Automated system for the lysis of microorganisms present in a sample, for extraction and for purification of the nucleic acids of said microorganisms for purposes of analysis

Abstract

A method for collecting microorganisms when contained in a fluid includes (i) introducing the fluid into a cavity of a collecting device via at least one admission duct, (ii) capturing the microorganisms when contained in the fluid with a set of beads retained in the cavity as the fluid passes through the set of beads, (iii) evacuating the fluid from the cavity via at least one evacuating duct, (iv) introducing a reaction liquid into the cavity via at least one admission channel, (v) collecting the microorganisms from the set of beads with the reaction liquid as the reaction liquid passes through the set of beads, and (vi) evacuating the reaction liquid from the cavity via at least one evacuating channel.

Claims

1. A method for collecting microorganisms when the microorganisms are contained in a fluid, comprising: introducing the fluid into a cavity of a collecting device via at least one admission duct; capturing the microorganisms when the microorganisms are contained in the fluid by adhesion of the microorganisms onto surfaces of a set of beads retained in the cavity as the fluid passes through the set of beads; evacuating the fluid from the cavity via at least one evacuating duct; introducing a reaction liquid into the cavity via at least one admission channel; detaching the microorganisms from the surfaces of the set of beads with the reaction liquid as the reaction liquid passes through the set of beads; and evacuating the reaction liquid from the cavity via at least one evacuating channel, wherein the admission duct is different from the admission channel and the evacuating duct is different from the evacuating channel, and wherein the set of breads includes beads coated with a substance to facilitate the adhesion of the microorganisms onto the surfaces of the set of beads.

2. The method as claimed in claim 1, wherein the admission channel and the evacuating channel and the admission duct and the evacuating duct are positioned as follows: the admission duct and the evacuating duct face one another along an axis; the admission channel and the evacuating channel are positioned respectively at two opposite ends of the cavity; and the admission channel and the evacuating channel are positioned in a plane perpendicular to the axis.

3. The method as claimed in claim 2, wherein the cavity has a “C” shape if the cavity is cut along the plane perpendicular to the axis in which the admission channel and the evacuating channel are positioned, to ensure that the reaction liquid passes completely through the set of beads between a moment when the reaction liquid is admitted to the cavity and a moment when the reaction liquid is evacuated from the cavity.

4. The method as claimed in claim 3, wherein the cavity has a quadrilateral shape if the cavity is cut along a radius passing through the center of the “C” shape and cut[ting] through the “C” shape.

5. The method as claimed in claim 3, wherein the admission channel is connected to one end of the “C” shape of the cavity, and the evacuating channel is connected to the other end of the “C” shape.

6. The method as claimed in claim 1, wherein at least one upper plate and at least one bottom plate of the collecting device [are configured to] close a reaction module including the cavity [to] and form an airtight confinement enclosure in order to isolate the fluid and the reaction liquid from outside of the collecting device.

7. The method as claimed in claim 1, wherein the beads have a diameter in the range from 200 to 600 μm and are retained in the cavity by retaining elements that comprise grids including pores having a diameter smaller than the diameter of the beads and having a diameter in the range from 100 to 500 μm.

8. The method as claimed in claim 1, wherein the fluid is a gas, the set of beads includes beads coated with glycerol, and the reaction liquid liquefies the glycerol upon contacting the reaction liquid to the beads coated with glycerol.

9. The method as claimed in claim 1, wherein the fluid is a liquid and passes through the set of beads and then a filter before it being evacuated from the cavity.

10. The method as claimed in claim 1, wherein the reaction liquid is filtered after it being evacuated from the cavity via the evacuating channel.

11. The method as claimed in claim 1, further comprising agitating the set of beads to mechanically lyse membranes of the microorganisms [in connection with detaching] during a process which the microorganisms are detached from the surfaces of the set of beads.

12. The method as claimed in claim 11, wherein [the beads are agitated] said agitating the set of beads is by ultrasound.

13. The method as claimed in claim 1, further comprising mechanically lysing membranes of the microorganisms to release nucleic acids from the microorganisms and separating the nucleic acids from cellular residues of the microorganisms in the collecting device.

14. The method as claimed in claim 13, further comprising obtaining amplicons by amplifying the nucleic acids [to obtain amplicons] and detecting the amplicons in the collecting device.

Description

(1) The aims and advantages of the device according to the present invention will be better understood in light of the following example, not in any way limiting, referring to the figures, in which:

(2) FIG. 1 shows an exploded top view of the card or cartridge used for collecting and lysing the microorganisms with all the elements that allow it to function, according to a particular embodiment of the invention.

(3) FIG. 2 shows a perspective top view of the main card forming the core of the device according to the embodiment of the invention shown in FIG. 1.

(4) FIG. 3 shows a perspective bottom view of the main card, still according to the embodiment of the invention shown in FIG. 1.

(5) FIG. 4 shows a detail of FIG. 2 at the level of the bed of beads and grid retaining them in the plane in contact with at least one fluid admission duct and allowing capture of the microorganisms. The two opposite ends of the C-shaped module are connected fluidically to the channels for admitting and evacuating the reaction liquid.

(6) FIG. 5A is a flowsheet of the filtering part of the main card where the beads are present when the confining plates are closed.

(7) FIG. 5B is a flowsheet similar to that of FIG. 5A, but in which the confining plates are open.

(8) FIG. 6 gives a detail A from FIG. 5B but in which the movement of the fluid within the capture beads and the movement of the confining plates are highlighted.

(9) FIG. 7 is identical to FIG. 6 but focuses on the new movement of the confining plates and the start of movement of the reaction liquid within the capture beads.

(10) FIG. 8 is identical to FIG. 7 and clearly shows the movement of the fluid within said capture beads.

(11) FIG. 9 is identical to FIG. 8 but emphasizes the extraction of the nucleic acids.

(12) FIGS. 10A to 10D show the effectiveness of filling of the chamber where the microorganisms are retained by the retaining means over the course of time.

(13) FIG. 11 shows a graph demonstrating the efficiency of lysis within a device according to the invention.

(14) The present invention relates to a microorganism collecting device 1. FIG. 1 shows an exploded view of one embodiment presented by the applicant in which the microorganism collecting device 1 is presented without the presence of any fluid 2 or liquid 12. Said collecting device 1 essentially comprises two main parts, a main card 4 positioned in a lower position relative to a secondary card 5, which is in an upper position. The main card 4 is L-shaped, and the secondary card 5 is integrated in this “L” shape to give a general parallelepipedal shape. The main card 4 and secondary card 5 are intended to be brought into contact with one another. In this way they will form a means 9 for retaining beads 6, essentially consisting of a cavity present in the horizontal portion of the “L” shape, this cavity 9 being closed by the bottom face of the secondary card 5 in contact with card 4. The two cards, the main card 4 and the secondary card 5, once assembled form the reaction module 3. However, the retaining means 9 does not simply consist of the edges of this reaction module 3 but also comprises one or more grids 27, not shown in FIG. 1, allowing passage of fluid 2 or liquids 12 but without allowing the beads 6, also not shown, to leave the reaction module 3. On the grid 27, shown in FIG. 4, the diameter of the holes 28 in the grid 27 depends on the diameter of the beads 6 used. The holes 28 in the grid 27 must be of smaller diameter than the diameter of the glass beads. In a preferred embodiment, the holes 28 in the grid 27 must be of significantly smaller diameter than the diameter of the glass beads, “significantly” meaning for example that the beads 6 have a diameter from 400 to 600 μm and that the grid 27 has holes 28 from 200 to 250 μm.

(15) The assembly of the two cards 4 and 5 as well as the ducts for admission and evacuation of the fluid 40 and 41 and channels for admitting and evacuating the reaction liquid 7 and 8, shown in FIG. 4, is confined using an airtight confinement enclosure 13, which consists, in the embodiment presented in FIG. 1, of a certain number of confining plates.

(16) Firstly, in an upper position, there are two top plates 14 and 15. Then in a lower position there are two bottom plates 16 and 17; plates 14 and 16 provide confinement of the reaction module 3 notably at the level of the fluidic channels of the analysis zone 26, which will be described later. Then there are the top and bottom ferromagnetic confining plates 15 and 17, which for their part are positioned above said reaction module 3.

(17) After the capture step, plates 15 and 17 are repositioned to close the consumable. Only the microorganisms 10 remain in the chamber 9. Channels 7 and 8 are then used for circulating the elution buffer before the ultrasonic lysis step.

(18) It should be noted that the top plate 15 and the bottom plate 17 are of a ferromagnetic nature as they enclose a certain number of magnets 21 positioned at the four corners of the reaction module 3 as well as at its center. The magnets 21 provide, on the one hand, pressure of the ferromagnetic plates 15 and 17 on the O-ring seals 18 and, on the other hand, hermeticity after capture of the microorganisms. These plates 15 and 17 provide access to the capture beads 6, and make it possible to capture the microorganisms 10 present in the fluid 2 on the beads 6. Plates 15 and 17 slide over the surface of the device 1 on slides 29, located on the sides, and in the present case they are of dovetail shape. On completion of air sampling, said plates 15 and 17 are repositioned to their original location by sliding along the slides 29. The liquid circuit is then closed. A “clamping” force is required on the plates in order to ensure perfect hermeticity on closure. This force is for example ferromagnetic, which has the advantage of being a uniform pressure force on the entire surface of the plates in question.

(19) Between the main card 4 and secondary card 5 and the top plate 15 and bottom plate 17, hermeticity is ensured by a set of hermetic O-ring seals 18. These O-ring seals 18 are three in number in the upper position and three in the lower position and are concentric. Of course, this number is not in any way limiting since the use of a single O-ring seal may suffice. Moreover, other sealing means exist between the top plate 14 and bottom plate 16 and the rest of the device 1. The means employed are notably gluing of each of these plates with a glue or a double-sided adhesive on the main card 4. Similarly, the secondary card 5 is glued permanently on the body 4 of the device. This gluing is performed ultrasonically, by laser or thermally, it is permanent and allows assembly of the collecting device 1.

(20) It should be noted that the main card 4 comprises two zones where there are channels. Firstly there is a storage zone referenced 25 near the reaction module 3. There is also an analysis zone 26, which was mentioned above. The various channels present at the level of zones 25 and 26 allow fluidic management of the whole card. The channel in the storage zone 25 is the reservoir for storage of the reaction liquid 12 (elution buffer). The channels in the analysis zone 26 are the fluidic channels for sample preparation for analysis. They circulate on the front and back of the card. Fluorescence reading also takes place in the channels in zone 26. Since these zones 25 and 26 are not the essence of the invention, they will not be described further.

(21) These types of channels and their functions are better explained in a previous patent application WO-A-2011/033231 filed by the applicant under French priority of Sep. 18, 2009. The reader is invited to refer to this for fuller information.

(22) After capture of the microorganisms 10, plates 15 and 17 are closed again. The buffer from chamber 25 is sent into the capture chamber 9 for resuspending the microorganisms. Then ultrasonic lysis is carried out in this same chamber 9 by a means external to the card. The lysate is then shared via the distributor valve 22, which slides in the hole 31, and is sent into the channels of zone 26 by means of the pump pistons 23, which slide in holes 32, for preparation for detection and amplification of the nucleic acids 11.

(23) However, it should be understood that various elements allow fluidic management and transfer of the reaction liquid 12 from compartment to compartment through the intervention of the distributor valve 22, as well as the pump pistons 23. Three of these pump pistons 23 are on the left of the main card 4, and the other three are on the right of this same card 4.

(24) Finally, to finish the description of FIG. 1, it is noted that there are desiccating stations 24, still within the main card 4. These stations 24 are located near the analysis zone 26, and contain drying agents for optimizing storage of the reagents integrated with the card in the form of beads of lyophilized reagent, also called “pellets”, obtained by lyophilization, by drying or else by gelation. The drying agents are not in direct contact with but are close to the pellets. These pellets contain the reagents necessary for amplification of the nucleic acids 11.

(25) The distributor valve 22 controls the distribution of the fluids in the collecting device 1. Accordingly, there are several positions, which provide: storage of the reaction liquid 12, transfer of the reaction liquid 12 to the chamber or retaining means 9 containing the beads 6 (after capture of the microorganisms 10), transfer of a total volume of 120 μl of the lysate, or 20 μl per channel, to the six channels of zone 26.

(26) Finally, the pump pistons 23 are similar to syringes and allow aspiration and transfer of the reaction liquid 12 throughout the process. Their particular feature is that they function in a closed air cycle (no air is aspirated from the exterior), although a simpler version with aspiration of external air is conceivable. The benefit of this closed vessel is maximum avoidance of communication with the exterior and thus risk of cross contamination from the interior to the exterior and vice versa.

(27) It can be seen in FIG. 1 that there is a spring plate 19 for locking the ferromagnetic plates 15 and 17. This plate 19 is attached to the reaction module 3 by a screw 20. Thus, it should not be possible to manipulate these detachable plates unintentionally. This spring plate 19 makes it possible to block and prevent unintended opening of them.

(28) FIG. 2 shows a more detailed view of the main card 4. The latter consists of two portions of different thickness; on the left the thickness is greater and comprises the analysis zone 26. We also note the presence of the drying stations 24, as well as a hole 31 for receiving the distributor valve 22, which is not shown in this figure. Finally there are also six holes 32 for receiving the six pump pistons 23, also not shown in this figure, within the main card 4. On the portion with small thickness of the main card 4, i.e. on the right of this figure, we note the presence of the storage zone 25 for the reaction liquid 12, after said reaction liquid 12 has: resuspended the captured microorganisms 10, provided lysis through ultrasonic agitation of the beads 6 via this liquid 12, transported the organisms of interest, and finally supplied the reaction mixture 12 necessary for amplification and detection of the nucleic acids 11.

(29) FIG. 3 shows the secondary card 4. It is noted that on this portion there are locations for receiving the O-ring seals 18 as well as locations 33 for receiving the magnets 21; seals 18 and magnets 21 are not shown in this figure.

(30) FIG. 4 shows an enlarged view of a detail of FIG. 2, at the level of the retaining means 9 of the beads 6 as well as channels 7 and 8. The two opposite ends of the C-shaped module are connected fluidically to the channels for admitting and evacuating the reaction liquid 7 and 8.

(31) In the present case, the means 9 consists of a grid 27, shown more clearly in FIG. 5A or 5B. It is noted that the space separating the holes 28 of the grid 27 is small; for example for beads of 500 μm, the holes will have a diameter from 200 to 300 μm and will be separated from one another by spaces of 200 to 300 μm, which leads to the fluids 2 or liquids 12 being forced into this space, which improves and increases the points of contact between the microorganisms, containing the nucleic acids of interest, and the surface of the beads 6 allowing better capture of said nucleic acids 11. The beads 6 are also coated with a substance that further facilitates adhesion of the microorganisms on the surface of the beads. This substance is notably glycerol in the case when the fluid used is a gas. Thus, the use of glycerol only functions in the case of collecting microorganisms in a gas, notably air. In this instance, the air passes over the glycerol and the microorganisms are captured on the latter. The glycerol is then dissolved by the reaction buffer 12. If bacteria are present, they will then be lysed in this buffer by ultrasound. When collecting microorganisms present in water, this does not function with glycerol, which is dissolved by water, and is driven out of the card. To achieve correct operation with a liquid medium, a replacement must be found for glycerol, for example a polymer coating that does not dissolve in water but only in a specific reaction buffer or allows “salting out” in this specific buffer, or a filter may be added downstream of the beads so that the fluid comes into contact with the beads before circulating through the filter.

(32) According to FIGS. 5A and 5B, at the level of the main card 4, with the chamber or retaining means 9, it is noted that there are two ducts referenced 40 and 41. Duct 40 allows admission of fluid 2 into the module 3, said fluid 2 containing the bacteria to be captured for subsequent analysis, whereas duct 41 allows evacuation of this same fluid 2 to the exterior of said module 3. Of course, between admission and evacuation, the fluid 2 will be passed through the module 3 where the beads 6 are present; it is at this level that collection of the microorganisms can be performed. If the fluid 2 is introduced and evacuated via ducts 40 and 41, the reaction liquid 12, which may consist of an elution buffer or other buffer, is introduced via an admission channel 7 into module 3, shown in FIG. 4, it is also evacuated from this same module 3 via an evacuating channel 8, also shown in FIG. 44.

(33) In fact this fluid 2, for example air, but it may also be a liquid different from the reaction liquid 12, circulates perpendicularly to the card. It flows through the grids 27.

(34) FIGS. 5 to 8 give a simplified description of the protocol for capture of a microorganism.

(35) FIG. 5A describes a portion of the device 1 according to the invention, at the level of treatment of the fluid 2, from which we wish to extract the microorganisms 10, in which there are: beads 6 present in the cavity, forming retaining means 9, an upper grid and a lower grid 27, perforated by several holes 28, which confine the beads 6 within the device 1 and therefore the retaining means 9 of the card 4, a ferromagnetic top plate for confinement 15, a ferromagnetic bottom plate for confinement 17, plates 15 and 17 not being glued to card 4 like the top and bottom plates for confinement 14 and 16. the fluidic channel, not shown in this figure. According to FIG. 5B, device 1 is more or less identical to the configuration in FIG. 5A, but the ferromagnetic top plate for confinement 15 and the ferromagnetic bottom plate for confinement 17 are translated along F1. This allows the fluid 2 to pass, according to F2, into the beads 6, held in position by the grids 27, by a movement perpendicular to the channels for the reaction liquid or liquids, not shown in the figure. This movement is performed via the admission duct 40 and evacuating duct 41 of the fluid 2 within module 3.

(36) FIG. 6 shows detail A from FIG. 5B. In this configuration, the detachable confining plates 15 and 17 are removed according to F1, and the fluid 2 circulates perpendicularly to the axis of the grids 27. The microorganisms 10 are captured on the beads 6 within the retaining means 9.

(37) In FIG. 7, the detachable plates 15 and 17 are returned to the closed position by a sliding motion according to F3. The elution buffer 12 is introduced into the chamber 9 via the admission channel 7. It detaches the microorganisms 10 from the beads 6.

(38) Finally in FIG. 8, once the chamber 9 is full, ultrasonic lysis is carried out to disrupt the microorganisms, for example bacteria, 10 and release the nucleic acids 11. The lysate containing the nucleic acids 11 is sent in the channels via the evacuating channel 8, for preparing amplification by resuspending the beads of lyophilized reagent, by optical detection, etc.

(39) The device therefore makes it possible to capture the microorganisms 10 notably from the air on glycerol-coated glass beads by circulating the air stream through a bed of beads 6.

(40) Capture of microorganisms by means of a dry surface offers many advantages, such as: absence of wear of the medium during capture (due to evaporation), no evaporation during storage, very high concentration of the microorganisms, very large developed capture surface (due to the microbeads) relative to a flat impacting surface, the capture surface does not saturate so easily, capture is therefore much more efficient than in an impaction system.

(41) Moreover, the device is designed to: offer a large surface area out of plane for capture of the microorganisms from the fluid. reduce the head losses and therefore the overall dimensions of the pump; thus, during capture of airborne microorganisms, the device is connected to a pump that takes in air through the capture grid 27. allow the microorganisms to be taken up in any liquid buffer simply by pipetting owing to the use of fluidic channels in the capture plane; thus, after capture of the microorganisms on the glycerol, the detachable plates 15 and 17 are closed again. The resuspension buffer (stored in chamber 25) is forced into the chamber containing the beads. The microorganisms are then taken in the glycerol; the latter dissolves in the buffer, so that the microorganisms captured are released. integrate lysis (notably with ultrasound) in the consumable or device for collecting microorganisms 1, obtain a high capture efficiency with a small head loss, as the air passes over a bed of glycerol-coated beads (and is not impacted on a bed of beads).

(42) The capture efficiency largely depends on the thickness of the bed of beads and the size of the beads, but the capture efficiency is significantly higher with capture in transmission through the beads, notably for particle sizes under 1 μm. This capture mode presents technical difficulties, such as: control of the head loss, keeping the beads in the capture chamber, taking up the microorganisms in a buffer, lysis.

(43) Dry capture is performed without bubbling or aqueous gel, without loss of efficiency over time (>4 m.sup.3 sample). The advantage of glycerol is that it does not dry out. Accordingly, the volume of air sampled can be very large, without notable loss of efficiency. Systems with capture on aqueous surfaces (agar, Petri dish) or aqueous liquid (Coriolis type) have the drawback of drying out as capture proceeds. This drying causes a decrease in capture efficiency. Capture in one defined liquid determines the type of analysis performed downstream of collection whereas with dry capture the microorganisms may be taken up in any buffer.

(44) Moreover, capture on a dry surface (glass beads+glycerol) allows the microorganisms to be released in a very small volume of buffer, less than 1 mL or even less than 100 μL. The advantage of increasing the concentration of the microorganisms is that it promotes detection of them. The systems for detection in molecular biology have a limit of detection of the order of 1 genome equivalent per microliter (μL).

(45) With the conventional devices, it is therefore necessary to capture a far larger quantity (from 15 to 30 times more, i.e. from 200 to 600 μL) of microorganisms as the capture volume is 15 mL (large volume that dilutes the microorganisms).

(46) Mechanical lysis is very effective and is compatible with all types of biological methods, such as ultrasonic lysis in any buffer required for the next steps of the protocol.

(47) Lysis of the captured microorganisms is integrated in the device. The lysis technique is purely mechanical (in contrast to chemical lysis) and, thus ensures an excellent lysis yield, regardless of the lysis buffer selected and the microorganism to be lysed.

(48) It is thus possible to use the lysate as it is, and then carry out amplification, for example NASBA or PCR.

(49) According to a preferred use of device 1 proposed by the invention, after the microorganisms 10 have been lysed and the nucleic acids 11 have been taken up in the reaction liquid or elution buffer 12, the collecting device 1 makes it possible to perform amplification as well as detection of said extracted nucleic acids 11. For this purpose, there are mainly four steps that are carried out after lysis.

(50) The first step consists of extracting the buffer 12 containing the nucleic acids 11 as well as a certain number of residues 30 of microorganisms 10, which are present in the liquid 12 after lysis, these residues not being taken into account subsequently in the analyses that will be carried out at the level of the analysis zone 26. In this first step, the liquid 12 laden with the nucleic acids 11 and the residues 30 will exit from the retaining means 9 via the evacuating channel 8. At the level of the evacuating channel 8, it is for example possible to have a filter that only allows biological elements to pass that have a size less than or equal to the nucleic acids extracted.

(51) In a second step this mixture is sent into a zone for taking aliquots 34, as clearly shown in FIG. 2. The volume thus obtained corresponds to 120 μl.

(52) This aliquot is then sent to a third step in a lysate separation zone referenced 35. In this figure, there are six zones that are well differentiated from one another to allow separation in six times 20 μl in the analysis channels of zone 26. In these channels of zone 26, amplification is carried out, by means of reagents for amplification, such as nucleotides, amplification primers and detection probes, which are positioned in the intermediate zone 36. It can be seen from FIG. 3 that there is a second intermediate zone 37 containing at least one enzyme (one enzyme if carrying out PCR, two if carrying out TMA and three if carrying out NASBA), which is located on the front of the main card 4.

(53) The fourth and last step takes place at the level of the channels of zone 26, as is clearly shown in FIG. 3 to the right of the intermediate zone 37 where detection may be performed.

(54) All of these movements are therefore performed by means of the distributor valve 22 and the pump pistons 23, which by their sliding motion, not shown in the figures, make it possible to direct the liquid from the various zones to other zones that will allow amplification of the nucleic acids 11 and their subsequent detection. The distributor valve 22 and the pump pistons 23 possess seals, clearly shown in FIGS. 1, 38 and 39 respectively, which ensure hermeticity of the system.

Example 1: Efficiency of Capture of Microorganisms (with a Particle Counter) as a Function of Bead Size and Thickness of the Bed of Beads

(55) For one and the same device, here is the protocol for capture efficiency as a function of bead size and thickness of the bed of beads.

(56) 1.A. Procedure:

(57) The sliding plates 15 and 17 are open throughout the experiment. For this experiment, a particle counter is used which evaluates the quantity of particles per cubic meter (m.sup.3) of aspirated air. The particles detected are classified in different size categories between 0.3 μm, 0.5 μm, 1 μm and 5 μm.

(58) Three cycles of measurements are carried out: 1: Blank reference measurement without the device. 2: Evaluation measurement with the device upstream of the particle counter. 3: A second reference measurement.

(59) 1.B. Experiment:

(60) The blank reference measurements are averaged. The capture efficiency is calculated as the ratio of the measurements with and without the device.

(61) The results are shown in Table 1 below:

(62) TABLE-US-00001 TABLE 1 Efficiency (in %) of capture of the microorganisms (with a particle counter) as a function of bead size and thickness of the bed of beads Bead diameter Particle sizes Thickness of the bed of beads 0.3 μm 0.5 μm 1 μm 5 μm 212-300 μm 50 87 95 98 1.5 mm 212-300 μm 65 85 92 95 2.5-3 mm 425-600 μm 18 65 87 95 1.5 mm 425-600 μm 45 85 90 89 2.5-3 mm WO-A-2009/001010 0 20 65 95

(63) 1.C. Analysis:

(64) For one and the same device 1, Table 1 shows that the capture efficiency is very effective regardless of the bead size and the thickness of the bed of beads relative to the prior art consisting of the device described in the PCT document. Particles smaller than 0.5 μm do not appear to give a sufficient yield, although it is still far higher than the prior art (minimum 18% yield versus zero). For a size between 0.5 and 5 μm the results are good (always above 65% and even, if we exclude the case at 65%, always above 85%). All the thicknesses of the bed of beads tested are acceptable.

Example 2: Optimization of the Head Loss of Each of the Devices Tested

(65) 2.A. Procedure:

(66) Each device is placed on a test bench, allowing the head loss to be measured as a function of the air flow rate. The objective is to find the best compromise between capture efficiency of the device 1 and its head loss. A high head loss signifies high electrical consumption of the sampling device (not favorable for portable applications).

(67) 2.B. Experiment:

(68) Table 2 is a table for aiding design of the device for capture of microorganisms. It compares the particle capture efficiency (by size range: 0.5-1 μm, 1-5 μm, 5-25 μm) with the head loss (mbar) of the device. The energy required for pumping a certain volume of air through the device is proportional to the head loss (for one and the same flow rate). For application with a portable pump it is necessary to find a good compromise between the capture efficiency and the head loss (see Table 2).

(69) TABLE-US-00002 TABLE 2 Efficiency of capture of microorganisms by particle size ranges and head loss of the device tested (at 50 L/min) as a function of bead size and thickness of the bed of beads Particle sizes Bead diameter 0.5- 1- 5- Head loss Thickness of the bed of beads 1 μm 5 μm 25 μm (mbar) 212-300 μm 75 95.5 95 15 1.5 mm 212 -300 μm 80 92 95 25-30 2.5-3 mm 425-600 μm 56 89 93 6-7 1.5 mm 425-600 μm 72 89 88 15 2.5-3 mm WO-A-2009/001010 43 80 94.5 6-11

(70) 2.C. Analysis:

(71) In light of this table and in comparison with patent application WO-A-2009/001010, constituting the prior art, it can be seen that the device according to the invention, regardless of the thickness of the bed of beads and the diameter of the beads used, always gives better performance than the solution proposed by the prior art. Moreover, with beads from 425 to 600 μm and a bed thickness of 1.5 mm, the results are the most efficient for capturing the particles than the device of patent WO-A-2009/001010 for an equivalent head loss.

Example 3: Filling and Emptying the Devices

(72) 3.A. Procedure:

(73) Only the chamber 9 of beads 6 is tested. The inlet of the bead chamber (full of beads but without liquid) is connected to a fluidic connector and a pipette. The liquid is forced into the bead chamber until it is full. Once filled, it is emptied in the same way.

(74) 3.B. Experiment

(75) FIGS. 10A to 10D show filling of the chamber 9 where the microorganisms 10 are captured. 500 μL is injected at the level of the admission channel 7 and 200 μL is recovered at the level of the evacuating channel 8.

(76) 3.C. Results:

(77) Filling takes place perfectly.

Example 4: Lysis of the Microorganisms

(78) 4.A. Procedure:

(79) A device 1 consisting of a lysis chamber is filled with a buffer 12 containing Staphylococcus epidermidis. The device is subjected to ultrasound, via a sonotrode, in order to lyse the microorganisms. The experiment is repeated with a lysis time of 0, 1, 5 and 10 minutes. The lysis yield is evaluated by growth and counting of the lysates on a Petri dish. The objective of this experiment is to find a suitable interface for transmission of ultrasound in the lysis step.

(80) 4.B. Experiment:

(81) In relation to FIG. 11, one and the same design of bead chamber device is used with some interface configurations, i.e.: 1st configuration: with plastic elements replacing the ferromagnetic plates 15 and 17 (sonotrode in direct contact with a modified device according to the invention), 2nd configuration: with a layer of silicone (present between the sonotrode and the device according to the invention), 3rd configuration: with metallic elements serving as ferromagnetic plates 15 and 17 added to the device.

(82) The three devices are tested as before and for two types of bead diameter (see Table 3).

(83) TABLE-US-00003 TABLE 3 Efficiency of ultrasonic lysis Silicone Device Standard Metal insert layer Diameter of 212-300 425-600 212-300 425-600 212-300 glass bead (μm) Lysis  90-100 50 100 98 40-55 efficiency (%)

(84) 4.C. Results:

(85) The results are quite good for all of the tests carried out. However, the solution using the metal inserts gives good results that are more or less identical for both types of beads.

Example 5: Lysis and NASBA Amplification without Purification of Microorganisms (S. epidermidis)

(86) 5.A. Procedure:

(87) A device is filled with buffer containing S. epidermidis bacteria as well as an internal control consisting of a bacterium different from the bacterium tested. Lysis is carried out in the device according to the 3rd configuration described in example 4. The lysate is then removed from the device and analyzed.

(88) 5.B. Experiment:

(89) The detection curves are presented in Tables 4 and 5 below.

(90) TABLE-US-00004 Reference (control range) (eq. CFU/μl) 0 0.1 1 10 100 1000 10000 Result Neg- Pos- Pos- Pos- Pos- Pos- Pos- ative itive itive itive itive itive itive

(91) TABLE-US-00005 Samples tested (eq. CFU/μl) 0 0.2 2 20 200 Result Negative Positive Positive Positive Positive

(92) This experiment shows the efficiency of the device described for lysing the microorganisms and for detecting them by NASBA analysis. The microorganisms are injected into the buffer 12 before the experiment (a different concentration of microorganism in each experiment). The metal flaps of the card are closed. The part containing the beads is filled with buffer 12 (which contains various concentrations of microorganisms). The device is placed on an ultrasound probe to perform lysis. At the end of the lysis step, the buffer 12 is collected manually and analyzed by NASBA away from the card. These results are to be compared with a control range.

(93) By first intention, the interpretation of the results is binary: Positive signifies that there is detection of the microorganism and negative signifies that the microorganism is not detected.

(94) 5.C. Analysis:

(95) It can be seen from the above tables that the samples tested are marked as positive for all concentrations above 0.2 CFU/μl.

REFERENCE SYMBOLS

(96) 1. Device for collecting microorganisms 10 2. Fluid 3. Reaction module 4. Main card 5. Secondary card 6. Beads 7. Reaction liquid admission channel 12 of module 3 8. Channel for evacuating the reaction liquid 12 from module 3 9. Retaining means of the beads 6 within module 3 10. Microorganisms present in the fluid 2 11. Nucleic acids present in the microorganisms 10 12. Reaction liquid 13. Airtight confinement enclosure consisting of plates 14 to 17 14. Top plate for confinement of device 1 15. Sliding ferromagnetic top plate for confinement of device 1 16. Bottom plate for confinement of device 1 17. Sliding ferromagnetic bottom plate for confinement of device 1 18. Hermetic O-ring seals between the enclosure 13 and the cards 4 and 5 19. Spring plate for locking the ferromagnetic plates 15 and 17 20. Fixing screw of the spring plate 19 21. Magnets 22. Distributor valve 23. Pump pistons 24. Desiccating stations 25. Storage zone of reaction liquid 12 26. Analysis zone on the front and back of the card 27. Upper and lower grids 28. Holes in grid 27 29. Slides 30. Residues of microorganisms present in the liquid 12 after lysis 31. Hole for receiving the distributor valve 22 32. Holes for receiving the pump pistons 23 33. Through-holes of cards 4 and 5 receiving the magnets 21 34. Aliquoting zone 35. Separation zone 36. First intermediate zone containing an enzyme 37. Second intermediate zone containing an enzyme 38. Seals on the distributor valve 22 39. Seals on the pump pistons 23 40. Fluid admission duct 2 within module 3 41. Duct for removing the fluid 2 within module 3 F1. Sliding motion of the ferromagnetic top plate 15 and ferromagnetic bottom plate 17 allowing opening for passage of fluid 2 F2. Movement of fluid 2 passing through beads 6 F3. Sliding motion of the ferromagnetic top plate 15 and ferromagnetic bottom plate 17 allowing closure to passage of liquid 12 F4. Movement of liquid 12 passing through beads 6 F5. Extraction of the nucleic acids 11