AU745247B2

AU745247B2 - Method for electroelution of a biological sample and implementing device

Info

Publication number: AU745247B2
Application number: AU31518/99A
Authority: AU
Inventors: Sophie Barral-Cadiere; Patrick Broyer; Bruno Colin; Marc Rodrigue; Lyse Santoro
Original assignee: Biomerieux SA
Current assignee: Biomerieux SA
Priority date: 1998-04-10
Filing date: 1999-04-09
Publication date: 2002-03-14
Anticipated expiration: 2019-04-09
Also published as: AU3151899A; FR2777293B1; CA2328298A1; EP1070246A1; JP2002511583A; WO1999053304A1; FR2777293A1

Description

METHOD FOR THE ELECTROSEPARATION OF A BIOLOGICAL SAMPLE AND IMPLEMENTING DEVICE The present invention relates to a method for the electroseparation of a nucleic fraction from a cell lysate, as well as any device, in particular for single use, for carrying out said electroseparation method.

In this specification, where a document, act or item of knowledge is referred to or discussed, this reference or discussion is not an admission that the document, act or item of knowledge or any combination thereof was at the priority date part of the common general knowledge; or known to be relevant to an attempt to solve any problem with which this specification is concerned.

Various methods, but also devices, have so far been provided or described, for the purpose of separating nucleic acids or a nucleic fraction from a cell lysate.

"Separation" is understood to mean generically any process which makes it possible to enrich or concentrate a medium, isolate or determine (qualitatively and/or quantitatively), in a complex medium, at least one nucleic acid, that is to say deoxyribonucleic acid (DNA) and/or ribonucleic acid (RNA).

In accordance with the document from the company ISCO, called ISCO Applications (Bulletin 54, "Electroelution of Nucleic Acids and Proteins from Gels, and Electrophoretic 20 Concentration of Macromolecules 1989), but also in accordance with US-C-4,164,464, there is described an electroelution, and not electroseparation, device comprising: a first reservoir for a first buffer solution, comprising two opposite compartments, each closed by a permeable membrane, one with a large section, and the other with a small section, andeach having a cut-off which makes it possible to retain the nucleic acid or protein material of o' 25 interest, a second reservoir for a second buffer solution, identical or different from the first buffer solution, separated from the first reservoir by said membranes, two electrodes in electrical contact with the second buffer solution, generating an electric field n_ r'l. -2going across the two permeable membranes, from the one having a large section to the one having a small section, and therefore going through the first buffer solution.

This device serves to concentrate a nucleic or protein fraction, which is already relatively pure but dilute, by placing this fraction on the membrane having a large section, and by collecting the same, but concentrated, fraction on the membrane having a small section. The concentration is obtained because of the transport of the biomnolecules by the electric field patterns, which become concentrated at the level of the membrane having a small section.

It is therefore neither a separation method nor a separation device within the meaning of the previous definition, that is to say within the meaning where, starting with a complex medium, a biological material of interest is separated or isolated.

In accordance with Figure 5 of the document US-C- 5,415, 758, a method of electroelution in parallel, inside a plurality of wells 42 of a microtitre plate 41, is described. The plate 41 is immersed in a buffer 52, without the latter overflowing into each well 42, and each well comprises a membrane 53 closing an opening 54 made at the bottom of it, such that an osmotic communication can be established between the buffer 52 and a buffer 55 placed in each well 42. An electric field is established in each well 42, by means of a cathode 24, distributed between the different wells 42, and the sole anode 14. The biological sample 44, comprising nucleic acids, for example a drop of blood placed on a porous support, is eluted by the electrolytic flow, such that the nucleic acids -:become dissolved or distributed in each well, for subsequent collection, for example with a pipette.

In general, the document US-C-5,415,758 describes a method of treating, under the action :of an electric field in a liquid medium, a biological sample comprising both nucleic acids and a nonnucleic, ~CtV-,~.~trSr, ~t -3for example a protein, material which are free and mobile in said liquid medium under the action of the electric field, method according to which: a) a buffer solution is provided; b) a permeable membrane is provided, in contact on one side with the buffer solution, and having a cut-off predetermined to stop the nucleic acids, on the side of said buffer solution, during its migration under the action of the electric field, c) said electric field is established, such that its electric field patterns pass inside the buffer solution, and cross the permeable membrane, d) the biological sample is placed in the buffer solution, upstream of the membrane, in the direction of circulation of the nucleic acids under the action of the electric field.

In practice, such a method does not make it possible to separate the nucleic acids in the biological sample to the point of practically removing all non-nucleic, for example protein, material.

On the other hand, for the purpose of separating nucleic acids, patent application published under the number WO 97/41219 is known, which discloses a method for capturing nucleic acids from a mixture consisting, for example, of lysed cells, The method requires placing an electrode in the mixture, applying to the electrode a voltage which makes it possible to attract the nucleic acids, and then removing the electrode coated with the nucleic acids. The electrode coated with the nucleic acids is then immersed in a solution which makes it possible, upon inversion of the current, to release the nucleic acids which are then directly amplifiable. The 'voltage applied in order to be able to carry out an amplification is preferably from 0.5 to 3 volts for about 30 seconds. This method, although useful for separating plasmids contained in E. coli cells lysed by heating, is not suitable for separating the DNA and/or l *o a a -4the RNA in a more complex cell lysate, in particular obtained from a biological sample.

In accordance with Figure 1 of the document WO 97/34908, there is described a method of separating nucleic acids from a biological sample, in two steps: a first step consists in bringing the lysed biological sample into contact with an adsorbent, so as to bind separately the nucleic; acids, a second step consists in releasing the nucleic acids from the adsorbent.

For the release, a buffer solution, subjected to an electric field between two electrodes and 20b, is provided in a container 10. A separate container 15, but immersed in the buffer solution, is placed in the container 10. This container communicates with the rest of the container 10 through an orifice 12 across which a membrane 30 is placed which makes it possible to stop the nucleic acids moving under the electric field. A well 60 for collecting the nucleic acids is situated near this orifice, and it is in the part 17 of the container 15 that the adsorbent component, from which it is desired to release the nucleic acids, is placed.

At the end of this inventory, no method appears to make it possible to separate, in a simple and effective manner, nucleic acids from a non-nucleic, in particular protein, material, starting with a complex cell lysate.

The present invention proposes to provide a solution to this unsolved problem.

In accordance with the present invention, it has been discovered that at least the nucleic acids could be electroseparated effectively from a biological sample by treating the biological sample directly under the action of an electric field in a liquid medium, and by choosing the following operating conditions: -the electric field is applied directly to the biological sample in the buffer solution, for a period 4~C~2'V'4z which is limited and defined, on the one hand, by the time which is sufficient to arrive on the membrane stopping the nucleic acids, and the presence of nucleic acids on the side of the buffer solution, and, on the other hand, by the fact that at the end of said period the non-nucleic material has not migrated and/or is still in the process of migrating toward said membrane, the nucleic acids are collected at the end of said period.

"Electroseparation" in the present invention is understood to mean that molecules present together in a medium, and which may have identical electrical charges, are distinguished from each other in said medium because of their respectively different kinetcs in the same electric field.

The method according to the invention provides the essential advantage of rapidly and easily obtaining a nucleic fraction free of other constituents, in particular proteins, which is directly amplifiable, from a cell lysate. The difficulties linked to amplification techniques are known, in particular for respiratory-type samples, in that the fraction to be amplified must be free of inhibitors.

Using the method according to the invention, the protein inhibitors in amplification techniques are practically eliminated.

The fraction enriched with nucleic acids, comprising DNA and/or RNA, which is obtained using the method according to the invention is directly amplifiable by the techniques commonly used, such as in particular the PCR technique for DNAs and the NASBA or TMA technique for RNAs.

In another embodiment according to the invention, the biological sample is placed on another membrane, on the side of the latter in contact with the buffer solution.

In a preferred embodiment according to the invention, the time for applying the electric field is lie 55.55.

S*lt •S 6 at most 30 minutes, and preferably between 5 and minutes.

In a highly preferred embodiment according to the invention, the duration of migration of the nucleic fraction is 10 to 20 minutes, more preferably minutes.

In a preferred embodiment according to the invention, the value of the molarity of the buffer solution is at least equal to 0.1 mol/l, and preferably between 0.1 and 5 mol/l.

The molarity of the buffer solution being between 0.1 and 5 mol/l, it is effectively possible either to put only the same buffer solution in both compartments, or to put two different buffer solutions in both compartments.

The molarity of the buffer solution may be the same in both compartments and will be between 0.1 and 1 mol/l, more preferably 0.1 mol/l.

The molarity of the buffer solution may be different in both compartments and will be respectively preferably 0.1 mol/l in the first compartment and 1 mol/l in the second compartment.

In a preferred embodiment according to the invention, the electric field has a value of between 100 and 250 V, and preferably equal to 150 V.

In another highly preferred embodiment according to the invention, the distance separating the biological sample from the permeable membrane, following the electric field patterns, is at least equal to 30 mm, and preferably to 75 mm.

The cell lysate from which the method according to the invention is carried out may be a cell lysate obtained from a simple culture or a complex culture, that is to say comprising different cells, or may also be a biological sample, such as in particular blood, urine and a respiratory-type sputum.

The cell lysate from which the nucleic fraction, which is then subjected to the method Saccording to the invention, is obtained may be obtained -7by different lysis techniques, in particular lysis using mechanical shock, lysis using electric shock and the like.

In a preferred embodiment according to the invention, the biological sample results directly from the lysis of a cellular sample, in particular by the mechanical or electrical route. This embodiment is performed in the same device as is described below.

In another preferred embodiment according to the invention, the cell lysate is obtained from a sample of body fluid, for example blood or respiratory sputum.

When the biological sample is blood, the pH of the buffer solution is preferably 7.

The membrane recovering the fraction enriched with nucleic acids has a predetermined cut-off, preferably of less than 100 kDa.

When the biological sample is blood, the porosity of the membrane at the cathode will be preferably greater than 10 kDa.

In a preferred embodiment according to the invention, a proteinase is added to the biological sample.

In yet another preferred embodiment according to the invention, the fraction enriched with nucleic acids is directly subjected to a subsequent amplification step.

The cell lysis will be more preferably carried out by electrical lysis in the presence of proteinase K and of a detergent.

A second subject according to the invention is a device for single use for carrying out a 20 method according to the invention, comprising a support or base, in which are assembled and integrated, as well as arranged between each other, the different means, in particular electrodes, required for the electroseparation, as well as means for interfacing said support with the outside, on the one hand for the transfer of the different fluids or liquids toward o goSS w r~ 7' F1~1~~~1 -8and/or out of the support, including the biological sample, the buffer solution and the fraction enriched with nucleic acids, and on the other hand for the supply and the control of the electrical means required at least for the electroseparation, including that of the electric field.

In a preferred embodiment according to the invention, the device comprises a means for receiving the biological sample; a reservoir for the buffer solution, comprising a compartment closed by the permeable membrane, communicating at one end opposite said compartment with the means for receiving the biological sample, said reservoir being intended to receive a first buffer solution; and a second reservoir for a second buffer solution, identical or different from the first buffer solution, separated from the first reservoir solely by said membrane; two electrodes for generating the electric field, one in contact with the first buffer solution, upstream of the membrane in the direction of circulation of the nucleic acids, and the other in contact with the second buffer solution; and a means for extracting the fraction enriched with nucleic acids from the compartment closed by the membrane.

In a highly preferred embodiment according to the invention, the reservoir comprises another compartment, for receiving at least one fraction obtained from the biological sample, placed upstream of said compartment in the direction of circulation of the nucleic acids, communicating with the means for receiving the biological sample.

In another preferred embodiment according to the invention, the two reservoirs communicate respectively with aeration vents, and with at least one filling channel.

20 In yet another preferred embodiment according to the invention, an intermediate well is placed and communicates between the means for receiving the biological sample and the first reservoir.

-9- In yet another preferred embodiment according to the invention, the intermediate well is provided with means which make it possible to lyse a cellular sample.

In yet another preferred embodiment according to the invention, the intermediate well is provided with electrodes which bring about a lysis of said cellular sample.

In yet another preferred embodiment according to the invention, the device comprises, communicating with each other, a compartment for receiving the lysate, an intermediate lysis well, and a compartment for receiving the fraction enriched with nucleic acids.

In yet another preferred embodiment according to the invention, the volume of the compartment for receiving the lysate is greater than the volume of the compartment for receiving the fraction enriched with nucleic acids.

In yet another preferred embodiment according to the invention, the ratio between the volumes of the compartments for receiving the lysate and for receiving the nucleic fraction is between and 1/50, in particular between 1/5 and 1/20.

A third subject according to the invention is the use of the device described above for electroseparating a nucleic acid fraction from a cell lysate.

A fourth subject according to the invention is the use of the fraction electroseparated using the method according to the invention for detecting and/or identifying nucleic acids directly after amplification.

Figure 1 schematically represents an apparatus for the electroelution of nucleic acids and 20 of proteins from a gel, and for the electrophoretic concentration of macromolecules (Isco, iNebraska, USA), which is used in a different method, namely electroseparation according to the invention. Compartments A and B, in which two electrodes, which are negative and positive 04 o •oo .o go oo o *o* .ooooo LT 1 ;~DmP-T(9/C~Lbn 10 respectively, are immersed are filled with lX TBE buffer; compartment C, comprising a portion C1 and a portion C2, and compartment Cf are filled with 0.lX TBE buffer. A dialysis membrane is placed at the interface of compartments Cf and B (porosity 100 kDa), and of compartments Cl and A (porosity 10 kDa).

Figure 2 indicates the percentage of S.

epidermidis nucleic acids recovered in compartment Cf, after migration from a cell lysate obtained by mechanical shocks, determined by analysis with a Vidas apparatus (BioMerieux, France), according to Example 2.

The migration time (minutes) is represented on the xaxis, and the percentage of nucleic acids recovered in compartment Cf, expressed as relative fluorescence units, is represented on the y-axis.

Figure 3 presents the kinetics of recovering the proteins in a cell lysate. The percentage of proteins in the bacterial lysate is recovered in compartment Cf, after migration from a cell lysate obtained by mechanical shocks, determined by Bradford assay, according to Example 2. The migration time (minutes) is represented on the x-axis, and the percentage of nucleic acids recovered in compartment Cf, expressed as optical density (OD) at 595 nm, is represented on the y-axis.

Figure 4 presents the kinetics of recovering nucleic acids from a cell lysate. The percentage of Staphycoccus epidermidis nucleic acids, recovered in compartment Cf after migration from a cell lysate obtained by electric shocks, is determined by analysis with a Vidas apparatus, according to Example 2. The migration time (minutes) is represented on the x-axis, and the percentage of nucleic acids recovered in compartment Cf, expressed as relative fluorescence units, is represented on the y-axis.

Figure 5 presents the amplification of nucleic acids purified from a cell lysate. The quantity of amplicons produced by PCR from 10 gl of DNA molecules Ih is recovered in compartment Cf, from a cell lysate -11obtained by mechanical shocks, after various migration times, according to Example 2. The migration time (minutes) is represented on the x-axis, and the percentage of nucleic acids recovered in compartment Cf, expressed as relative fluorescence units, is represented on the yaxis.

Figure 6 presents the quantity of amplicons produced by PCR amplification, after lysis of various initial concentrations of bacteria by mechanical shocks, and migration of their nucleic acids for 15 minutes under an electric field, according to Example 2. The number of initial bacteria per 200 [d of lysate is presented on the x-axis, and the signal obtained after PCR, expressed as relative fluorescence units, is represented on the y-axis.

Figure 7 illustrates the quantity of amplicons produced by TMA amplification, after lysis of carious initial concentrations of bacteria by mechanical shocks, and migration of their nucleic acids for 15 minutes under an electric field, according to Example 2. The number of initial bacteria per 200 pl of lysate is represented on the x-axis, and the signal obtained after TMA, expressed as OD X 1000, is represented on the y-axis.

Figure 8 indicates the percentage of proteins in blood recovered in compartment Cf as function of the migration time of a clinical blood sample lysed by mechanical shocks, according to Example 3. The migration time (minutes) is represented on the x-axis, and the percentage of proteins recovered in compartment Cf, expressed as OD at 595 nm, is represented on the y-axis.

Figure 9 indicates the percentage of proteins in a sputum recovered in compartment Cf as 20 a function of the migration time of a respiratory-type clinical sample lysed by mechanical shocks, according to Example 2. The migration time (minutes) is presented on the x-axis, and the percentage of proteins recovered in goa _i 12 compartment Cf, expressed as OD at 595 nm, is represented on the y-axis.

Figure 10 shows the elimination of the inhibitors of PCR initially present in the respiratorytype clinical sample. It indicates the quantity of DNA amplicons produced by PCR from various initial quantities of S. epidermidis DNA, according to Example The number of initial copies of DNA per 10 p1 of sample is represented on the x-axis, and the signal obtained after PCR, expressed as relative fluorescence units, is represented on the y-axis.

Figure 11 indicates the quantity of amplicons produced from various initial quantities of bacteria lysed and deposited in compartment Cl. Each point represented on the graph represents a mean value obtained from 13 different bronchial aspirations and sputa, according to Example 5. The number of initial copies of DNA per 200 g1 of sample is represented on the x-axis, and the signal obtained after PCR, expressed as relative fluorescence units, is represented on the y-axis.

Figure 12 indicates the quantity of amplicons produced from various initial quantities of bacteria lysed by mechanical shocks and deposited in compartment Cl, according to Example 5. The study was carried out in parallel on 4 different sputa. The number of initial copies of DNA per 200 g1 of sample is represented on the x-axis, and the signal obtained after TMA, expressed as OD x 1000, is represented on the y-axis.

Figure 13 represents a perspective view of a device for single use according to the invention.

Figure 14 represents a bottom view of the device represented in Figure 13.

Figure 15 represents represents a top view of the device represented in Figure 13.

Figure 16 represents represents a sectional view, along the sectional line represented in Figure of the device according to Figure 13.

13 Example i: General procedure for carrying out the method according to the invention.

The procedure is based on the use of an apparatus for the electroelution of nucleic acids according to Figure i. A volume of biological sample is carefully deposited at the bottom of compartment Cl.

The two electrodes in the circuit are connected to an electric generator. A constant voltage of 150 V is applied to the generator terminals. The circuit intensity varies from 15 to 20 mA and the temperature of the buffer in compartment C varies from 23 to 30 0

C.

The biological molecules initially deposited at the bottom of compartment C1 migrate in the electric field thus created, at a defined speed as a function of their charge and their size. After various migration times, the voltage at the generator terminals is stopped and the molecules which have migrated toward the cathode and which have an apparent molecular weight of less than or equal to 10 kDa are recovered in compartment Cf, in a final volume of 200 p1. The sample thus recovered is stored on ice before analysis.

The biological sample deposited in compartment C1 may be a nucleic and/or protein material, purified or otherwise, a cell lysate, obtained from a biological sample such as blood, urine and a respiratory-type sputum. The cell lysates studied were obtained from S.

epidermidis, a Gram+ wall-containing bacterium (A054).

These bacteria are cultured in HBB (heart-brain broth, bioMerieux 41019). Before lysis, they are suspended either in a clinical sample (sputum which has been liquefied and decontaminated, blood, plasma, serum and the like), or in lysis buffer (30 mM Tris-HCl, 5 mM EDTA, 100 mM NaCl pH 300 g1 of this cellular suspension were lysed according to essentially two protocols: Lysis protocol using mechanical shocks: 90 .1 of glass beads having a diameter of between 90 and 150 pun, 3 iron beads having a diameter of 2 mm and glass beads having a diameter of 3 mm are placed in a 14 Falcon tube in the presence of 300 Li of the cellular suspension, as described in patent (FR-A-2,768,743) 6 mg/ml final of proteinase K (PK) 3.4.21.14 Boehringer-Mannheim, ref. 1092766) may be added to the cellular suspension. The closed tube is vortexed for two minutes at the maximum power of the apparatus (Reax 2000, Heidolph). The bacterial suspension thus treated and recovered is deposited immediately into compartment C1 of the device described in Figure i. If PK is present in the cellular solution, an additional incubation of 15 minutes is carried out at 370C.

Lysis protocol using electric shock: 300 i of the cellular suspension are deposited in an electroporation tank characterized by a distance between the two electrodes of 2 mm, in the presence of 0.01 to 6 mg/ml final of PK. The tank is placed in an electric circuit in which the parameters are chosen as follows: voltage 500 V, resistance 186 Ohms, capacitance of 500 to 1500 gFD. After generating an electrical pulse, the electrical discharge is produced within a few milliseconds between the two electrodes.

The lysate thus recovered is deposited immediately in compartment A of the device described in Figure 1 (FR- A-2,763,957) The percentage of nucleic acids recovered on the side of the cathode, after migration in compartment Cf, is determined by measuring the OD (optical density) of the sample recovered at 260 nm. The percentage recovered is equal to the ratio of the OD value at 260 nm after migration, to the OD value at 260 nm before migration. Likewise, the quantity of proteins recovered at the cathode after migration is determined by the Bradford assay and OD reading at 595 nm. The percentage of proteins recovered is equal to the ratio of the protein quantity recovered to the initial quantity.

The quantity of nucleic acids recovered on the side of the cathode after migration is checked on 0.8% agarose gel, 10 p1 of the sample are deposited per I n~ i 15 well, the electrophoretic migration is carried out at constant voltage (150 V) and the gel is stained with ethidium bromide (EtBr) before visualization under ultraviolet radiation. In parallel, the proteins recovered at the cathode after migration may be visualized on polyacrylamide gel in the presence of SDS SDS-PAGE); 5 to 10 gl of the sample are deposited per well, the electrophoretic migration is carried out at constant intensity (25 mA), and the gel is stained with Coomassie blue.

The quantity of nucleic acids recovered on the side of the cathode is determined by specific detection according to a so-called sandwich hybridization technique using the Vidas apparatus marketed by bioM6rieux (France). Capture and detection oligonucleotide probes specific for S. epidermidis nucleic acids (EP-A-0,632,269) were chosen. The capture and detection oligonucleotides have respectively as sequence: 5'-GACCACCTGTCACTCTGTCCC-3' (SEQ ID No. :1) and 5'-GGAAGGGGAAAACTCTATCTC-3' (SEQ ID No. The detection probe is labeled by coupling with alkaline phosphatase (AKP). The specific hybridization of these probes with the nucleic acids released in the lysate depends on the quantity of nucleic acids present, but also on their accessibility for the probes used.

Two protocols for specific amplification of S.

epidermidis DNA and RNA molecules were carried out starting with samples recovered after migration: a PCR protocol for the amplification of the DNA and a NASBA protocol for the amplification of the 16S rRNA and a TMA protocol for the amplification of the 16S rRNA.

PCR protocol: the PCR technique followed is that described by Goodman in PCR Strategies, Ed: Innis, Gelford and Sninsky Academic press 1995, pp. 17-31. Two amplification primers were used; they have the following sequences: Primer 1: 5'-ATCTTGACATCCTCTGACC-3' (SEQ ID No.: 3) 16 Primer 2: 5'-TCGACGGCTAGCTCCAAAT-3' (SEQ ID No.: 4) The following temperature cycles were used: Once 3 minutes at 94 0

C

2 minutes at 65 0

C

times 1 minute at 72°C 1 minute at 94 0

C

2 minutes at Once 5 minutes at 72°C TMA protocol: the TMA technique followed is that described by US-A-5,554,516. Two amplification primers were used; they have the following sequences: Primer 1: 5'-TCGAAGCAACGCGAAGAACCTTACCA-3' (SEQ ID No.: Primer 2: AATTCTAATACGACTCACTATAGGGAGGTTTGTCACCGGCAGTCAACTTAGA-3' (SEQ ID No.: 6) gl or 50 gl of recovered sample are used for each PCR and TMA assay, respectively. The amplicons produced by PCR are visualized on 0.8% agarose gel and quantified on Vidas according to the protocol described above. The amplicons produced by TMA are detected and quantified on microplate by hybridization with a capture probe and a detection probe, which are specific for S. epidermidis, according to the method described by P. Cros et al., Lancet 1992, 240 870. The detection probe is coupled to "horseradish peroxidase" (HRP). The two probes have the following sequences: Capture probe: 5'-GATAGAGTTTTCCCCTTC-3' (SEQ ID No.: 7) Detection probe: 5'-GACATCCTCTGACCCCTCTA-3' (SEQ ID No.: 8) Example 2: Kinetics of recovery of nucleic acids from a cell lysate.

A suspension of Staphylococcus epidermidis x 109 b./300 gl) is lysed by mechanical shock or by electrical shock, as described in the general protocol above. 200 il of each lysate are deposited at the Sbottom of compartment Cl. The electric field is applied -17for various times between the two electrodes. The quantities of nucleic acids or of proteins present in the sample recovered in compartment Cf are determined by analysis with a Vidas apparatus or Bradford assay, respectively. The quality of the molecules recovered was assessed on agarose or acrylamide gel in the presence of SDS.

Cell lysate obtained by mechanical shock: The nucleic acids in the lysate migrated into compartment Cf gradually over time, as shown in Figure 2. After 60 minutes, the entire nucleic acids in the lysate is recovered, regardless of the presence or otherwise of proteinase K during the lysis step. This result is in favor of a good release and accessibility of the nucleic acids in the lysate. The DNA molecules recovered have the same migration profile on 0.8% agarose gel as before migration in the lysate. In the presence of proteinase K during the lysis step, as shown in Figure 3. only a negligible percentage of proteins is recovered after 60 minutes, whereas in the absence of protease, about 50% of proteins are recovered.

Cell lysate obtained by electric shock: In the presence of PK during the lysis step, as shown in Figure 4, the nucleic acids in the lysate migrate gradually up to the cathode. After 60 minutes, a large percentage of nucleic acids is recovered. The recovery of the nucleic acids initially present in this lysate is similar to that of the nucleic acids present initially in a lysate obtained by mechanical shock (cf. above). On the other hand, in the absence of PK during the lysis step, no nucleic acid can be detected in compartment 20 Cf, even after 60 minutes of migration (Vidas analysis and 0.8% agarose gel). On agarose gel, the SDNA and RNA molecules in the lysate which are recovered are visualizable separately after migration, whereas they are not initially visualizable in the lysate.

c -18- Amplification of the nucleic acids purified from cell lysate: The S. epidermidis bacteria diluted in 300 pl of lysis buffer are lysed by mechanical shock in the absence of PK, as described in the general protocol of Example 1. 200 pi of the lysate are deposited at the bottom of compartment C1. An electric field is applied between the two electrodes for various times. The nucleic acids which have migrated into compartment Cf is amplified by PCR pl per assay) or TMA (50 pl per assay). The quantity of amplicons produced is analyzed by specific hybridization of Vidas or microplate, respectively.

PCR amplification of the DNA molecules recovered after migration: For 10 4 initial bacteria lysed and deposited in compartment C1, various quantities of amplicons are produced, as a function of the migration of time. For 15 minutes of migration, as represented in Figure 5, the DNA molecules recovered allow optimum production of amplicons by PCR. Before 15 minutes, a smaller quantity of DNA molecules are recovered. For 20 minutes and above, a larger quantity of DNA molecules are recovered, but the quality is less suited to optimum PCR amplification. The longer the migration, the more the structure of nucleic acid may be impaired. In order to obtain optimum PCR amplification, a compromise between quantity and quality of DNA molecules recovered at the cathode must be observed. This result was confirmed using more concentrated initial suspensions of S. epidermidis (10-106 initial bacteria/200 pi).

As represented in Figure 6, the DNA of a number greater than or equal to 1x10 3 initial bacteria (/200 pi) may be amplified and detected, after mechanical lysis of the cells and migration 20 of the constituents of the lysate under an electric field; that is 102 molecules of DNA or bacteria per i PCR assay, given that the migrated material is recovered in 200 pi and that 10 pi of this material is S used for each PCR 9 o a a.

z 19assay. 10 2 molecules of DNA correspond to the limit of sensitivity of the PCR protocol in general.

This result demonstrates a high sensitivity of recovery of the molecules of bacterial DNA after cell lysis by mechanical shock and purification of the intracellular DNA molecules by an electric field in solution.

MA amplification of the RNA molecules recovered after migration: As represented in Figure 7, the RNA of at least 40 initial bacteria (/200 pl) may be detected after lysis of the bacteria, migration of the cellular constituents under an electric field and specific amplification of the S. epidermidis 16S rRNA; that is 10 initial bacteria per TMA assay, given that the migrated nucleic acids are recovered in 200 pi final and that 50 pl are added per TMA assay.

This result reflects a high sensitivity of recovery of the molecules of bacterial 16S rRNA, after lysis by mechanical shock and purification of the nucleic acids of the lysate by an electric field.

Example 3: Kinetics of recovery of nucleic acids from a blood sample.

The blood sample is used without prior pretreatment. 200 pl of this clinical sample are deposited at the bottom of compartment C1. A voltage of 150 V is applied between the two electrodes for various times, and then the material recovered on the side of the cathode in compartment Cf is analyzed by protein assay according to the Bradford method, and on polyacrylamide gel in the presence of SDS (10% SDS-PAGE).

In this example, the pH of the TBE buffer used for the migration was set at pH 7.0, the isoelectric point of hemoglobin being equal to 7.0. Membranes of 50 or 100 kDa were placed at the 20 interface of compartments Cf and B. As is represented in Figure 8, after 60 minutes of migration, a negligible percentage of proteins is recovered with the membrane of 100 kDa, and 10% of the proteins are recovered with the membrane of 4 4 t 4 l 9. 20 kDa. At pH 7.0, only 10% of the blood proteins which are negatively charged at pH 7.0 have an apparent molecular weight greater than 50 kDa and less than 100 kDa. A negligible percentage of proteins are recovered after 15 minutes of migration, using either a membrane of 50 or 100 kDa. These conclusions were verified on polyacrylamide gel in the presence of SDS-PAGE. The porosity of the membrane will be preferably greater than 10 kDa, so as not to obtain an excessively high percentage of proteins.

Example 4: Kinetics of recovery of nucleic acids from a respiratory-type sample.

Before using, the respiratory sample is rendered fluid, decontaminated according to the standard N-acetyl-L-cysteine and sodium hydroxide (NALC/NaOH) protocol, and it is inactivated for minutes at 95 0

C.

In this example, the TBE buffer used for the migration has a pH equal to 8.3 or 7.0, and membranes with different pore sizes were placed at the interface of compartments Cf and B: 10, 50 or 100 kDa. As is represented in Figure 9, at pH 8.3, 10% of the proteins are recovered after 30 minutes, and 70-80% after minutes with membranes of 50 or 10 kDa, which suggests that 70-80% of the proteins in the sputum are negatively charged at these pH values and have an apparent molecular weight greater than 50 kDa. At pH 7.0, about 0% of the proteins are recovered after or 60 minutes of migration, regardless of the size of the pores of the membrane used (within the limit of sensitivity of the detection techniques used). At this pH, 55-60% of the proteins previously recovered at pH 8.0 are no longer negatively charged. With the membranes of 10 or 50 kDa, a negligible percentage of the proteins in the sputum is recovered after minutes of migration. This point was verified on polyacrylamide gel in the presence of 10% SDS-PAGE.

f~i~ c~ 21 Example 5: Lifting of inhibition of amplification protocols by respiratory-type samples after migration.

The respiratory-type samples are inhibitors of PCR and TMA reactions. 200 p1 of these samples were deposited at the bottom of compartment C1 of the system described in Figure 1. A constant voltage of 150 V was applied between the two electrodes for 15 minutes. A separation membrane between Cf and B of 10 kDa was chosen. After migration, the sample recovered in compartment Cf (200 1) was supplemented with various quantities of purified S. epidermidis nucleic acids.

p1 or 50 1 of this solution were used for each PCR or TMA assay, respectively. As represented in Figure 10, up to 102-103 initial copies of DNA may be amplified in 10 g1 of migrated sample. This result demonstrates that the migrated sputum lost its PCR-inhibiting character. Similar results were obtained with TMA amplification.

l-5x10 9 S. epidermidis were inoculated into 300 p1 of sputum rendered fluid, decontaminated and inactivated as described in Example 4 above. These cells in suspension were lysed by mechanical shock in the absence of PK, and the lysate migrated for minutes under 150 V with a membrane of 10 kDa between compartments Cf and B, according to the scheme presented in Figure i. The quantity of bacterial nucleic acids recovered in compartment Cf after migration was quantified by Vidas analysis specific for the bacterial species studied. The experiment was carried out using various sputa on average. 80% of S.

epidermidis nucleic acids are recovered. This result indicates that the molecules of the matrix in the sputum do not hamper the migration of the DNA and RNA molecules in the bacterial lysate in the electric field, under these conditions. On the contrary, it appears that the environment is suitable for a better recovery and/or migration of these molecules.

4PCR amplification: =~T~III--YT~T~1 22 The S. epidermidis bacteria were inoculated into 300 p 1 of sputum rendered fluid, decontaminated, inactivated and then lysed by mechanical shock in the absence of PK as described in Example 1. 200 pI of the lysate migrated for 15 minutes under 150 V with a membrane of 10 kDa between compartments Cf and B. The nucleic acids present in the sample were amplified by PCR (10 p per assay), and the amplicons produced were quantified by Vidas analysis. As represented in Figure 11, up to 10 4 -10 3 lysed initial bacteria (/200 pI of lysate) can be detected after migration and amplification of the DNA molecules (that is 100-10 molecules of p of PCR assay). This result confirms, on the one hand, a very good yield of recovery of the molecules of bacterial DNA of the lysate in the sputum, and, on the other hand, demonstrates a very good elimination of the PCR inhibitors initially present in the sputa.

TMA amplification: As for the PCR amplification study described above, the S. epidermidis bacteria were inoculated into 300 pl of sputum rendered fluid, decontaminated, inactivated and then lysed by mechanical shock in the absence of PK. 200 p of lysate migrated for 15 minutes under 150 V with a membrane of 10 kDa between compartments Cf and A. The rRNA molecules present in the sample recovered were amplified by TMA (50 p per assay), and the amplicons produced were quantified by specific sandwich hybridization on microplate. As represented in Figure 12, at least 106_105 lysed initial bacteria (/200 p of lysate) can be detected after migration and amplification of the RNA molecules (that is at least 5x1 0 4 -5x1 03 initial bacteria/50 p1 of TMA assay). This result 20 demonstrates the elimination of the inhibitors of TMA initially present in the sputa.

Example 6: Electroseparation after lysis by electric shocks.

1-5x10 9 S. epidermidis were inoculated into 300 pl of sputum rendered fluid, decontaminated and *od.:b

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-23 inactivated as described in Example 4. These cells in suspension were lysed by electric shock in the presence of 10- 2 mg/ml final of PK and 2% final of LLS (lithium lauryl sulfate, Sigma). 200 pl of lysate migrated for 15 minutes under 150 V with a membrane of 10 kDa between compartments Cf and B, according to the scheme presented in Figure 1. The nucleic acids recovered after migration (200 pl) were amplified by PCR specific for S. epidermidis (10 pl/assay), and the amplicons produced were quantified by Vidas analysis. Up to 10 4 -10 5 lysed initial bacteria can be detected after migration and amplification of the DNA molecules (that is 100-10 molecules of pl of PCR assay, which represents the limit of sensitivity of the PCR technique used).

In accordance with Figures 13 to 16, a device for single use, that is to say consumable, or disposable after use, which makes it possible to carry out the electroseparation method described and exemplified above, is described below.

Such a device comprises a support 1, or base, generally having a parallelepipedal shape, comprising in particular a top face 1b, a bottom face 1c and a lateral flank la, which are shown in Figures 13 and 14.

In general, in this support 1, are assembled and integrated, as well as arranged between each other, the different means, in particular electrodes, required for the electroseparation, as well as means for interfacing corresponding to the flank la of the support 1 with the outside, on the one hand for the transfer of different fluids or liquids toward and/or out of the support 1, including the biological sample treated, the aqueous liquid medium (buffers) and the fraction enriched with 20 nucleic acids, and on the other hand for the supply and the control of the electrical means required 0 •at least for the electroseparation, including that of the electric field.

In a manner which is not represented, by way of example, the two faces la and 1b of the device, or map,

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0 00 0 t* 00 00OO 0 0 -24are coated with a leaktight transparent film adherent to the support and closing the various conduits and cavities represented at the surface in Figures 13 and 14.

This devise further comprises: a means 2 for receiving the biological sample treated a first reservoir 3 for a first aqueous liquid medium (TBE buffer diluted tenfold), itself comprising a compartment 31 closed by a permeable membrane 4, having for example a capacity of the order of 100 pi, as well as another compartment 32, having a capacity of 1 ml, for receiving at least one fraction obtained from the biological sample this other compartment is placed upstream of compartment 31 in the direction of circulation of the nucleic acids, under the effect of the electric field, and communicates itself with the means 2 for receiving the biological sample a second reservoir 5 for a second aqueous liquid medium (for example TBE buffer, concentrated one-fold), identical or different from the first aqueous liquid, separated from the first reservoir 3 solely by the membrane 4 two electrodes 61 and 62 for generating an electric field, communicating with two blocks 63 and 64 for electrical contact on the flank la; one of the electrodes, namely 61, or cathode, is in contact with the first aqueous liquid medium in the first reservoir 3, and the other electrode 62, or anode, is in contact with the second aqueous medium in the second reservoir a means 9 for extracting the fraction enriched with nucleic acids in compartment 31 closed by the membrane 4.

The two reservoirs 3 and 5 communicate respectively with the aeration vents 33 and 53, and with at least one filling channel 34 communicating, for its part, with the second reservoir _--47P 25 An intermediate well 7 is placed and communicates between the means 2 for receiving the biological sample and the first reservoir 3. This well is not obligatory since the lysate can be placed directly into compartment 32. This well 7 is provided with means which make it possible to lyse a cellular sample in order to obtain a fraction which is then subjected to electroseparation; these means are electrodes 81 and 82, which make it possible to expose the sample to one or more electrical pulses, in accordance with the method described elsewhere in French patent application FR-A-2 763 in the name of the applicant. These electrodes 81 and 82 communicate respectively with blocks for electrical contact 83 and 84 which are provided on the flank la. Two electric circuits therefore exist, one associated with the electrodes 81 and 82, and the other associated with the electrodes 61 and 62.

I Preparation of the device Before starting any reaction, the reservoirs 3 and 5 are filled with buffer. The latter may consist of a TBE (Tris-Borate-EDTA) buffer whose concentration varies from one reservoir to another. The first, top, reservoir 3 is filled, by the receiving means 2, with buffer diluted tenfold, and the second, bottom, reservoir 5, by the channel 34, with buffer concentrated onefold. The aeration vents 33 and 53 are pierced at the level of the leaktight films covering the faces la and lb, so as to allow air to escape during the filling of the reservoirs.

1) Lysis The sample is introduced into the receiving means 2 with the aid of a pipette, manually or by an automated device. The volume of the sample is between 0 and 1 ml. It then arrives in the intermediate well 7 for lysis. The lysis is performed by means of an electrical discharge of 500 V for about 1 second, between the electrodes 81 and 82.

i. 26 In the case where the sample volume is greater than 50 Ill, several lysis steps are performed one after another, the lysed fractions being transferred progressively into the receiving compartment 32.

2) Electroseparation Once the sample has been lysed and completely transferred into the compartment 32 for initiation of the electroseparation, the first electric circuit of the electrodes 81 and 82 is opened, and the second of the electrodes 61 and 62 is closed. The negatively charged constituents of the lysate will then migrate up to the anode 62. The nucleic acids will migrate faster, being highly negative. It will therefore be possible to recover them selectively on top of membrane 4, at the level of the receiving compartment 31 by the collecting channel 9.

The current imposed for this migration is between 0 and 30 mA, under a voltage of 150 V. The optimum duration of migration is about 15 minutes.

In order to recover the nucleic acids, the top reservoir 3 is emptied until there is no longer fluidic communication between compartment 32 and the receiving compartment 31. The 100 gl of buffer contained in compartment 31 are then collected with a pipette, through the channel 9. The collection can be made manually or by means of an automated device.

It is possible to envisage also recovering the purified proteins. For that, it is necessary to carry out two successive runs of electropurification, the first making it possible to recover the fraction enriched with nucleic acids and the second the fraction enriched with proteins. In this precise case, the nucleic acids should be recovered between the two runs, and without emptying the top reservoir 3 of buffer which contains the proteins which have not finished migrating.

It is possible for a device as described above to comprise only three electrodes. Indeed, the two >h cathodes 61 and 81, respectively assigned to the A .Zf .W -27electroseparation circuit and to the lysis circuit, become the cathode common to the two circuits. In this case, the lysis of the biological sample is performed in compartment 32. Such a device may be used on the edge-section. It will then be easy to integrate it into an automated device by virtue of this gain in space.

The word 'comprising' or forms of the word 'comprising' as used in this description and in the claims do not limit the invention claimed to exclude any variants or additions.

e* u u

Claims

2. Method according to Claim 1, characterized in that the biological sample comprises a cell lysate.
3. Method according to Claim 1 or 2, characterized in that the biological sample is placed on 25 another membrane, on the side of the latter in contact with the buffer solution.
4. Method according to Claim 1 or 2, characterized in that the time for applying the electric field is at most 30 minutes, and preferably between 5 and 30 minutes.
5. Method according to Claim 1 or 2, characterized in that the value of the molarity of the buffer solution is at least equal to 01 mol/l, and preferably between 0.1 and 5 mol/l.
6. Method according to Claim 1 or 2, characterized in that the electric field has a value of between 100 and 250 V, and preferably equal to 150 V.
7. Method according to Claim 1 or 2, characterized in that the distance separating the biological sample from the permeable membrane is at least equal to 30 mm, and preferably to mm.
8. Method according to Claim 7, characterized in that the biological sample is obtained from a sample of a body fluid, for example blood or respiratory sputum.
9. Method according to Claim 1 or 2, characterized in that a proteinase is added to the biological sample.
10. Method according to Claim 1 or 2, characterized in that the fraction enriched with nucleic acids is directly subjected to a subsequent amplification step.
11. Device for single use for carrying out a method according to any one of the preceding claims, characterized in that it comprises a support or base, in which are assembled and integrated, as well as arranged between each other, the different means, in particular electrodes, required for the electroseparatio, as well as means for interfacing (la, ib, Ic) said support with the outside, on the one hand for the transfer of the different fluids or liquids toward and/or out of the support, including the biological sample, the buffer solution and the fraction enriched with nucleic acids, and on the other hand for the supply and the control of the electrical means required at least for the electroseparation, including that of the electric field.
12. Device according to Claim 11, characterized in that it comprises a means for receiving the biological sample; a reservoir for the buffer solution, comprising a compartment (31) closed by the permeable membrane communicating at one end opposite said compartment (31) with the means for receiving the biological sample, said reservoir being intended to receive a first buffer solution; and a second reservoir for a second buffer solution, identical or different from the first buffer solution, separated from the first reservoir solely by said membrane; two electrodes (61, 62) for generating the electric field, one (61) in contact with the first buffer solution, upstream o*oP*. of the membrane in the direction of circulation of the nucleic acids, and the other (62) in contact with the second buffer solution; and a means for extracting the fraction enriched with nucleic S°acids from the compartment (31) closed by the membrane. O* O* S: 25 13. Device according to Claim 12, characterized in that the reservoir comprises another S:-o compartment for receiving at least one fraction obtained from the biological sample, placed upstream of said compartment (31) in the direction of circulation of the nucleic acids, communicating with the means for receiving the biological sample.
14. Device according to Claim 12, characterized in that the two reservoirs 5) communicate respectively with aeration vents (33, 53), and with at least one filling channel (34). Device according to Claim 12, characterized in that an intermediate well is placed and communicates between the means for receiving the biological sample and the first reservoir
16. Device according to Claim 15, characterized in that the intermediate well is provided with means which make it possible to lyse a cellular sample. I
17. Device according to Claim 16, characterized in that the intermediate well is provided with electrodes (81, 82) which bring about a lysis of said cellular sample.
18. Device according to Claim 11, characterized in that it comprises, communicating with each other, a compartment (32) for receiving the lysate, an intermediate lysis well and a compartment (31) for receiving the fraction enriched with nucleic acids.
19. Device according to Claim 18, characterized in that the volume of the compartment (32) for receiving the lysate is greater than the volume of the compartment (31) for receiving the fraction enriched with nucleic acids. Device according to Claim 19, characterized in that the ratio between the volumes of the compartments for receiving the lysate and for receiving the nucleic fraction is between 12 and 1/50, in particular between 1/5 and 1/20.
21. Use of the device according to Claims 11 to 20, for electroseparating a fraction or all of the nucleic acids from a cell lysate.
22. Use of the fraction electroseparated using the method according to Claims 1 to 10 for detecting and/or identifying nucleic acids directly after amplification. c *e C e* e C tn~~,