FR3032719A1 - DIAGNOSTIC DEVICE BY ISOTHERMAL AMPLIFICATION OF DNA - Google Patents

Abstract

The device (10) diagnostic isothermal amplification of DNA comprises: - a substrate (105) porous comprising: - a zone (110) comprising at least one compound (115) reagent reacting with a compound (120) target of a sample, called "diagnostic zone", - a zone (125) for deposition of fluid transported, by capillarity, in the sample deposition zone and in the diagnostic zone, - the zone (130) of sample deposition positioned between the fluid deposition zone and the diagnostic zone, and two channels (135, 140) positioned in the thickness of the porous substrate, respectively between, on the one hand, the fluid deposition zone and the zone sample deposit, on the other hand, the sample deposit area and the diagnostic area; and - means (205) for local isothermal heating the diagnostic zone.

Description

TECHNICAL FIELD OF THE INVENTION The present invention relates to a diagnostic device by isothermal amplification of DNA. It applies, in particular, to the diagnosis of Ebola virus from drops of blood, urine or saliva.

STATE OF THE ART In the field of medical diagnosis, there exist, inter alia, two families of biological tests: On the one hand, the family of immunoassay tests, of which are included the "enzyme immunoassays" and abbreviated ELISA tests (for "Enzyme-linked immunosorbent assay", translated as "bound enzyme immunoadsorption assay"). The purpose of these tests is to detect the presence of an antibody or an antigen in a sample. The formation and spatial localization of the antibody-antigen complex is detectable by several techniques: grafting of colored or fluorescent species, nanoparticles, enzymes, etc. The signal characterizing the presence of the desired species can be detected by the eye or by spectroscopy. This family includes strip, dipstick or lateral flow test. These Point-of-Care tests (translated as "at the bedside") are very suitable for field situations with little material available. But immunoassay diagnosis is restricted to detecting species involved in antibody-antigen complexes. For example, in the case of a virus, the detection is indirect: the immunoassays make it possible to probe the presence of characteristic proteins or antibodies emitted during the presence of the virus.

On the other hand, the family of DNA amplification tests that can recognize and multiply a specific sequence of DNA or RNA. In the case of a virus, it is therefore a much more direct test: we recognize the genetic material of the desired species. But these tests require expensive equipment and know-how to be implemented. These tests do not make it possible to perform tests at the bedside of the patient, and particularly in the geographical areas remote from the laboratories. For all these reasons, these isothermal DNA tests do not make it possible to quickly and inexpensively carry out diagnostics outside laboratories.

OBJECT OF THE INVENTION The present invention aims to remedy all or part of these disadvantages. For this purpose, the present invention provides a diagnostic device by isothermal amplification of DNA, which comprises: a porous substrate comprising: a zone comprising at least one reactive compound reacting with a target compound of a sample, called diagnostic zone "; - a fluid deposition zone transported by capillarity in the sample deposition zone and in the diagnostic zone; - the sample deposition zone positioned between the fluid deposition zone and the diagnostic zone, and - two channels, positioned in the thickness of the porous substrate, respectively between, on the one hand, the fluid deposition zone and the sample deposition zone, on the other hand, the zone of sample depot and diagnostic zone; and - local isothermal heating means of the diagnostic zone. With these arrangements, a sample of blood, urine or saliva is placed on the sample deposit area and this sample is transported by the fluid deposited in the fluid deposition zone to the diagnostic zone. This transport 25 is made by capillarity through the porous substrate. The diagnostic zone is heated to separate the double strands of DNA, activate existing enzymes, promote mixing by thermal agitation. The function of the enzymes is to replicate the complementary DNA strands of the primers, ie short DNA strands present in the mixture of reactive compounds. The choice of primers defines the target of the diagnostic test. In the case where the sample contains RNA strands, a first enzyme carries out the transcription step, that is to say the copy of the RNA into DNA, before allowing the replication of the DNA. The same enzyme can perform both transcription and replication steps.

3032719 3 The local heating of the diagnostic zone allows in particular an energy saving in the case of an autonomous device. In embodiments: the sample deposition zone and the diagnostic zone and the two channels are respectively merged. These embodiments make it possible to limit the risks of sample loss in zones and channels that do not comprise a reactive compound. In embodiments, at least one reactive compound is present in the diagnostic zone in dried form. These embodiments allow easy transport of the device from a production site to a site of use. In embodiments, the device of the present invention comprises, in the area of deposition of a sample, a compound for preparing the sample by chemical lysis of the cells. The advantage of these embodiments is that they make the DNA or RNA in the sample more accessible in addition to decontaminating the sample. In embodiments, the device that is the subject of the present invention comprises means for local isothermal heating of the sample deposition zone zo. The advantage of these embodiments is that they make the DNA or RNA in the sample more accessible in addition to decontaminating the sample. In embodiments, the porous substrate is a sheet of paper. These embodiments allow easy and low cost manufacture of the device. In embodiments, the local isothermal heating means is a conductive electrical circuit facing the porous substrate. The advantage of these embodiments is that they make it possible to heat up using a battery, which allows the device to be mobile and made at a low cost. In embodiments, the device of the present invention comprises: a support surface of the conductive electrical circuit and a sealed surface, the impervious surface being interposed between the porous substrate and the support surface. These embodiments make it possible to contain the sample in the porous substrate while allowing the local isothermal heating means to ensure an optimal yield due to the proximity with the substrate. In embodiments, the conductive electrical circuit is supported by a sheet of paper. The advantage of these embodiments is that they allow a low cost realization of the heating means, this heating means occupying more of a limited space. In embodiments, at least one channel is formed by depositing molten wax penetrating the thickness of the substrate and then solidifying. These embodiments have the advantage of presenting a great simplicity of manufacture.

In embodiments, at least one channel is formed by hydrophilic-hydrophobic contrast in the thickness of the substrate. The advantage of these embodiments is that they allow an optimal realization of the channel. In embodiments, at least one channel is formed by photolithography in the thickness of the substrate. These embodiments have the advantage of allowing an optimal realization of the channel. In embodiments, the device which is the subject of the present invention comprises an additional zone comprising at least one reactive compound of the diagnostic zone connected by a channel to the zone of deposition of a fluid. The advantage of these embodiments is that they make it possible to verify that the fluid is not contaminated. In embodiments, the device of the present invention comprises an additional zone comprising the reactive compound of the diagnostic zone, and at least one target compound, connected by a channel to the deposition zone of a fluid.

These embodiments make it possible to verify that the reagent of the device reacts with the target compound so as to make the diagnosis of the diagnostic zone reliable. In embodiments, the local isothermal heating means is configured to heat the sample and diagnostic deposition areas to a temperature between 20 ° C and 120 ° C. These embodiments make it possible to heat the sample deposition and diagnostic zones to a temperature at which the reagent mixture functions.

In embodiments, at least one reactive compound changes color upon contact with the target compound. The advantage of these embodiments is that they make it easy to detect the reaction between the mixture of reactive compounds and the target compound. In embodiments, at least one reactive compound emits a fluorescence signal upon contact with the target compound. The advantage of these embodiments is that they make it easy to detect the reaction between the mixture of reactive compounds and the target compound. BRIEF DESCRIPTION OF THE FIGURES Other advantages, aims and particular characteristics of the invention will emerge from the following nonlimiting description of at least one particular embodiment of the device which is the subject of the present invention, with reference to the appended drawings, in which: 1 shows schematically and in perspective, a first embodiment of the device object of the present invention, Figure 2 shows, schematically and in perspective, a particular embodiment of the device object of the present invention, FIG. 3 schematically and in perspective shows a particular embodiment of the assembled system which is the subject of the present invention, FIG. 4 schematically represents a particular flow diagram of the process which is the subject of the present invention, FIG. , schematically and in perspective, a second particular embodiment of the dispo 6 is a schematic and perspective view of a third particular embodiment of the device that is the subject of the present invention; and FIG. 7 is a diagrammatic perspective view of a fourth embodiment of the present invention. FIG. In particular, FIG. 8 is a diagrammatic perspective view of a fifth particular embodiment of the device that is the subject of the present invention. DESCRIPTION OF EXAMPLES OF THE INVENTION This description is given in a nonlimiting manner, each feature of an embodiment being combinable with any other feature of any other embodiment in an advantageous manner. Moreover, each parameter of an exemplary embodiment can be implemented independently of other parameters of said exemplary embodiment. It is noted that the term "one" is used in the sense of "at least one".

It is already noted that the figures are not to scale. FIGS. 1 to 3 show a sectional view of an embodiment of the device 10 which is the subject of the present invention. This diagnostic device by isothermal amplification of DNA comprises: a porous substrate 105 comprising: a zone 110 comprising at least one reactive compound 115 reacting with a target compound 120 of a sample, called a "diagnostic zone", a zone 125 for deposition of fluid transported, by capillarity, in a zone 130 of sample deposition and in zone 110 of diagnosis, the zone 130 of sample deposition, positioned between the zone 125 of fluid deposition and the diagnostic zone 110, which comprises a compound 145 for preparing the sample by chemical lysis of the cells, two channels, 135 and 140, positioned in the thickness of the porous substrate, respectively between, on the one hand, the 125 and the sample depositing zone 130, on the other hand, the sample deposition zone 130 and the diagnostic zone 110, an additional zone 155 comprising at least one compound 115 responsive zon 110 of the diagnosis connected via a channel 160 to the deposition zone 125 of a fluid, and an additional zone 165 comprising at least one reagent 115 of the diagnostic zone 110, and at least one target compound 120, connected by a channel 170 to the zone 125 for depositing a fluid; means 205 for local isothermal heating of the diagnostic zone 110 and additional and additional control zones 155; means 150 for local isothermal heating of the sample deposition zone 130; a surface 210 for supporting the conductive electrical circuit; and a sealed surface 215, the sealed surface 215 being positioned between the porous substrate 105 and the support surface 210. In particular, FIG. 1 shows the porous substrate 105 used by the device 10. This porous substrate 105 is, for example, a sheet of paper made from cellulose fibers, nitrocellulose membranes or paper filter constituting a porous medium capable of allowing the passage of a fluid. In contact with the porous substrate 105, a drop of water or a drop of blood diffuses at the surface and in the thickness of the porous substrate 105 around the point of contact between the droplet and the porous substrate 105. In order to channel the flow of the drops in this porous substrate 105, a set of zones and channels are defined in the porous substrate 105. These zones and channels are delimited by barriers passing through the porous substrate 105 in thickness. These barriers are formed, for example: by depositing molten wax penetrating into the thickness of the porous substrate 105 and then solidified, by a hydrophilic-hydrophobic contrast in the thickness of the porous substrate 105 by laser cutting, by cross-linking of polymers and / or photolithography in the thickness of the porous substrate 105. A "zone" is a closed volume of the porous substrate 105 having at least one opening and "channel" a conduit connecting at least two zone openings.

The device 10 implements several zones in the porous substrate 105: A first zone, called the diagnostic zone 110, comprises at least one reactive compound 115 reacting with a target compound 120. For example, a mixture of reactive compounds 115 is consisting of an enzyme, a mixture of the four deoxyribonucleotides and a selection of primers allowing the realization of an isothermal amplification technique of DNA among: PCR ("Polymerase Chain Reaction", translated into French by "Reaction chain by polymerase "), RT-PCR (" Reverse Transcriptase - PCR ", translated in French by" PCR after reverse transcription ") and its derivatives, LAMP (" Loop-mediated isothermal amplification ", translated into French as" amplification isothermal by loop structures) and RT-LAMP ("Reverse transcription - LAMP", translated into French as "LAMP after reverse transcription"), RPA ("Recombinase polymerase amplification", t raduit in French by "Amplification by polymerase, assisted by recombinases") and RT-RPA ("Reverse transcription - RPA", translated into French by "RPA after reverse transcription"), SDA ("Strand displacement amplification", translated into French by "strand displacement amplification") and RT-SDA ("reverse transcription 20 - SDA"), HDA ("Helicase-dependent amplification"), translated into French as "amplification assisted by helicases ") and RT-HDA (" Reverse transcription HDA ", translated into French as" HDA after reverse transcription ") and - NEAR (" Nicking enzyme amplification reaction ", translated into French as" 25 Enzyme amplification reaction cutoff And RT-NEAR ("Reverse transcription - NEAR") translated into French by "NEAR after reverse transcription") At least one reactive compound is present in dried form in the diagnostic zone In variants, at least one reactive compound is present in the form of a hydrogel. In other embodiments, at least one reactive compound is present in a wet form. In these variants, the diagnostic zone 110 is isolated from the external environment by plastic sheets, for example.

When at least one reactive compound is in dried form, each said reactive compound is rehydrated by the fluid deposited in the fluid deposition zone 125. When at least one reactive compound comes into contact with the target compound 120, under If adequate thermal conditions are maintained, the primers hybridize to the complementary piece of DNA, the enzyme can then attach to the primers and replicate the DNA strand (use the available deoxyribonucleotides to create the complementary strand of the DNA strand. DNA). During this replication, a colorimetric or fluorescent intercalator is generally integrated and is at the origin of the detection signal.

At least one reactive compound 115 changes color upon contact with the target compound 120, thereby detecting the presence of the target compound. If a target compound is detected, a user of the device 10 can deduce a medical diagnosis such as infection of an individual with a virus whose DNA is the target compound 120 of the reactive compound 115 of the device 10.

In variants, at least one reactive compound 115 emits a fluorescence signal in contact with the target compound 120. This diagnostic zone 110 is connected to the sample deposition zone 130 by a straight channel 135. The sample depositing zone 130 comprises a sample preparation compound 145 by chemical lysis of the cells present in dried form in the thickness of the porous substrate 105. This compound 145 is, for example, a detergent agent which destroys the plasma membrane of the cells present in the sample, which makes it possible to extract the genetic material from the cells. The sample deposition zone 130 is connected to the deposition area of a fluid 125 via a channel 140. The fluid deposition area 125 is configured to receive water, for example. This fluid deposition zone 125 is connected by a channel 160, different from the channel 140, to an additional zone 155 comprising at least one reactive compound 115. This additional zone 155 has the function of verifying that the fluid deposited in the deposition zone fluid 125 does not exhibit the target compound 120, which would lead to an erroneous diagnosis with respect to the sample deposited in the sample deposition zone 130. The deposition zone of a fluid 125 is also connected by a channel 170 different from channel 140 and channel 160, to an additional area 165 comprising the reactive compound 115 and at least one target compound 120 in dried forms. When the fluid hydrates the additional zone 165, the reactive compound 115 reacts with the target compound 120. This additional zone 165 serves to verify the operation of the device 10.

In variants, the fluid deposition zone 125 has an area substantially greater than the areas of the other zones of the device 10. Thus, as understood, the deposition of a sample in the deposition area of a sample 130 and then the deposition of water in the deposition zone of a fluid 125 causes, by a capillary pump mechanism, a displacement of the water in the verification zones, 155 additional and 165 additional, and in the zone of depositing a sample 130. The water thus conveys the sample in the sample deposition zone 130 and in the diagnostic zone 110. The zones and channels of the porous substrate 105 act as a microfluidic system. The porous substrate 105 is, for example, wrapped in a plastic sheet covering all the surfaces of the porous substrate 105 with the exception of the deposition zones 125 and 130. In this way, the porous substrate 105 is protected and each reactive compound 115 is isolated to prevent contamination. FIG. 2 shows a particular embodiment of the local isothermal heating means 205 and the support surface 210.

The support surface 210 is, for example, a sheet of paper or cellulose fibers similar to the material used to make the porous substrate 105. This support surface 210 has dimensions that are preferentially identical to those of the porous substrate 105. A circuit Electrical conductive 205 is positioned on this support surface 210 so that, when a current flows through the circuit, this circuit locally heats the porous substrate 105 by Joule effect. The positioning of the conductive electrical circuit 205 is such that when the support surface 210 and the porous substrate 105 are brought into contact, the local isothermal heating means 205 is confronted with the diagnostic zones 110 and 30. zones, 155 additional and 165 additional. The local isothermal heating means 205 is configured to heat the diagnostic zone 110 and the further and further control zones 155 to a temperature between 20 ° C and 120 ° C. Preferably, the temperature is between 45 ° C and 65 ° C. A secondary electrical conductor circuit 150 is positioned to face the sample deposition area 130 of the porous substrate 105 to act as a means 150 for preparing the sample by thermal denaturation of the sample. These electronic circuits, 205 and 150, are powered by a battery 220 for example. This battery 220 has, for example, a voltage of 9 volts, which allows the substrate system 105, surface 205 and battery 220 to be portable.

In variants, the local isothermal heating means 205 is configured to perform a set of thermal cycles in order to perform a PCR reaction. In these variants, at least one reactive compound 115 is adapted to carry out this reaction. FIG. 3 shows the device 10 assembled to perform a diagnosis. In this embodiment, a sealed surface 215 is interposed between the porous substrate 105 and the support surface 210. The local isothermal heating means 205 is positioned under the diagnostic zones 110 and the additional and additional zones 155 while the thermal preparation means 150 is positioned below the sample deposition zone 130. In this manner, the sample transported to the sample deposition zone 130 and the diagnostic zone 110 is heated, allowing the enzymes to function and the isothermal amplification function of DNA to take place. On the other hand, the local isothermal heating of the additional zone 165 makes it possible to check the operation of each reactive compound 115. The thermal preparation means 150 makes it possible to purify and disinfect the sample deposited in the deposition zone of As can be seen from the description of FIGS. 1 to 3, the device 10 can be manufactured in a controlled manner, standardized in the laboratory or by industrial processes. After manufacture, the device 10 is stored in an airtight plastic bag and must be stored away from heat, light and moisture to allow the storage of the devices 10.

FIG. 4 shows a logic diagram of steps of the method that is the subject of the present invention. This method comprises: a sampling step 305 of a sample; a deposit deposition step 310 of the sample in a sample deposition zone 5 of the device 10 as described with reference to FIGS. 1 to 3; sample preparation step 315, a transport step 320 of the prepared sample, an isothermal amplification step 325 of the sample, and a detection step 330 of the presence of a target compound in the sample. the sample. The sampling step 305 is performed, for example, by a syringe or a needle pricking an individual at the end of the finger to extract a drop of blood. In variants, a drop of urine or saliva is removed. In preferred embodiments, a drop of blood drawn from a blood test is used. For simplicity of use, a drop of blood taken at the fingertip of the individual is often used. The deposition step 310 is carried out, for example, by contacting the sample with the sample deposition zone of the device as described with reference to FIGS. 1 to 3.

The preparation step 315 is carried out by chemical lysis of the sample cells and / or by thermal denaturation of the sample. The chemical lysis is carried out by a detergent agent, for example, while the thermal denaturation is carried out by local isothermal heating of the sample deposition zone by a conductive electrical circuit positioned opposite said zone. The purpose of this preparation step 315 is to extract the DNA or RNA material from the cells of the sample and to decontaminate the sample by deactivating the virus for example. The transport step 320 is carried out, for example, by depositing a standardized amount of sterile water on the deposition zone of a fluid of the device 10. This water, by a capillary pump mechanism, carries the sample in the sample deposit area and in the diagnostic area of the device 10. The amount of water deposited is, for example, thirty times greater than the amount of sample deposited.

The isothermal amplification step 325 is performed, for example, by heating the reagent medium, comprising the prepared sample and each reactive compound, in the diagnostic zone. This heating allows enzymes, initially present in dried, rehydrated form to amplify the DNA of the target compound. The isothermal amplification 325 is less restrictive than the PCR methods (for "Polymerase Chain Reaction", translated into French as "polymerase chain reaction") and the thermal cycles associated with these methods. The local isothermal heating means is supported by a surface which, associated with the porous substrate of the device 10 forms a three-ply chip: a porous substrate thickness comprising the microfluidic system of channels and zones; to the porous substrate and - a surface supporting the electric heating circuit.

The detection step 330 is performed, for example, by detecting the color or fluorescence of the porous substrate at the diagnostic zone. This spectrometry is performed directly by the user or, for example, by a communicating portable terminal, such as a mobile phone having an image sensor for example. An image processing software embedded in said terminal 20 or in a remote computer server determines the color of the porous substrate and derives diagnostic information therefrom. In variants, the method comprises a step of checking the purity of the water by detecting the color in an area of the porous substrate in connection with the deposition zone of a fluid. If this zone, provided with only each reactive compound, has a color or fluorescence characteristic of the presence of the target compound in said zone, the water used is infectious and the diagnosis made by the device 10 can not be exact. In variants, the method includes a step of verifying the operation of the device 10 by detecting color or fluorescence in an area of the substrate having, in dried forms, each reactive compound and the target compound. When the deposited water hydrates this zone, the compounds are contacted and the color of said zone becomes representative of the detection of the target compound. If this zone does not take this color, then the diagnosis made can not be considered correct. Once the diagnosis is made, the chip can be burned to avoid generating infectious waste. The heating system can be extracted for reuse in connection with another porous substrate. FIG. 5 shows a particular embodiment of the device 40 which is the subject of the present invention. This device 40 differs from the device 10 as described with reference to FIG. 1 in that: - the sample deposition zone 410 and the diagnostic zone 455 are merged and - the two channels are merged into a single channel 430 Thus, this device 40 comprises a porous substrate 405 similar to the porous substrate 105 described with reference to FIG. 1, a set of channels 430, 440 and 450, and zones 410, 425, 435, 445 and 455, being delimited on and in this porous substrate 405. Zone 410 corresponds to a sample deposition zone. This sample deposition zone 410 is connected to a deposition zone 425 of fluid similar to the deposition zone 125 of fluid described with reference to FIG. 1. In this embodiment, the fluid is mainly used to hydrate the zone of deposition. sample 410 and diagnostic 455 to prevent drying of the compounds before a diagnosis is made. The additional areas 435 and 445 respectively correspond to the areas 155 and 165, described with reference to FIG. 1, and provide similar functions.

In variations, the sample and diagnostic 455 deposition zones 410 comprise a sample preparation compound similar to the sample preparation compound 165 described with reference to FIG. 1. In FIG. 6, a particular embodiment of the device 50 object of the present invention. This device 50 comprises: a porous substrate 505, a fluid deposition zone 525 connected to a channel 530 carrying the fluid by capillarity to a zone 560 comprising at least one reactive compound 515, three channels, 535 , 545 and 555, for transporting the fluid and each reactive compound 515 by capillarity respectively from zone 560 to: - a sample deposition zone 510 acting as a diagnostic zone at the contact between the sample and each reactive compound 515 under the action of a local isothermal heating means (not shown), an additional zone 540 comprising no reactive compound 515 or target compound 520, making it possible to verify that the fluid does not contain any compound target 520 and 10 - an additional zone 550 comprising the target compound 520 to check the operating capacity of the device 50. FIG. 7 shows a particular embodiment of the device Object of the present invention. In this Figure 7, there are three components of the reactive compounds described above. These reactive compounds are formed of: primers, i.e., small strands of DNA which specifically recognize a desired sequence in a sample, this sequence being dubbed "target compound" in the above figures, deoxyribonucleic bases that is, the elemental bricks that form the DNA and enzymes capable of assembling the deoxyribonucleic bases to form the complementary DNA strand of the target that it seeks. The enzyme only works if the primer has found a complementary sequence. This device 60 comprises a porous substrate 605 comprising a fluid deposition zone 610 connected to three channels, 615, 620 and 625, for conveying the fluid by capillarity. Via channel 615, this fluid is transported to a zone 685 comprising a type of primer 665, deoxyribonucleic bases and enzymes 645, in dried form for example, to verify that the fluid does not contain the target compound 630. Target compound 630 is present, primers 665 and enzymes 645 react and indicate to a user, through fluorescence for example, that target compound 630 is present in the fluid. Via channel 625, the fluid is transported to an area 680 having a primer type 665, deoxyribonucleic bases, 645 enzymes, and the target compound 630 in dried forms, for example. In contact with the fluid, primers 665 and enzymes 645 react and fluoresce to indicate to a user that device 60 is functioning properly in the presence of target compound 630.

Via the channel 620, the fluid is transported to a sample deposition zone 625, which sample here comprises the target compound 630. This sample deposition zone 625 is connected by a channel 635 to an area 640 of storage of 645 enzymes in dried form. The fluid transports the stored sample and enzymes 645 into three distinct zones, 650, 660 and 670, each connected to the storage zone 640 by a separate channel. In each zone, 650, 660 and 670, a different primer, 655, 665 and 675 is placed. Each primer, 655, 665 and 675, corresponds to a different diagnosis, each primer, 655, 665 and 675, reacting with a different target compound 630 or with a different part of a target compound 630.

FIG. 8 shows a particular embodiment of the device 70 which is the subject of the present invention. In this figure 8, there are three components of the reactive compounds described above. These reactive compounds are formed of: - primers, ie small strands of DNA which specifically recognize a desired sequence in a sample, this sequence being nicknamed "target compound" in the figures above, - Deoxyribonucleic bases, ie the elementary bricks that form the DNA and enzymes capable of assembling the deoxyribonucleic bases to form the DNA strand complementary to the target that seeks it. The enzyme only works if the primer has found a complementary sequence. This device 70 comprises a porous substrate 705 comprising a fluid deposition zone 710 connected to a channel 720 for conveying the fluid by capillarity. Via channel 720, the fluid is transported to an enzyme storage area 725 745 in dried form. This storage zone 725 is connected to three channels, 715, 725 and 735. Via channel 715, the enzyme-containing fluid 745 is transported to a zone 785 having a type of primer 765 and deoxyribonucleic bases, in dried form. for example, to verify that the fluid does not have the target compound 730. If the target compound 730 is present, primers 765 and enzymes 745 react and indicate to a user, through fluorescence for example, that target compound 730 is present in the fluid. Via channel 725, the enzyme-containing fluid 745 is transported to an area 780 having a primer type 765, deoxyribonucleic bases and the target compound 730 in dried forms, for example. In contact with the fluid, primers 765 and enzymes 745 react and cause fluorescence indicating to a user that device 70 is functioning properly in the presence of target compound 730.

Through channel 735, the enzyme-containing fluid 745 is transported to a sample deposition zone 740, which sample here comprises the target compound 730. The fluid carries the stored sample and enzymes 745 into three distinct zones, 750 , 760 and 770, each connected to the storage area 740 by a separate channel. In each zone, 750, 760 and 770, a different primer 755, 765 and 775 is placed. Each primer, 755, 765 and 775, corresponds to a different diagnosis, each primer, 755, 765 and 775, reacting with a different target compound 730 or with a different part of a target compound 730.

Claims (17)

REVENDICATIONS1. Diagnostic device (10, 40) by isothermal amplification of DNA, characterized in that it comprises: - a porous substrate (105, 405) comprising: - a zone (110, 410) comprising at least one compound (115, 415) reagent reacting with a target compound (120, 420) of a sample, called a "diagnostic zone", - a region (125, 425) of fluid deposition transported, by capillarity, in the sample deposition zone and in the diagnostic zone, the sample deposition zone (130, 410) positioned between the fluid deposition zone and the diagnostic zone, and two channels (135, 140, 430) positioned in the thickness of the porous substrate, respectively between, on the one hand, the fluid deposition zone and the sample deposition zone, on the other hand, the sample deposition zone and the diagnostic zone; and - means (205) for local isothermal heating the diagnostic zone. 2. Device (10) according to claim 1, which comprises a zone (155) additional comprising at least one compound (115) reactive diagnostic zone (110) connected by a channel (160) to the deposition zone (125). ) of a fluid. 3. Device (10) according to one of claims 1 or 2, which comprises a zone (165) additional comprising at least one compound (115) reactive diagnostic zone (110), and at least one compound (120) target, connected by a channel (170) to the zone (125) for depositing a fluid. 4. Device (10) according to one of claims 1 to 3, wherein at least one compound (115) reagent is present in the diagnostic zone (110) in dried form. 25 3032719 19 5. Device (10) according to one of claims 1 to 4, which comprises, in the region (130) of depositing a sample, a compound (145) for preparing the sample by chemical lysis of the cells. 5 6. Device (10) according to one of claims 1 to 5, which comprises means (150) for local isothermal heating of the sample deposition zone. 7. Device (40) according to one of claims 1 to 6, wherein: - the zone (410) of sample deposition and the zone (455) diagnostic and 10 - the two channels (430) are respectively confused . 8. Device (10) according to one of claims 1 to 7, wherein the substrate (105) porous is a sheet of paper. 15 9. Device (10) according to one of claims 1 to 8, wherein the means (205) isothermal local heating is a conductive electrical circuit facing the substrate (105) porous. 20 10. Device (10) according to claim 9, which comprises: a surface (210) for supporting the conductive electrical circuit and a sealed surface (215), the sealed surface being positioned in an intermediate manner between the porous substrate (105) and the support surface. 11. Device (10) according to one of claims 9 or 10, wherein the conductive electrical circuit is supported by a sheet of paper. 12. Device (10) according to one of claims 1 to 11, wherein at least one channel (135, 140) is formed by deposition of molten wax penetrating into the thickness of the substrate (105) and then solidified. 3032719 20 13. Device (10) according to one of claims 1 to 12, wherein at least one channel (135, 140) is formed by a hydrophilic-hydrophobic contrast in the thickness of the substrate (105). 5 14. Device (10) according to one of claims 1 to 13, wherein at least one channel (135, 140) is formed by photolithography in the thickness of the substrate (105). 15. Device (10) according to one of claims 1 to 14, wherein the means (205) for local isothermal heating is configured to heat the deposition zones (130) and diagnostic (110) at a temperature of between 20 ° C and 120 ° C. 16. Device (10) according to one of claims 1 to 15, wherein at least one reagent (115) changes color in contact with the target compound (120). 15 17. Device (10) according to one of claims 1 to 16, wherein at least one reactive compound (115) emits a fluorescence signal in contact with the target compound (120).