematodes from human and animal feces|

---

CN108784749A|2018-06-19|2018-11-13|耿全好|A kind of stool and urine collector of clinical laboratory's health|

---

RU191895U1|2019-07-03|2019-08-26|Федеральное государственное бюджетное образовательное учреждение высшего образования Санкт-Петербургская государственная академия ветеринарной медицины ФГБОУ ВО СПбГАВМ|Device for collecting larvae and small nematodes from feces of animals and humans|

法律状态:
2015-08-17| PLFP| Fee payment|Year of fee payment: 2 |

---

2016-03-04| PLSC| Search report ready|Effective date: 20160304 |

---

2016-08-25| PLFP| Fee payment|Year of fee payment: 3 |

---

2017-08-25| PLFP| Fee payment|Year of fee payment: 4 |

---

2018-08-27| PLFP| Fee payment|Year of fee payment: 5 |

---

2019-08-26| PLFP| Fee payment|Year of fee payment: 6 |

---

2020-08-25| PLFP| Fee payment|Year of fee payment: 7 |

---

2021-08-25| PLFP| Fee payment|Year of fee payment: 8 |

优先权:

---

申请号 | 申请日 | 专利标题

---

FR1458143A|FR3025315B1|2014-08-29|2014-08-29|DEVICE FOR OBTAINING BIOLOGICAL MATERIAL AND / OR BIOLOGICAL INFORMATION FROM A HETEROGENEOUS MATRIX SAMPLE|

---

FR1458143|2014-08-29|FR1458143A| FR3025315B1|2014-08-29|2014-08-29|DEVICE FOR OBTAINING BIOLOGICAL MATERIAL AND / OR BIOLOGICAL INFORMATION FROM A HETEROGENEOUS MATRIX SAMPLE|

---

JP2017507798A| JP6817932B2|2014-08-29|2015-08-28|A device for acquiring biomaterials and / or bioinformation from a sample containing a heterogeneous matrix.|

---

CN201580046677.1A| CN107072642B|2014-08-29|2015-08-28|Device for obtaining biological material and/or biological information from a sample having a heterogeneous substrate|

---

US15/507,187| US10856854B2|2014-08-29|2015-08-28|Device for obtaining biological material and/or biological information from a sample with a heterogeneous matrix|

---

EP15771673.9A| EP3185782B1|2014-08-29|2015-08-28|Device for obtaining biological material and/or biological information from a sample with a heterogeneous matrix|

---

PCT/FR2015/052292| WO2016030642A1|2014-08-29|2015-08-28|Device for obtaining biological material and/or biological information from a sample with a heterogeneous matrix|