EP2318539A2

EP2318539A2 - Method for purifying nucleic acids from microorganisms present in liquid samples

Info

Publication number: EP2318539A2
Application number: EP09784273A
Authority: EP
Inventors: Stéphane COUPE; Dorothée JARY
Original assignee: Commissariat a lEnergie Atomique CEA; Commissariat a lEnergie Atomique et aux Energies Alternatives CEA
Current assignee: Commissariat a lEnergie Atomique et aux Energies Alternatives CEA
Priority date: 2008-07-16
Filing date: 2009-07-16
Publication date: 2011-05-11
Also published as: WO2010007255A2; FR2933989B1; US20120009560A1; FR2933989A1; WO2010007255A3

Abstract

The present invention relates to a method for treating liquid samples with a view to detecting any possible pathogenic microorganisms in a very small amount. More specifically, this method comprises a generic step of capturing and concentrating microorganisms on an ion exchange surface, followed by an in situ lysis treatment carried out on the microorganisms and capture of the nucleic acids released during the lysis. The implementation of this method enables an extremely concentrated and purified solution of nucleic acids to be obtained. This method is suitable for a continuous treatment of liquid samples. The invention also relates to a device for analysing liquid samples for biology, health or the environment, which is suitable for the implementation of the method according to the invention.

Description

Process for purifying nucleic acids from microorganisms present in liquid samples

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.

It also relates to devices for analyzing liquid samples for biology, health or the environment; in particular cellular and molecular collector-concentrator devices, and, more generally, integrated sample processing devices. Problem of the quality of the 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.

Thus it is common that 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.

In many cases, 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.

In the case of a complex sample due to, for example, a very high total microbial load, or a sample rich in colloids, etc. the detection of a very diluted micro-organism is made difficult.

Need for microbiological monitoring Given the possible contaminations of water by infectious agents, it is necessary to be able to monitor the load of pathogenic micro-organisms with sufficient frequency. In the case of environmental waters, the controls make it possible to initiate, if necessary, preventive measures in terms of human or animal use, by limiting access to sites for example, and in terms of withdrawal to treatment for health networks. drinking water, among others.

In the case of industrial water networks, 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.

Monitoring the microbiological quality of the air is subject to the same problems with the same health consequences as those defined for water.

Optimizing Sample Processing Methods Most of the conventional techniques used to process large volume liquid samples have several disadvantages. They treat point samples, are not very sensitive because of the low purification yields and the high dilution required before the biological analyzes. Finally, they are long, require heavy equipment and come back expensive in human and material resources.

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.

Finally, other techniques allow more specific answers. This is the case of microbial captures using specific antibodies. However, the response time is still often long and the technique may have defects in sensitivity and specificity due to cross reactions. In the same way, the use of labeled nucleic probes allows a very specific detection after hybridization with their targets. The disadvantage lies in the low number of targets per cell which makes this technique insensitive and poorly adapted to the detection of low-concentrated microorganisms. In recent years, this disadvantage has been overcome by the use of gene amplification techniques in real time, for example by PCR or NASBA.

However, most of the techniques first require heavy treatment by filtration, centrifugation or precipitation. However, 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.

Finally, the samples are made punctually over time with frequencies that can be variable. The 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.

Analysis of miniaturized integrated system development work up to the micrometric scale shows that the sample preparation stage is crucial and very difficult to integrate, especially when all the preparative steps are taken. supported by the same microsystem, from the capture of the elements to look for until obtaining the pure and ultra concentrated molecules.

It therefore appears that a system capable of capturing and concentrating microorganisms continuously over a relatively long time would be more informative of the state of contamination of an environment than a one-off sampling, and would allow both a better concentration. and better purification. Globally, this would make it possible to work with typical laboratory-on-a-chip systems and avoid, for example, the necessary dilutions before the gene amplifications as they are practiced today. Therefore, it is useful to develop an integrated system that meets the following criteria:

1. a wide range of use allowing simple and generic strategies of sample processing,

2. a significant change in scale of the sample to be processed that can be integrated into microsystems,

3. a strong purification and concentration capacity to allow the analysis of the whole sample and to allow the detection of trace microorganisms (the number of which may be less than 10 in 100 ml),

4. fast preparation time, less than 20 minutes. These different requirements are met by the process according to the invention.

Miniaturized microarray detection devices have been developed. 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). By immunomagnetic capture of the target bacteria, 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).

It has been shown that certain ion exchange polymers, such as polyethyleneimine (PEI), can adsorb bacteria or viruses (Deponte et al.

Anal Bioanal Chem. 2004; 379 (3): 419-26; Yamaguchi et al. J Virol Methods. 2003

Dec; 114 (1): 11-9.); cellulose matrices carrying sulfate ester functions have also been described for the capture of viruses or viral particles

(EP 1 808 607). Such ion exchange polymers also allow the capture of chemical molecules (Boom et al., Clin Microbiol 1990 Mar; 28 (3): 495-

503; United States Patent US 5,342,931 "Process for purifying DNA on hydrated silica"; United States Patent US 5,503,816 "Silicate compounds for DNA purification") with good performance in terms of kinetics, purification and saturation.

Some systems have been developed for the extraction of DNA from blood samples with a volume of a few milliliters. The DNA extract is purified and then eluted in at least 100 μl (King Fisher, Easy Mag).

However, none of these devices or methods allows both a capture of microorganisms that is of a generic nature, the possibility of performing lysis of microorganisms in situ and a capture of the nucleic acids released during said lysis.

The Applicant has now demonstrated the possibility of applying pretreatment to liquid samples that may contain microorganisms for subsequent analysis of the nucleic acids of said liquid samples.

The method implemented in this invention relies on the use of ion exchange surfaces to perform all steps of nucleic acid preparation, from capture of microorganisms to purification and concentration of acids. nucleic acid (DNA and RNA).

In summary, 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:

(A) capturing the microorganisms contained in said liquid sample by adsorption of said microorganisms on a first active ion exchange surface, anions and / or cations;

(B) the lysis of said microorganisms in the presence of said first active surface on which said microorganisms are adsorbed or not:

(C) adsorbing the nucleic acids released in step (B) onto a second anion exchange active surface. Preferably, 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.

The generic nature of the process comes from its ability to be implemented for all of the microorganisms defined below and nucleic acids without distinguishing particular specificity that can a priori differentiate them from each other.

'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. Preferably, these are aqueous or high aqueous component samples.

In order to avoid any exogenous contamination, the samples are preferably made under conditions of great cleanliness with sterile material. In the case of analysis of the microbiological quality of the air, 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. Preferably, the liquid sample is such:

- it does not contain, or a negligible amount, that is to say less than 5% by weight relative to the total weight of the sample, ionic detergents;

that its saline concentration does not exceed 2 M and is preferably less than 200 mM;

- Its pH does not exceed 10, preferably, its pH is close to neutral.

"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.

By "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. Preferably, the active surface is selected from strong ion exchange resins. Preferably, the active surface will be an anion exchange surface or anionic resin.

By "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. For example, polyethyleneimine (PEI) is a strong anion exchanger since a fraction of the amines is quaternary while diethylaminoethyl (DEAE) is an exchanger low since no amine is quaternary. Thus, 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.

Table I

Cationic resins (cation exchange) can be classified according to Table II below.

Table II 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. Said 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. In addition, 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. In particular, the volume of the sample can be reduced to a volume of between 1 and 10 μl. In addition to allowing the capture of microorganisms, 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. Preferably, 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 μl.

The 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. 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.

When 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.

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.

Finally, the sample is brought into contact with the active surfaces in fractions, at speeds depending on the flow rate applied to the device.

According to a particular embodiment of the process according to the invention, step (A) of capture of the microorganisms contained in a liquid sample is carried out on an active surface, ie anion exchange. when it comes to capturing microorganisms with negative net surface charge, or cation exchange when it comes to capturing microorganisms with net positive surface charge; preferably, 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.

Regular stirring of the sample, for example by means of a vortex, can be carried out. This agitation is advantageous when beads are used as a support for the active surface.

Following the capture, 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. In the case where the active surface is mobile carried by magnetic beads, a magnetic attraction of the beads for 30 seconds to 2 minutes, or until clarification of the sample, is recommended. 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.

Concretely, 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.

By way of example, 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.

According to one variant, 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. According to another variant of the invention, 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. For example, a salt solution may be employed, the characteristics of which are dependent on the active surface employed.

By "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.

Thus, after the opening or lysis of the 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). This makes it possible to segregate and quickly concentrate the material of interest, here the nucleic acids. Preferably, 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. In this case, 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.

According to a variant of 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.

According to another variant of step (C) of the process, 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. Preferably, the second active surface is the same anion exchange surface as used in step (A).

Again, 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.

Finally, the method according to the invention may also comprise a nucleic acid elution step (C2) following step (C) or step (C1).

Depending on the experimental conditions, 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.

After a series of washings based on salts and possibly alcohol, 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. In fact, 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. Depending on whether the active surface is carried by beads or on a fixed support, 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. Once the nucleic acids have been collected either on active surface or in solution, it is possible to purify them and to collect them according to a similar process consisting in the adsorption of the nucleic acids on an active surface of the PEl or Silanol type, according to the stage (D).

The step (D) of capturing the nucleic acids on an active surface, which is similar to or different from the first active surface for capturing the microorganisms, can be carried out under physicochemical conditions adapted to the type of active surfaces. To do this, the active surface is incubated with a saline concentration solution of less than 0.8 M NaCl, preferentially less than

200 mM, devoid of detergent, and pH lower than the pKa of the group involved in the adsorption, preferably around the neutrality, in the case of an active surface similar to the PEI.

Incubation of the active surface with a solution of guanidine HCl with a concentration greater than 3 M, Tris-HCl at 10 mM, ethanol at 80% (v / v), pH 4.5, in the case of an active surface Silanol is preferred.

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.

Finally, 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.

In 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.

According to another of its aspects, 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.

According to a particular embodiment of said device, it comprises a laboratory on a chip consisting of moving balls supporting the active surfaces. In this case, the optimal inter-ball distance for the microorganism capture step is about 10 μm. This distance is also optimal for the capture of nucleic acids. In addition, the developed surfaces given per unit volume based on kinetic studies of capture of microorganisms (see the theoretical model of Deponte et al.) Correspond well to the experimental data obtained.

Those skilled in the art can then size a lab-on-a-chip based on prior art data (e.g., Microfabήcated reaction and separation Systems, Madhavi Krishnan et al., In "Analytical Biotechnology", Elsiever Science ed.). .

In the first place, these three data should be defined:

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. By way of example, when using a chamber of dimension 20 mm × 5 mm × 0.01 mm; 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, then the SA / ratio is 200 m ² / l, which is in the state very greater than the active surface developed by the 1 μm 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.

According to the kinetic model of Deponte et al. (see above), 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.

According to Figure 1, there is described a method of preparing nucleic acids according to the invention for microorganisms present in liquid samples. In this Figure 1, 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;

in fluidic microsystems (Laboratory on Chip (LOC) or in Microsystems of Total Analysis (μTAS)), in which the integration of modified surfaces (fixed or mobile magnetic ball-type) is controlled, and 00872

17

- in all integrated systems requiring sample preparation, extraction type - purification of nucleic acids.

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;

- the possibility of differential elution of I ¹ DNA and RNA; the high concentration of nucleic acids; the implementation of the process in a short time, of the order of 15 to 20 minutes.

The invention is now described in more detail in connection with the following figures:

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.

The experimental protocol is generically described for all the microorganisms tested.

1-A. Protocol of step (A)

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). 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. 1-B. Protocol of step (B)

According to 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.

Alternatively, microorganisms adsorbed to the beads are purified by 500μl of a solution of NaCl 0.5M, Tris-HCl 10 m M ₁ 15% ethanol (v / v), pH 8, 0, before being lysed (A1).

According to one variant, 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). Finally, according to a variant, after elution of the microorganisms, 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).

1-B.1. 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 μl of a solution of 50 mM Tris-HCl, 50 mM NaCl, EDTA at 5 mM M, pH 7.5 called "low knows".

1-B.2. Lyse in conditions "high knows"

According to one variant, the beads may be contained in 200 μl 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.

According to another variant, the lysis is performed by two successive enzymatic digestions (with lysozyme and proteinase K), as described above. 1 C. Protocol of step (C)

1-C.1. Conditions "low knows"

In the case of a lysis performed under "low knows" conditions, 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 μl 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 μl 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 μl 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)

According to one variant, 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. 3M Guanidine HCI, 20mM Tris-HCl, 80% (v / v) ethanol pH 4.5. 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 μl 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 μl 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 μl 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).

2. Results

The variants described above have each been validated independently. 2-A. Capture and elution of model microorganisms

Table III summarizes the elution results of model microorganisms (β 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

(Conditions) β. s E. c Cp Ad2

PBS <0.05 <0.05 <0.05 H2O (di)

PBS ₁ SDS 0.1% <0.05 0.18 <0.05

PBS, SDS 0.5% <0.05 0.27 <0.05 PBS, SDS 1% <0.05 0.52 0.21

1 M NaCl, pH 9 <0.05 3.16 3

2M NaCl, pH9 0.31 0.2 0.15

NaCl 3M ₁ pH9 <0.05 0.17 <0.05

4M NaCl, pH9 <0.05 0.22 0.05

50 mM NaOH> 2.14 0.84

10 mM NaOH> 3> 2> 2

200 mM NaOH> 3> 2> 2

100 mM NaOH, 0.01% to 1% SDS> 3> 2> 2

Table III

This Table III shows that the conditions PBS and H2O do not allow the elution of previously adsorbed species to the active surface.

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). 2-B. Ball reuse

The capture capacity after purification, elution of microorganisms and regeneration of the active surface (polyethylene imine (PEI) at 9.2 m ² / liter of sample, supported by super paramagnetic beads of 1 μm in diameter (Chemicell)) has has been evaluated after 10 cycles of the process comprising the purification / elution / regeneration steps, the graph of this experiment is shown in FIG.

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.

This test confirms that a device adapted to the implementation of the method according to the invention can be reused after regeneration of the active surface.

2-C. Enzymatic and physical lysis in the presence of active surfaces

The tests carried out also show that the lysis of the microorganisms does not affect the functionality of the active surfaces. 2-D. Adsorption of DNA and RNA

The result obtained in the following point implicitly demonstrates the ability of the active surface to properly capture the DNA and RNA I ^1. 2-E. Purification and elution of DNA and RNA on active surface (Silanol or PEH

Nucleic acids (NAs) were prepared according to two sample preparation variants: using only PEI or PEI + silanol. 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.

2 F. Evaluation of the complete process

Finally the complete process has been validated in artificial and real conditions by the detection of model microorganisms in different real samples.

The graph of Figure 4 shows the detection of nucleic acids of model microorganisms by PCR.

At each amplification cycle, 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.

It is determined automatically by the software associated with the thermal cycler (Stratagene).

Example 2 Device Implementing the Process Described Principle of the Test

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.

It has been validated on various types of liquid samples: industrial water, aerial samples (4 m ³ ) containing various pollutants such as dust, hair, and human cervical samples, to which a high bacterial contamination artificially carried out. Device

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;

zones of segregation processing of the balls;

- a computer, which allows the piloting of the steps. Protocol

The process was carried out according to the method described in Example 1 according to all the variants that are explained therein.

Claims

A method of treating a liquid sample for nucleic acid analysis of the microorganisms likely to be contained in said sample, characterized in that it comprises at least the following steps:

(A) capturing the microorganisms contained in said liquid sample by adsorption of said microorganisms on a first anion exchange active surface;

(B) lysing said microorganisms in the presence of said first active surface on which said microorganisms are adsorbed or not; and

(C) adsorbing the nucleic acids released during step (B) on said first anion exchange active surface.

2. Method according to claim 1, characterized in that the lysis step (B) is carried out in situ while said microorganisms are adsorbed on said first active surface.

3. Method according to claim 1 or 2, characterized in that said first active surface is selected from the group consisting of resins having groups selected from quaternary amino groups, tertiary amine groups, secondary amino groups, amino groups primary; and hydroxylapatite, di-ethyl-amino-ethyl (DEAE), poly-lysine and poly-ethylene-lmine (PEI) resins.

4. Method according to any one of the preceding claims, characterized in that said first surface is on a support selected from silica and polycarbonate.

5. Method according to any one of the preceding claims, characterized in that the lysis of step (B) is carried out by sonication, abrasion by glass beads, enzymatic digestion, thermal shock, osmotic shock, light irradiation, electroporation and / or microwave action.

6. Method according to any one of the preceding claims, characterized in that it further comprises a step (A1) for purifying the microorganisms by washing said first surface implemented prior to step (B).

7. Method according to any one of claims 1 and 3 to 6, characterized in that it comprises, prior to step (B), a step (A2) of elution of said microorganisms.

8. Method according to the preceding claim, characterized in that it comprises after step (A2) a step (A3) for regenerating said first active surface.

9. Method according to any one of the preceding claims, characterized in that it comprises, after step (C), a step (C1) for purifying nucleic acids adsorbed by washing with a salt-based solution.

10. Method according to any one of the preceding claims, characterized in that it comprises, after steps (C) or (C1), a step (C2) of elution of said nucleic acids by changing the charge of said second active surface.

11. The method of claim 10, characterized in that it comprises, following step (C2), the following additional steps: step (D) of capturing nucleic acids on a surface selected from the PEI or the silanol and the step (E) of purification of said nucleic acids and the step (F) of concentration of said nucleic acids by elimination of the liquids and elution of said nucleic acids.

12. Integrated liquid sample treatment device capable of containing microorganisms, characterized in that it contains at least one active anion exchange surface selected from the group consisting of resins having groups selected from quaternary amino groups, tertiary amine groups, secondary amine groups, primary amino groups; and the hydroxylapatite, Di-ethyl-amino-ethyl (DEAE), poly-lysine and poly-ethylene-lmine (PEI) resins; said active surface being disposed in a chamber such that the ratio of said active surface per unit volume of sample which may be contained in said chamber is between 1 and 200 nf7l.