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
  • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
  • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor

Definitions

• the invention relates to the field of global detection of pathogenic microorganisms possibly poorly represented in the samples to be analyzed and whose impact on public health, whether human or animal, can be considerable.
• the present invention more specifically relates to a method of treating liquid samples for the detection of possible pathogenic microorganisms in very small quantities. More specifically, this process consists of a generic step of capture and concentration of microorganisms, followed by in situ lysis of microorganisms and capture of the nucleic acids released during lysis. The implementation of this method makes it possible to obtain an extremely concentrated and purified nucleic acid solution. This method is further adapted for continuous processing of liquid samples.
• the samples to be treated may be of complex biological and physicochemical composition.
• the characteristics that make the analysis of the sample difficult lie mainly in the variability of total biomass, ionic strength, pH, the presence of colloids, small molecules of decomposition of organic matter, or artificial chemical substances. in said sample.
• the samples to be analyzed contain a ubiquitous microbiological flora more or less concentrated, with no real impact on public health or the scientific studies carried out. This is for example the case of water samples (industrial, environmental) and conforms they may contain, these bacteria being pathogenic only at high concentrations; this is also valid for samples obtained from air, sludge or stool.
• the search for microorganisms with little or no concentration in a sample may be necessary, for example in the case of the detection of pathogens likely to colonize drinking water circuits, industrial or environmental waters or air. This is most often viruses, bacteria, protozoa, amoebae, fungi, yeasts, algae, worms ... This detection is essential since these pathogens can then be responsible for serious diseases including cholera, legionella, typhoid , in which one can observe the following symptoms: diarrhea, dysentery, gastroenteritis.
• microbiological monitoring makes it possible to initiate disinfection or treatment of water or industrial installations before discharge into the environment, and thus to comply with the regulatory microbiological standards.
• the reference technique is based on identification and enumeration of microorganisms by culture on different selective media. This is a technique that takes a long time to implement, often hours and which can be biased by both commensal flora but also by the presence of microorganisms stunted or even viable but not culturable and yet pathogenic. In addition, this method may not take into account slow growing microorganisms.
• Another technique allows rapid quantification of the total microbial population by measurement of enzymatic activity. This technique has the advantage of taking into account viable but non-culturable bacteria. On the other hand, it does not allow the specific identification of microorganisms.
• the filters or pellets generated are respectively often saturated or compact, contain many molecules and particles that are difficult to characterize, and which are known to interfere strongly with biological methods of analysis, such as antigen-antibody interactions or gene amplifications. It is therefore often necessary to dilute the samples very strongly to avoid inhibitions of the detection reactions, and to avoid false negative results.
• results obtained therefore correspond to instantaneous images of a state of contamination of a particular environment. They can not take into account the variability of contamination related to the environment.
• Liu et al. describe a biochip for the detection of bacteria in which the following steps are performed: the immunomagnetic capture of the target bacteria; pre-concentration of bacteria and their purification; lysis of bacteria; PCR amplification of the nucleic acids obtained and detection based on electrochemical DNA chips (Liu et al Anal Chem (2004) 76, 1824-1831).
• this device allows only a specific detection, targeting a particular bacterium.
• Cheng et al. disclose a biochip for the detection of bacteria in blood samples having a fluid chamber in which a dielectrophoretic separation of bacterial cells and blood cells is performed; electronic lysis (by application of a series of electrical pulses) of the captured bacteria; digestion of proteins with proteinase K; and an analysis of RNA and DNA released with DNA chips.
• the dielectrophoretic separation step allows the capture of only a small fraction of the bacteria thus limiting the applications of this device; in particular, it is not suitable for processing samples that may contain small amounts of microorganisms (Cheng et al., Nature Biotechnology (1998) 16, 541-546).
• PEI polyethyleneimine
• nucleic acid DNA and RNA
• the advantages of the described method and device are: generic capture; a high degree of concentration of microorganisms and possibly nucleic acids; a high degree of purification of microorganisms and possibly nucleic acids; simple integration into automated systems. More specifically, the present invention relates to a method for treating a liquid sample for analysis of the nucleic acids of the microorganisms likely to be contained in said sample consisting of:
• step (C) adsorbing the nucleic acids released in step (B) onto a second anion exchange active surface.
• the lysis step (B) is carried out in situ while said microorganisms are adsorbed on said first active surface and / or said first active surface is anion exchange surface and said second active surface is the same as said first active surface.
• 'Liquid sample means an industrial water withdrawal (eg from the cooling circuit), environmental water, or drinking water intended for human or animal consumption and, by extension, any sample in which the element or elements to be detected are in solution or in suspension.
• This sample may itself have been obtained from a sample or other sample containing the elements of interest, for example a body fluid, a sample of air, obtained by physical and / or chemical treatment, and / or biological according to any method adaptable by the skilled person.
• these are aqueous or high aqueous component samples.
• the samples are preferably made under conditions of great cleanliness with sterile material.
• the liquid samples are prepared according to techniques known to those skilled in the art (see in particular the publication of Stachowiak JC, Shugard EE, BP Mosier, Renzi RF, Caton PF Ferko SM, Van de Vreugde JL, Yee DD, Haroldsen BL, VanderBoot VA Autonomous microfluidic sample preparation system for protein profile-based detection of aerosolized bacterial cells and spores Anal Chem 2007 Aug. 1; 79 (15): 5763 -70).
• the volume of the liquid samples is between 1 and 100 ml, preferably it is 10 ml.
• the liquid sample is such:
• Micro-organisms means enveloped or non-enveloped viruses, Gram-positive and negative vegetative bacteria, spore-forming bacteria, protozoa, microscopic fungi and yeasts, microplankton, pollens, animal cells and the plant cells that we want to capture, and / or concentrate, and / or purify, and / or detect.
• active ion exchange surface is meant any ion exchange surfaces (anions or cations), more or less strong, allowing adsorption of microorganisms, or their constituents up to the molecular level.
• the active surface is selected from strong ion exchange resins.
• the active surface will be an anion exchange surface or anionic resin.
• anion exchange surface or anionic resin is meant a surface carrying charged chemical functions whatever the pH conditions. This is for example the case of quaternary amines, all of whose bonds are engaged with radicals other than protons.
• PEI polyethyleneimine
• DEAE diethylaminoethyl
• a strong anion exchange surface is all the stronger as the proportion of quaternary amine is important.
• the anionic resins (anion exchange) can be classified according to Table I below.
• Cationic resins (cation exchange) can be classified according to Table II below.
• the anion or cation exchange surfaces may be fixed or mobile, arranged within a device for extraction and purification of nucleic acids of microorganisms, they are chosen from charged polymers whose branching with a carbon chain allows the reinforcement of the load; in particular, they are selected from the group consisting of resins having groups selected from quaternary amino groups, tertiary amine groups, secondary amine groups, primary amino groups, sulphonic groups, phosphorus groups, carboxymethyl groups or carboxylic acid; or the hydroxylapatite, Di-ethyl-amino-ethyl (DEAE), poly-lysine and poly-ethylene-lmine (PEI) resins.
• DEAE Di-ethyl-amino-ethyl
• PEI poly-ethylene-lmine
• ion exchange surfaces in particular anions, described above allow the generic adsorption of microorganisms, and can be adapted to a very broad spectrum of applications.
• the elimination of the liquid medium initially containing the microorganisms to be sought can be easily achieved without risking picking up microorganisms from the surface.
• the elimination of the liquid medium allows the concentration of the microorganisms captured before lysis.
• the volume of the sample can be reduced to a volume of between 1 and 10 ⁇ l.
• ion exchange surfaces useful for carrying out the method of the invention resist the physical, chemical or enzymatic processes of opening microorganisms to release their nucleic acids.
• these surfaces also allow the purification and concentration of the nucleic acids in a solvent compatible with the biological reactions, in very low volumes, from 1 to 10 ⁇ l.
• anion or cation exchange surfaces advantageously rest on a support material enabling the active groups to be received vis-à-vis the process described, and whose integrity is not or very little altered by the opening treatments of the microorganisms.
• a support material enabling the active groups to be received vis-à-vis the process described, and whose integrity is not or very little altered by the opening treatments of the microorganisms.
• silica and polycarbonate By way of example and without limitation, mention may be made of silica and polycarbonate.
• the surfaces and their support can be either mobile, as in the case of magnetic beads for example, or fixed, as in the case of a lab on a chip.
• Lab-on-a-chip means any fluidic and / or microfluidic device comprising adequately sized, structured and functionalised sample passage zones (by surface modification or filling of functional reagents) completely or partially automated or not automated.
• the active surface and its support are movable in the form of balls, they are added to the whole of the liquid sample and are distributed in such a way that they behave like a net, with the smallest mesh size distance between the balls.
• the device must then be able to collect the beads to retain the captured elements (microorganisms, nucleic acids) and concentrate the sample.
• the device When the medium is fixed, there is no risk of aggregation.
• the device must, however, be sized and designed to allow effective capture of microorganisms for example by promoting the probabilities of meeting active surface - sample.
• the sample is brought into contact with the active surfaces in fractions, at speeds depending on the flow rate applied to the device.
• step (A) of capture of the microorganisms contained in a liquid sample is carried out on an active surface, ie anion exchange.
• the active surface is an anion exchange surface selected from quaternary amino groups, tertiary amine groups, secondary amino groups, primary amino groups; or the resins hydroxylapatite, Di-Ethyl-Amino-Ethyl (DEAE), poly-Lysine and Poly-Ethylene Imine (PEI).
• Step (A) can be carried out with an ion exchange surface such that:
• the active surface per unit volume of liquid sample that may be contained in the chamber in which the capture of the microorganisms is carried out is between 1 and 200 m 2 / l of sample, preferably between 9 and 100 m 2 / l ; the distance between the active surface and the elements to be captured is of the order of 10 to 100 micrometers, preferably 10 micrometers;
• the time for contacting the liquid sample with the active surface is of the order of 30 seconds to 10 minutes, preferably 10 minutes;
• the contacting temperature is between 4 ° C. and 40 ° C., preferably 25 ° C. ⁇ 5 ° C.
• an optional concentration step (A ') is advantageously carried out by the physical separation of the capture surface (fixed support or mobile support) and the liquid solvent.
• the active surface is mobile carried by magnetic beads
• the opening step (B) (lysis) of the microorganisms allowing the release of the nucleic acids is carried out directly on the microorganisms retained on the active surface; it can be carried out by any method known to those skilled in the art, in particular by sonication, abrasion by glass beads, enzymatic digestion, thermal shock, osmotic shock, light irradiation, electroporation, microwave action, etc. depending on the sample considered and the intended application following this step.
• An advantage of this method is to perform the lysis in situ, ie while the microorganisms are adsorbed on the active surface. To omit a stage of elution of microorganisms is advantageous; in fact, the fewer stages of the process, the more fluid transfers, potential contamination factors and loss of material are avoided; in addition, this makes the process more easily automatable.
• the step (B) of lyse microorganisms directly in the presence of the active surface can be achieved by different chemical and / or physical and / or biological and not altering, or very little, the structural properties and functional surfaces and / or their supports.
• the lysis can be carried out by enzymatic digestion with lysozyme alone or with lysozyme and then proteinase K.
• Preferential digestion with lysozyme followed by proteinase K will be preferable under the respective lysis conditions in a medium of composition: Tris-HCl at 50 mM, NaCl at 50 mM, EDTA at 5 mM, pH 7.5, for 10 minutes at room temperature, then in a medium of composition: Tris-HCl 10 mM, EDTA at 2 mM, pH 8.0 or 4M NaCl, 10 mM Tris-HCl, 2 mM EDTA, 0.1% sarcosyl, pH 9.0, for 15 minutes at 70 ° C.
• step (B) is carried out by ultrasonication of the sample under optimum conditions as a function of the geometry of the chamber containing the sample and whether the support of the active surface is fixed or mobile.
• the present process may comprise an additional step (A1), which is intercalated between step (A) and step (B), for purification of the microorganisms.
• This purification step (A1) corresponds to a washing of the surface on which the microorganisms are fixed with a solution chosen so as not to disturb the attachment of the microorganisms to the exchange surface.
• a salt solution may be employed, the characteristics of which are dependent on the active surface employed.
• purification is generally meant the elimination of unnecessary or troublesome compounds which have attached to the active surfaces under the same conditions as the microorganisms or nucleic acids of interest released at course of step (B), but whose elution can be achieved by suitable solutions, without the risk of eluting, or at least very partially, said microorganisms and nucleic acids which remain adsorbed on the active surface.
• the optional purification step (A1) may be carried out by incubating the active surface with a washing solution based on salts, preferably having the following composition: 0.8M NaCl, 10 mM Tris-HCl, 15% ethanol % (v / v), pH 8.
• the process according to the invention also comprises a step (C), following step (B), of adsorption of the nucleic acids released by the lysis of said microorganisms.
• the nucleic acids released are, during step (C), either immediately adsorbed on the first active surface that has allowed the capture of microorganisms, or adsorbed on a second electrostatic force anion exchange surface similar to that of step (A).
• a second electrostatic force anion exchange surface similar to that of step (A).
• the same first anion exchange surface is used as the first and second active surfaces.
• a possible step (A2) of elution of the microorganisms of the first active surface can be carried out, following step (A) or step (A1) and preceding step (B), with a solution which promotes the separation of the microorganisms of the first active surface by competition with an ion of the same charge, or by chemical modification of the surface charge density of the balls and / or microorganisms.
• Step (A2) can be carried out by incubating the active surface with a solution of 100 mM sodium hydroxide, containing or not detergents, optionally supplemented by physical methods such as heating, stirring with a vortex or ultrasound.
• the microorganisms contained in the eluate may be isolated from the first active surface to undergo the lysis provided in step (B) or undergo lysis in the presence of the first active surface but without being adsorbed.
• a possible step (A3), following step (A2), of regeneration of the first active surface can also be carried out with a solution of sodium hydroxide (NaOH) so as to continue with another capture cycle in accordance with step (A) for a predetermined number of cycles.
• Regeneration of the active capture surface of the microorganisms can be carried out in the following conditions: incubation of the active surface with a solution of sodium chloride, preferably 100 mM, then its removal or incubation of the active surface with deionized water, and its removal.
• step (C) of the process the adsorption of the nucleic acids is carried out on the same ion exchange surface as that used in step (A) (first active surface), which is a surface exchange surface. anion.
• the adsorption is carried out on a second active surface, corresponding to an anion exchange surface useful for this nucleic acid adsorption step (C), a prelude to the purification and concentration of nucleic acids.
• This second anionic active surface can contain groups bearing different lengths of carbon chains, C 1 -C 18 for example, but also of silica, DNA, RNA I 1 and synthetic analogue of nucleic acids such as PNA and LNA.
• the second active surface is the same anion exchange surface as used in step (A).
• the second surface used may rest on a support and said second active surface and its support may be fixed or movable.
• the method may comprise an optional step (C1), following step (C), of washing purification of the adsorbed nucleic acids.
• This step (C1) is carried out by adding a salt-based solution with preferably, but not necessarily, alcohol in well-defined concentrations, depending on the active surface; it allows the elimination of many chemical and biological contaminants, such as sugars, proteins, lipids, small RNAs, so that only the DNAs and most of the RNAs remain. sample.
• a salt-based solution with preferably, but not necessarily, alcohol in well-defined concentrations, depending on the active surface; it allows the elimination of many chemical and biological contaminants, such as sugars, proteins, lipids, small RNAs, so that only the DNAs and most of the RNAs remain. sample.
• the method according to the invention may also comprise a nucleic acid elution step (C2) following step (C) or step (C1).
• this step (C2) can allow the separation of DNAs and RNAs.
• the retained RNA can be selectively eluted with a saline solution of lower concentration than that required for DNA.
• the elution is carried out by modifying the surface charge of the active surfaces, either by varying the pH and / or by varying the ion concentration. in ranges of concentrations compatible with biochemical and biological reactions, according to methods known to those skilled in the art.
• the overall process for preparing nucleic acids on active anion exchange surfaces is particularly well suited to devices of the laboratory-on-a-chip type.
• the surface capture makes it possible to envisage a very significant downscaling as soon as the first steps of the sample processing are carried out.
• the sample of interest will ultimately be circumscribed to the capture surface, overall the microorganisms and / or DNA and / or RNA will ultimately be stored in a two-dimensional structure, that is to say surface, instead of being stored in solution in a three-dimensional reservoir.
• the invention can be declined in several variants, in particular, subsequent steps of sample processing can be added.
• the step (D) of capturing the nucleic acids on an active surface can be carried out under physicochemical conditions adapted to the type of active surfaces.
• the active surface is incubated with a saline concentration solution of less than 0.8 M NaCl, preferentially less than
• the method according to the invention can be further completed by a step (E) of purification of said nucleic acids.
• This step (E) of purifying the nucleic acids is carried out by passing a washing solution via the incubation of the active surface with a solution based on salts, preferably of composition: 0.5M NaCl, tris-HCl 10 mM, 15% (v / v) ethanol, pH 8 for a PEI type surface or via incubation of the active surface with a salt-based solution, preferably of composition: 2 mM NaCl, 5 mM EDTA, 80% ethanol (y N), pH 7.0.
• a washing solution via the incubation of the active surface with a solution based on salts, preferably of composition: 0.5M NaCl, tris-HCl 10 mM, 15% (v / v) ethanol, pH 8 for a PEI type surface or via incubation of the active surface with a salt-based solution, preferably of composition: 2 mM NaCl, 5 mM EDTA, 80% ethanol (y N), pH 7.0.
• the method may further include a final concentration step (F) via a drying step of removing residual liquids followed by elution to a small volume of liquid.
• step (F) elution of nucleic acids in small volume is carried out by a solution promoting the separation of nucleic acids from the active surface, according to the same principles as those described in (A2); with, preferably, the incubation of the active surface with a NaOH solution not exceeding 100 mM, preferably 50 mM, for 20 seconds at room temperature, for an active surface of the PEI type, or the incubation of the active surface with a 10 mM Tris-HCl solution at 60 ° C. for 2 minutes for a silanol surface.
• the invention relates to a liquid sample treatment device capable of containing microorganisms, characterized in that it contains at least one ion exchange surface, preferably anion, such as as defined above arranged in a chamber such that the ratio of said active surface per unit volume of sample that can be contained in said chamber is between 1 and 200 m 2 / l, preferably between 9 and 100 m 2 / l.
• the device comprises a laboratory on a chip consisting of moving balls supporting the active surfaces.
• the optimal inter-ball distance for the microorganism capture step is about 10 ⁇ m. This distance is also optimal for the capture of nucleic acids.
• the developed surfaces given per unit volume based on kinetic studies of capture of microorganisms Correspond well to the experimental data obtained.
• the surface area per unit volume (S / V) ratio of the chamber through which the sample will pass is calculated with the value of the active area ion exchange present in the chamber and the value of the sample volume that may be contained in the chamber;
• the volume of sample that may be contained in the capture chamber and - the flow rate of the sample.
• Each input data influences the other two.
• the volume of said chamber is 1 .mu.l
• the surface of the two rectangles (20 mm x 5 mm) 200.10 "6 m 2
• the SA / ratio is 200 m 2 / l, which is in the state very greater than the active surface developed by the 1 ⁇ m beads used in the description of the process which follows and can therefore bring even more efficiency to the process.
• the active surface can be further increased by adding a structuring of the chamber, for example by pillars, or a parallelization of the fluidic circuits.
• the adsorption rates should be very fast and therefore allow high flow rates.
• Suitable dimensioning and structuring of the chambers containing the active surface in contact with the liquid sample must therefore make it possible to treat sample volumes with, at least, the same performances as those described in the examples presented below.
• Figure 1 there is described a method of preparing nucleic acids according to the invention for microorganisms present in liquid samples.
• the dashed arrows represent possible steps, or alternative possibilities of sample processing, the term "AN" means nucleic acids.
• the path combinations shown in the figure are as many variants of possible sample preparations.
• This invention finds application in very varied fields: - all the biological analyzes where the elements sought are very diluted and where the whole of the sample must be analyzed;
• This sample preparation method described in the present invention offers several advantages: the simple and generic capture of microorganisms without distinction of sizes; capture of microorganisms and their constituents even at very low concentrations; the capture of microorganisms and their constituent even very concentrated; capture of poorly concentrated microorganisms in a high concentration of total biomass; the preparation of a liquid sample with a high concentration of microorganisms making the process incorporable in standard devices lab-on-a-chip; capturing and continuously concentrating the liquid sample, which may be the whole sample or a modified or unmodified fraction of the whole sample; the possibility of purifying captured microorganisms; the ability to elute and regenerate active surfaces to process large volumes of samples (modified fractions or not); • the possibility of lysing microorganisms in the presence of the active surface; "Capture and purification of nucleic acids;
• FIG. 1 presents a general flowchart detailing the different steps of the method according to the present invention, it is the method implemented in example 1.
• Figure 2 details the capture and regeneration performance of the active surfaces according to the invention.
• Figure 3 shows the capacity in terms of purification yield of DNA or RNA samples according to the nature of the surfaces used, reference to a commercial process.
• Figure 4 shows the detection of model microorganisms used after capture.
• Example 1 Process for preparing nucleic acids 1. Materials and methods
• the method according to the invention was tested during the preparation of nucleic acids from model microorganisms, here Escherischia coli and Bacillus subtilis for vegetative bacterial forms, Bacillus subtilis for sporulated bacterial forms, human adenovirus type 2 (group C) for viruses, Cryptospomoidium parvum for protozoa, and using polyethyleneimine coated (PEI) coated active surface for capture - concentration and purification of microorganisms, and possibly nucleic acids, and silanol for nucleic acids.
• model microorganisms here Escherischia coli and Bacillus subtilis for vegetative bacterial forms
• Bacillus subtilis for sporulated bacterial forms
• human adenovirus type 2 (group C) for viruses
• Cryptospomoidium parvum for protozoa
• PEI polyethyleneimine coated
• the experimental protocol is generically described for all the microorganisms tested.
• a liquid sample of 10 milliliters is previously collected, it may be the whole sample, modified, for example by a treatment such as an ultrafiltration or not, or a fraction of the total sample, modified for example by a treatment such as whether ultrafiltration or not.
• This sample is contacted with an active surface of polyethylene imine (PEI), here 9.2 rVVIitre sample, and supported by super paramagnetic beads of a micrometer diameter (Chemicell).
• PEI polyethylene imine
• the contacting is carried out for 10 minutes with stirring using a vortex, in order to keep the beads well dispersed, and at room temperature.
• the beads are collected using a magnet until clarification of the sample and the liquid phase is eliminated.
• step (B) the beads, containing on their surface the microorganisms, are subjected to a lysis step to allow the opening of said microorganisms and the release of nucleic acids.
• microorganisms adsorbed to the beads are purified by 500 ⁇ l of a solution of NaCl 0.5M, Tris-HCl 10 m M 1 15% ethanol (v / v), pH 8, 0, before being lysed (A1).
• the purified microorganisms are eluted with a solution of 100 mM NaOH at ambient temperature for 2 minutes and then separated from the active surface (here supported by the beads) to be stored (A2).
• the active surface can be regenerated by addition of NaOH to 100 mM and then deionized water, and return to the initial capture step (A3).
• Lyse under "low knows” conditions The lysis of the microorganisms is carried out in the presence of the active surface supported by the beads, contained in 200 ⁇ l of a solution of 50 mM Tris-HCl, 50 mM NaCl, EDTA at 5 mM M, pH 7.5 called "low knows”.
• the beads may be contained in 200 ⁇ l of a solution of 4M NaCl, 50 mM Tris-HCl, 5 mM EDTA, 0.1% sarcosyl, pH 8.5 called "high know. Lysis is performed by ultrasonication.
• the lysis is performed by two successive enzymatic digestions (with lysozyme and proteinase K), as described above. 1 C. Protocol of step (C)
• the beads that adsorbed the nucleic acids are collected and the aqueous phase removed.
• the PEI-adsorbed nucleic acids are purified with 0.5M 1 NaCl solution of 10 mM Tris-HCl, 15% ethanol (v / v), pH 8.0 according to step (C1).
• the beads are collected and the purification solution evacuated.
• the nucleic acids are then eluted by incubating the PEI beads in 100 ⁇ l of a 100 mM NaOH solution at room temperature for 10 seconds.
• the beads are collected, the nucleic acids recovered and the 100 mM NaOH solution neutralized by adding 100 ⁇ l of a 100 mM HCl solution according to step (C2).
• the nucleic acids in solution are then brought into contact with the active surface, here 1, 18 m 2 / l of sample, for 30 seconds with stirring at room temperature, according to (D).
• the beads are collected and the aqueous phase removed.
• a purification step according to (E) is performed by incubation of the beads in a solution of 0.5M NaCl 1 Tris-HCl 10 mM, ethanol 15% (v / v), pH 8.0. Finally, an elution step is carried out with 2 to 10 ⁇ l of a solution of 100 mM NaOH at room temperature for 10 seconds.
• the beads are collected, the supernatant recovered and neutralized by adding the same volume of HCl to 100 mM, according to step (F)
• the nucleic acids eluted from the PEI beads at the end of the lysis stage and neutralized can be mixed with 5 volumes of solution.
• Balls of one micrometer in diameter developing a silanol active surface area of 9.2 m 2 / l of sample are then added according to (D).
• the beads are collected, the aqueous phase removed.
• the adsorbed nucleic acids are purified twice by adding 500 ⁇ l of a 2 mM NaCl solution, 10 mM Tris-HCl, 75% (v / v) ethanol, according to (E).
• the beads are collected, the aqueous phase removed.
• the nucleic acids are then eluted in 5 to 10 ⁇ l of Tris-HCl at 10 mM, pH 8.0 with stirring and at 60 ° C., according to step (F).
• 1-C.2. "High knows” conditions In the case of a lysis performed under "high knows” conditions, the experimenter collects the beads that adsorbed the nucleic acids and recovers the aqueous phase that he mixes with 5 volumes of Guanidine HCl solution. 3M, 20 mM Tris-HCl, 80% (v / v) ethanol pH 4.5. Balls of one micrometer in diameter developing a silanol active surface of 0.92 m 2 / l of sample are then added according to (D).
• the beads are collected, the aqueous phase removed.
• the adsorbed nucleic acids are purified twice by adding 500 ⁇ l of a 2 mM NaCl solution, 10 mM Tris-HCl, 75% (v / v) ethanol, according to step (E). ).
• the beads are collected, the aqueous phase removed.
• the nucleic acids are then eluted in 5 to 10 .mu.l of 10 mM Tris-HCl, pH 8.0 with stirring and at 60.degree. C., according to the principle of concentration in a very small volume of step (F).
• Table III summarizes the elution results of model microorganisms ( ⁇ for Bacillus subtilis, E. c for Escherichia coli, Cp for Cryptospomoidium parvum and Ad2 for human Adenovirus type 2). Solutions used L-Og 10 reduction
• the most favorable condition for elution is an incubation in a solution of 100 mM NaOH, performed here by incubating the active surfaces for 2 minutes at room temperature (25 ° C.) and with stirring (650 rpm).
• This graph shows, on 3 separate samples, that the capture of N microbial elements is perfectly possible by the capture of N / 10 microbial elements repeated 10 times.
• NAs Nucleic acids
• the ANs eluted at the end of each of the two variants were purified once again on a Qiagen column dedicated to either the purification of DNA or of RNA on a silica column.
• the amount of RNA or DNA was measured spectrophotometrically and the yield calculated relative to the reference.
• the graph of Figure 3 shows that the sample preparation method described herein allows for good purification of nucleic acids (NAs), in particular by using the same ion exchange surface to capture both microorganisms and viruses. nucleic acids.
• the graph of Figure 4 shows the detection of nucleic acids of model microorganisms by PCR.
• the initial copy number is doubled.
• the increase in the number of copies is monitored in real time by measuring the specific increase in fluorescence released during the reaction.
• the Ct is directly proportional to the concentration of targets in the amplification reaction medium and corresponds to the first cycle of the linear phase of the amplification.
• the method described has been optimized with magnetic beads as support for active surfaces dedicated to the capture of microorganisms and nucleic acids. It has been validated on recognized model microorganisms: gram-negative and gram-positive bacteria, sporulated bacteria, viruses and protozoa.
• the device comprises:
• a peristaltic pump to allow the displacement of the sample and of all the liquid reagents;
• - a magnet, which allows the collection of magnetic beads;
• a heating ultrasound device which allows the resuspension of the beads collected by the magnet and the opening of the microorganisms;

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• 2008
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  • 2009-07-16 WO PCT/FR2009/000872 patent/WO2010007255A2/fr active Application Filing
  • 2009-07-16 US US13/054,294 patent/US20120009560A1/en not_active Abandoned
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