Method for treating at least one biological sample containing a target microorganism

Abstract

One embodiment provides a device for processing at least one biological sample capable of containing at least one target microorganism within at least one container. The device having at least one displacement device for generating the displacement of the contents of the at least one container and at least one site for receiving the at least one container. Additionally, the at least one container can receive the at least one biological sample within the at least one container, the container being delimited by a wall fixed on a base. Further, the at least one displacement device may be movable with respect to the base, and the at least one container may include a flexible material which allows the at least one container to be compressed against said wall.

Claims

1. A device for processing at least one biological sample capable of containing at least one target microorganism within at least one container, said device comprising: at least one displacement device for generating the displacement of the contents of the at least one container; at least one site for receiving the at least one container, the at least one container being configured to receive the at least one biological sample within the at least one container wherein the site is delimited by a wall fixed on a base and the at least one displacement device, movable with respect to the base, wherein the at least one container comprises a flexible material to allow the at least one container to be compressed against the wall, wherein the at least one displacement device is movable with respect to the wall to exert a pressure onto the outer surface of the at least one container comprising the flexible material to impose on the at least one container a deformation for generating at least two displacements of the contents of the at least one container at at least two different intensities comprising: a weakest displacement intensity allowing the homogenization of the at least one biological sample wherein the at least one displacement device is displaced from it/their first position(s) to it/their second position(s), and vice versa, leading the contents of the at least one container to be displaced from a level n, corresponding to a level of the contents at rest, to a homogenization level nh, distinct from the level n, and vice versa and a strongest displacement intensity in a direction of the wall allowing a generation of an increased displacement of the contents to a level n+1, which is different from levels n and nh, such that the contents come into contact with at least one culture, at least one analysis device, or a combination thereof, positioned inside the at least one container, between level n+1 inclusive and level nh exclusive, wherein the at least one displacement device is selected from a movable arm, a movable blade, a movable wall and a moveable applicator.

2. The device according to claim 1, said device comprising an optical detection device configured to detect a presence of said at least one target microorganism.

3. The device according to claim 1, said device comprising a control element configured to alter the at least two different intensities of displacement of the contents.

4. The device according to claim 1, said device comprising at least one heating element configured to incubate the at least one container.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) The invention, its functionality, its applications and its advantages shall be better understood by reading the present description, made with reference to the figures, in which:

(2) FIG. 1 depicts a perspective view of a device according to the invention (improved “carrier”), suitable for incubating the contents of a container,

(3) FIG. 2 shows the device from FIG. 1, in lateral view,

(4) FIGS. 3, 4 and 5 show the functionality of the movable arms (blades) which make it possible to exert a pressure onto a container,

(5) FIG. 6 depicts an alternative embodiment of an applicator which makes it possible to exert a force onto a container,

(6) FIG. 7 shows a container containing an assembly consisting of a sample and a culture medium before the recovery of a culture means such as a selective agent (for example an antibiotic),

(7) FIG. 8 depicts the container according to FIG. 7 during the recovery of a culture means,

(8) FIG. 9 shows the container according to FIGS. 7 and 8 after the recovery of a culture means,

(9) FIG. 10 depicts a container, equipped with a detection means such as a biosensor, before this biosensor is put into the presence of the contents of the container,

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

(11) FIG. 12 depicts a container equipped with an assembly consisting of a sample and a culture medium, comprising a first and a second detection means consisting of two biosensors,

(12) FIG. 13 shows the container according to FIG. 12 after elevation of the level of the contents and placing said contents into contact with the first biosensor, positioned in the chamber of the container, below the second biosensor,

(13) FIG. 14 depicts the container according to FIGS. 12 and 13 after an additional elevation of the level of the contents (of greater magnitude than that depicted in FIG. 13) and placing said contents into contact with the first biosensor, positioned in the chamber of the container, above the first biosensor,

(14) FIG. 15 depicts, schematically, a capture support sensitised (at its capture zone) by a binding partner specific to the target bacteria to be detected (in this case an anti-Salmonella recombinant phage protein),

(15) FIG. 16 depicts the sensitised capture support from FIG. 15 after placing in contact with an enrichment medium supplemented with TTC (2,3,5-Triphenyltetrazolium chloride; ref. T8877 SIGMA-ALDRICH), the red coloration at the capture zone revealing the presence of the target bacteria to be detected,

(16) FIG. 17 aims to illustrate Example 2 infra and schematically depicts the immersion at t.sub.0 of a first capture support of which an example is illustrated in FIG. 15 in two food samples A and B,

(17) FIG. 18, also aiming to illustrate Example 2 presented below, depicts, schematically, the placing in contact of a second sensitised capture support with the contents by automatic compression of the bag with the aid of the device according to the invention after 10 h of incubation (t.sub.0+10 h).

DETAILED DESCRIPTION OF THE INVENTION

(18) The detailed description below aims to set out the invention in a manner which is sufficiently clear and complete, notably by means of examples and references to figures, but must by no means be regarded as limiting the scope of protection to the particular embodiments which are the subject of said examples and figures.

(19) For the purposes of clarity, FIG. 1 depicts only a part of a device 1 according to the invention. The device 1 is equipped with a base 2 onto which a wall 10 has been fixed. The wall 10, according to the embodiment from FIG. 1, comprises a wall with a fixed position relative to the base 2.

(20) Furthermore, the device 1 comprises a first and a second movable arm (similar to two blades) 11 and 12, which are movable in relation to the base 2. The movement of said arms 11 and 12 relative to the base 2 may be generated by any suitable means, such as an electric motor.

(21) The movable arms 11 and 12 may be displaced from a first position, indicated using the line 22, to a second position, indicated using the line 21. According to this FIG. 1, the arm 11 is in the second position 21 and the arm 12 is in the first position 22.

(22) The arms 11 and 12 are movable in order to be able to exert a pressure onto the outer surface of a container 40 positioned within the device 1, in a site 30 envisaged for this purpose. This site 30 is delimited on one side by the fixed wall 10 and on the other by the set of arms 11 and 12. This container 40 may be of the Stomacher® bag type. The container 40, such as shown in FIG. 1, comprises a bag made of flexible material able to receive, inside it, contents comprising or consisting of the assembly of a sample 51 (depicted in the drawing much larger than in reality) and a culture medium 52. This culture medium 52 is, for example, in the liquid state.

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

(24) The culture medium 52, present inside the container 40 aims to ensure the enrichment of the biological sample with target/sought microorganism(s). In other words, the culture medium 52 offers the microorganisms sought the ideal conditions which make it possible, if they are present, to grow inside this container 40. Of course, as is known to the person skilled in the art, the culture medium or media used may vary depending on the microorganisms sought.

(25) When the container 40 is positioned in the site 30, the assembly formed by the device 1 and the container 40 may, for example, be placed in incubation inside an incubator (not depicted). These conditions may be optimised inside said incubator to allow the growth of the microorganisms sought. Characteristics, such as the temperature, may be regulated so as to be optimal to promote the growth of the target microorganisms. When the assembly formed by the device 1 and the container 40, positioned in the site 30, is introduced into the incubator, the functioning of the arms 11 and 12 may be activated.

(26) Alternatively, and as indicated supra, the device 1 comprises advantageously at least one heating means (3 of FIG. 2), for example at least one contact heating means.

(27) The arms 11 and 12 may be displaced from their first position 22 to their second position 21 (and vice versa). This movement makes it possible to exert a force onto 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 serves to homogenise, inside the container 40, its contents comprising the sample 51 and the culture medium 52. This homogenisation has the objective of guaranteeing the accessibility of the nutrients in the culture medium to the microorganisms present in the sample, more particularly to the target microorganisms. As indicated previously, the arms 11 and 12 exert a weak-to-moderate force onto the flexible wall of the container in order to enable a homogenisation (also called “soft homogenisation”) of the contents and to avoid brutal “kneading”, as is the case in the prior art. There results from the weak-to-moderate force exerted onto the flexible wall of the container by the arms 11 and 12 a slight elevation of the level of the contents within the container to a level called the homogenisation level n.sub.h (not depicted in FIG. 1).

(28) The activation of the arms 11 and 12 makes it possible to guarantee a constant movement of said arms 11 and 12 (in a back-and-forth motion), with the aim of continuously homogenising the assembly comprising the sample 51 and the culture medium 52. The arms 11 and 12 may be displaced periodically. The frequency may be chosen and adapted to the sample type and/or to the culture medium type present inside the container 40.

(29) As explained above, the arms 11 and 12 may be displaced in opposite directions and/or in phase opposition (phase alternation) so as to homogenise the assembly consisting of the sample 51 and the culture medium 52. Furthermore, the arms 11 and 12 may also be displaced together (jointly) to modify the level of the fluids present inside the container 40. The functionality of this movement is described with reference to FIG. 5 (cf. infra).

(30) FIG. 2 depicts a lateral view of the device 1. The device 1 is depicted with a base 2, a heating element 3, a control element 4, and a first site 30 suitable for receiving a first container 40, a second site 31 suitable for receiving a second container 41 and a third site 32 making it possible to receive a third container (not shown).

(31) The site 30 is delimited by the wall 10 and the set of arms 11 and 12. The site 31 is, in the same way, delimited by a wall 10 and arms 11 and 12. Similarly, the site 32 is delimited by a wall 10 and arms 11 and 12, as is the case for the sites 30 and 31.

(32) The device 1, as depicted in FIG. 2, therefore comprises three sites 30, 31 and 32. One container 40 is received in the site 30, a second container 41 is received in the site 31, the site 32, for its part, being unoccupied.

(33) It should be noted that the device 1 according to the invention comprises at least one site 30 but may advantageously benefit from a large number of sites 30, 31, 32. The device may include 5, 10, 15, 20 or any other quantity of sites 30, 31, 32, depending on the desired use.

(34) FIGS. 3, 4 and 5 describe more clearly the functioning of the assembly composed of a wall 10 and the arms 11 and 12. For reasons of clarity, no container is depicted on these FIGS. 3, 4 and 5.

(35) FIG. 3 shows the arms 11 and 12, respectively in their first and second positions. The arms 11 and 12 may be displaced from their respective position to the position as depicted in FIG. 4. This means that the arm 11 is displaced from its first position to its second position. Simultaneously, the arm 12 is displaced from its second position to its first position. The arms 11 and 12 are displaced in opposite directions (in phase opposition), which makes it possible for the liquid in a container, which is enclosed between the wall 10 and the arms 11 and 12, to stay substantially at the same level during the movement of said arms 11 and 12. Thus, the level of the liquid varies little during the homogenisation step c) and remains substantially at the height of the homogenisation level n.sub.h. However, the movement of the wall 10 of the container imposed by the arms 11 and 12 enables the contents of the container, interposed between the wall 10 and the arms 11 and 12, to be homogenised “softly”, and thus enables the violent kneading step, and its disadvantages, to be avoided.

(36) As depicted in FIG. 2, the device 1 makes it possible to homogenise several samples concomitantly, during the first minutes of incubation, preferably between 10 and 60 minutes. Thus, as the homogenisation duration is more significant, the intensity of kneading is considerably reduced. The corresponding advantages are detailed supra.

(37) A better oxygenation of the sample may also be obtained due to this homogenisation, thus allowing an increase in the biomass of the sample.

(38) As depicted in FIG. 5, the arms 11 and 12 may also be displaced together to their second position 21 in order to generate, within the container 40, an elevation of the level of the contents greater than that observed during the homogenisation step c). The technical effect of this embodiment, as well as its advantages, are described with reference to FIGS. 7 to 13.

(39) As mentioned above, the arms 11 and 12 may be displaced to their second position—indicated by reference number 21—but may also be positioned in any other suitable position, indicated by lines 23 and 24. The distance between the wall 10 and the assembly made up of the arms 11 and 12 must be defined with the aim of imposing upon the container 40 a liquid level suitable for the objective sought.

(40) 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 forms, provided that they are capable of exerting a certain force on the exterior of a container 40. The arms 11 and 12 may, for example, be replaced by movable walls.

(41) One alternative embodiment is depicted in FIG. 6, wherein a single applicator 13 may pivot around an axis of rotation 14. By performing a pivot movement, the element 13 may be displaced from the first position as indicated in FIG. 6 to a second position indicated by a dotted line, whilst obtaining the same result as the movement obtained with the arms 11 and 12, and such as explained with reference to FIGS. 3 and 4.

(42) In the position such as shown in FIG. 6, the applicator 13 may be displaced in the direction of the wall 10, for example in the direction of the line 25. Through this movement, the applicator 13 may bring about the modification of the level of the liquid present inside a container 40 situated between the wall 10 and said applicator 13.

(43) A container 40 is described in FIG. 7, said container including, inside it, the assembly consisting of a sample 51 and a culture medium 52. Furthermore, the container 40 is equipped with a selective agent (for example an antibiotic) 60 having been positioned inside said container 40, at a level higher than the level 53 of the assembly consisting of the sample 51 and the culture medium 52 (and higher than the homogenisation level n.sub.h; not depicted). The container 40 may be placed in the device 1 according to the invention so as to be incubated.

(44) During the period of incubation, the contents of the container 40, that is to say the assembly consisting of the sample 51 and of the culture medium 52, may be, firstly, homogenised with the aid of the arms 11 and 12. In other words, the arms 11 and 12 move between the first and second positions as shown in FIGS. 3 and 4, in phase opposition. After a predefined period of time (depending on the wishes of the user), the arms 11 and 12 may be used so as to exert, together (jointly), a force onto the external wall of the container 40 made of flexible (deformable) material. This force brings about the deformation of the exterior 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 such as shown in FIG. 7 to the position such as shown in FIG. 8. In this configuration, the two arms 11 and 12 are both positioned at their second position 21, such as is depicted in FIG. 5. This embodiment is particularly advantageous because it makes it possible to defer the contacting of the contents (comprising a small quantity of target microorganisms, if the latter are present) with the selective agent intended for orienting the growth of the microorganisms toward that of the microorganisms sought. Thus the microorganisms in microbial stress phase are not directly placed in contact with the selective agent, since, at this stage, this risks either slowing their growth and therefore increasing the time necessary for analysis, or totally inhibiting their growth and, thus, impeding their detection/identification.

(45) Indeed, the target microorganisms are referred to as “stressed” when they are present in the sample to be analysed. The microorganisms (including the target microorganisms) need a certain period of time to adapt to the existing conditions inside the container 40. In their so-called “stressed” state, the target microorganisms are particularly sensitive, notably to the presence of selective agents such as antibiotics.

(46) During the incubation phase b), subsequent to the homogenisation step c) and after a predefined period of time sufficient to ensure adequate growth of the microorganisms—and thus to overcome the initial stress phase—an elevation of the level of the contents is generated by the simultaneous displacement of the arms 11 and 12 from their first position 22 to their second position 21 (as depicted in FIG. 5). This level elevation is depicted in FIG. 8. The contents reach a level 53 (n+1), which is higher than the homogenisation level n.sub.h. Via this level elevation (from rest level n to level n+1 or from the homogenisation level n.sub.h to this level n+1, depending on the wishes of the user), of the assembly consisting of the sample 51 and of the culture medium 52, the contents of the container 40 may come into contact with the selective agent 60. This means that the selective agent 60 is added to the assembly consisting of the sample 51 and of the culture medium 52, at an opportune moment, i.e. when the microorganisms have been able to overcome the initial stress phase and reproduce by using the nutrients at their disposal in the culture medium.

(47) Concerning the recovery of the element 60, several motions of the liquid to and fro may prove to be advantageous/necessary.

(48) When the assembly consisting of the sample 51 and of the culture medium 52 has been placed in contact with the selective agent 60, the arms 11 and 12 may be displaced to their first position 22 in order to resume a rest level 53 (level n), such as shown in FIG. 9. Alternatively, a new homogenisation step may be engaged directly after this step, in which case the arms 11 and 12 are displaced in opposite directions and/or in phase opposition (phase alternation) so as to homogenise the assembly consisting of the sample 51, of the culture medium 52 and of the selective agent 60. In this embodiment the level of the contents therefore passes directly from level n+1 to the homogenisation level n.sub.h.

(49) Subsequently, and where applicable still during the incubation phase, other steps, for example one or more detection steps may be carried out by elevation of the contents level above level n+1, such that the assembly consisting of the sample 51, of the culture medium 52 and of the selective agent 60 comes into contact with a detection means positioned within the container, above level n+1. This or these detection steps make it possible to reveal the presence or the absence of the target microorganisms.

(50) The device, such as described within FIGS. 1 to 6, is particularly suitable for a microbiological analysis of a food-type sample and, in particular, for a use which makes it possible to detect the presence or the absence of one or more pathogenic microorganisms such as bacteria.

(51) Thus, the functionality which makes it possible to modify the level 53 of the assembly consisting of the sample 51 and the culture medium 52 may also be harnessed to put said assembly into the presence of a sensor-type detection means, such as a biosensor, located inside a container 40. This functionality is described with reference to FIGS. 10 and 11.

(52) FIG. 10 depicts a container 40 comprising, inside it, an assembly consisting of a sample 51 and of a culture medium 52. As shown in this FIG. 10, the detection device (e.g., an optical detection device, a biosensor, etc.) 70 is located at a level situated above the level 53 of the assembly consisting of the sample 51 and of the culture medium 52. More precisely, the biosensor is positioned above the rest level of the contents n and the homogenisation level nh, such that it does not come into contact with the contents during the homogenisation 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 may serve to homogenise the contents of the container 40. At a predetermined moment, the arms 11 and 12 may be used to raise the level 53 to the level as shown in FIG. 11, and thus to place the contents and the biosensor in contact.

(53) As depicted in FIG. 11, the level 53 is sufficient to enable the biosensor 70 to come into contact with the assembly consisting of the sample 51 and of the culture medium 52.

(54) The biosensor is, for example, introduced into the container at the start of the incubation phase. This incubation phase extends, for example, over 24 hours during which the target microorganism concentration will increase progressively. In the first ten hours for example, the target microorganism concentration is too low to interact with the biosensor. Thus, during these first ten hours, and as indicated previously, it is preferable to keep the biosensor apart from the assembly composed of the sample and of the culture medium in order to preserve its integrity and prevent a deterioration of the capacities of said biosensor due to non-specific compounds contained in the sample.

(55) Furthermore, it is possible to place, 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 the sample 51 and of the culture medium 52, as long as a certain pressure is exerted onto the container 40 containing this selective agent or this reagent. The latter may 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, in order to allow the mixing of the selective agent or of the reagent, and of the unit consisting of the sample 51 and the culture medium 52.

(56) FIGS. 12, 13 and 14 illustrate an alternative use of the method and of the device according to the invention.

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

(58) The container 40 contains a first biosensor 121 (consisting for example of a solid phase functionalised by an antibody specific to a given bacterial species) and a second biosensor 122 (consisting for example of a solid phase functionalised by a bacteriophage protein specific to said bacterial species or to a different bacterial species), positioned within the container 40, above said first biosensor 121.

(59) Said first and second biosensors are positioned inside the container 40 such that they are out of reach of the contents during the homogenisation step (not depicted), during which the level rises from the rest level n to the homogenisation level n.sub.h. When the contents are “at rest” and during the homogenisation step or steps, said first and second biosensors are therefore preserved, namely they are not “polluted”/“degraded” notably by the matrix debris of the contents.

(60) As depicted in FIG. 13, the level elevation generated by the device according to the invention, from the rest level n to level n+1, situated above levels n and n.sub.h (not depicted), results in the contents being placed in contact with the first biosensor 121, the second biosensor 122 being preserved since it is not immersed. Thus the first biosensor 121 is placed in contact with the microorganisms from the biological sample to be analysed and, if the target bacteria are present amongst said microorganisms, the latter bind to their specific binding partner present on said first biosensor 121, for example to a functionalised antibody. The target bacteria are therefore (immuno) concentrated on the first biosensor 121 and may be identified in situ, for example by immuno-detection techniques well known to the person skilled in the art, implementing revelation systems also well known to him. According to a variant, the identification step is carried out outside the container 40, for example by employing a machine of the VIDAS® type.

(61) In any event, at the end of the identification step, the analysis reveals itself to be either positive (detection and identification of the target bacteria), or negative (absence of detection and identification of said target bacteria).

(62) The level elevation depicted in FIG. 13 may, in practice, consist in a succession of level elevations and falls, from level n or n.sub.h to level n+1 and vice versa. In other words, the contents “lap” the first biosensor 121 in waves, in a pounding and recoiling motion.

(63) Subsequent to the level elevation depicted in FIG. 13, a new level elevation (of greater intensity than that shown in FIG. 13) may be generated by the device according to the invention, from level n, n.sub.h, or n+1 to a higher level n+2. This new level elevation step is shown in FIG. 14. During this new level elevation to level n+2, the contents 40 come into contact with the second biosensor 122 (the first biosensor 121 being de facto also immersed). If the target bacteria are present amongst said microorganisms, the latter bind to their specific binding partner present on said second biosensor 122, for example a bacteriophage protein specific to a given bacterial species. The target bacteria are therefore (immuno)concentrated on the second biosensor 122 and may be identified in situ, for example by immuno-detection techniques well known to the person skilled in the art, implementing revelation systems also well known to him. According to a variant, the identification step is performed outside the container 40, for example by employing a machine of the VIDAS® type.

(64) Thus, if a first biosensor 121 comprising a binding partner specific to bacterial species X of a first type, for example an antibody directed against the bacteria X, and a second biosensor 122 comprising a binding partner specific to the bacterial species X of a second type, for example a bacteriophage protein specific to the bacterial species X, are used, the level elevation step depicted in FIG. 13 is carried out in order to attempt to detect and identify the bacteria X after (immuno)concentration on the first biosensor 121. Once a result—positive or negative—has been obtained, the additional level elevation step depicted in FIG. 14 is carried out in order to confirm or to overturn the result obtained after (immuno)concentration on the first biosensor 121. This so-called “confirmation” step is carried out in order to attempt to detect and identify the bacteria X after (immuno) concentration on the second biosensor 122.

(65) It should be noted that a confirmation step of this type may be carried out several hours after the level elevation step depicted in FIG. 13 and proves to be particularly useful when the result obtained after (immuno)concentration on the first biosensor 121 is negative.

(66) Just like the level elevation depicted in FIG. 13, the one depicted in FIG. 14 may, in practice, consist of a succession of level elevations and falls, from level n, n.sub.h or n+1 to the higher level n+2 and vice versa. In other words, the contents “lap” the second biosensor 122 in waves, in a pounding and recoiling motion.

(67) Of course, numerous alternatives can be envisaged, amongst which it is possible to cite (as a non-exhaustive list): the employment of a first biosensor 121 comprising a binding partner specific to the bacterial species X and a second biosensor 122 comprising a binding partner specific to the bacterial species Y (both binding partners being able to be of the same type or of a different type), the substitution of the first biosensor 121 and/or of the second biosensor 122 with culture means such as an antibody intended for orienting the growth of one or more target microorganism(s).

(68) According to a particular embodiment of the invention, the first biosensor 121 is replaced by at least one antibiotic-type selective agent in the FIGS. 12, 13 and 14 and the reference number 122 still designates a biosensor. According to this particular embodiment, the level elevation step depicted in FIG. 13 makes it possible to place the contents in contact with the selective agent, preferably after a step of homogenising the medium and after incubation (or during incubation) of the contents 40. During this level elevation step, the biosensor 122 is preserved.

(69) Subsequent to this level elevation step (and possibly after a new homogenisation step), the additional level elevation step depicted in FIG. 14 is performed. The contents 40 are therefore in contact with the biosensor 122. As previously, if the target bacteria are present amongst said microorganisms, the latter bind to their specific binding partner present on the second biosensor 122, (for example a bacteriophage protein specific to a given bacterial species). The target bacteria are then (immuno)concentrated on the second biosensor 122 and may be identified in situ, for example by immuno-detection techniques well known to the person skilled in the art, employing revelation systems also well known to him. According to a variant, the identification step is carried out outside the container 40, for example by employing a machine of the VIDAS® type.

(70) Generally, it should be noted that several alternatives are possible for introducing, within the assembly consisting of a biological sample 51 and a culture medium 52, a culture means such as a selective agent.

(71) According to a preferred embodiment, the analysis method according to the invention is a detection method which may be implemented by visually or optically reading a capture support sensitised by a binding partner specific to the microorganism to be detected (for example phage protein, antibody, etc.).

(72) A preferred example of sensitised capture support is depicted schematically in FIGS. 15 and 16, under the reference 150. The lower part may, advantageously and according to a preferential embodiment, be divided into two zones. The zone labelled 1501 (called the “capture zone”) may be sensitised with a solution of binding partners (polyclonal antibodies, monoclonal antibodies, Fab′ or Fab′2 fragments, phage proteins, etc.), whereas zone 1502 (called the “control zone”) remains free of any binding partner and thus acts as a negative control.

(73) By way of non-limiting example, an appropriate capture support may be made of irradiated polystyrene such as marketed by Nunc/Thermo Scientific (Cat. No. 472230).

(74) The capture support is sensitised (functionalised) by at least one specific binding partner, selected by way of example from antibodies, aptamers, phages, recombinant phage proteins, or any equivalent means which is known to the person skilled in the art and which enables the specific capture of the target bacteria.

(75) Said target bacteria can be coloured simultaneously with their growth due to the revelation system contained in the culture medium. According to a particular example, the revelation system is based on the reduction of TTC (2,3,5-Triphenyltetrazolium chloride; ref. T8877 SIGMA-ALDRICH) by the microorganisms. Simultaneously to the growth, the TTC (colourless in its non-reduced form) is internalised by said microorganisms, then reduced by the latter into triphenyl-formazan (red), thus colouring said microorganisms red and then enabling their revelation on the sensitised capture support, and more precisely in its capture zone referenced 1501 on FIGS. 15 and 16.

(76) The method of detecting microorganisms in a food sample is thus performed by automated or non-automated (preferably automated) visual or optical reading of a sensitised capture support.

(77) Once a certain quantity of coloured target microorganisms has been effectively captured (in case of a positive sample), a change of the optical properties of the support is produced by the appearance of a red coloration thereon (transduction of the biological signal). This coloration of the capture support is therefore detectable to the naked eye and/or via a reading machine such as a camera. When the sensitised capture support 150 (cf. FIGS. 15 and 16) is placed in contact with a medium comprising the target microorganisms, the capture zone 1501 appears coloured (in red) due to the fixation of the target microorganisms onto the specific binding partners. The control zone 1502, which, as its name indicates, acts as a negative control, remains, for its part, the initial colour of the capture support.

(78) In order to facilitate reading, it is preferable that the sensitised capture support is no longer in contact with the contents during the visual or optical reading step. To this end, the device according to the invention is utilised to generate a “fall in level” to the rest level n or homogenisation level nn, such that the capture support emerges during the visual or optical reading step.

(79) According to a preferred embodiment, the device such as described above is suitable for introduction into an incubator, i.e. this may be used in place of the carriers from the prior art with the aim of introducing one or more samples inside this incubator. Compared to these conventional carriers, the device according to the invention—which may be regarded as an “improved carrier” or “intelligent carrier”—makes it possible to carry out a succession of enrichment and/or analysis steps in an automated or semi-automated manner, without superfluous human intervention, all or part of these steps being carried out during the incubation period, hitherto unharnessed.

(80) In a particular embodiment, and as mentioned previously, the device according to the invention comprises means which make it possible to regulate 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, potentially on the wall 10 and potentially on the blades (arms) 11 and 12, with contact heating means which make it possible to heat said base 2, which in turn heats the container 40 positioned in a site such as the sites 30, 31, 32, etc., thus heating the assembly consisting of a sample 51 and a culture medium 52.

(81) Obviously, a device of this nature equipped with such heating means does not require/no longer requires incubation within an incubator. The device may, for example, be left on a laboratory bench potentially equipped with a cover to prevent heat dispersion. Furthermore, and as explained previously, it will be possible to alter the temperature during incubation and thus benefit from an advantage on the selectivity or on the sensitivity of a test (cf. supra).

(82) The container 40, used in combination with the device 1, may be a container furnished with a transparent outer wall, which facilitates the analysis of the biological processes in progress inside said container. If the container 40 comprises walls made of a transparent material, part of the analysis may be automated with the aid of optical means such as cameras and/or spectrometers.

(83) According to a particular embodiment, after a given incubation period, an aliquot of the contents 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 target microorganism(s).

(84) It should furthermore be noted that it is possible, at the end of incubation, to place the contents in contact with one or more dialysis case(s) comprising at least osmotic compound (for example polyethylene-glycol (PEG)), which will absorb a quantity of water through the dialysis case(s) in order to concentrate the quantity of analytes in solution. According to a particular embodiment, the dialysis case(s) are situated at a higher level than the rest level n and homogenisation level n.sub.h, such that the contents are placed in contact with the dialysis case(s) via at least one level elevation step.

(85) The functionality of the invention is illustrated with the (non-limiting) examples presented hereafter.

EXAMPLE 1

Development of a Capture Support Sensitised with at Least One Binding Partner Specific to the Target Microorganism (S. Napoli) for the Purposes of Optical Detection

(86) An irradiated polystyrene capture support, marketed by Nunc/Thermo Scientific (Cat. No. 472230), is depicted on FIGS. 15 and 16.

(87) The sensitisation of the capture support is carried out in three steps, as follows: 1) the polystyrene support is immersed at 37° C. overnight in a BSA (Bovine Serum Albumin) solution-biotinylated at 5 μg/mL; 2) the support is then immersed at 37° C. for two hours in a streptavidin solution 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 to 40 μg/mL; the specific binding partner being an anti-Salmonella recombinant phage protein).

(88) The sensitised support thus produced may be used for the optical detection of the microorganisms or stored at 2-8° C. with a view to subsequent use.

EXAMPLE 2

Preservation of the Sensitised Capture Support from Example 1 During the First Phase of the Incubation due to Deferred Contacting of Said Capture Support

(89) The applicant has discovered, surprisingly, that the deferred placing in contact of the capture support leads to a higher signal being obtained. Indeed the degradation of said capture support (such as the soiling, the loss of bioreceptors, etc.) is manifestly reduced when the latter is immersed for a shorter period of time. When the placing in contact of the sensitised capture support from Example 1 with the cultured biological sample is deferred, this degradation is reduced. Further, the target flora level is particularly high when the capture support is in contact with the sample-culture medium mixture. In consequence, the capture of the target microorganism(s) is then at a maximum.

(90) For the purposes of the present example, the device according to the invention was used. As detailed hereafter, the detection is performed during the incubation period by placing in contact, thanks to the device according to the invention, the lower part of the capture support 150 from Example 1 (sensitised with an anti-Salmonella recombinant phage protein) and the contents of a closed container which contains a food sample, diluted to 1/10th in the reaction medium. As mentioned previously, the lower part of the capture support 150 comprises the capture zone 1501 and the control zone 1502.

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

(92) This colourless water-soluble substrate is reduced inside bacteria into an insoluble red compound, formazan. The intensity of the red coloration observed in the capture zone 1501 of the sensitised capture support from Example 1 will therefore be proportional to the number of target bacteria fixed onto said sensitised capture support 150, in the capture zone 1501 (the control zone 1502 remaining, in principle, free from any coloration).

(93) Protocol:

(94) Step 1: Suspension of the Samples in the Reaction Medium

(95) Two samples are prepared as described hereafter.

(96) Samples A. In one container (Stomacher® bag) 25 g of ground beef 15% FM contaminated by 10 colony forming units (CFU) of S. Napoli are suspended in 225 ml of BPW (“buffered peptone water”, bioMérieux, Cat. No. 42043), supplemented by 0.01 g/l of vancomycin (Sigma, Cat. No. 75423) and 0.4 g/l of TTC (bioMérieux, Cat. No. 04568088).

(97) Samples B. In one container (Stomacher® bag) 25 g of ground beef 15% FM contaminated by 10 colony forming units (CFU) of S. Napoli are suspended in 225 ml of BPW (bioMérieux, Cat. No. 42043), supplemented by 0.01 g/l of vancomycin (Sigma, Cat. No. 75423) and 0.4 g/l of TTC (bioMérieux, Cat. No. 04568088).

(98) For each sample, two repetitions have been performed.

(99) Step 2: Immersion of the Sensitised Capture Supports in the Container before incubation

(100) The sensitised capture supports are placed in each Stomacher® bag (samples A and B). The Stomacher® bags are then resealed with the aid of a sealing clip then placed in the device according to the invention and incubated in an oven at 37° C. for 24 h. Thus one of the sensitised capture supports is immersed directly at to in the food sample (as depicted in FIG. 17) and the second is placed in contact with the reaction medium by automatic compression of the bag with the aid of the device according to the invention after 10 h of incubation (t.sub.0+10 h), as described below and depicted in FIG. 18.

(101) Step 3: Reading the Capture Supports at the End of the Incubation Period

(102) At the end of the incubation (24 h at 37° C.), and following the non-specific reduction of TTC by all of the bacteria present in the sample (i.e. the additional flora and the target flora), the reaction medium has been coloured red. To be able to observe the sensitised capture supports which reveal the positivity or negativity of the analysed 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 of the sensitised capture supports placed in contact with the reaction medium at t.sub.0+10 h. For the one immersed at t.sub.0, the bag is taken out of the device according to the invention then inclined so as to isolate the support from the reaction medium.

(103) Thus, for the food samples A and B, a low to non-existent red coloration is observed in the capture zone 1501 of the capture supports immersed directly in the food sample at to (contact time 24 h). No red coloration appears in control zone 1502.

(104) On the other hand, the capture supports immersed (placed in contact with the contents) after 10 h (t.sub.0+10 h) in each Stomacher® bag (samples A and B) through the device of the invention (cf. FIG. 18) exhibit in their capture zone 1501, a uniform and intense red coloration, thus revealing the presence of salmonellas (S. Napoli) in the capture zone 1501 of said sensitised capture supports. No red coloration appears in the control zone 1502.

(105) Thus, the deferred immersion of the sensitised capture support, implemented by the device of the present invention (and depicted in FIG. 18), has made it possible to preserve said capture supports (and in particular their capture zone 1501) against degradation and/or soiling due to prolonged immersion. These capture supports therefore conserved their integrity, thus increasing the quantity of germs captured per surface area unit.

EXAMPLE 3

Example of Use of the Method and of the Device According to the Invention—Immuno-concentration of Listeria within the Container (Homogenisation Pouch) then Transfer of the Solid Phase to the VIDAS Machine (Detection of the Presence of Listeria in a Food Sample)

(106) 3.1 VIDAS Listeria LPT Protocol (Prior Art)

(107) The VIDAS Listeria LPT protocol is the following: 25 g of food sample are weighed in a plastic pouch, then homogenised with the aid of a Stomacher® for 1 min in 225 ml of enrichment medium (bioMérieux LPT broth ref. 410848). The mixture is then incubated at 30° C. for 26 to 30 hours. At the end of incubation, the sample is manually homogenised and 0.5 ml are sampled and introduced into the VIDAS strip for analysis. As the analysis volume is 0.5 ml, it is necessary to wait for at least 26 hours so that the Listeria concentration is sufficient to enable revelation by the VIDAS technique. Furthermore, the sample is mainly composed of non-specific bacteria and matrix debris which interfere with the test sensitivity by generating background noise.

(108) 3.2 Protocol According to the Invention

(109) 25 g of food sample are weighed in a plastic pouch, then 225 ml of enrichment medium (bioMérieux LPT broth ref. 410848) are added as well as a solid phase functionalised by antibodies and/or recombinant phage proteins directed against Listeria. The functionalised solid phase is kept above the level of the liquid. The plastic pouch is directly placed in incubation, at 30° C., in the device according to the present invention which manages the soft homogenisation of the sample during the first hour of incubation. Approximately one hour before the sampling, i.e. after 15 h of incubation, a phase of immuno-concentration of the Listeria on the solid phase is triggered by successive “level elevations”, namely top-down movements of liquid “lap” the functionalised surface. At the end of 16 hours of incubation, the operator transfers the solid phase directly into the VIDAS machine strip for analysis.

(110) 3.3 Comparison of the Two Protocols

(111) The protocol (method) according to the invention has proven to be doubly advantageous. Indeed, it has made it possible: to considerably reduce the duration of incubation due to the immuno-concentration step from the full volume of the sample (and not from a volume of 500 μL, as is the case in the VIDAS Listeria LPT protocol), and to reduce the background noise of the test due to the transfer of a solid phase and no longer of a volume.

(112) The “level elevation(s)” immuno-capture protocol during the incubation may vary depending on the desired duration of contact of the solid phase with the sample.

(113) Furthermore, it may prove advantageous to add to the protocol according to the invention an incubation step subsequent to the “level elevation(s)” immuno-capture protocol in order to enable a colonisation of the solid phase and thus increase the quantity of germs per surface area unit.

(114) Moreover, after immuno-capture, the solid phase can be processed by other methods of detection/analysis such as for example PCR or on an agar medium in a Petri dish.

EXAMPLE 4

Suppression of the Matrix Interference for the Capture and Concentration of Target Microorganisms from a Food Sample

(115) It is widely described in the literature that food particles are, amongst others, a limiting factor for the techniques for capturing and concentrating target microorganisms before detection.

(116) The present example aims to establish a comparison between: i) food samples homogenised with the aid of the device according to the invention (FIG. 1), and ii) the same samples kneaded by implementing a reference method employing a specific instrument, the SMASHER™, marketed by AES (reference AESAP1064), in order to disperse the target bacteria in the culture broth.

(117) After a predefined period of incubation, an immuno-capture step was carried out on a fraction of the culture broth followed by a detection of the pathogen of interest (here Escherichia coli O157H7; ref: ATCC 43888) by the PCR (Polymerase Chain Reaction) amplification technique.

(118) Protocol:

(119) Step 1: Suspension of the Samples in the Reaction Medium and Incubation

(120) Eight samples, namely the samples T1 (negative control), A, B, C, T2 (negative control), D, E, F are prepared in the following manner: in a Stomacher® bag-type container, 75 g of ground beef 15% FM (fatty material) are placed in suspension in 225 ml of BPW (bioMérieux, Cat. No. 42043) supplemented by 0.01 g/l of vancomycin (Sigma, Cat. No. 75423).

(121) The four samples (A, B, C, T1) are directly introduced into the device according to the invention (FIG. 1) for “soft” homogenisation for 5 hours at 41° C. The device was programmed with the following parameters: Speed: 100 m/s Frequency 1.3 Hz Amplitude: 10 mm (from 22 mm to 12 mm between the mobile elements (numerical references 11 and 12 in FIG. 1) and the fixed element (numerical reference 10 in FIG. 1))

(122) These parameters ensure displacement of the container's liquid from the rest level n to the homogenisation level n.sub.h. The displacement of liquid is thus less than 30%.

(123) The four other samples (D, E, F, T2) are, for their part, kneaded violently for 1 min, by implementing the above-mentioned reference method in force, using the SMASHER™, marketed by AES (reference AESAP1064). Subsequent to this “violent” kneading step, the four samples D, E, F, T2 are introduced into an incubator at 41° C. for 5 hours.

(124) Step 2: Artificial Contamination of Samples A, B, C, D, E and F by Escherichia coli O157H7 Ref: ATCC 43888.

(125) The six samples A, B, C, D, E and F are post-contaminated with pathogenic bacteria so as to monitor the concentration before immuno-concentration. The concentration of Escherichia coli O157H7 targeted is 10 CFU/ml (the acronym “CFU” signifies “Colony Forming Unit”). Thus, 2250 CFU of Escherichia coli O157H7 are introduced into three samples out of four for each of the two experimental conditions, namely in: samples A, B, C (introduced into the device according to the invention, as depicted in FIG. 1), and samples D, E, F, (kneaded using the SMASHER™ then incubated at 41° C. for 5 hours).

(126) Of course, the “negative control” samples T1 and T2 were not contaminated with E. coli O157H7.

(127) Step 3: Immuno-concentration of Escherichia coli O157H7.

(128) Concerning the immuno-capture step, a capture support, namely a 5 cm.sup.2 non-woven poly(ethylene terephthalate) filter, from a filter bag for a kneader marketed by AES (reference 111 425), was functionalised by a specific binding partner by adapting the three-step protocol described in Example 1.

(129) After incubation, 10 ml of each of the eight samples mentioned above are taken and placed in contact with the capture support for 30 minutes at 41° C. under agitation.

(130) Step 4: Detection of Escherichia coli O157H7.

(131) After the capture step, the capture support is rinsed once in EasyMag buffer (bioMérieux ref. 280132) before being heated to 100° C. in order to release the DNA of the lysed cells. The extract is then analysed by PCR by means of the Adiafood E. coli O157 kit (ref: DFS6210a).

(132) Step 5: Results.

(133) Table 1 infra, 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 hereafter:

(134) TABLE-US-00001 TABLE 1 Homogenisation/ CY5 FAM Inter- kneading mode Sample (Ct) (Ct) pretation Device according T1 −1 −1 − to the invention (negative (“soft” homogenisation) control) A 34.41432 34.96523 + B 34.46399 34.75306 + C 34.45198 35.29125 + Smasher ™ T2 −1 −1 − (“violent” kneading) (negative control) D −1 −1 − E −1 −1 − F −1 −1 −
In the column headed “Interpretation”, the “+” sign denotes a positive result (detection of Escherichia coli O157H7), whereas the “−” sign denotes a negative result (absence of detection of Escherichia coli; O157H7).
Conclusion

(135) Table 1 supra clearly shows that it was possible to perform the detection of Escherichia coli O157H7 at a concentration of 10 UFC/ml only by means of the device according to the invention. Without being bound by the theory, the matrix interference generated by “violent” kneading for 1 minute appears to have impeded the capture—and therefore the detection—of the target pathogen.