EP2916939A1

EP2916939A1 - Method for treating at least one biological sample

Info

Publication number: EP2916939A1
Application number: EP13786519.2A
Authority: EP
Inventors: Florent BARIL; Bruno Colin; Jean-Pierre Flandrois; Thomas JUNILLON; Benoît MALLEN; Edgard MINASSIAN; David Mosticone; Antoine Vimont
Original assignee: Biomerieux SA
Current assignee: Biomerieux SA
Priority date: 2012-11-07
Filing date: 2013-11-07
Publication date: 2015-09-16
Also published as: US9784654B2; US20170370812A1; FR2997703A1; US20150292994A1; WO2014072438A1; US10352834B2; FR2997703B1

Abstract

A method for treating at least one biological sample likely to contain at least one target microorganism, corresponding analysis and enrichment methods, device for implementing said methods and use of said device.

Description

Process for treating at least one biological sample

Technical area

The present invention relates generally to the processing of biological samples (for enrichment and / or analysis), as well as the analysis of these biological samples, for example in the field of microbiology and more particularly that of industrial microbiology.

State of the art Raw products used and processed products marketed by the food industry (meat products, dairy products, seafood, plants, etc.), as well as pharmaceutical and cosmetic products and water intended for the beverage, are subjected to numerous microbiological tests to ensure their safety (absence of pathogenic bacteria or degradation, absence of bacteria markers of contamination dangerous to health, absence of toxins).

The microbiological analysis of these products requires precise techniques whose time of obtaining the result must be as short as possible.

In the medical field, it is necessary to predict and diagnose the risk of infection: the faster and more accurate the diagnosis, the more effective the management of the patients and the reduced risk of transmission. The approach is similar for animal health in the veterinary field.

In the food sector, the problem is the same. It distinguishes however: pathogenic microorganisms (such as Salmonella or E. Coli 0157: H7 strain) and their toxins, the research of which applies to raw materials, intermediate products, finished products marketed,

- non-pathogenic microorganisms, used as indicators-quality of the production process, from raw materials to finished products, all along the chain,

- the bacteria of technological interest such as ferments,

- the micro-organisms markers of contamination. The rapid and precise detection of presumed contaminations (within the food lots) makes it possible to control them and to initiate corrective actions in the short term.

Technically, the microbiological analysis generally involves one or more pre-enrichment and / or enrichment phases, one or more detection phases, one or more enumeration phases of the microorganisms. For particular applications, such as food microbiological control, a confirmation phase may also be required to meet the standards in force in this area.

The detection phase is historically based on the growth (culture) of microorganisms on essentially agaric media, and by the demonstration of the metabolic characteristics of the microorganisms sought. Typical enzyme substrates are conventionally used. These substrates can be compounds used in bacterial metabolism and leading to a modification of the medium detected by indicators (pH variation, reduction, precipitation, etc.). In other cases these enzymatic substrates are composed of two parts, a first specific part of the enzymatic activity to be revealed, also called target part, and a second part serving as a marker, called the marker part, generally constituted by a chromophore or a fluorophore. By the choice of these substrates, depending on whether or not there is a reaction, it is possible to characterize the nature of a microorganism or to discriminate between different groups of microorganisms. organizations. Thus, the appearance or disappearance of a coloration or a fluorescence will be the signature of a kind or a type of microorganisms. In this respect, the use of chromogenic media enables the simultaneous detection and identification of the desired microorganisms. It simplifies the process and significantly reduces the time to get the result. As a concrete example, the ChromlD ^® media of the applicant. These chromogenic media are based on the detection of specific metabolic characteristics of the desired seeds, for example the enzymatic activity beta-glucuronidase for Escherichia coli. Immunoassays are another technology used for the detection test. They use the immunogenic characteristics of the microorganisms sought. Non-exhaustive, we can mention ELISA techniques ("Enzyme Linked Immuno Sorbent Assay"), by competition or sandwich type. Finally, molecular biology techniques, based on the genomic characteristics of the microorganisms sought, are also used to detect and identify target microorganisms. These molecular biology techniques offer extremely interesting perspectives. By way of example, mention may be made of conventional amplification techniques such as PCR ("Polymerase Chain Reaction" in English) and NASBA ("Nucleic Acid Sequence Based Amplification" in English), which can be coupled with real-time detection known to those skilled in the art.

The confirmation phase, in turn, is more particularly attached to microbiological analysis in the agri-food sector. Indeed, when the result of the previously developed methods is positive, it is necessary to confirm the presence of the pathogen sought. This imposes a complementary test and the use of a detection principle different from that used during the first analysis. The techniques described above are used at leisure for confirmation.

The complete and precise identification of a microorganism in a sample therefore requires the sequence of several stages: enrichment, detection and, the where appropriate, confirmation. The standardization of routinely used tests has enabled the automation of detection methods, which however remain long to implement. A disadvantage of the traditional methods of analysis used in the state of the art lies in the fact that the stages of enrichment, detection and, where appropriate, confirmation are carried out sequentially and require a large number of time-consuming manipulations. , thus impacting the time required to render the results.

As indicated above, the detection phase is generally preceded by at least one pre-enrichment and / or enrichment phase (more generally referred to as phase or enrichment step for the purposes of the present application). The latter is essential insofar as, at present, there is no method for detecting a target microorganism in a biological sample, present in a minimal amount, for example of the order of a few cells in the body. sample, without resorting to a prior stage of enrichment. This enrichment phase requires the use of culture media, selective or not (depending on the purpose), which aims to promote the growth of target microorganisms in biological or environmental samples, while limiting growth. non-target flora. The culture media are frequently used in containers of the sterile plastic bag type, in which they are brought into contact with food or environmental samples, for the purpose of resuspension and enrichment of the desired microorganisms. As mentioned above, this enrichment phase is necessary in particular in order to reveal the presence of at least one target microorganism in a very variable and possibly very large sample quantity, for example from 25 g (g) to 375 g. diluted in a volume of culture medium of between 225 and 3375 milliliters (mL). At the end of this enrichment step, an aliquot (generally of a volume between 5 microliters (μί) and 5 ml) is traditionally taken to implement the target microorganism detection step. However, in this aliquot, it is necessary to have a sufficient quantity of target microorganisms to ensure their systematic detection. This enrichment step requires not only an ad hoc culture medium but also an incubation of the assembly formed at least by the biological sample and the culture medium at an optimum temperature to allow the growth of the microorganism ( s) target (s). Incubation is generally carried out at a temperature ranging from 25 to 45 ° C. for a predetermined period of time (for example from 6 to 48 hours). During this incubation period, no additional action is performed on the sample. This time is not used, it is somehow "lost". However, this goes against the problem presented above, aiming at developing a precise and rapid analysis technique. Indeed, during this enrichment step, the sample is immobilized in an oven without intervention means because this step is generally performed during the night. In addition, especially when it is desired to analyze a biological sample whose molecules have a significant cohesion between them, for example a solid or semi-solid sample, it is necessary to provide, before the enrichment step, a step so-called "mixing" (high power homogenization), during which the structure of the biological sample is partially or completely impaired. This kneading step of the sample aims to homogenize the sample to be analyzed in a diluent (the culture medium) and to release the bacteria into the liquid. This step guarantees the accessibility of the nutrients from the culture medium to the microorganisms present in the sample and in particular to the target microorganisms (eg Salmonella spp, Listeria spp, etc.). This step is ensured by the use of kneading machines (also called kneaders) whose three main types are as follows:

Stomacher,

mixer and

Pulsifier®.

However, the use of such kneaders has a number of disadvantages, among which: - need to "knead" one sample at a time,

- presence of several devices to reduce the total analysis time (problem of surface space of the laboratory),

the mixing time is short (from 30 seconds to 1 minute) but extremely violent, this mixing violence being liable to cause a deterioration of the sample matrix, which is likely to cause interference in the detection means , depending on the detection method used (for example PCR inhibitors in molecular biology). Moreover, the violence of traditional "mixing" generates a lot of noise in the laboratory, which disrupts the work of the laboratory staff,

- In the presence of certain samples (seeds, pasta), the violence of mixing sometimes causes a perforation of the plastic bag.

In addition, traditional methods of microbiological analysis frequently require the use of selective media to target the microorganism (s) of interest. In general, the selective agents are arranged at the beginning of the enrichment and in low concentration therefore at a concentration which is not well adapted to allow both the inhibition of the annex flora and the optimal growth of microorganisms. organisms to detect. However, as is known to those skilled in the art, the microorganisms - including the target microorganisms - are said to be "stressed" when they are present in the sample to be analyzed. This is particularly the case after the kneading step referred to above. In this state of stress, the target bacteria are particularly fragile and sensitive, especially with the presence of selective agents. It is therefore difficult - and even inappropriate - to directly contact a biological sample to be analyzed with a selective medium. In addition, the microorganisms stressed directly in contact with the selective agents have a lag phase before growth, which affects their growth and therefore possibly their detection. In order to ensure the presence or absence of the target microorganisms, it is therefore necessary to increase the enrichment / incubation time, thereby delaying detection and / or identification. microorganisms of interest. Consequently, to obtain the desired level of selectivity, a method comprising at least two stages has been developed in the state of the art. This includes:

a primary enrichment phase, during which the biological sample is introduced into a primary enrichment medium free of a selective agent (for example peptone water),

a secondary enrichment phase of transplanting an aliquot of the primary enrichment medium into a second enrichment medium (secondary enrichment medium) containing selective agents.

In general, when it is necessary to perform a secondary enrichment, it is performed only the day after (d + 1), when the technician returns to his workstation. This method considerably increases the incubation time and therefore delays the detection time and, where appropriate, identification of the target microorganism (s). In addition, the step of subculture of the primary enrichment medium in a secondary enrichment medium is likely to give rise to handling errors and also represents a source of error for the traceability of the sample. In order to reduce the total incubation time and reduce the risks inherent in additional manipulations, certain methods provide for the use of a selective pre-enrichment medium, less dosed with selective agent (s) than the medium of secondary enrichment (to preserve stressed germs). Unfortunately, this reduction in the concentration of selective agent (s) does not fail to affect the selectivity of the analysis as a whole.

In an attempt to limit this harmful phenomenon of bacterial stress, the company Oxoid has developed a system (SPRINT Salmonella) which consists in introducing, at the same time as the pre-enrichment medium, capsules containing the selective agent of interest. These capsules were made to release their contents after 6 hours of incubation so as to leave sufficient time for the target germs stressed to strengthen and therefore resist the contribution of this selective agent. However, this principle does not work optimally, in all likelihood because of the impossibility of homogenizing the reaction medium after release of the active ingredient. In addition, at the end of incubation and despite the use of selective media, the concentration of target microorganisms remains in some cases insufficient and / or the concentration of the annexed flora remains too important to perform an effective detection of target microorganisms . In this configuration, one skilled in the art generally uses sample processing methods with the objective of increasing the ratio between the target microorganism concentration and the annex flora concentration. For example, after enrichment, a fraction of the sample (preferably between 1 and 10 ml) is treated by an immuno-concentration method using magnetic beads functionalized with antibodies specific for the target microorganisms. The product of this treatment is then analyzed via a detection method, preferably of the molecular biology type, making the detection method more specific and more sensitive. However, this method of treating the sample is tedious because completely manual and also presents a risk of contamination of the user because the sample, potentially containing pathogens, is handled after enrichment.

In view of the sum of the problems developed above, one of the objectives of the present invention aims to enhance / optimize the incubation time during the enrichment phase of the biological sample.

Another objective is to overcome the disadvantages of "traditional" mixing of biological samples, as indicated above. In view of this "traditional" kneading problem, the object of the invention is also to make it possible to treat several biological samples simultaneously during this step. The invention also aims to improve the speed of the analysis protocols comprising contacting selective agent (s) or selective medium (s) comprising them with a biological sample, not stressing the phenomenon of microbial stress by directly contacting the microorganisms with one / such selective agent (s). This aims to limit - or avoid - the latency phase in the growth cycle of microorganisms sought or even, in the worst case, the total inhibition of their growth.

Another object of the present invention is to provide a method for increasing the rate of sample analysis and / or reducing the total time required for the analysis of the biological sample.

Another object of the present invention is to limit the handling of the sample contained in the container, thereby limiting the risk of contamination, either of personnel handling the sample or of the sample itself. In this regard, the present invention also relates to the development of a biological sample analysis method (s) automated or semi-automated. Another object of the present invention is to provide a method for multi-detection.

When a confirmation step is desirable or necessary (see above), one of the objectives of the present invention aims to achieve the latter in the continuity of the detection and / or identification step, preferably within the same container.

Another object of the present invention is to improve the traceability of the analysis due to the drastic reduction of the sample handling steps.

The invention also aims to improve the gas exchange and in particular the exposure of the target microorganism (s) to the dissolved oxygen at within the mixture constituted by the biological sample and at least one culture medium.

The invention also aims to produce a dispersion of microorganisms by preventing the formation of localized colonies or localized clusters, in particular biofilms on the surfaces of the container but also on the sample fragments and within the mixture constituted by the biological sample and at least one culture medium. Another object of the present invention is to perform, during the incubation / enrichment and growth period of the microorganisms, a treatment of the sample (preferably in an automated manner) on the total volume of the sample (and not just an aliquot), said treatment consisting of immunoconcentrating target microorganisms (of interest) to improve the sensitivity and specificity of the detection method used post-treatment.

Other objectives will appear on reading the present application.

The present invention therefore aims to achieve all or part of the aforementioned objectives.

Presentation of the invention

Accordingly, the subject of the present invention is a method of treating at least one biological sample that is susceptible (or suspected) of containing at least one target microorganism, said method being carried out within a container and comprising the following steps: :

a) optionally bringing said biological sample into contact with at least one culture medium within said container, the mixture of the biological sample and said culture medium forming all or part of the contents, b) optionally incubate the container at a temperature and for a period of time sufficient to allow the growth of said at least one target microorganism,

c) at least one step of homogenizing the biological sample, during which the content moves from a level n, corresponding to the level of the content at rest, to a level of homogenization ¾, distinct from the level n and vice versa

said process comprising, previously, subsequently or during all or part of the homogenization step c), preferably after said homogenization step c), the following step: c ') generating a displacement of the content towards a level n + 1, different from the levels n and ¾, so that the content comes into contact with at least one culture means and / or at least one analysis means positioned in the enclosure of the container, between the level n + 1 included and level ¾ excluded.

Thus, the process according to the invention makes it possible to effectively treat a biological sample (for the purpose of enrichment and / or analysis) while limiting the handling of the sample contained in the container, thereby limiting the risks of contamination, either personnel handling the sample or the sample itself. In addition, this method can easily be automated in whole or in part and can treat several biological samples simultaneously.

As mentioned in the preamble of this application, steps a) and b) above - aimed at enriching the biological sample of interest in target micro-organisms - are only necessary when they are present in a minimal amount, for example of the order of a few cells in the sample. In this case, the method according to the invention comprises the above steps a) and b).

On the other hand, when the biological sample of interest is naturally rich in target micro-organisms, as may be the case especially with regard to certain biological samples of food origin (for example cow's milk) or clinical (liquid stools taken from a patient suffering from cholera), the method according to the invention does not require the implementation of steps a) and b).

According to a preferred embodiment, this biological sample treatment method is used as part of a process for enriching and / or analyzing a biological sample that may contain at least one target microorganism, said process being carried out within a container and comprising the following steps:

a) optionally bringing said biological sample into contact with at least one culture medium within said container, mixing the biological sample and said culture medium forming all or part of the contents, b) optionally incubating the container at a temperature and for a period of time sufficient to allow the growth of said at least one target microorganism,

said process comprising, before, after or during all or part of the homogenization step c), the following step:

c ') generating a displacement of the content to a level n + 1, different from the levels n and ¾, so that the content comes into contact with at least one culture means and / or at least one analysis means positioned (s) ) in the enclosure of the container, between the level n + 1 included and the level ¾ excluded.

Step c ') above allows in particular to obtain an effective treatment of the biological sample of interest (for enrichment and / or analysis) while limiting the risk of contamination (ie personnel handling the sample, ie from the sample itself).

'Process for the enrichment of a biological sample which is susceptible (or suspected) of containing at least one target micro-organism' means, within the meaning of the present invention, a method for allowing the growth of at least one target microorganism preferably in the presence of at least one culture medium / enrichment broth (especially when it is suspected that the micro (s) -organism (s) sought is / are present (s) in a minimal amount, for example of the order of a few cells, in the biological sample), so that said at least one target microorganism either present at the end of the enrichment process at a concentration such that the user can possibly detect it in a systematic or quasi-systematic manner by resorting to traditional methods of detection (culture on agar media, immunoassays, molecular biology techniques, etc.).

By "a method of analysis of a biological sample susceptible (or suspected) of containing at least one target microorganism", is meant in the sense of the present invention, a method for analyzing said micro-or micro-organisms. target organism (s) and / or all or part of their properties. The analysis can consist in particular of a method of direct detection - and possibly identification - of said microorganism (s) or of a detection method - and if appropriate of indirect identification -, for example linked detection of nucleotide and / or protein information specific to a type of microorganism to be detected and / or identified. This detection and / or indirect identification may also result from the detection of specific bacteriophage proteins of said microorganism (s) to be detected. The presence of a target microorganism can also be detected by resistance to a given antibiotic or a set of antibiotics, the resistance profile of this or these antibiotic (s) being, in this case, characteristic of the microorganism (s) to be detected.

This "method of analysis of a biological sample susceptible (or suspected) of containing at least one target microorganism" may also have the purpose of determining properties of potential resistance of said at least one target microorganism (for example a target bacterial species) to at least one antimicrobial (eg one or more antibiotics). In addition, said "method of analysis of a biological sample susceptible (or suspected) of containing at least one target microorganism" may make it possible to measure one or more biological and / or physico-chemical parameters of said sample, to evidence of a particular contaminant or marker within that sample.

The analysis method according to the present invention also makes it possible to carry out sterility checks, in particular within the food and environmental samples. To this end, generic means for detecting microorganisms, such as capture supports functionalized with generic anti-Gram-, anti-Gram + type binding partners, are used as means of analysis. etc. The type of analysis carried out with the analysis method of the invention can therefore be not only qualitative (detection and identification of specific micro-organism (s)) but also quantitative or semi-quantitative.

According to the present invention, the biological sample can be of various origins, for example of food, environmental, veterinary or clinical origin. Among the samples of food origin, there may be mentioned, non-exhaustively, a sample of dairy products (yogurt, cheese ...), meat, fish, egg, fruit, vegetable, water, drink (milk, fruit juice, soda, etc.). Of course, these food-based samples may also come from sauces or more elaborate dishes or non-(or partially) processed raw materials. A food sample may finally be derived from a feed intended for animals, such as cakes, animal meal. As examples of biological samples, it is also worth mentioning biological samples related to the environment such as surface samples, water samples, air samples, etc.

Clinical biological samples may be samples of biological fluids (whole blood, serum, plasma, urine, cerebrospinal fluid, etc.), stool, nose, throat, skin, wounds organs, tissues or isolated cells. This list is obviously not exhaustive. In general, the term "sample" refers to a part or quantity (more particularly a small part or a small quantity) taken from one or more entities for analysis. This sample may possibly have undergone prior treatment, involving for example mixing, dilution or grinding steps, in particular if the starting entity is in the solid state.

According to a preferred embodiment, the biological sample to be analyzed (ie the biological sample as taken or pre-treated, as indicated above) is solid or semi-solid (the molecules present within said biological sample have a relatively large cohesion between them). The biological sample analyzed is, in general, likely to - or suspected of - containing at least one target microorganism. In most cases, the latter is a pathogenic micro-organism (such as Salmonelld) that must be detected for health purposes. The term "microorganism" has the same meaning as that generally accepted in microbiology and particularly includes gram-positive or gram-negative bacteria, yeasts, molds and, more generally, unicellular organisms, invisible to the naked eye, which can be manipulated and multiplied in the laboratory.

According to a preferred embodiment, the microorganism (s) to be detected are bacteria, for example enterobacteria such as E. coli.

If the micro-organism enrichment of a biological sample is carried out in order to carry out the detection and, where appropriate, the identification of one or more target micro-organisms, this detection - and, where appropriate, identification - can be carried out directly (by contact of microorganisms with a carrier of capture having a very good affinity for the latter) or indirect (for example by the detection of proteins secreted by the target microorganisms). By way of illustration, the detection of toxins secreted by Staphylococcus aureus may be mentioned.

Advantageously, the biological sample is brought into contact with at least one culture medium allowing the growth of microorganisms and, in particular, of the target microorganism (s). "Culture medium" means a medium comprising all the elements necessary for the survival and / or growth of the microorganisms and, in particular, the microorganisms sought (for example peptone-buffered water) . The culture medium may contain any additives, for example: peptones, one or more growth factors, carbohydrates, one or more selective agents, buffers, one or more gelling agents, one or more vitamins, etc. This culture medium may be in liquid or gel form ready for use, namely ready to be seeded in a tube, in a bottle or on a petri dish. The expression "culture medium" obviously includes enrichment media and broths.

The container used for the purposes of the present invention is an open or closed enclosure (for example hermetically sealed or sealed) optionally provided with a vent system, preferably closed (possibly equipped with a vent system), the wherein the biological sample of interest is contacted with one or more culture media. According to a particularly preferred embodiment, the container is a container comprising a base and walls. It may be, for example, a rigid container such as a bottle, a bottle or a pillbox. According to a preferred embodiment, the container is a bag having a flexible envelope of the homogenization bag type. Preferably, at least one wall of the container is transparent in order to be able to perceive the volume occupied by the liquid inside the container. The compound content of the mixture comprising the biological sample and optionally at least one culture medium may, of course, comprise additional elements, such as vitamins or other nutrients useful for the culture of microorganisms, selective agents, specific substrates and other elements well known to those skilled in the art.

The method according to the present invention however requires that this content is mobile, in particular to allow homogenization with slight stirring. Preferably, this content is a fluid, preferably a liquid.

Regarding the incubation step b), when the method according to the invention comprises this step, the skilled person will know, from his experience, his general knowledge and / or bibliographic data at his disposal, adapt the temperature and time necessary to allow sufficient growth of the target microorganism, depending on the type of microorganism sought. For information, and as mentioned in the preamble of the present application, the incubation is generally carried out at a temperature ranging from 25 to 45 ° C for a predetermined period of time (for example from 6 to 48 hours) . Preferably, unlike the traditional mixing carried out using Stomacher ^® type mixers, mixers and other Pulsifier ^® , the homogenization of the biological sample is carried out, according to the present invention, in a less violent manner than the kneading of the prior art and for a longer period of time, in particular in order to avoid excessive deterioration of the sample matrix (liable to cause interference at a detection means), as well as possible risks of perforation of the pouch of the homogenisation bag. This is particularly advantageous in the context of the treatment of a biological sample of relatively firm consistency, for example of solid or semi-solid nature.

Preferably, during this homogenization step (called "soft homogenization"), the displaced volume of level n (content level at rest) to the level ¾ (homogenization level) is less than or equal to 50%, advantageously less than or equal to 40%, preferably less than or equal to 30%, and the frequency of displacement of the content is less than or equal to 2 Hertz (Hz), preferably less than 2 Hz, preferably between 0.1 and 1 Hz, advantageously between 0.45 and 0.7 Hz. By way of comparison, a Stomacher ^® type mixer causes, during the step mixing, a volume displacement of the content greater than 100% and kneading at a frequency ranging from 2 to 5 Hz, generally between 3 and 4 Hz.

In general, and at crushing pressure of the biological sample equivalent to that of kneaders of the prior art, the homogenization is carried out at the abovementioned frequency, namely less than or equal to 2 Hz, preferably less than 2. Hz, preferably between 0.1 and 1 Hz, advantageously between 0.45 and 0.7 Hz. By way of example, at crushing pressure equal to that of commercially available Stomacher ^® type mixers (variable between 3 and 30 kilograms on the bag containing the food sample), and considering a traditional homogenization bag (or bag) whose wall is made of flexible plastic (for example PVC, polyethylene or polyester), the frequency is such that previously indicated, namely less than or equal to 2 Hz, preferably less than 2 Hz, preferably between 0.1 and 1 Hz, preferably between 0.45 and 0.7 Hz.

According to a particular embodiment, it is possible to carry out at least two homogenization steps of the biological sample, namely a first step intended to disperse the biological sample (for example of food origin) analyzed, generally of a duration of between 2 and 90 minutes, preferably between 10 and 60 minutes, advantageously between 15 and 50 minutes, then a second homogenization step that can continue until the end of the incubation period, this second step of homogenization make it possible to obtain a good oxygenation of the medium as well as an ad hoc dispersion of the micro-organisms (in particular bacteria) in the medium, and this in order to avoid the interactions with the biological particles (for example food particles) resulting from the biological sample (eg food). This second homogenization step may continue after the end of the incubation step.

Specifically, and when using a device with blades (or arm), said blades from compressing a flexible wall homogenization bag (for example plastic PVC, polyethylene, polyester), the bag of homogenization is compressed by at least one blade at the abovementioned frequency, namely less than or equal to 2 Hz, preferably less than 2 Hz, preferably between 0.1 and 1 Hz, advantageously between 0.45 and 0.7 Hz with a stroke expressed in centimeters of the blades from an adjustable distance of 0.1 to 2.7 cm, preferably 2.3 cm leaving a 0.4 cm gap between the blades and the fixed wall against which the pocket is compressed homogenization.

This homogenization of the biological sample, which can be described as "soft" homogenization with respect to the "violent" kneading of the prior art makes it possible to overcome all or part of the problems inherent to kneading devices of the prior art.

For the purposes of the present invention, the term "culture medium" is intended to mean a means of favoring and / or orienting the culture of the target / desired microorganism (s). This culture means may be, for example, a selective agent, such as one or more antibiotic (s), intended (s) to improve the selectivity of the analysis (by eliminating all or part of the unwanted microorganisms) . The culture means may also be a nutrient element, for example selected from vitamins, peptones, carbohydrates, etc., intended to promote the primary function of enriching target microorganisms of the biological sample tested. "Analytical means" means any means for directly or indirectly measuring (in association with at least one other means of analysis) one or more biological and / or physico-chemical parameters of a biological sample (eg pH variation), to demonstrate the presence of a contaminant or a particular marker in said sample. Preferably, such a means of analysis allows the direct or indirect analysis of the target microorganism (s), as well as all or part of their properties and any change in the medium generated by said micro or micro -organism (s) (such as a change in pH). A means for indirect analysis (also called "indirect analysis means") may, for example, consist of an indicator, marker or other tracer that will be subject to one or more subsequent analyzes, or in a way concentration such as an immunoconcentration means. In the latter case, the function of the concentration means is to ensure that the desired analyte (s) is / are in sufficient concentration for subsequent analysis steps (performed in situ or ex situ).

Preferably, the one or more means of analysis consist of one or more means of detection - direct or indirect (in association with at least one other means of detection) - of microorganisms. In other words, the detection means used may be any means for detecting, directly or indirectly (in association with at least one other detection means) the presence or absence of target microorganisms in a biological sample and allowing, where appropriate, to identify them directly or indirectly.

In a nonlimiting manner, the aforesaid detection means may be selected from the electrical detection means (in particular electrochemical), the optical detection means, the acoustic detection means, the thermal detection means, the mechanical detection means, the magnetic detection means.

An exemplary detection means that can be used according to the present invention consists of a capture support functionalized / sensitized by a specific or non-specific (preferably specific) binding partner of a target microorganism. According to a preferred embodiment, the specific binding partner is selected from the group consisting of antibodies, Fab fragments, Fab 'fragments, recombinant or non-recombinant phage proteins, phages or any other ligand well known to those skilled in the art. The revelation of the presence of the target microorganisms at the level of the capture medium may be carried out by means of any appropriate revelation system, that is to say capable of allowing the detection of the microorganism (s) target (s). By "revealing system" is meant any molecule capable of coupling with the microorganisms or binding partners of said microorganisms and allowing, by their transduction properties (fluorescence, coloring, radioactivity, etc.), to reveal the presence of said microorganisms. This revelation of the presence of the target microorganisms can be obtained in particular by visualization (with the naked eye) or optical reading (via a camera-type optical reading device) of a coloration (such as a red coloration due to TTC reduction in formazan by microorganisms) or fluorescence on all or part of the capture medium.

It is important to note that said at least one culture means and / or said at least one means of analysis is / are disposed inside the container so as not to be in contact with the contents when the latter is at level n or moves from this level n to level ¾, and

- come into contact with the content during the step of variation (preferably of elevation) of level c ').

According to a particular embodiment, instead of a culture means or a means of analysis, a means of culture and analysis is used.

The displacement of the content can be obtained by any means known to those skilled in the art. In particular, this displacement can be generated by the application of a force or a set of forces on the container or the change in the balance of forces applying to the container (for example if the container is held in two points by two holding forces and if one of the two forces ceases, the container will tilt and the level of the content will vary, at least a portion of the inner surface of the container, the rest level to a level above the latter and will possibly come into contact with the culture means and / or the analysis means).

Another embodiment for obtaining a variation of the level of the content inside the container can be observed when the container is positioned on a plateau stirrer (trivially called "rocking floor"), the latter regularly varying the level. contents inside the container, in a back-and-forth movement (comparable to a bag-like movement and surf).

The displacement of the contents can also be achieved by applying a force or a force system inside the container, for example by inflating and deflating an inflatable flexible bag (such as a balloon or an inflatable bead) placed in the enclosure of the container.

If we consider the upper part of the container as the "high" part and the lower part of this container as the "low" part, the level of the content will very slightly increase during the homogenization step c) to a level of homogenization ¾ but not sufficiently to be brought into contact with the culture means and / or the analysis means positioned in the enclosure of the container, above the level ¾. When a selective agent-type culture means is used, this is quite desirable as indicated above, so as not to bring this selective agent into contact with the biological sample at an early stage, namely before that the target microorganism (s) have overcome the microbial stress phenomenon and are sufficiently "viable" to support the contribution of this selective agent.

When detection means are used as the means of analysis, this is also very desirable in order to preserve the integrity of the detection means before the detection / identification step takes place. This is particularly true when it comes to a biosensor, in order to avoid a deterioration of the biosensor capacities because of the non-specific compounds contained in the sample (in particular after the homogenization step). By schematizing, the method according to the invention makes it possible to increase, precisely and at will, the level of the content (preferably in the liquid state) in the container. This function allows the delayed recovery of a reagent (for example a selective agent of the antibiotic type) and its homogenization in the content, thus ensuring its optimal efficiency. This solves a major problem in terms of selectivity.

According to a preferred embodiment, the container is a bag of flexible or semi-flexible material (analysis bag type or homogenization bag) and the rise of the liquid within the latter is obtained through a pressure system. and depression applied to the pocket by mechanical means, preferably by the movement of arms or blades. It is quite possible - and in some cases desirable - to parameterize a new homogenization step subsequent to step c ') (as indicated above), for example in order to homogenize the content comprising the biological sample. , the culture medium and the culture medium resumed following the elevation level achieved in step c '), within the container.

According to a preferred embodiment, said level n + 1 is situated above the level of homogenization ¾, which level ¾ is situated above rest level n, so that the step of "level variation" c ') is preferably a step of "leveling up", during which the content rises from level n or ¾ to the higher level n + 1 so as to come into contact with at least one means culture and / or at least one analysis means positioned in the enclosure of the container above the level of homogenization ¾ and below the level n + 1 or height thereof. Thus, during variations in the level of the content occurring during the homogenization step (from level n to level ¾ and vice versa), said at least one culture means and / or at least one means of analysis is in somehow "preserved", which means that it does not come into contact with the content during a homogenization step. In other words, this prevents said culture means and / or said analysis means from being "polluted / deteriorated" during a homogenization step.

According to a preferred embodiment, the displacement of the content of the level n to the level ¾ and the displacement of the content of the level n to the level n + 1 are generated by the same displacement means, at two different intensities, preferably the intensity of the displacement is greater for the displacement towards the level n + 1 than towards the level ¾. This allows the easy and cost-effective development of a method making it possible to perform at least one homogenization step and at least one so-called "level variation" step, (preferably "step"). level raising "), to take at least one culture means and / or at least one means of analysis, during the incubation step or after it.

According to a particular embodiment, said method according to the invention comprises, after step c '), at least the following step:

c ") generating a displacement of the content to a level n + 1 + x so that the content comes into contact with at least one culture means and / or at least one additional analysis means, positioned (s) in the enclosure of the container, between the levels n + 1 + x inclusive and the level (n + 1 + x) -l excluded, wherein x is an integer, preferably between 1 and 10.

Preferably, step c ') and / or step c ") (preferably both) is / are performed during the incubation step b). at least one homogenization step is performed after step c ') and / or after step c ").

This embodiment makes it possible to perform several enrichment steps in culture means and / or several analysis steps (for example direct or indirect detection) at different levels of the container.

Preferably the level n + 1 + x is located above the level (n + 1 + x) -1. By way of example, if x represents the integer 1, step c ") consists in generating the displacement of the content to a level n + 2, distinct from the levels n, η _¾ and n + 1 (preferably located at above these), in order to come into contact with a culture medium and / or an analysis means positioned within the container, between the levels n + 1 (inclusive) and n (excluded). Thus, according to this example, it is possible, for example, to position a selective agent between the level n (excluded) and the level n + 1 (inclusive) and a detection means such as a biosensor between the level n + 1 (excluded ) and the level n + 2 (inclusive) In a first step, step c ') allows the selective agent to be taken back into the content comprising the biological sample (or its residues) and at least one culture medium Optionally, a homogenization step can be carried out after step c '), in order to optimize the efficiency of the selective agent. electivity being sufficient, step c ") is carried out in order to detect and, where appropriate, identify target microorganisms that have survived the selective agent.

The method according to the invention can be declined to envy, according to the desiderata of the user, different culture and / or analysis means that can be positioned within the container, between the levels n + 1 + x ( inclusive) (n + l + x) -l (excluded). In addition, no manipulation is required between the different steps. This process is therefore very easily automated.

According to a preferred embodiment, the method according to the invention comprises a step of generating the displacement of the content to a transfer level n _t , so that the transfer of all or part of said content takes place from this transfer level. n _t of the container to another part of said container or at least one other container.

This transfer of all or part of the content may be of interest especially in view of a detection - and possibly an identification - of the target microorganism or "deported" to another container, this other container may be an analysis device, for example of the VIDAS ^® type. According to an alternative, this transfer makes it possible to perform - either in another part of the same container, or in another container - additional enrichment steps, where appropriate.

According to a particularly preferred embodiment, the homogenization step c) is carried out at least partly during the incubation step b), preferably for a period of time greater than 2 minutes. Thus, the homogenization step c) can for example:

start before the start of incubation step b) and continue during all or part of it; or

- begin during incubation stage b) and continue during all or part of the latter; or

begin before the start of incubation step b) or during incubation step b) and continue after the end of said incubation step b). In any case, this embodiment is particularly advantageous since it makes it possible to use all or part of the generally "lost" incubation time in the processes of the prior art.

According to a particular embodiment, the homogenization step c) begins before the start of the incubation step in order to allow an aliquot to be sampled after homogenization and before incubation to perform, for example, a micro count. -organisms.

According to another particular embodiment, the homogenization step c) begins concomitantly with the incubation step b) or in the first minutes following the start of the incubation step b), preferably in a period of time between 1 and 10 minutes from the start of said incubation step b).

The fact that the homogenization step c) starts concomitantly with the incubation step b), or in the first minutes after the start of the incubation b), does not represent in any case an arbitrary parameter since, on the contrary, and against all odds, the latter made it possible to obtain the best results in terms of enriching the biological sample tested while reducing the background noise generated during conventional homogenization.

Another subject of the present invention relates to a process for enriching at least one biological sample capable of containing at least one target microorganism, said method implementing the biological sample treatment method according to the invention, wherein said at least one culture medium and / or at least one means of analysis is a culture medium such as a selective antimicrobial agent.

The invention also relates to a method for analyzing at least one biological sample capable of containing at least one target microorganism, said method implementing the method for treating at least one biological sample according to the invention, said at least one culture means and / or at least one analysis means being at least one analysis means such as a functionalized capture support or a biosensor, and said method comprising an additional step d) of analyzing said at least one means of analysis; least one biological sample, preferably to analyze (directly or indirectly) said at least one target microorganism, using said analysis means.

Advantageously, this analysis method comprises, before and / or after the analysis step d) (preferably before the latter), at least one step of transferring all or part of the mixture comprising said biological sample, optionally the medium culture, said at least one means of analysis, the container, then said principal, to a second container called secondary. According to a preferred embodiment, said method comprises, before and / or after the analysis step d) (preferably before the latter) the transfer of the means (s) of analysis to said secondary container (consisting, for example, in a VIDAS ^® analysis device).

According to a preferred embodiment, the above analysis method comprises, after the analysis step d), a confirmation step e) aimed at confirm or invalidate the analysis results obtained at the end of the analysis step d). This confirmation step may be performed either in situ, namely in the enclosure of the aforesaid main container, or ex situ, ie for example within the secondary container mentioned above (such as a VIDAS analysis device).

Another subject of the present invention relates to a method for enriching a biological sample capable of containing at least one target microorganism, said method being carried out within a container and comprising the following steps:

a) contacting said biological sample with at least one culture medium within said container, mixing the biological sample and said culture medium forming all or part of the contents,

b) incubating the container at a temperature and for a period of time sufficient to allow the growth of said at least one target microorganism,

c) homogenizing the biological sample during at least part of the incubation step b), preferably for a period of time greater than 2 minutes. Advantageously, during this step c), the volume displaced from the level of the content at rest n to the homogenization level ¾ is less than or equal to 50%, advantageously less than or equal to 40%, preferably less than or equal to 30%, and the frequency of displacement of the content, during said homogenization step c), is less than or equal to 2 Hz, preferably less than 2 Hz, preferably between 0.1 and 1 Hz, advantageously between 0.45 and 0.7 Hz.

The fact that the Applicant has discovered, surprisingly, that a homogenization step (which may be called "soft" homogenization, see definition above) could be carried out during all or part of the incubation step b) (for example at the beginning of incubation) makes it possible, besides obtaining a good oxygenation of the medium, to provide nutrients of the target microorganism (s), etc., to obtain a biological sample less altered than a biological sample "kneaded" violently, according to the traditional methods of the state of the art. This makes it possible in particular to avoid in the end the risks of interference of the matrix residues resulting from the destruction of the biological sample at the means used in detection (this is particularly true for the detection means used in molecular biology, such as PCR probes). In general, we obtain a clearer supernatant, less particles in suspension, so less background noise. In addition, the device implemented in this process is significantly less noisy than those used in the prior art.

According to a particularly preferred embodiment, the subject of the invention is also a method for analyzing at least one target microorganism, said method implementing the enrichment method according to the invention, said process comprising, subsequently in step c), an analysis step d) using at least one analysis means (for example a step of identification of microorganism (s) via at least one detection means such as a capture support functionalized by a link partner), said analysis step being performed within the container or outside thereof.

Preferably, the analysis method is a detection method - and if appropriate identification - comprising, after step c) a detection step d) using a detection means, said step detection being performed within the container or outside thereof.

Preferably, the detection step - and if necessary identification - referred to above is performed within the container (in the enclosure of the latter), this for the sake of practicality and especially in an automation optics.

In the event that the detection of the microorganism (s) sought is carried out in the container, the latter may comprise, within it, any revelation system capable of detecting the presence of this or these microorganisms and, where appropriate, appropriate, their identification.

By "revealing system" is meant any molecule capable of coupling with the microorganisms or the binding partners of said microorganisms and allowing by their transduction properties (fluorescence, coloring, radioactivity in particular) to reveal the presence of said microorganisms.

According to a preferred embodiment, the revelation system is based on the reduction of certain tetrazolium salts by the microorganisms, in particular 2,3,5-triphenyltetrazolium chloride (whose acronym is TTC) by the microorganisms. At the same time as growth, the TTC (colorless in its unreduced form) is internalized by said microorganisms, then reduced by the latter to triphenyl-formazan (of red color), thus coloring said micro-organisms in red and thus allowing their revealing on a capture medium, preferably positioned within the container. The direct and real-time detection of microorganisms in a food sample, during the incubation period, is in this case carried out by optical reading of the capture medium, in an automated manner or not, preferably in an automated manner by means of an optical detection device.

Advantageously, at least one binding partner, specific or non-specific, of the one or more microorganisms is fixed on a capture medium. A capture medium may be any support capable of allowing the revelation of microorganisms. In this regard, it is particularly appropriate to mention particulate media, possibly magnetic or monobloc supports, possibly porous. The capture medium may simply be an inert carrier, such as a plate of plastic material or glass fiber or, advantageously, may be sensitized with a possibly specific binding partner. The capture medium may also consist of a compressible monoblock support. According to a particular embodiment, the capture medium can be one with the detection means. This is the case, for example, when the capture medium is constituted by an electrochemical biosensor or an optical fiber. As regards the binding partner, when such a binding partner is attached to a capture medium, it is advantageously selected from antibodies, Fab fragments, Fab 'fragments, recombinant phage proteins or no, phages, lectins, aptamer-type nucleic acids or any other ligand well known to those skilled in the art.

Advantageously, the detection means, preferably present in the enclosure of the container, is selected from the group consisting of: the electrical and especially electrochemical detection means, the optical detection means, the acoustic detection means, the detection means thermal, the mechanical detection means, the magnetic detection means (non-exhaustive list) or their combination.

When the detection of the target microorganism (s) is carried out in the actual enclosure of the container, and according to a particular embodiment, it is quite possible to couple the detection means in order to realize on the one hand the detection and on the other hand, to carry out the confirmation simultaneously or subsequently, if the latter is desired or necessary (which is generally the case in the agri-food sector). For example, it is possible to detect the target microorganism (s) by means of an electrochemical biosensor. If the binding of the target microorganisms is carried out by means of specific binding partners, the detection step then constitutes an identification step. An optical analysis of the microorganisms specifically attached to the biosensor at the analysis zone by an optical detection device then makes it possible to confirm the identification of the microorganisms. If the optical detection device is a Raman spectrometer, a Raman spectrum analysis by comparison with a database of reference spectra corresponding to the different target microorganisms, then allows to confirm the identification of said microorganism.

According to another particular embodiment, it is possible to perform the detection and confirmation with the same technology. Thus, if the detection means is an optical means such as an intrinsic fluorescence measurement means, it is particularly advantageous to carry out the detection of the target microorganisms by appearance of intrinsic fluorescence and possibly their identification. The response is then a yes response (presence of fluorescence) / no (no fluorescence). If there is fluorescence, a spectral analysis of the fluorescence signal by comparison with a database of reference spectra corresponding to the different target microorganisms, then makes it possible to identify said microorganism and, thereby, to confirm the detection. the presence of said microorganism.

Preferably, the detection of the microorganism (s) is carried out in real time. Nevertheless, alternatively, the detection of the microorganism (s) can be carried out, in end point, at the end of the step of growth of said microorganism (s).

According to a particular embodiment, the detection means present within the container is connected with a data analysis system.

Advantageously, the connection between the detection means and the data analysis device is a wired connection or a wireless connection.

According to a particular embodiment, the detection means used, preferably within the container, is an electrochemical biosensor for the detection of at least one microorganism present in the biological sample placed within the container. This biosensor comprises a support comprising:

at least one detection electrode, coated with at least one electroactive polymer, on which is fixed by one of its ends at least one single-stranded or double-stranded oligonucleotide, the second end of said oligonucleotide being linked to at least one binding of the microorganism (s) to be detected, specific or nonspecific;

at least one counter-electrode.

Advantageously, the electroactive polymer is taken from the group comprising polypyrrole, polyacethylene, polyazine, poly (p-phenylene), poly (p- phenylene vinylene), polypyrene, polythiophene, polyfuran, polyselenophene, polypyridazine, polycarabazole, polyalinine.

According to a particular embodiment, the electroactive polymer comprises at least one electrochemical mediator. Such an electrochemical mediator is taken from the group comprising ferrocene, quinone and derivatives thereof or any other mediator well known to those skilled in the art.

According to an alternative embodiment, the electrochemical mediator is found in free form in the culture medium. Such a mediator may be, for example, ferricyanide / ferrocyanide [Fe (CN) 6] 3/4 ^" pair, the iridium chloride [IrCl ₆ ] 3 / 4- pair, ruthenium hexamine [Ru (NH ₃ ) ₆ ] 3 ⁺ / 2 ⁺ .

Preferably, the linkage between the oligonucleotide and the binding partner of the microorganism (s) is carried out using at least one biotin-streptavidin or biotin-avidin linkage pair.

When the oligonucleotide is single-stranded, a biotin is attached to the 3 'end of said nucleotide, the 5' end allowing the attachment of the latter to the electroactive polymer, in particular by covalent bonding. By using an also biotinylated binding partner, it is then easy to attach it to the 3 'end of the oligonucleotide using a streptavidin or avidin molecule.

When the oligonucleotide is double-stranded, the first strand is fixed, in particular by covalent bonding to the electroactive polymer at its 5 'end. The second strand, for its part, is biotinylated at its 5 'end, allowing attachment of the also biotinylated binding partner, by means of a streptavidin or avidin molecule.

Advantageously, the binding partner is taken from the group comprising: antibodies, Fab fragments, Fab 'fragments, recombinant or non-recombinant phage proteins, complete phages or bacteriophage fragments. Thus, the method according to the present invention may comprise an additional step of detecting microorganisms or the protein secreted by the latter through the binding partner. According to another particular embodiment, the revelation system is a non-specific substrate internalized by the microorganism (s) to be detected. Thus, once the effective capture of a certain quantity of colored target microorganisms (case of a positive sample), there is a change in the optical properties of the capture medium (generally consisting of a solid phase, for example in a compressible support, possibly porous, as indicated above) by appearance of a coloration (for example a red coloration in the case of the TTC) on this one (transduction of the biological signal). This coloration of the capture medium is then detectable to the naked eye or measurable via the use of a reading automaton such as a camera. For ease of reading, it is preferable that the capture medium is no longer in contact with the culture medium.

According to another embodiment, the revelation system is a cellular dye of the microorganism (s) to be detected. The detection step may be performed using a means selected from the optical detection means, the magnetic detection means, the electrochemical detection means, the electrical detection means, the acoustic detection means, the means thermal detectors or their combination.

Another object of the present invention relates to a device for implementing the method according to the invention, said device comprising at least one location for receiving at least one container, at least one moving means for generating the displacement of the content, in wherein the moving means is adapted / adapted to generate at least two displacements of the content at at least two different intensities, the lowest displacement intensity allowing the homogenization of the biological sample and the highest displacement intensity. generating a displacement of the content so that it comes into contact with at least one culture medium and / or less a means of analysis. In other words, said at least one container comprises, within it, at least one culture means and / or at least one analysis means arranged so as to:

not to be in contact with the content when the latter is at level n or moves from this level n to level ¾, and

come into contact with the content during the step of variation (preferably elevation) of level c ').

As indicated above, the use of a displacement means adapted to / adapted to generate at least two displacements of the content at said at least two different intensities makes it easy to develop, and at a lower cost, a device enabling performing both at least one homogenization step and at least one so-called "level variation" step (preferably "level-up"), in order to allow the content to come into contact with at least culture means and / or at least one means of analysis during or after the incubation step.

According to an advantageous embodiment, said device comprises: Preferably, the device comprises at least two locations in order to treat several containers simultaneously.

This device can be considered as an improved rack capable not only of receiving one - and preferably several containers - but also to act on this or these containers, generating for example a homogenization of its / their content or a level increase of way to bring the content into contact with a culture medium and / or a means of analysis.

According to a particular embodiment, the container may be a container comprising a base and walls. According to this embodiment, a force or a set of forces can be applied to at least one wall and / or the base of the container by the one or more displacement means. According to a particularly preferred embodiment, the container comprises at least one flexible or semi-flexible wall. Preferably, this flexible or semi-flexible wall is made of translucent or transparent material, advantageously transparent.

Advantageously, the container is of the bag or flexible pouch type (such as a bag or homogenization bag), constituted in whole or in part by a flexible (or deformable) membrane. Thus, the means for displacing the device will act on this flexible membrane - for example by pressure - in order to deform it and thus cause the level of its content to be raised to a predefined level, for example a homogenization level ¾ or a higher level (for example the level n + 1) in order to bring the content into contact with a means of culture and / or detection positioned in the container, between the levels nh and n + 1. Preferably, the device comprises optical detection means for detecting the presence of said target microorganism.

This makes it possible to go in the direction of automating the process according to the invention. This optical detection means may be a camera or a camera. It may also include advanced features such as optical density (OD) reading or fluorescence.

Still with a view to enabling easy automation of the method according to the invention, a preferred embodiment of the invention relates to a device comprising a control means making it possible to vary the intensity of displacement of the content, for example by adapting the intensity and / or the frequency of the force, or of all the forces, applied to the container in order to generate the displacement of the contents (for example towards the level of homogenization ¾ and / or towards a higher level such as the level n 1). This integrated control means may, advantageously, provide management and coordination of the other means of the device, namely in particular optical detection means and / or heating means. The invention also relates to the use of the aforementioned device for implementing the method according to the invention. In order to allow the incubation of the content (step b) of the method), the device can either be placed in a traditional incubator, adapted for the occasion, or comprise at least one heating means for performing the incubation step b) (for example a contact heating means). This option is particularly preferred because it avoids a handling step, namely the transport of the device to and in the incubator.

In addition, the integration of this heating function is particularly interesting since it makes it possible to increase the heating rate of the media in order to reach very rapidly the optimal growth temperature of the microorganisms and thus to reduce the lag phase.

In addition, the integration of the heating means with the device of the invention offers an additional advantage in that it allows temperature variations during the incubation and thus possibly makes it possible to gain in selectivity and / or sensitivity of detection. By way of example, mention should be made of the ELISA method for the detection of Listeria spp, based on the use of anti flagellar antibodies. In fact, the optimal growth temperature of Listeria spp is greater than 35 ° C, whereas the optimum temperature for flagella production is 30 ° C. One solution is therefore to favor the growth of the microorganism by incubating the sample at a temperature above 35 ° C, usually 37 ° C at first, and then to favor the production of flagella by lowering the temperature to 30 ° C, which allows the device according to the invention in its version incorporating at least one heating means. These thermal variations can be repeated several times, be progressive or rapid and follow or not a nonlinear evolution. These thermal variations may accompany a change in the height of culture medium obtained according to the methods described above. Thus, the possibility of automatically changing the temperature conditions during the incubation step b) offers a real advantage. The invention also relates to a rack for incubating and homogenizing the contents of at least one container, said content being formed by mixing a biological sample and at least one growth medium intended to promote growth. microorganisms present within said biological sample, said rack comprising at least one and preferably at least two locations for receiving said at least one container and at least one homogenizing means for homogenizing said contents, said rack being adapted for receiving at least one container comprising at least one wall of flexible material and said at least one homogenizing means comprising at least one applicator adapted to exert a force on said at least one flexible material wall in order to allow the deformation of said wall flexible material for changing the shape of said at least one container to homogenize the content.

Preferably, the applicator is adapted to exert, periodically, a force on the flexible material wall of the container (consisting for example of a container).

Preferably, the applicator is connected to a management / control element for adjusting / managing the intensity of the force exerted on the flexible material wall of the container and the frequency with which said force is applied.

According to a particular embodiment, the location for receiving the container is delimited by at least a first and a second support member, adapted to contact opposite sides of the container, wherein at least the first support member is removable relative to the second support member to change the distance between the first and second support member to change the force exerted on the flexible material wall of the container. According to a preferred embodiment, the location for receiving a container is delimited by at least one removable first and second arms, adapted to contact a first side of the container and at least one fixed support member adapted to come into contact with each other. with a second side of the container; said at least first and second movable arms being adapted to exert a force to urge said container against said at least one fixed support member.

According to a preferred embodiment, said at least first and second movable arms are adapted to move alternately.

According to another preferred embodiment, said at least first and second removable arms are adapted to move together. According to this preferred embodiment, the fact that the movable arms move together makes it possible to generate a "level elevation" of the content to a level n + 1, located above the level of homogenization ¾.

According to a particularly preferred embodiment, said at least first and second movable arms are adapted to move alternately or together. Preferably, and still according to this particularly preferred embodiment, if it is desired to achieve homogenization (soft) of the content, it generates - advantageously via the control means - an alternating movement of said at least a first and second arm removable, preferably at a frequency less than or equal to 2 Hz, preferably less than 2 Hz, preferably between 0.1 and 1 Hz, preferably between 0.45 and 0.7 Hz. However, when it is desired inducing a "raising level" of the content to a level n + 1, located above the level of homogenization ¾, it is preferably generated - advantageously via the control means - a joint displacement (simultaneous) of said at least first and second removable arms. The invention also relates to the use of the abovementioned carrier for implementing the method according to the invention. According to a particular embodiment, the device according to the invention comprises means for opening and closing the container (for example a means for opening and closing the sealing bag). Preferably, in the context of an automated process, this means of opening and closing the container is controlled by the aforementioned control means.

The invention also relates to the use of the aforementioned device for implementing the method according to the invention. Another subject of the invention relates to an incubator adapted for incubating at least one biological sample capable of containing at least one target microorganism at a temperature and for a period of time sufficient to allow the growth of the target microorganisms, said incubator comprising the device according to the invention.

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: FIG. 1 represents a perspective view of a device according to the invention ( "improved" rack), adapted to incubate the contents of a container,

FIG. 2 shows the device of FIG. 1, in side view,

Figures 3, 4 and 5 show the functionality of the removable arms (blades) for exerting pressure on a container, Figure 6 shows an alternative embodiment of an applicator for exerting a force on a container, - FIG. 7 shows a container containing a set consisting of a sample and a culture medium before taking up a culture means such as a selective agent (for example an antibiotic), FIG. 8 represents the container according to FIG. 7 during the recovery of a culture means,

FIG. 9 shows the container according to FIGS. 7 and 8 after taking up a culture means,

FIG. 10 represents a container, provided with a detection means such as a biosensor, before bringing this biosensor into contact with the contents of the container,

FIG. 11 shows the container according to FIG. 10 in which, following an elevation of the level of the contents, the latter comes into contact with the biosensor,

FIG. 12 shows a container provided with a set consisting of a sample and a culture medium, comprising first and second detection means consisting of two biosensors, FIG. 13 shows the container according to FIG. 12 after elevation. the level of the content and bringing said content into contact with the first biosensor, positioned in the enclosure of the container, below the second biosensor,

FIG. 14 represents the container according to FIGS. 12 and 13 after an additional elevation of the level of the content (larger than that represented in FIG. 13) and bringing said content into contact with the second biosensor, positioned in the enclosure of the container , above the first biosensor,

FIG. 15 represents, in a schematic manner, a sensitized capture support (at its capture zone) by a specific binding partner of the target bacteria to be detected (in this case a recombinant phage protein Salmonella), the Fig. 16 shows the sensitized capture support of Fig. 15 after contact with an enrichment medium supplemented with TTC (2,3,5-Triphenyltetrazolium chloride, reference T8877 SIGMA-ALDRICH), the red coloration at the level of capture zone revealing the presence of the target bacteria to be detected, FIG. 17 is intended to illustrate Example 2 infra and schematizes the immersion of a first capture medium of which an example is illustrated in FIG. 15 in two food samples A and B, FIG. 18, also intended to illustrate the Example 2 below shows schematically the bringing into contact of a second sensitized capture medium with the contents by automatic compression of the bag with the aid of the device according to the invention after 10 h of incubation ( to + 10h). 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 by means of examples and references to figures, but must in no way be regarded as limiting the scope of the invention. protection to the particular embodiments object of said examples and figures.

For the sake of clarity, FIG. 1 represents only a part of a device 1 according to the invention. The device 1 is provided with a base 2 on which a wall 10 has been fixed. The wall 10, according to the embodiment of FIG. 1, comprises a wall with a fixed position relative to the base 2.

In addition, the device 1 comprises a first and a second removable arms (similar to two blades) 11 and 12, which are removable relative to the base 2. The movement of said arms 11 and 12 relative to the base 2 can be generated by any suitable means, such as an electric motor.

The arms 11 and 12, removable, can move from a first position, indicated with the line 22, to a second position, indicated using the line 21. According to this Figure 1, the arm 11 is in the second position 21 and the arm 12 is in the first position 22. The arms 11 and 12 are removable so as to exert pressure on the outer surface of a container 40 positioned within the device 1, in a location 30 provided for this purpose. This location 30 is delimited on one side by the fixed wall 10 and on the other by all of the arms 11 and 12. This container 40 may be of the Stomacher ^® bag type. The container 40, as shown in FIG. 1, comprises a bag of flexible material able to receive, within it, a content comprising or constituted by the set of a sample 51 (represented in a much larger design than in reality ) and a culture medium 52. This culture medium 52 is, for example, in the liquid state.

The biological sample 51 may be any sample for which the user wishes to control the presence of microorganisms of interest. As indicated above, the sample may be of food, environmental or clinical origin (non-exhaustive list) and the microorganisms sought may be pathogenic microorganisms, for example of the Salmonella or E. coli type.

The culture medium 52, present inside the container 40 is intended to ensure the enrichment of the biological sample target microorganism (s) / sought (s). In other words, the culture medium 52 offers the microorganisms sought ideal conditions allowing, if present, to grow inside this container 40. Of course, as is known to those skilled in the art the culture medium (s) used may vary according to the desired microorganisms.

When the container 40 is positioned in the location 30, the assembly formed by the device 1 and the container 40 may, for example, be incubated inside an incubator (not shown). The conditions can be optimized within said incubator to allow growth of the desired microorganisms. Features, such as temperature, can be adjusted to be optimal for promoting the growth of target microorganisms. When the assembly formed of the device 1 and the container 40, positioned in the location 30, is introduced into the incubator, the operation of the arms 11 and 12 can be activated. Alternatively, and as indicated above, the device 1 advantageously comprises at least one heating means, for example at least one contact heating means (not shown). The arms 11 and 12 can move from their first position 22 to their second position 21 (and vice versa). This movement makes it possible to exert a force on the outer surface of the flexible wall of the container 40 and thus to impose on said container 40 a deformation of this flexible wall. This deformation has the effect of homogenizing, inside the container 40, its contents comprising the sample 51 and the culture medium 52. This homogenization aims to ensure the accessibility of the nutrients of the culture medium to the microorganisms present in the sample, more particularly to the target microorganisms. As indicated above, the arms 11 and 12 exert a weak to moderate force on the flexible wall of the container to allow a homogenization (also called "soft homogenization") of the contents and to avoid a "kneading" brutal, as is the case in the state of the art. It results from the weak to moderate force exerted on the flexible wall of the container by the arms 11 and 12 a slight rise in the level of the content within the container to a so-called level of homogenization ¾ (not shown in Figure 1).

The activation of the arms 11 and 12 makes it possible to guarantee a constant movement of said arms 11 and 12 (in a round-trip movement), with the aim of continuously homogenizing the assembly comprising the sample 51 and the medium The arms 11 and 12 can be moved periodically. The frequency can be chosen and adapted to the type of sample and / or the type of culture medium present inside the container 40.

As explained above, the arms 11 and 12 can move against the direction and / or in phase opposition (alternation of phase) to homogenize the assembly consisting of the sample 51 and the culture medium 52. On the other hand, the arms 11 and 12 can also move together (jointly) to change the level fluids present inside the container 40. The functionality of this movement is described with reference to Figure 5 (see below).

Figure 2 shows the device 1 in side view. The device 1 is shown with a base 2, and a first location 30 adapted to receive a first container 40, a second location 31 adapted to receive a second container 41 and a third location 32 for receiving a third container (not shown). The location 30 is delimited by the wall 10 and the set of arms 11 and 12. The location 31 is, in the same way, delimited by a wall 10 and arms 11 and 12. Similarly, the location 32 is delimited by a wall 10 and arms 11 and 12, as is the case for the locations 30 and 31. The device 1, as shown in Figure 2, therefore comprises three locations 30, 31 and 32. A container 40 is received in the location 30, a second container 41 is received in the location 31, the location 32 is unoccupied.

It should be noted that the device 1 according to the invention comprises at least one location 30 but may advantageously benefit from a large number of locations 30, 31, 32. The device may comprise 5, 10, 15, 20 or any other amount of locations 30, 31, 32, depending on the desired use.

Figures 3, 4 and 5 more clearly describe the operation of the assembly consisting of a wall 10 and arms 11 and 12. For the sake of clarity, no container is shown in these Figures 3, 4 and 5.

Figure 3 shows the arms 11 and 12 respectively in their first and second positions. The arms 11 and 12 can move from their respective positions to the position as shown in Figure 4. This means that the arm 11 moves from its first position to its second position. Simultaneously, the arm 12 moves from its second position to its first position. The arms 11 and 12 move against the direction (in opposition of phase), this allows the liquid of a container, sandwiched between the wall 10 and the arms 11 and 12, to remain substantially at the same level during the movement of said arms 11 and 12. Thus, the level of the liquid varies little during the homogenization step c) and remains essentially at the level of the level of homogenization ¾. However, the movement of the wall 10 of the container imposed by the arms 11 and 12 allows the contents of the container, interposed between the wall 10 and the arms 11 and 12 to be homogenized "gently", and thus avoid the step of "kneading" violent and its disadvantages.

As represented in FIG. 2, the device 1 makes it possible to homogenize several samples concomitantly during the first minutes of incubation, preferably between 10 and 60 minutes. Thus, as the homogenization time is longer, the mixing intensity is considerably reduced. The corresponding benefits are detailed above.

A better oxygenation of the sample can also be obtained thanks to this homogenization, thus allowing an increase in the biomass of the sample.

As shown in FIG. 5, the arms 11 and 12 can also move together towards their second position 21 in order to generate, in the container 40, a rise in the level of the content greater than that observed during the homogenization step vs). The technical effect of this embodiment, as well as its advantages, are described with reference to FIGS. 7 to 13.

As mentioned above, the arms 11 and 12 can move to their second position - indicated with the reference numeral 21 - but can also be positioned in any other suitable position, indicated with the help of the lines 23 and 24. The distance between the wall 10 and the assembly composed of the arms 11 and 12 must be defined in order to impose the container 40 a liquid level adapted to the desired objective. The arms 11 and 12 have been described with reference to FIGS. 1 to 5. It should be noted that the arms 11 and 12 may be replaced by applicators of various shapes, provided that they are capable of exerting a certain force. on the outside of a container 40. The arms 11 and 12 may, for example, be replaced by removable walls.

An alternative embodiment is shown in FIG. 6, in which a single applicator 13 can pivot about an axis of rotation 14. By performing a pivoting movement, the element 13 can move from the first position as indicated in FIG. 6 to a second position indicated with the aid of dots, while obtaining the same result as the movement obtained with the arms 11 and 12 and as explained with reference to FIGS. 3 and 4. In the position such that shown in Figure 6, the applicator 13 can move towards the wall 10, for example towards the line 25. With this movement, the applicator 13 can cause the modification of the level of the liquid present inside. a container 40 located between the wall 10 and said applicator 13. A container 40 is described in FIG. 7, said container comprising, within it, the assembly consisting of a sample 51 and a culture medium 52. In addition, the container 4 0 is provided with a selective agent (for example an antibiotic) 60 having been positioned inside said container 40, at a level higher than level 53 of the set consisting of the sample 51 and the culture medium 52 (and higher than the level of homogenization not shown). The container 40 may be placed in the device 1 according to the invention in order to be incubated.

During the incubation period, the contents of the container 40, that is to say the set consisting of the sample 51 and the culture medium 52, can be, at first, homogenized using the arm 11 and 12. In other words, the arms 11 and 12 move between the first and second positions as shown in Figures 3 and 4, in phase opposition. After a predefined time (depending on desiderata of the user), the arms 11 and 12 can be used to exert, together (jointly), a force on the outer wall of the container 40 of flexible material (deformable). This force causes deformation of the outer wall of the container 40 and the level 53 of the assembly consisting of the sample 51 and the culture medium 52 increases, from the position as shown in Figure 7 to the position as shown in FIG. FIG. 8. In this configuration, the two arms 11 and 12 are both positioned at their second position 21, as shown in FIG. 5. This embodiment is particularly advantageous since it makes it possible to delay the contact between the content (including a small amount of target microorganisms, if present) with the selective agent to direct the growth of microorganisms to that of the desired microorganisms. Thus microorganisms in the microbial stress phase are not directly put in contact with the selective agent, the latter risking, at this stage, either to slow down their growth and thus to increase the time required for the analysis, either to completely inhibit their growth and thus prevent their detection / identification.

Indeed, the target microorganisms are said to be "stressed" when they are present in the sample to be analyzed. The microorganisms (including the target microorganisms) need a certain amount of time to adapt to the existing conditions inside the container 40. In their "stressed" state, the target microorganisms are particularly sensitive, especially to the presence of selective agents such as antibiotics. During the incubation phase b), after the homogenization step c) and after a predefined time period and sufficient to ensure adequate growth of the microorganisms - and thus overcome the initial stress phase - an increase in the content level is generated by the simultaneous movement of the arms 11 and 12 from their first position 22 to their second position 21 (as shown in Figure 5). This elevation of level is represented in FIG. 8. The content reaches a level 53 (n + 1), higher than the level of homogenization ¾. Through this level elevation (from rest level n to level n + 1 or level homogenization ¾ to this level n + 1, depending on the desiderata of the user), the assembly consisting of the sample 51 and the culture medium 52, the contents of the container 40 can come into contact with the selective agent 60. This means that the selective agent 60 is added to the set consisting of the sample 51 and the culture medium 52, at a convenient time, that is to say when the microorganisms were able to overcome the initial stress phase and multiply using the nutrients available to them in the culture medium.

Regarding the recovery of the element 60, several back and forth of the liquid can be advantageous / necessary.

When the set consisting of the sample 51 and the culture medium 52 has been brought into contact with the selective agent 60, the arms 11 and 12 can be moved to their first position 22 in order to return to a rest level 53 ( level n), as shown in FIG. 9. Alternatively, a new homogenization step can be started directly after this step, in which case the arms 11 and 12 move against the direction and / or in opposition of the phase (alternation of phase) in order to homogenize the set consisting of the sample 51, the culture medium 52 and the selective agent 60. In this embodiment, the level of the content therefore passes directly from the n + 1 level to the level of the sample. homogenization ¾.

Subsequently, and if necessary still during the incubation phase, other steps, for example one or more detection steps can be performed by raising the level of the content above the level n + 1, so that the set consisting of the sample 51, the culture medium 52 and the selective agent 60 comes into contact with a detection means positioned within the container, above the level n + 1. This or these detection steps make it possible to detect the presence or absence of the target microorganisms. The device, as described in FIGS. 1 to 6, is particularly suitable for microbiological analysis of a food-type sample and, in particular, to a use for detecting the presence or absence of one or more pathogenic microorganisms such as bacteria.

Thus, the functionality for modifying the level 53 of the set consisting of the sample 51 and the culture medium 52 can also be used to bring said assembly together with a sensor type of detection means, such as a biosensor, being inside a container 40. This feature is described with reference to FIGS. 10 and 11. FIG. 10 shows a container 40 comprising, within it, a set consisting of a sample 51 and a A culture medium 52. As shown in this FIG. 10, the biosensor 70 is at a level above the level 53 of the set consisting of the sample 51 and the culture medium 52. More specifically, the the biosensor is positioned above the rest level of the content n and the level of homogenization ¾, so that it does not come into contact with the content during the homogenization step c). This makes it possible to preserve the integrity of the biosensor and to prevent biological sample residues from interfering unduly with this biosensor. At this stage, the arms 11 and 12 can serve to homogenize the contents of the container 40. At a predetermined time, the arms 11 and 12 can be used to raise the level 53 to the level as shown in FIG. in contact with the contents and the biosensor.

As shown in FIG. 11, the level 53 is sufficient to allow the biosensor 70 to come into contact with the set consisting of the sample 51 and the culture medium 52.

The biosensor is, for example, introduced into the container at the beginning of the incubation phase. This incubation phase extends, for example, over 24 hours during which the concentration of target microorganisms will gradually increase. In the first ten hours, for example, the concentration of target microorganisms is too low to interact with the biosensor. Thus, during these first ten hours, and as indicated above, it is preferable to maintain the biosensor away from the set composed of the sample and the culture medium to preserve its integrity and avoid deterioration of the capabilities of said biosensor because of non-specific compounds in the sample. In addition, it is possible to have, inside a container 40, a device comprising a culture means such as a selective agent or a reagent, the contents of this device being added to the assembly consisting of sample 51 and culture medium 52 when a certain pressure is exerted on the container 40 comprising this selective agent or reagent. The latter can be, for example, available in a device such as a compartment or a drawer, closed in a first position, which opens under the pressure of one or both arms 11 or 12, to allow mixing the selective agent or reagent and the assembly consisting of the sample 51 and the culture medium 52. Figures 12, 13 and 14 illustrate an alternative use of the method and the device according to the invention.

FIG. 12 shows a container 40 comprising a content consisting of a biological sample 51 (for example of food origin) and a culture medium 52. The content reaches a level 53 inside the container. This level 53 corresponds, in this figure 12, to the content "at rest" n.

The container 40 contains a first biosensor 121 (consisting for example of a solid phase functionalized with an antibody specific for a given bacterial species) and a second biosensor 122 (consisting for example of a solid phase functionalized with a specific bacteriophage protein of said bacterial species or of a different bacterial species), positioned within the container 40, above said first biosensor 121. Said first and second biosensors are positioned such that inside the container 40 they are out of position. reaching the content during the homogenization step (not shown), during which the level rises from rest level n to homogenization level ¾. When the content is "at rest" and during the step or steps of homogenization, said first and second biosensors are preserved, namely that they are not "polluted" / "degraded" in particular by the matrix debris content.

As represented in FIG. 13, the level elevation generated by the device according to the invention, from the rest level n to the level n + 1, situated above the levels n and ¾ (not shown), results in the setting contacting the content with the first biosensor 121, the second biosensor 122 being preserved since not immersed. Thus the first biosensor 121 is brought into contact with the microorganisms from the biological sample to be analyzed and, if the target bacteria are present among said microorganisms, the latter bind to their specific binding partner present at the level of said microorganism. first biosensor 121, for example to a functionalized antibody. The target bacteria are then (immuno) concentrated at the level of the first biosensor 121 and can be identified in situ, for example by immuno-detection techniques well known to those skilled in the art, implementing revelation systems equally well. known to this one. According to one variant, the identification step is carried out outside the container 40, for example by implementing a VIDAS ^® type automaton.

Be that as it may, at the end of the identification step, the analysis is either positive (detection and identification of the target bacteria) or negative (no detection and identification of said target bacteria). The elevation level represented in FIG. 13 may, in practice, consist of a succession of elevations and drops in level, from the level n or down to the level n + 1 and vice versa. In other words, the contents "lick" the first biosensor 121 in waves, in a bag-like movement and surf. After the elevation level represented in FIG. 13, a new elevation of level (of intensity greater than that shown in FIG. 13) can be generated by the device according to the invention, of the level n, ¾, or n + 1. to a level higher n + 2. This new stage of elevation is shown in FIG. 14. During this new elevation towards the level n + 2, the content 40 comes into contact with the second biosensor 122 (the first biosensor 121 being de facto also immersed) . If the target bacteria are present among said microorganisms, the latter bind to their specific binding partner present at said second biosensor 122, for example a specific bacteriophage protein of a given bacterial species. The target bacteria are then (immuno) concentrated at the level of the second biosensor 122 and can be identified in situ, for example by immuno-detection techniques well known to those skilled in the art, implementing revelation systems equally well. known to this one. According to one variant, the identification step is carried out outside the container 40, for example by implementing a VIDAS ^® type automaton. Thus, if using a first biosensor 121 comprising a specific binding partner of the bacterial species X of a first type, for example an antibody directed against the bacteria X, and a second biosensor 122 comprising a specific binding partner of the bacterial species X of a second type, for example a bacteriophage protein specific for the bacterial species X, the level-raising step represented in FIG. 13 is carried out in order to attempt to detect and identify the bacteria X after (immuno) concentration at the first biosensor 121. Following obtaining a result - positive or negative - the additional leveling step shown in FIG. 14 is carried out in order to confirm or refute the result obtained after (immuno) concentration at the first biosensor 121. This so-called "confirmation" step is carried out in order to try to detect and identify the bacteria X apr s (immuno) concentration at the second biosensor 122.

Note that a confirmation step of this type can be performed several hours after the leveling step shown in Figure 13 and is particularly useful when the result obtained after (immuno) concentration at the first biosensor 121 is negative. Just like the elevation level represented in FIG. 13, that represented in FIG. 14 can, in practice, consist of a succession of elevations and drops in level, from the level n, ¾ or n + 1 to the higher level n + 2 and vice versa. In other words, the content comes "lick" the second biosensor 122 waves, in a bag-like movement and surf.

Obviously many alternatives are possible, among which we can cite (non-exhaustive list):

the use of a first biosensor 121 comprising a specific binding partner of the bacterial species X and a second biosensor 122 comprising a specific binding partner of the bacterial species Y (the two binding partners can be of the same type) or of a different type),

the substitution of the first biosensor 121 and / or the second biosensor 122 by a culture means such as an antibody intended to guide the growth of one or more target microorganism (s).

According to a particular embodiment of the invention, the first biosensor 121 is replaced by at least one selective agent of the antibiotic type in FIGS. 12, 13 and 14 and the reference numeral 122 always designates a biosensor. According to this particular embodiment, the leveling step represented in FIG. 13 allows the content to be brought into contact with the selective agent, preferably after a step of homogenization of the medium and after incubation (or in progress). Incubation) of the content 40. During this leveling step, the biosensor 122 is preserved.

After this leveling step (and possibly after a new homogenization step), the additional leveling step shown in FIG. 14 is performed. The content 40 is then in contact with the biosensor 122. As previously, if the target bacteria are present among said microorganisms, the latter bind to their specific partner of binding present at the level of the biosensor 122 (for example a bacteriophage protein specific for a given bacterial species). The target bacteria are then (immuno) concentrated at the level of the second biosensor 122 and can be identified in situ, for example by immuno-detection techniques well known to those skilled in the art, implementing revelation systems equally well. known to this one. According to one variant, the identification step is carried out outside the container 40, for example by implementing a VIDAS ^® type automaton. In general, it should be noted that several alternatives are possible to introduce, within the set consisting of a biological sample 51 and a culture medium 52, a culture means such as a selective agent. . According to a preferred embodiment, the analysis method according to the invention is a detection method that can be implemented by visual or optical reading of a capture medium sensitized by a specific binding partner of the microorganism to be detected ( for example phage protein, antibodies, etc.). A preferred example of a sensitized capture medium is shown diagrammatically in FIGS. 15 and 16, under the reference 150. The lower part can be advantageously and according to a preferred embodiment divided into two zones. The zone 1501 (referred to as "capture zone") may be sensitized with a solution of binding partners (polyclonal antibodies, monoclonal antibodies, Fab 'or Fab'2 fragments, phage proteins, etc.), whereas the 1502 zone (called "control zone") remains untouched by any liaison partner and thus plays a negative control role.

By way of nonlimiting example, a suitable capture medium may be irradiated polystyrene, such as that marketed by Nunc / Thermo Scientific (Cat No. 472230). The capture medium is sensitized (functionalized) with at least one specific binding partner, selected for example from antibodies, aptamers, phages, recombinant phage proteins, or any equivalent means known to those skilled in the art and allowing the specific capture of the target bacteria.

Said target bacteria can be stained simultaneously with their growth thanks to the revelation system contained in the culture medium. In a particular example, the disclosure system is based on the reduction of TTC (2,3,5-Triphenyltetrazolium chloride, T8877 SIGMA-ALDRICH) by microorganisms. At the same time as growth, the TTC (colorless in its unreduced form) is internalized by said microorganisms, then reduced by the latter to triphenyl-formazan (red) thus dyeing said microorganisms in red and then allowing their revelation on the sensitized capture support, and more precisely at its capture zone referenced 1501 in FIGS. 15 and 16.

The method for detecting microorganisms in a food sample is thus carried out by visual or optical reading of a sensitized capture support, in an automated manner or not (preferably in an automated manner).

Once the effective capture of a certain quantity of colored target microorganisms (case of a positive sample), there is a change in the optical properties of the support by appearance of a red coloration on it (signal transduction organic). This coloration of the capture medium is then detectable with the eye and / or via a reading automaton such as a camera. When the sensitized capture support 150 (see FIGS. 15 and 16) is brought into contact with a medium comprising the target microorganisms, the capture zone 1501 appears colored (in red) because of the fixation of the target microorganisms. specific liaison partners. The control zone 1502, playing, as its name suggests, the role of negative control remains, for its part, the initial color of the capture medium. In order to facilitate reading, it is preferable that the sensitized capture medium is no longer in contact with the content during the visual or optical reading step. For this purpose, the device according to the invention is used to generate a "drop in level" towards the level of rest n or of homogenization ¾, so that the capture medium has emerged during the reading step. visual or optical.

According to a preferred embodiment, the device as described above is adapted to be introduced into an incubator, that is to say that it can be used in place of prior art racks in the purpose of introducing one or more samples into this incubator. Compared to these traditional racks, the device according to the invention - which can be regarded as an "improved rack" or "intelligent rack" - allows for a succession of enrichment steps and / or analysis of automated or semi-automated, without unnecessary human intervention, all or part of these steps being performed during the incubation period, hitherto not used.

In a particular embodiment, and as mentioned above, the device according to the invention comprises means for adjusting the temperature of the assembly consisting of a sample 51 and a culture medium 52. For example, and with reference to FIG. 2, the device is provided, at its base 2, possibly at the level of the wall 10 and possibly at the level of the blades (arms) 11 and 12, of contact heating means (s) allowing heating said base 2, which in turn heats the container 40 positioned in a location such as locations 30, 31, 32, etc., thereby heating the assembly consisting of a sample 51 and a culture medium 52.

Of course, a device of this nature equipped with such means (s) heating does not require / more incubation within an incubator. The device may, for example, be left on the bench of a laboratory possibly equipped with a hood to prevent heat dispersion. In addition, and as explained above, it will be possible to vary the temperature during incubation and benefit from an advantage over the selectivity or sensitivity of a test (see above).

The container 40, used in combination with the device 1, may be a container provided with a transparent outer wall, which facilitates the analysis of the biological processes in progress inside said container. In the case where the container 40 comprises walls of a transparent material, part of the analysis can be automated using optical means such as cameras and / or spectrometers.

According to a particular embodiment, after a given incubation period, an aliquot of the content is transferred into at least one other compartment of the device according to the invention, said at least one other compartment containing one or more selective agents depending on the or targeted microorganism (s).

Note, in addition, the possibility at the end of incubation, to bring the contents into contact with one or more dialysis casings (x) comprising at least one osmotic compound (for example polyethylene glycol (PEG)), which will absorb a quantity of water through the dialysis casing (s) to concentrate the amount of analytes in solution. According to a particular embodiment, the dialysis casing (s) are situated at a level higher than the resting and homogenization levels ¾, so that the content is brought into contact with the casing (s). analysis via at least one leveling step. The functionality of the invention is illustrated with the (non-limiting) examples presented below.

EXAMPLE 1 Elaboration of a Sensitized Capture Carrier with at least one Specific Binding Partner of the Target Microorganism (S. Napoli) for Optical Detection Purposes An irradiated polystyrene capture medium marketed by Nunc / Thermo Scientifc (Cat No. 472230) is shown in Figures 15 and 16.

The capture medium is sensitized in three stages as follows:

1) the polystyrene support is immersed at 37 ° C overnight in a solution of BSA (Bovine Serum Albumin) -Biotinylated at 5μg / mL;

2) the support is then immersed at 37 ° C. for two hours in a solution of streptavidin at 10 μg / ml;

3) the support is then immersed for two hours at 37 ° C. in a solution of specific binding partners (1 μg / ml at 4 μg / ml, the specific binding partner being a recombinant phage protein against Salmonella).

The sensitized support thus produced can be used for the optical detection of microorganisms or stored at 2-8 ° C for later use.

Example 2 Preservation of the sensitized capture support of Example 1 during the first phase of the incubation by delayed contacting said capture medium.

The applicant has discovered, surprisingly, that the delayed contacting of the capture medium leads to obtaining a higher signal. Indeed the degradation of said capture medium (such as fouling, loss of bioreceptors, etc.) is obviously less when the latter is immersed for a shorter period of time. During delayed contacting of the sensitized capture support of Example 1 with the cultured biological sample, this degradation is less. In addition, the target flora level is particularly high at the moment when the capture medium is in contact with the sample-culture medium mixture. As a result, the capture of the target microorganism (s) is then maximal. For the purposes of this example, the device according to the invention has been used. As detailed below, the detection is carried out during the incubation period by contacting, thanks to the device according to the invention, the lower part of the capture medium 150 of Example 1 (sensitized with a phage protein recombinant anti-Salmonella) and the contents of a closed container which contains a food sample, diluted 1 / 10th in the reaction medium. As previously mentioned, the lower portion of capture medium 150 includes capture area 1501 and control area 1502.

In order to measure the relative amount of target bacteria captured at capture zone 1501, the enrichment medium is supplemented with a cell marker. The label used is a tetrazolium salt, 2,3,5-Triphenyltetrazolium chloride (TTC, T8877 SIGMA-ALDRICH).

This water-soluble and colorless substrate is reduced inside the bacteria into an insoluble red compound, formazan. The intensity of the red coloration observed on the capture zone 1501 of the sensitized capture support of Example 1 will therefore be proportional to the number of target bacteria fixed on said sensitized capture support 150, at the level of the capture zone 1501 ( the control zone 1502 remaining, in principle, virgin of any coloration).

Protocol: Step 1: Resuspension of the Samples in the Reaction Medium Two samples are prepared as described below.

Samples A. In a container (Stomacher® bag) 25g of Ground Steak 15% MF contaminated with 10 colony-forming units (CFU) of S. Napoli are resuspended in 225ml of EPT (Buffered Peptone Water), bioMérieux, Cat No. 42043), supplemented with 0.01 g / l of vancomycin (Sigma, Cat No. 75423) and 0.4 g / l of TTC (bioMérieux, Cat No. 04568088).

Samples B. In a container (Stomacher® bag) 25g of 15% MG Ground Steak contaminated with 10 colony-forming units (CFU) of S. Napoli are resuspended in 225ml of EPT (bioMérieux, Cat # 42043), supplemented with 0.01 g / l of vancomycin (Sigma, Cat No. 75423) and 0.4 g / l of TTC (bioMérieux, Cat No. 04568088). For each sample, two repetitions were made.

Step 2: Immersion of sensitized capture supports in the container before incubation Sensitized capture supports are placed in each Stomacher® bag (samples A and B). The Stomacher® bags are then closed with a closing bar and placed in the device according to the invention and incubated in an oven at 37 ° C for 24 hours. Thus, one of the sensitized capture supports is immersed directly in the food sample (as shown in FIG. 17) and the second is brought into contact with the reaction medium by automatic compression of the bag using the device according to the invention. invention after 10 h of incubation (to + 10h), as described below and shown in Figure 18.

Step 3: Reading the capture media at the end of the incubation period

At the end of the incubation (24h at 37 ° C.), and following the nonspecific TTC reduction by all the bacteria present in the sample (ie the annex flora and the target flora), the reaction medium is colored in red. In order to be able to observe the sensitized capture supports revealing the positivity or the negativity of the analyzed sample, there is a decompression of the device according to the invention (lowering of the level of the fluid inside the container) making it possible to observe the surface sensitized capture media contacted with the reaction medium at + 10h. For that immersed in to, the bag is removed from the device according to the invention and then inclined to isolate the support of the reaction medium.

Thus, for the food samples A and B, there is a weak red to nonexistent coloration on the capture zone 1501 of the immersed capture media directly in the food sample at (contact time 24h). No red coloring appears in control zone 1502.

On the other hand, immersed capture media (brought into contact with the contents) after 10 h (to + 10 h) in each Stomacher® bag (samples A and B) by virtue of the device of the invention (see FIG. level of their capture zone 1501, a uniform and intense red coloration, thus revealing the presence of salmonella (S. Napoli) on the capture zone 1501 of said sensitized capture supports. No red coloring appears in control zone 1502.

Thus, the deferred immersion of the sensitized capture support, implemented thanks to the device of the present invention (and shown in FIG. 18), made it possible to preserve said capture supports (and in particular their capture zone 1501) against degradation and / or fouling due to prolonged immersion. These capture media have retained their integrity, thus increasing the amount of captured germs per unit area.

Example 3 Example of Use of the Method and the Device According to the Invention - Listeria Immuno Concentration Within the Container (Homogenization Pouch) and Transfer of the Solid Phase to the VIDAS Automaton

(detection of the presence of Listeria in a food sample)

3.1 VIDAS Listeria LPT protocol (state of the art) The VIDAS Listeria LPT protocol is as follows: 25 g of food sample are weighed in a plastic bag then homogenized, using a Stomacher® for 1 min in 225 ml of medium enrichment (bioMérieux LPT broth ref. 410848). The mixture is then incubated at 30 ° C for 26 to 30 hours. At the end of the incubation, the sample is homogenized manually and 0.5 ml are taken and introduced into the VIDAS bar for analysis. As the analysis volume is 0.5 ml, it is necessary to wait at least 26 hours for the concentration of Listeria to be sufficient to allow revelation by the VIDAS technique. In addition, sampling is mainly composed of non-specific bacteria and matrix debris that interfere with the sensitivity of the test by generating background noise. 3.2 Protocol according to the invention

25 g of food sample are weighed in a plastic bag, then 225 ml of enrichment medium (bioMérieux LPT broth No. 410848) are added as well as a solid phase functionalized with antibodies and / or recombinant phage proteins directed against Listeria. The functionalized solid phase is maintained above the level of the liquid. The plastic bag is directly incubated at 30 ° C in the device according to the present invention which manages the smooth homogenization of the sample during the first hour of incubation. About one hour before the sampling, ie after 15 hours of incubation, a phase of immuno-concentration of Listeria on the solid phase is triggered by successive "elevations of level", ie upward movements of liquid from " lick the functionalized surface. After 16 hours of incubation, the operator transfers the solid phase directly into the bar of the VIDAS PLC for analysis.

3.3 Comparison of the two protocols

The protocol (process) according to the invention has been found to be of dual benefit. Indeed, it allowed to:

to considerably reduce the incubation time by means of the immuno-concentration step from the entire volume of the sample (and not from a volume of 500 μί, as is the case in the VIDAS Listeria LPT protocol), and

to reduce the background noise of the test by transferring a solid phase and not a volume.

The "elevation level (s)" immuno-capture protocol during incubation may vary depending on the desired duration of contact of the solid phase with the sample. In addition, it may be advantageous to add to the protocol according to the invention an incubation step subsequent to the immuno-capture phase by "elevation (s) level" to allow colonization of the solid phase and thus increase the amount of germs per unit area. Moreover, after immuno-capture, the solid phase can be treated by other detection / analysis methods such as, for example, PCR or on Petri dish agar medium.

Example 4: Suppression of matrix interference for capture and concentration of target microorganisms from a food sample.

It is widely described in the literature that food particles are, inter alia, a limiting factor for capture and concentration techniques of target microorganisms before detection.

The purpose of this example is to compare:

i) food samples homogenized using the device according to the invention (FIG. 1), and

if) the same samples mixed by implementing a reference method using a specific instrument, the SMASHER ™, marketed by the company AES (reference AESAP1064), in order to disperse the target bacteria in the culture broth.

After a predefined incubation period, an immuno-capture step was performed on a fraction of the culture broth followed by detection of the pathogen of interest (in this case Escherichia coli O157H7, ref: ATCC 43888) by the PCR amplification technique (English "Polymerase Chain Reaction").

Protocol:

Step 1: Resuspend the samples in the reaction medium and incubate.

Eight samples, namely samples T1 (negative control), A, B, C, T2 (negative control), D, E, F are prepared in the following manner: in a container of the Stomacher® bag type, 75 g of ground beef 15% fat (MG) is resuspended in 225 ml of EPT (bioMérieux, Cat No. 42043) supplemented with 0.01 g / l of vancomycin (Sigma, Cat No. 75423). The four samples (A, B, C, Tl) are directly introduced into the device according to the invention (FIG. 1) for "soft" homogenization for 5 hours at 41 ° C. the device has been programmed with the following parameters:

Speed: 100 m / s

Frequency: 1.3 Hz

- Amplitude: 10 mm (from 22 mm to 12 mm between moving parts

(Numerals 11 and 12 in Figure 1) and the fixed element (reference numeral 10 in Figure 1)) These parameters ensure a displacement of the container liquid from the level at rest n to the level of homogenization nh. The displacement of liquid is thus less than 30%. The four other samples (D, E, F, T2) are, for their part, violently mixed for 1 min, by implementing the reference method in force referred to above, using SMASHER ™, marketed by AES (reference AESAP1064). ). After this "violent" mixing step, the four samples D, E, F, T2 are introduced into an incubator at 41 ° C. for 5 hours.

Step 2: Artificial Contamination of Samples A, B, C, D, E and F by Esche chia coli Q157H7 Ref: ATCC 43888. The six samples A, B, C, D, E and F are post-contaminated with pathogenic bacteria to to control the concentration before immuno-concentration. The target concentration of Esche chia coli 0157H7 is 10 CFU / ml (the acronym "CFU" stands for "Colonial Forming Unit"). Thus, 2250 CFU of Esche chia coli 0157H7 are introduced in three out of four samples for each of the two experimental conditions, namely in:

samples A, B, C (introduced into the device according to the invention, as represented in FIG. 1), and

samples D, E, F, (kneaded using SMASHER ™ and then incubated at 41 ° C for 5 hours).

Of course, the "negative control" samples T1 and T2 were not contaminated with E. coli O157H7.

Step 3: Immuno-concentration Esche chia coli Q157H7.

Regarding the immuno-capture step, a capture medium, namely a 5 cm ² non-woven polyethylene terephthalate filter, obtained from a filter bag for a kneader marketed by AES (reference 111,425), was functionalized by a specific binding partner by adapting the three-step protocol described in Example 1.

After incubation, 10 ml of each of the eight samples referred to above are taken and brought into contact with the capture medium for 30 minutes at 41 ° C. with stirring. Step 4: Detection of Escherichia coli Q157H7.

After the capture step, the capture medium is rinsed once in EasyMag buffer (bioMérieux ref 280132) before being heated to 100 ° C in order to release the DNA from the lysed cells. The extract is then analyzed by PCR using the Adiafood E. coli 0157 kit (ref: DFS6210a).

Step 5: Results.

Table 1 below, indicating the "cycle threshold" values, namely the number of cycles necessary for the fluorescence value of the probes (CY5 and FAM) to be above the positivity value of the test, is presented below. after:

Table 1

In the "Interpretation" column, the "+" sign denotes a positive result (detection of Escherichia coli 0157H7), while the "-" sign denotes a negative result (no detection of Escherichia coli 0157H7).

Conclusion Table 1 above clearly shows that the detection of Escherichia coli O157H7 at a concentration of 10 CFU / ml could only be carried out via the device according to the invention. Without being bound by the theory, the matrix interference generated by the "violent" mixing for 1 minute appears to have prevented the capture - and thus the detection - of the target pathogen.

Claims

1. Method for treating at least one biological sample capable of containing at least one target microorganism, said method being carried out within a container and comprising the following steps:

a) optionally bring said biological sample into contact with at least one culture medium within said container, the mixture of the biological sample and said culture medium forming all or part of the contents, b) optionally incubate the container at a temperature and for a period of time sufficient to allow the growth of said at least one target microorganism,

c) carry out at least one step of homogenization of the biological sample, during which the content moves from a level n, corresponding to the level of the content at rest, towards a homogenization level ¾, distinct from level n , and vice versa,

c') generate a movement of the content towards a level n+1, different from levels n and ¾, so that the content comes into contact with at least one culture means and/or at least one positioned analysis means(s ) within the container, between level n+1 inclusive and level ¾ excluded.

2. Method according to claim 1, said method comprising, before step c), the following steps:

a) bringing said biological sample into contact with at least one culture medium within said container, the mixture of the biological sample and said culture medium forming all or part of the contents,

b) incubate the container at a temperature and for a period of time sufficient to allow the growth of said at least one target microorganism.

3. Method according to claim 1 or 2, in which the movement of the content from level n towards level ¾ and the movement of the content from level n towards level n+1 are generated by the same means of movement, at two different intensities .

4. Method according to one of claims 1 to 3, said method comprising, subsequent to step c'), at least the following step:

c") generate a movement of the content towards a level n+l+x so that the content comes into contact with at least one culture means and/or at least one additional analysis means(s), positioned within the enclosure of the container, between levels n+l+x inclusive and level (n+l+x)-l excluded, in which x is an integer, preferably between 1 and 10.

5. Method according to one of claims 1 to 4, said method comprising a step consisting of generating the movement of the content towards a transfer level n _t , so that the transfer of all or part of said content takes place from this level transfer n _t from the container to another part of said container or at least one other container.

6. Method according to one of claims 2 to 5, in which the homogenization step c) is carried out at least partly during the incubation step b), preferably for a period of time greater than 2 minutes .

7. Method according to claim 6, in which the homogenization step c) begins concomitantly with the incubation step b) or in the first minutes of the incubation step b), preferably within a period of time between 1 and 10 minutes from the start of said incubation step b).

8. Method for enriching at least one biological sample capable of containing at least one target microorganism, said method implementing the method according to one of claims 1 to 7, in which said at least one culture means and/or at least one analysis means is a culture means such as a selective agent of antimicrobial type.

Method for analyzing at least one biological sample capable of containing at least one target microorganism, said method implementing the method according to one of claims 1 to 7, said at least one culture means and/or at least one analysis means being an analysis means such as a functionalized capture support or a biosensor, and said method comprising an additional step d) consisting of analyzing said at least one biological sample, preferably said at least one micro -target organism, via said means of analysis.

10. Method according to claim 9, said method comprising, before and/or after the analysis step d), at least one step of transferring all or part of the mixture comprising said biological sample, optionally the culture medium and said means of analysis, from the container, then called primary, to a second container called secondary.

11. Method according to claim 9 or 10, said method comprising, subsequent to the analysis step d), a confirmation step e) aimed at confirming or denying the analysis results obtained at the end of the step analysis d).

12. Method according to one of claims 1 to 11, in which, during the homogenization step c), the volume displaced from the level of the content at rest n towards the homogenization level ¾ is less than or equal to 50 %, advantageously less than or equal to 40%, preferably less than or equal to 30%, and the frequency of movement of the contents, during said homogenization step c), is less than or equal to 2 Hz, preferably less than 2 Hz, preferably between 0.1 and 1 Hz, advantageously between 0.45 and 0.7 Hz.

13. Device making it possible to implement the method according to one of claims 1 to 12, said device comprising at least one location for receiving at least one container, at least one movement means for generating the movement of the contents, in which the displacement means is capable of generating at least two displacements of the contents at at least two different intensities, the lowest displacement intensity allowing the homogenization of the biological sample and the highest displacement intensity making it possible to generate a moving the content so that it comes into contact with at least one culture means and/or at least one analysis means.

14. Device according to claim 13, said device comprising optical detection means making it possible to detect the presence of said target microorganism.

15. Device according to claim 13 or 14, said device comprising control means making it possible to vary the intensity of movement of the content.

16. Device according to one of claims 13 to 15, said device comprising at least one heating means making it possible to carry out the incubation step b).

17. Use of a device according to one of claims 13 to 16 for implementing the method according to one of claims 1 to 12.

18. Incubator suitable for incubating at least one biological sample likely to contain at least one target microorganism at a temperature and for a period of time sufficient to allow the growth of said at least one target microorganism, said incubator comprising a device according to one of claims 13 to 16.

19. Method for enriching at least one biological sample capable of containing at least one target microorganism, said method being carried out within a container and comprising the following steps: a) bringing said biological sample into contact with at least one at least one culture medium within said container, the mixture of the biological sample and said at least one culture medium forming all or part of the contents,

b) incubate the container at a temperature and for a period of time sufficient to allow the growth of said at least one target microorganism,

c) carry out homogenization of the biological sample during at least part of the incubation step b), preferably for a period of time greater than 2 minutes, said homogenization step c) being characterized by a displaced volume from the level of the content at rest n towards the homogenization level ¾ less than or equal to 50%, advantageously less than or equal to 40%, preferably less than or equal to 30%, and a frequency of movement of the content less than or equal to 2 Hz , preferably less than 2 Hz, preferably between 0.1 and 1 Hz, advantageously between 0.45 and 0.7 Hz.

20. Method for analyzing at least one target microorganism, said method implementing the enrichment method according to claim 19, said method comprising, subsequent to step c), an analysis step d) using at least one analysis means, said analysis step being carried out within the container or outside it.