Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6844—Nucleic acid amplification reactions
  • C12Q1/6846—Common amplification features

Definitions

• the present invention belongs to the field of molecular biology and, more particularly, to the field of nucleic acid amplification.
• the present invention provides a miniaturized device, transportable, easy to implement and use and a method implementing such a device, both for the amplification of nucleic acids from samples including low volume samples.
• the polymerase chain reaction technique or PCR (for "Polymerase Chain Reaction") technique is a molecular biology technique for the in vitro amplification of nucleic sequences. This technique based on thermal cycles with a denaturation step and a hybridization - elongation step began to be used at the end of the 80s. It is now a classic and unavoidable technique that applies to all fields of analysis in which the nucleic acids play a direct or indirect role. These areas include academic research; clinical and diagnostic research and analysis, with tests conducted on blood samples or cell biopsies or tissues; environmental monitoring; civilian surveillance; food safety and especially quality control; criminal analyzes on traces ...
• the current tools for routine PCR including a well plate, a heating system and a detection system are relatively bulky and do not allow to develop fully portable systems for the analysis of ground. They also do not allow to develop very fast analysis systems because the constituent materials such as plastic and the volume of each analysis often of the order of ten ⁇ l lead to kinetics, during temperature changes, incompressible .
• the first preamplification step has at most 10 cycles of denaturation and hybridization-elongation, and implements the two specific primers of the nucleic acid to be amplified in a small amount (less than 0.0625 ⁇ M).
• the second amplification step uses the same primers in larger amounts for 30 to 35 cycles.
• the method of the international application WO 02/20845 only addresses the problem of sensitivity without addressing the problem of multiple analyzes for the same sample of reduced volume.
• the device comprises a compartment for 1 st step the amplification which may be a multiplexed amplification and several other compartments for the 2 nd amplification step. Liquid samples pass 1 compartment to other compartments by centrifugation, the 1st compartment being connected directly or indirectly to other compartments.
• the international application WO 2008/106719 solves the problem of contaminating the samples by avoiding manipulation between the two amplification steps, but the device described remains heavy to use and can not be portable since it requires means to enable it to be used. centrifuge, to bring it to different temperatures and possibly valves of wax for interrupting fluidic communication between compartments.
• the present invention overcomes, at least in part, the disadvantages and technical problems listed above. Indeed, the latter proposes a method and a device for amplification of nucleotidic molecules, based on electrowetting that make it possible to implement the amplification method in the field, to make a large number of parallel analyzes from of the same sample and in particular of the same "micro-sample", ie a sample of low or reduced volume, and without losing sensitivity.
• the present invention provides a method of amplifying at least one nucleotide molecule contained in a sample, hereinafter referred to as sample (E), comprising the following successive steps of: a) subjecting said sample (E) to the st step amplification on a first zone of a carrier, hereinafter designated area (Zi); b) bringing by electrowetting at least a portion of the sample obtained after step (a), (hereinafter referred to the sample (E) of the zone (Zi) on at least one 2 nd area of said support , separate from the area
• sample (E) comprising the following successive steps of: a) subjecting said sample (E) to the st step amplification on a first zone of a carrier, hereinafter designated area (Zi); b) bringing by electrowetting at least a portion of the sample obtained after step (a), (hereinafter referred to the sample (E) of the zone (Zi) on at least one 2 nd area of said support , separate from the area
• zone (Z 2 ) and hereinafter referred to as zone (Z 2 ); c) subjecting the sample (E) to a 2 nd amplification step on said zone (Z 2).
• amplification is meant, in the context of the present invention, both a conventional polymerase chain reaction (or “PCR") amplification and any PCR variant known to a person skilled in the art such as a PCR asymmetric, interleaved thermal asymmetric PCR, temperature gradient PCR, end-point PCR, multiplex PCR, real-time PCR, RT-PCR (Reverse Transcription - Polymerase Chain Reaction), or Quantitative PCR.
• st (or 2 nd) amplification step means a step in a preview ere (or 2 nd) amplification ie a l st (or 2 nd) amplification.
• RT-PCR is meant reverse transcription followed by polymerase chain reaction.
• An RT-PCR thus comprises two steps: a reverse transcription step, ie a synthesis of a single-stranded DNA complementary to an RNA sequence, implementing a reverse transcriptase followed by a chain amplification step by polymerase.
• a reverse transcription step ie a synthesis of a single-stranded DNA complementary to an RNA sequence
• implementing a reverse transcriptase followed by a chain amplification step by polymerase.
• multiplex PCR is meant an amplification method for amplifying more than one amplicon at a time. This technique uses a set of amplification primer pairs, each primer pair being designed or adapted to amplify a different nucleotide molecule.
• multiplex RT-PCR is meant a multiplex PCR as defined above preceded by reverse transcription as previously defined.
• real-time PCR is meant an amplification method that makes it possible to detect and / or quantify the presence of the amplicons during the PCR cycles, in particular by means of a fluorescent marker. Amplification of the amplicons or the signal related to the amount of amplicons formed during the PCR cycles is used for the detection and / or quantification of a given nucleotide sequence in the solution subjected to PCR.
• amplicon is meant a target nucleotide molecule resulting from the PCR amplification of a nucleotide molecule present in the sample (E).
• the size of the amplicons in the context of the present invention, can be between 40 and several thousands of base pairs (bp), in particular between 50 and 1000 bp, in particular between 60 and 500 bp and, especially, between 60 and 150 bp.
• nucleotide molecule used herein is equivalent to the following terms and expressions: "nucleic acid”, “Polynucleotide”, “nucleotide sequence”, “polynucleotide sequence”.
• nucleotide molecule is meant, in the context of the present invention, a chromosome; a gene ; a regulatory polynucleotide; DNA, single-stranded or double-stranded, genomic, chromosomal, chloroplast, plasmidic, mitochondrial, recombinant or complementary; total RNA; messenger RNA; ribosomal RNA; a transfer RNA; a portion or a fragment thereof.
• sample (E) used in the context of the present invention may be very varied in nature.
• This sample (E) is a sample of an element advantageously chosen from the group consisting of a liquid solution containing at least one nucleotide molecule as defined above; a biological fluid; a vegetable fluid such as sap, nectar and root exudate; one or more animal or plant cells; an animal or plant tissue; a food matrix; city water, river water, sea water, air-cooled towers; an air sample; a sample of earth; etc. or a mixture thereof.
• the biological fluid is preferably selected from the group consisting of blood such as whole blood or anti-coagulated whole blood, blood serum, blood plasma, lymph, saliva, sputum, tears, sweat, sperm, urine, stool, milk, cerebrospinal fluid, interstitial fluid, isolated spinal fluid bone, mucus or fluid of the respiratory tract, intestinal or genitourinary tract, cell extracts, tissue extracts and organ extracts.
• the biological fluid may be any fluid naturally secreted or excreted from a human or animal body or any fluid recovered from a human or animal body by any technique known to those skilled in the art such as extraction. , a sample or a wash. The steps of recovery and isolation of these different fluids from the human body or animal are performed prior to the implementation of the method according to the invention.
• the sample (E) used in the context of the present invention has a volume of between 0.01 and 1000 ⁇ l, in particular between 0.02 and 500 ⁇ l, in particular between 0.05 and 100 ⁇ l, and more particularly, between 0.1 and 20 ⁇ l.
• the amount of nucleotide molecules in the sample (E) is very variable: it may be between 1 and 10 10 nucleotide molecules / sample (E).
• the nucleotide molecules contained in the sample (E) can be synthetic or natural, originating from the same organism or from different organisms.
• the method according to the present invention implements a device comprising:
• a support having at least two distinct zones (Z 1 ) and (Z 2 ); - said first zone (Zi), hereinafter referred to as the "large reservoir”, being adapted to a first amplification step;
• the method according to the present invention may comprise a preliminary step of providing such a device or any variant of this device which will be presented below.
• the sample (E) is brought into contact with the reaction mixture necessary for the amplification of the step
• the reaction mixture used in step (a) can be any mixture of commercially available PCR reagents such as kits sold by Roche Applied Science or Applied companies.
• reaction mixture used during the amplification of step (a) may have any of all the reaction mixtures described in the state of the art for PCR.
• the reaction mixture comprises one or more of a thermostable polymerase such as Taq polymerase and possibly reverse transcriptase; a salt such as TRIS (for "trishydroxymethylaminomethane"), KCl, NaCl or MgCl 2; deoxyribonucleotide triphosphates such as dATP, dGTP, dTTP, dCTP and optionally dUTP; at least one pair of primers, specific or degenerate, which may comprise from 15 to 35 base pairs; and optionally at least one oligo-dT or specific primer useful for reverse transcription.
• a thermostable polymerase such as Taq polymerase and possibly reverse transcriptase
• a salt such as TRIS (for "trishydroxymethylaminomethane"), KCl, NaCl or MgCl 2
• deoxyribonucleotide triphosphates such as dATP, dGTP, dTTP, dCTP and optionally dUTP
• reaction mixture may contain other additives and in particular additives known to improve the efficiency of a PCR such as BSA (for "Bovine Serum Albumin"), betaine, formamide, dimethylsulfoxide , Acetamide or Amide C2, Tween-20, polyethylene glycol (PEG) 6000, proteins such as the "Single Strand binding protein from E. CoIi” marketed by Sigma Aldrich (increase in the specificity of the reaction) , or the "T4 Gene32 Protein from E.
• BSA for "Bovine Serum Albumin”
• betaine betaine
• formamide dimethylsulfoxide
• Acetamide or Amide C2 Acetamide or Amide C2
• Tween-20 polyethylene glycol (PEG) 6000
• proteins such as the "Single Strand binding protein from E. CoIi” marketed by Sigma Aldrich (increase in the specificity of the reaction) , or the "T4 Gene32 Protein from E.
• CoIi B infected with T4aml34 phage / amBL292 / amE219" marketed by Roche Applied Science increase production yield of long PCR products, or the reaction yield in the presence of inhibitors such as Humic acid), Rnase inhibitors such as Ribonuclease Inhibitor or Diethyl pyrocarbonate which improve the yield of the reverse transcription step in RT-PCR.
• This reaction mixture may contain, when the amplification of step (a) is an RT-PCR multiplexed or multiplexed PCR, from 2 to 100 different primer pairs, each primer pair being specific or degenerate and may comprise from 15 to 35 base pairs.
• An example of a reaction mixture that can be used for the amplification of step (a) of the process according to the present invention comprises between 50 and 100 mM Tris, between 10 and 100 mM and, advantageously, 50 mM KCl (or NaCl).
• each primer is advantageously present in this reaction mixture at a concentration of between 1 and 200 nM and in particular between 10 and 100 nM.
• concentration of 0.5 U / ⁇ l is used instead of the 0.1 U / ⁇ l concentration recommended by ABI.
• the sample (E) which is electrically conductive is brought to said zone (Z 1 ) by electrowetting and this, in the form of one (or more) drop (s).
• the sample (E) can be brought directly to the zone (Z 1 ) or to a zone (Z 10 ), distinct from (Z 1 ) then to the zone (Z 1 ).
• the contact between the sample (E) and the reaction mixture prior to step (a) of the process according to the invention can be done in different ways: it can take place before the disposal of the sample ( E) on the zone (Zi) of the support, in this case, it is the sample (E) associated with the reaction mixture which is disposed on said zone (Zi) by electrowetting; it can take place on the zone (Zi) of the support, after or simultaneously at the disposal of the sample (E) on said zone (Zi).
• the reaction mixture is brought, by electrowetting, advantageously in the form of a drop, to the zone (Zi) where it mixes with the sample (E) by coalescence.
• the reaction mixture may be present: either on the support and in particular on a zone (Z 3 ) of the support, said zone (Z 3 ) being distinct from the previously defined zones (Z 1 ) and (Z 2 ), either in a reaction mixture tank located outside the device used in the context of the present invention, and can be brought onto the support via an orifice passing through a substrate disposed opposite said support, said orifice being connectable to the reservoir reaction mixture directly or indirectly by any input device such as a capillary or a hose.
• the reaction mixture or at least some of its components has (have) been previously dried (s) or freeze-dried (s) on the zone (Zi) of said support, during its manufacture or following it.
• Additives known in the state of the art can be implemented for the drying or lyophilization stage of the reaction mixture or of some of its constituents such as primers or probes on the zone (Zi) of the support.
• These additives are chosen in particular from sugars or sugar alcohols such as mannitol, sucrose, trehalose, glucose, etc. ; polymers such as dextran, PEG, polyvinylpyrrolidone (PVP) or polyvinyl acetate (PVA) ...; or else EDTA.
• the quantity of reagents deposited for drying or lyophilization can be increased to avoid a loss of reagents during amplification of the step
• step (a) due to the loss of reagents on the surfaces due to drying or lyophilization. It has been found that, without increasing the amount of dried reaction mixture constituents, the efficiency of the amplification during step (a) is decreased compared to that obtained under standard conditions, ie without dried reagents.
• the amount of the constituents of the reaction mixture to be dried must be multiplied by a factor of about 2 relative to the amount used in the absence of any drying or lyophilization step.
• the amplification implemented during step (a) of the method according to the invention is chosen from the group consisting of a multiplex PCR and a multiplexed RT-PCR.
• This step constitutes a pre-amplification step of the nucleotide molecules contained in the sample (E).
• step (a) of the method the multiplex PCR is carried out with a first denaturation step at 95 ° C, lasting from 1 second to 10 minutes depending on the size of the nucleotide molecules present in the sample (E) and the nature of the employed enzyme, followed by 5 to 15, advantageously 10 thermal cycles with a denaturation step at 95 ° C for 1 to 30 seconds and a hybridization-elongation step at a lower temperature, especially included between 50 and 70 ° C., advantageously 60 ° C., for 5 to 300 seconds and, in particular, for 5 to 30 seconds.
• reverse transcription step can last from 5 to 90 minutes at 37 ° C and cycles of this multiplex PCR following the st step There can be 5 to 20, advantageously 10 in number.
• the thermal cycles during the PCR can: comprising two temperatures with a hybridization-elongation stage of between 50 and 70 ° C., preferably 60 ° C., maintained for 5 to 300 seconds and a denaturation plateau at 95 ° C. held for 1 to 30 seconds; comprising three temperatures with a hybridization plateau of between 50 and 65 ° C., advantageously 60 ° C., maintained for 5 to 300 seconds, an elongation stage between 65 ° C.
• step (a) of the process according to the invention are achieved, maintained and controlled using means for heating the support used and in particular the zone
• the concentration for each pair of primers during the multiplex PCR cycles during step (a) of the process is between 10 and 200 nM, advantageously of the order of 50 nM, and may be different for the different nucleotide molecules. to amplify. In particular, it can be adjusted if it makes it possible to correct the biases amplification due to competition phenomena well known for multiplex amplification.
• the duration of the cycles may also be adapted to correct all competition bias during the multiplex PCR.
• the number of sequences amplified in parallel during this first step may be between 2 and several hundred, advantageously being of the order of several tens.
• Step (b) of the process according to the present invention consists more particularly in forming at least one drop from the sample (E a ) obtained after step (a) of the electrowetting method and moving said drop of said zone (Zi) to said zone (Z 2 ) by electrowetting.
• step (b) consists of forming, by electrowetting, x drop (s) from the sample (E a ) obtained after step (a) of the process and to move, by electrowetting, said (or said) drop (s) of said zone (Zi) to y zone (s) (Z 2 ) with x ⁇ y and x and y representing an integer between 1 and 500, advantageously between 1 and 50, in particular between 2 and 30 and, in particular, between 2 and 20.
• the zones (Z 2 ) of the support are also referred to herein as "small reservoirs".
• x is equal to y.
• the (or) drop (s) formed during step (b) of the process has (s) an extremely small volume, of the order of one nanoliter.
• This volume is in particular between 0.1 and 100 nor, in particular, between 1 and 80 ⁇ l and more particularly a volume of 10, 30, 50 or 65 ⁇ l.
• the latter may have an identical volume (drops of calibrated volume) or different.
• sample (E a ) corresponds to the sample (E) supplemented with the reaction mixture used for the amplification of step (a) and the amplicons produced during this first step. amplification.
• the amplification during step (c) of the process according to the present invention is advantageously a simplex PCR, insofar as there is mainly a PCR implementing at least one pair of primers specific for a given nucleotide molecule and optionally at least one labeled probe specific for said nucleotide molecule.
• labeled probe is meant, in the context of the present invention, a fluorescent probe capable of binding either to double-stranded DNA (SYBR technology) or to a specific DNA sequence (Taqman and Beacon technology) and which is fluorescent only once attached to the DNA.
• SYBR technology double-stranded DNA
• Tiqman and Beacon technology a specific DNA sequence
• the specific labeled probes can be of any kind among the probes known and described in the state of the art.
• specific labeled probes that may be used in the context of the present invention, mention may be made of TaqMan probes, molecular beacons, Scorpion probes and LNA probes.
• step (c) employs from 1 to 4 pairs of primers specific for a given nucleotide molecule (or a given organism) and optionally from 1 to 4 labeled probes specific for said nucleotide molecule (or said organism), provided with 1 to 4 different markers.
• the method according to the invention can take place on the zone (Z 2 ) of the support, after or simultaneously at the disposal of the sample (E a ) on said zone (Z 2 ).
• an electrically conductive solution (S) containing the specific primer pair (s) and optionally the specific labeled probe (s) is supplied by electrowetting. , advantageously in the form of a drop, to the zone (Z 2 ) where it mixes with the sample (E a ).
• said solution (S) may be present: - Or on the support and in particular on a zone (Z 4 ) of the support, said zone (Z 4 ) being distinct from the zones (Zi), (Z 2 ) and (Z 3 ) previously defined,
• a solution tank (S) located outside the device implemented in the context of the present invention, and can be brought onto the support via a hole passing through the substrate disposed opposite said support, said orifice being be connected to the solution tank (S) directly or indirectly by any input device such as a capillary or a hose.
• the concentration of primers and probes, specific, dried, after mixing with the sample drop (E a ) will be between 100 and 2000 nM, preferably of the order of 600 nM. That the pair (s) of specific primers and possibly the probe (s) specific label (s) occurs in liquid form or in dried or freeze-dried form, their mixing with the sample (E a ) at the level of the
• (Z 2 ) is realized by a round-trip movement on electrodes adjacent to said zone (Z 2 ).
• step (c) of the process of the present invention all temperature and duration conditions for the various cycles (denaturation, hybridization and elongation), known to those skilled in the art and suitable for PCR amplification with primers and possibly specific probes, are usable.
• the amplification implemented during step (c) of the process according to the invention comprises from 20 to 40 cycles, in particular 30, each cycle including a denaturation step and a hybridization-elongation step.
• the amplification implemented during step (c) of the method makes it possible to obtain amplicons of reduced size comprising from 50 to 200 bp and, in particular, from 60 to 150 bp.
• the thermal cycles during the amplification implemented during step (c) of the process according to the invention may be between 50 and 70 ° C. for hybridization-elongation, advantageously of the order of 60 ° C. , and be of the order of 95 ° C for denaturation. They can last between 5 and 180 seconds, advantageously between 5 and 30 seconds, for the hybridization-elongation and between 1 and 30 seconds, advantageously between 1 and 5 seconds for the denaturation.
• step (c) of the process according to the invention The temperatures used at each cycle during the amplification of step (c) of the process according to the invention are achieved, maintained and controlled using means for heating the support used and in particular the (or zone (s) (Z 2 ) of the latter, and possibly means for controlling the temperature of the support used and in particular the (or) zone (s) (Z 2 ) thereof.
• step (c) of the method according to the invention and when the latter implements at least one specific labeled probe, the fluorescence of the drops can be measured at each thermal cycle for a real-time analysis or at the end of step (c) for end-point analysis. Fluorescent detection of the amplified DNA uses, during the step
• a fluorophore such as Sybr green or a conventional fluorophore for labeling PCR probes such as Alexa Fluor488, Alexa Fluor546, Carboxy-Rhodamine 6G, Cy3, Cy5, 6-FAM, Fluorescein , HEX, JOE, TAMRA, TET, Texas Red.
• step (c) of the method according to the invention when the latter implements no specific labeled probe, no detection of the amplicons is performed during this step.
• the amplicons may require further analysis by another technique such as DNA hybridization or electrophoresis.
• the product obtained after step (c) can be recovered on each zone (Z 2 ) manually or by electrowetting.
• At least one drop is formed by electrowetting from the product obtained after step (c) and the latter is, by electrowetting, fed to a structure such as an orifice passing through the substrate disposed facing the support. , an orifice passing through said support or any hybrid device adaptable by those skilled in the art for handling discrete drops and / or transforming the sample in the form of drops in sample fluidic vein.
• the method according to the present invention may comprise a preliminary step of purification of the sample (E), the latter involving the preparation of said sample and optionally the isolation of the nucleotide molecules contained in the latter.
• This prior step can be carried out in different ways and can implement detergents leading to lysates, enzymes such as lysosyme or proteinase K, sonication or mechanical agitation in the presence of beads.
• nucleotide molecules it may be necessary to purify the extracted nucleotide molecules in order to rid them of possible contaminants such as nucleases or PCR inhibitory molecules.
• This purification of the nucleotide molecules may be carried out by phenol-chloroform extraction, by chromatography, by ion exchange, by electrophoresis, by equilibrium centrifugation and / or by hybridization capture on a solid support.
• Commercially available purification and extraction kits or kits may be used in the prior purification step of the sample (E).
• the device used in the context of the present invention may optionally be interfaced with an automatic sample preparation module making it possible to obtain a solution of pure nucleotide molecule (s) of volume adapted to said device, ie a volume of between 0.1 and 20 ⁇ l.
• This sample preparation module can be directly present on the device.
• it will preferentially be used a technique for purifying nucleotide molecules based on the use of magnetic particles such as magnetic beads on lab-chip operating by electrowetting, as described in the literature (Fouillet et al. microfluidic design and optimization of classic and new fluidic functions for Systems lab on a chip "3rd Microfluidics French Conference ⁇ Flu2006-23;.
• This purification technique uses a magnet placed at proximity of the device implemented in the context of the present invention during the steps of magnetic beads capture. This magnet is removed during the redispersion steps of the balls. The redispersion is done by stirring the fluid by displacements of the drops by activation of the electrodes.
• the method according to the present invention comprises a preliminary step of purifying the sample (E) comprising the steps of: i) contacting the sample (E) with a lysis solution ; ii) contacting the sample (E) lysed obtained after step (i) with at least one magnetic ball capable of adsorbing at least one nucleotide molecule; iii) washing said magnetic ball; iv) contacting said washed ball obtained after step (iii) with an elution solution and optionally heating the solution thus obtained; v) isolating the nucleotide molecule (s) obtained after step (iv).
• the magnetic beads are advantageously in a saline solution in which the salt concentration and the pH are optimal for the capture of nucleotide molecules on the beads.
• the magnetic beads used are advantageously commercially available and may be generic for the capture of nucleotide molecules (Chemicell silanol beads,) or specific (Dynabeads Streptavidin beads), or any other type of beads that can be functionalized with an oligonucleotide.
• compositions of the lysis, washing and elution solutions are well known in the state of the art and those skilled in the art can prepare them without any inventive effort.
• the lysis, washing or elution solutions can be obtained from commercial kits.
• Commercial kits for purification of genomic or plasmid DNA, messenger RNA or total or ribosomal can be used
• Bilatec Among the advantages of nucleic acid purification based on commercial kits on chip may however require adaptations: (1) possibly modification of the recommended volumes and (2) optionally addition of surfactant (s) in certain solutions to allow the introduction of the latter on the support implemented in the context of the present invention.
• surfactants there may be mentioned Tween20 or Triton X-100, more particularly, used at final concentrations of 0.05% to 2%.
• the latter may be placed on the support, advantageously by electrowetting, ⁇ ) prior to step (i) (sample
• step (ii) following step (ii) and prior to step (iii) (sample (E) lysed and mixed with magnetic ball (s)).
• the sample (E) can be deposited either on the zone (Zi) as defined above, or on a zone (Z 5 ) distinct from the zones (Zi), (Z 2 ), (Z 3 ) and
• the sample can be brought onto the support via an orifice passing through the substrate disposed opposite said support, said orifice being connectable to the sample reservoir (E) directly or indirectly by any input device such as a capillary or a flexible.
• the sample (E) deposited on the zone (Zi) or on a zone (Z 5 ) is mixed with the electrically conductive lysis solution, brought to said zone by electrowetting and this, in the form of one (or more) drop (s).
• Said lysis solution may be present on the support on a zone (Z 6 ) distinct from the zones (Zi) to (Z 5 ) as previously defined or may be brought onto the support via an orifice passing through the substrate disposed opposite said support said orifice being connectable to the lysis solution reservoir directly or indirectly by any input device such as a capillary or a hose.
• Said solution of magnetic beads may be present on the support on a zone (Z 7 ) distinct from the zones (Zi) to (Z 6 ) as previously defined or may be brought onto the support via an orifice passing through the substrate disposed opposite said support, said orifice being connectable to the magnetic bead solution tank directly or indirectly by any input device such as a capillary or a hose.
• the magnetic beads are captured by means of a magnet to form a pellet of magnetic beads and the supernatant is removed from the device by electrowetting and this, in the form of a ( or several) drop (s).
• the supernatant is advantageously directed towards a "trash can" present on the device.
• the latter is advantageously as an orifice passing through the substrate disposed opposite the support, an orifice passing through said support or an enclave in the cover.
• enclave means that the hood is hollowed in its thickness so as to provide a larger space between the hood and support.
• the electrically conductive washing solution can be brought to the magnetic bead end, by electrowetting and this, in the form of one (or more) drop ( s).
• Said washing solution may be present on the support on an area (Z 8 ) distinct from zones (Zi) to
• the magnetic beads may be redispersed in the washing solution by a back-and-forth movement of the latter, induced by electrowetting.
• the magnetic beads can again be recaptured into a compact base by bringing the magnet closer to the zone (Zi) or (Z 5 ).
• the supernatant is again evacuated via the bin as previously defined.
• a new washing solution identical or different to the previous one may be brought into contact with the pellet of magnetic beads according to the previously defined protocol variants and this, to repeat the washing step (iii) at least once.
• the washing step (iii) may be repeated as many times as necessary with the same wash solution or with one or more other wash solutions.
• Step (iv) consists of bringing into contact a solution of elution of the nucleotide molecules on the pellet of magnetic beads obtained following the last washing step.
• the introduction of said electrically conductive elution solution advantageously uses electrowetting.
• Said washing solution may be present on the support on a zone (Z 9 ) distinct from the zones (Z 1) to (Z 8 ) as previously defined or may be brought via a orifice through the substrate disposed opposite said support, said orifice being connectable to the elution solution reservoir directly or indirectly by any input device such as a capillary or a hose.
• the magnetic beads can be redispersed in the elution solution by a back-and-forth movement of the latter, induced by electrowetting.
• the elution solution in contact with the dispersed magnetic beads may be heated in order to more effectively "unhook" the nucleotide molecules retained on said beads.
• This heating may involve the use of means for heating the support used and in particular the zone (Zi) or (Z 5 ) thereof, and possibly means for controlling the temperature of the support used and in particular of the zone (Zi) or (Z 5 ) of the latter.
• Step (v) consists in capturing the magnetic beads freed of the nucleotide molecules in a compact base using a magnet.
• the supernatant thus obtained is the pure solution containing the nucleotide molecules which constitutes the sample
• the present invention also relates to a device that can be implemented in a method as defined above, said device comprising a support having at least two distinct zones (Z 1 ) and (Z 2 ),
• said 2 nd zone (Z 2) being adapted for 2 nd amplification step; - Means for bringing a fluid from said zone (Zi) to said zone (Z 2 ) by electrowetting.
• said zone (Z 2 ) of the support has at least one pair of specific primers and optionally at least one specific labeled probe such as previously defined, freeze-dried or dried (s).
• the support may have at least one additional zone, distinct from the zones (Zi) and (Z 2 ) and chosen from the group consisting of:
• the support also referred to herein as "chip” comprises, in a sectional view, a substrate having an electrode matrix, an insulating layer (ie dielectric) covering substrate and electrodes and a hydrophobic layer covering said dielectric layer .
• the different zones (Zi) to (Z 9 ) as previously defined are therefore located on the surface of said hydrophobic layer.
• zones is therefore mainly meant an arrangement of electrodes defined on the surface of the chip, each of these electrodes having a particular function and possibly a form adapted to the function.
• the support may be carried out according to the methods described in the state of the art (Fouillet Y et al., "Microfluidic Design and Optimization of Classical and New Fluidic Functions for the Labeling of Microfluid Systems.” Nanofluid. (2008) 4 : 159-165).
• the support has not only means for bringing a fluid from the zone (Zi) to the zone (Z 2 ) but also:
• a fluid of a selected zone into the group formed by the zones (Z 3 ), (Z 6 ), (Z 7 ), (Z 8 ) and (Z 9 ) to a zone chosen between zone (Zi) and zone (Z 5 ).
• These means make it possible to manipulate and move drops of fluid and implement series of adjacent electrodes in a plane, which can be arranged linearly but also in two dimensions, and this, to define a plane of displacement of a fluid. in the form of drops.
• the displacement of the drops also involves a counter electrode maintaining electrical contact with the drop during this movement and a voltage generator for applying a potential difference between the electrodes and the counter electrode.
• the electrodes may be made by depositing a metal layer such as an Au, Al, ITO, Pt, Cr or Cu layer by photolithography.
• the dielectric layer may be Si 3 N 4 , parylene or SiO 2 .
• the device of the invention may have an "open" configuration. In this case, it consists only of the support as defined above and the counter-electrode is advantageously in the form of a catenary wire.
• the latter advantageously has a "closed” configuration.
• the support of the device as previously defined is associated with a substrate disposed opposite said support.
• This substrate also referred to herein as the "bonnet”
• the hydrophobic layers of the support and the cover will preferentially deposited according to the method described in the article of Thery et al., "SiOC as a hydrophobic layer dielectric applications for electrowetting is” ll th International Conference on Minaturized Systems for Chemistry and Life Sciences ( MicroTas - 07-11 / 10/2007 - Paris France).
• the drops disposed on the support or displaced on the support are confined between the support and the substrate disposed opposite said support, the space between the support and the substrate being between 10 and 1000 nm, advantageously between 50 and 100 nm. Given the small volume of liquid displaced via the drops, we can add an oil aimed at contain the drops and prevent any risk of evaporation.
• Said oil may be, for example, a mineral oil such as an oil M3516 from Zigma-Aldrich or any other oil adaptable by those skilled in the art.
• a mineral oil such as an oil M3516 from Zigma-Aldrich or any other oil adaptable by those skilled in the art.
• the oil also has a passive role of protecting against sample contamination.
• the substrate has, as already explained, at least one orifice or suction hole that may advantageously have a shape converging towards the space between the support and the substrate and allowing the deposition of a liquid on the support or its suction from this last. Indeed, the suction of a liquid on a given area of the support is possible when the electrodes connected to the orifice through the hood are activated. Similarly, the deposition of a liquid on a given area of the support is possible via said orifice when the latter is connected to an injection device.
• the device according to the present invention may comprise means making it possible to carry the support or at least a given zone of this support and, more particularly, the zones (Zi), (Z 2 ) and (Z 5 ) as previously defined in a given temperature in particular during step (a), during the step
• These means for carrying the support at a given temperature can be either a Peltier-type heating element in contact with the face of the support opposite to the face carrying the zones (Zi), (Z 2 ) and any other zones (Z 3 ) to (Z 9 ), one (or more) resistance ( s) heating (s) integrated into the device.
• These integrated heating resistors can be made from a thin metal layer such as a thin layer of platinum, aluminum, doped silicon, etc., by defining patterns by conventional microfabrication techniques (photolithography and etching). for example) or for example by screen printing a conductive material such as platinum.
• the design of these heating resistors (thickness, section, length) will be performed to generate a given heating power for a fixed voltage, obtaining a well-defined electrical resistance value.
• these heating resistors can make it possible to locate the heating under the areas of interest, such as the zones (Z 1 ), (Z 2 ) and (Z 5 ), etc., in the context of the process carried out. in the invention.
• the device according to the present invention may also comprise one (or more) temperature probe (s) which makes it possible to control the temperature of the samples and reagents as close as possible to the zones (Zi), (Z 2 ) and (Z 5 ) as previously defined.
• the method of manufacturing such temperature probes is identical to the method of manufacturing the previously exposed heating resistors. It is, for example, It is possible to slave the probes to the Peltier block in order to precisely control the temperature of the support.
• the manufacturing technologies of devices using electrowetting involve patterning steps in metal layers.
• the production of heating resistors and temperature probes therefore does not add complexity to the overall manufacturing process of the device according to the invention.
• the device according to the present invention differs from the devices of the prior art, in that: it is portable because there is miniaturization of the volumes and the bulk of the analysis consumable;
• the device may have a large number of zones such as zones (Z 5 ) to (Z 9 ) previously defined, and the organization of zones (Z 1) to (Z 9 ) previously defined on the surface of the support and their provisions relative to each other are freer and are not imposed contrary to the case of the device described in the international application WO 2008/106719, in which the disposition is imposed by the centrifugal force which moves the liquids of a compartment to another.
• the device once the sample (E) introduced on the latter may no longer be in contact with the contaminant potential environment. This is particularly the case of a closed device on which the reagent (s) necessary for the different PCR amplification steps and the possible purification step of the sample to be analyzed have been previously embedded in liquid form, dried or freeze-dried.
• the device according to the invention offers the possibility of amplifying and analyzing at least two different samples, or even 3, 4, 5 or more different samples.
• Figures 1 and 2 propose optimal architectures for the amplification of nucleotide molecules for two samples (El) and
• FIG. 1 proposes a device seen from above, in "closed" configuration of which only the support is materialized, said device presenting:
• FIG. 2 proposes a device seen from above, in "closed" configuration, said device having: two zones (Zio-i) and (Z10-2) on which the samples (Ei) and (E 2 ) are respectively deposited;
• the present invention also relates to an amplification kit comprising: a device as previously defined, and
• thermostable polymerase a reverse transcriptase, deoxyribonucleotide triphosphates, a primer pair, specific or degenerate, an oligo-dT or specific primer useful for reverse transcription and a labeled probe.
• a thermostable polymerase a reverse transcriptase, deoxyribonucleotide triphosphates, a primer pair, specific or degenerate, an oligo-dT or specific primer useful for reverse transcription and a labeled probe.
• the method, the device and the amplification kit according to the present invention allow and are useful in field analyzes such as environmental monitoring, civilian surveillance, food safety, medical analyzes that can pass from the blood test with syringe to capillary sampling, oncologic markers analyzes on microbiopsies, criminal analyzes on traces, agrifood quality controls, veterinary analyzes including the search for viral, bacterial, parasitic or mycological pathogens on hair samples or microbiopsies.
• FIG. 1 proposes an optimized architecture device for performing the analysis of a sample by two-step gene amplification with pre-amplification of the sample by closed microcomponent functioning by electrowetting.
• FIG. 2 proposes an optimized architecture device for performing the nucleic acid purification of two samples in parallel, followed by the two-step gene amplification analysis with pre-amplification of the closed microcomponent sample using electrowetting.
• FIG. 3 provides an illustration of the realization of the amplification protocol in two steps on a micro device operating by electrowetting.
• FIG. 4 shows the increase in fluorescence obtained in 3 small reservoirs during the second amplification step of the protocol making it possible to detect the presence of E. coli, Bacillus subtilis and adenovirus 2 pathogens in the starting sample.
• Figure 5 shows the reagent feed, the position of the magnet and the electrodes connecting the reagent feed zone, the purification zone and the bin for the purification of nucleic acids on an EWOD chip from a blood sample.
• Figure 6 shows the PCR analysis of the EWOD purified DNA on an EWOD chip operating by electrowetting from a blood sample.
• a sample from a nucleic acid preparation containing 10 6 copies of the bacterium Escherichia coli gram 10 3 copies of virus Adenovirus Type II and 10 3 copies of the bacterium Bacillus subtilis is mixed with the PCR reagents (BSA 0.8 mg / ml, buffer of AmpliTaq GoId enzyme without MgCl 2 Ix, MgCl 2 3 mM, nucleotides (dATP, dTTP, dCTP, dGTP) 200 ⁇ M, betaine 450 mM, AmpliTaq GoId (0.5 U / ⁇ l) and pathogen specific primers investigated at different concentrations (Escherichia CoIi 20 nM / Adenovirus 20 nM / Bacillus subtilis 80 nM).
• the sample is first mixed in tube with the PCR reagents for the first amplification step and heated for 10 minutes at 95 ° C. in a tube (step of denaturation of the DNA and activation of Taq GoId). This solution is then introduced on the chip through a hole of the cover (Ti) and by activation of the electrodes.
• the first step of the cascade PCR is carried out in the large reservoir (Zi) (FIG. 3): 10 thermal cycles with a denaturation step at 95 ° C. (5 seconds) and a hybridization-elongation step at 60 ° C. during 20 seconds.
• the sample is dispensed in drops of 65 ni and each drop is sent to the small storage tanks Z 2 -I, Z2-2, Z 2 - 3 and Z2-4.
• the mixing of the drop of sample and specific reagents previously shipped is achieved by a movement of round-trip drops on electrodes adjacent to the small tank (10 A / R).
• the second step of cascade PCR is performed in small reservoirs. It consists of 40 thermal cycles with a denaturation step at 95 ° C. (5 seconds) and a hybridization-elongation step at 60 ° C. for 20 seconds. During this second PCR step, the fluorescence of the drops is measured at each thermal cycle at the end of the hybridization-elongation step. The fluorescence signal obtained during this second amplification step is given in FIG. 4.
• the first wash solution (Bilatest Genomic DNA kit wash buffer A) is then deposited in the hole (Ti) using a micro-pipette. It is introduced on the chip by activating the electrodes 1, 2 and 3 and then deactivating the electrode 1.
• the beads are redispersed in the washing solution by moving the magnet (A) away from the cover and activating the electrodes 2 and 3 for 2 minutes.
• the magnet (A) is then again approached the chip cover to reform a compact ball pellet.
• the supernatant is then brought into the bin constituted by the hole (T 2 ) by activating the electrodes 3, 4, 5, 6, 7 and 8 and then successively deactivating the electrodes 3, 4, 5, 6, 7 and 8. L all of these steps for washing the magnetic beads with washing solution A is repeated three times ( Figure 5).
• the second washing solution (washing buffer B of the Bilatest Genomic DNA kit) is then deposited in the hole (Ti) using a micro-pipette. It is introduced on the chip by activating the electrodes 1, 2 and 3 and then deactivating the electrode 1. The beads are not redispersed in this washing solution and the supernatant is brought into the bin constituted by the hole (T 2 ) by activating the electrodes 3, 4, 5, 6, 7 and 8 and then successively deactivating the electrodes 3, 4, 5, 6, 7 and 8.
• the DNA elution solution (Bilatest Genomic DNA kit) is then deposited in the hole (Ti) using a micro-pipette. It is introduced on the chip by activating the electrodes 1, 2 and 3 and then deactivating the electrode 1.
• the beads are redispersed in the elution solution by moving the magnet (A) away from the cover, activating the electrodes 2 successively. and 3 for 10 minutes and heating the chip to 55 ° C.
• the magnet (A) is then again approached the chip cover to reform a compact ball pellet.
• the supernatant is the purified DNA solution.

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