FR2672301A1 - Process and device for amplifying the number of a defined sequence of nucleic acid in a biological sample - Google Patents

Abstract

The process and device relate to the amplification of the number of at least one defined sequence of nucleic acid or its complementary sequence, hereinafter called target sequences, in a biological sample, characterised by the fact that, on the one hand, the process comprises a supply of active ingredients to the said sample and at least one preliminary step, designed to increase the accessibility of the nucleic acids present in the sample, followed by an amplification step, on the other hand, that the said sample, as well as the active ingredients, become confined inside a hermetically closed space during the entire period for carrying out the process.

Description

 The present invention applies to the field of diagnosis in biology, whether it is human health, animal health or food industry. In human and animal health, such a diagnosis applies to the detection of an infection by a pathogen and to the detection of genetic diseases. In the food industry, this diagnosis applies to the detection in food of a contaminant such as a pathogenic micro-organism.

 For example, a viral infection in an individual can only be routinely detected using immunological tests. Indeed, in most cases it is the antibodies directed against the virus which are sought, which is indirect proof of the presence of the virus. In certain specific cases where viremia is high, viral antigens can be directly tested for in the blood of individuals. This is the case, for example, for the diagnosis of an infection with the hepatitis B virus (HBV). In general, immunoassays have average performance due to their lack of sensitivity and specificity.

 The most efficient immunological tests can detect 105 to 106 viral particles per milliliter. However for HBV, a serum is considered infectious up to 102 viral particles per milliliter (Prince et al., 1983). The number of false negative results is therefore important, since the infectivity threshold is at least 1000 times lower than the sensitivity threshold of immunological tests.

 The lack of specificity of these tests is directly linked to the immunological reactions involved. This results in false positive results which we try to demonstrate using a second immunological test, commonly called "confirmatory test". ".

This approach is used in particular for the detection of viruses responsible for AIDS (Acquired Immunodeficiency Syndrome)
There are today in the field of research methods of molecular biology making it possible to diagnose an infection by a pathogen with a sensitivity and specificity much higher than those obtained with immunological tests. These methods based on molecular hybridization of nucleic acids also allow direct proof of the presence of the pathogen, since it is its own genetic material that is detected.

 The purpose of such a diagnosis, based on molecular hybridization, is to demonstrate at least one defined sequence of deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) in a biological sample. For infections with a pathogen (virus, bacteria, parasite, etc.) it is a precise sequence of the genetic material of the pathogen that is highlighted. For genetic diseases (more than 3000 in humans), it is an anomaly in a precise sequence of genomic DNA which is revealed. To reach a very high sensitivity it is necessary to amplify in vitro the number of DNA or RNA molecules which must be demonstrated. The DNA or RNA sequences thus amplified are detected in a second step by molecular hybridization using nucleic probes.

The best results are currently obtained using the technique of amplification by chain reaction of a DNA polymerase, more commonly called PCR technique ("Polymerase Chai n Reaction") (Saiki et al., 1985).

 The in vitro enzymatic amplification technique or PCR amplification technique was described in December 1985 and applied to the diagnosis in humans of sickle cell anemia.

Unlike all the amplification systems described so far, the PCR technique makes it possible to specifically amplify the quantity of target double-stranded DNA sequences before they are detected by molecular hybridization. The principle of PCR is to repeatedly use the activity of a DNA polymerase to copy the DNA sequence to be amplified. The technological bases of PCR rest on two fundamental points. The first point concerns the use of two oligonucleotide primers distant from n nucleotides, one complementary to the DNA strand (+), the other to the DNA strand (-) and whose 3 'ends are opposite. The second point concerns the repetitive use of the activity of a DNA polymerase. The immediate consequence of these two points is that, under suitable conditions, all the neosynthesized sequences in cycle (n) can be used as template by DNA. ati cycle polymerase (n + 1). Thus all the specific sequences present after cycle (n) can be copied to cycle (n + 1). This results in an exponential amplification of the number of specific sequences as a function of the number of cycles.

 Several successive steps are necessary for the course of a PCR cycle. First of all, it is necessary to allow the specific hybridization of an oligonucleotide with a single-stranded target DNA sequence to be amplified. From the hybridization complex thus formed, a DNA polymerase performs the synthesis of the complementary DNA strand using the oligonucleotide as primer and the target DNA strand to be amplified as template. More precisely, an amplification cycle is made up of three stages. The first step allows denaturation of the double-stranded DNA sequences at 90-100 C. The second step allows the specific hybridization of the oligonucleotides to be achieved by lowering the temperature during the hybridization phase. primers. The third step authorizes the extension of the hybridized oligonucleotide to a temperature close to the optimal operating temperature of the DNA polymerase (37 "C for the Klenow fragment and 720C for a heat-resistant DNA polymerase extracted from Thermus aquiticus).

 During the amplification cycle (n + 1), the single-stranded DNA sequences are synthesized using as matrices the neosynthesized sequences in cycle (n), as well as the sequences present at the end of the cycle (n-1). A neosynthesized sequence in the n th cycle can thus serve as a primer for the cycle (n + 1) only if its length is at least equal to the distance in bases separating the two primers. Under these conditions, the number of DNA sequences amplified specifically varies exponentially with respect to the number of cycles.

 Amplification from RNA sequences requires carrying out a preliminary step of synthesis of complementary DNA (cDNA).

This approach is important for the detection of the genome of retroviruses such as the human immunodeficiency virus (HIV) responsible for
AIDS in humans (Ou et al., 1988).

 The molecular hybridization reaction between two nucleic acid sequences is directly related to temperature. This hybridization based on the complementarity of the bases between two sequences can occur, between two DNA molecules, between two RNA molecules, or between a DNA molecule and an RNA molecule.

Molecular hybridization therefore makes it possible to carry out either the pairing by hydrogen bond between two distinct molecules, or the intra-molecular pairing between two complementary sequences. In the latter case, there is formation of a so-called secondary structure of DNA or RNA molecules. The influence of temperature on the hybridization reaction is essential and each DNA (or RNA) sequence is defined by its Tm, i.e. the temperature at which 50% of the sequences are paired with the complementary sequences . The Tm of a precise sequence can be evaluated experimentally by following spectrophotometry the hyperchromicity at 260 nanometers which accompanies the mismatch (or denaturation) of two complementary DNA sequences. All the DNA sequences are in single stranded form. high temperature (100 "C) and in double strand form at low temperature (1 0-200C).

 The Tm of a DNA sequence ultimately depends on the following two parameters: the base sequence of the DNA in question and the ionic strength of the medium. The Tm generally encountered vary from 20 to 85 "C. Thus, all of the molecular hybridization reactions must be carried out under perfectly defined and controlled thermal conditions.

 For biological diagnosis (human health, animal health, food industry) a defined DNA or RNA sequence can be detected using the complementary DNA or RNA sequence used as a molecular probe. The probe is previously marked, either using a radioactive isotope, or using a chemical modification. After hybridization between the target sequence and the probe sequence, the latter can be detected by means of its labeling.

 In the current state of knowledge, it is possible to detect the presence of a single DNA (deoxyribonucleic acid) or RNA (ribonucleic acid) molecule in a biological sample by associating the PCR technique and molecular hybridization with using a nucleic probe. In other words, tests based on molecular hybridization allow extreme sensitivity to be reached since only one pathogen can be detected in a sample (Wong et al., 1987). By comparison with an immunological test, the gain in sensitivity is approximately 10,000 times.

 The specificity of the molecular hybridization reactions is excellent since it is possible to demonstrate a minute variation in the sequence of the bases composing the genetic material of an individual. The modification of a single base on the 3,500,000,000 bases that make up the human genome can thus be highlighted.

 Despite this great specificity of hybridization reactions, an ultra-sensitive diagnostic test, combining the use of PCR and molecular hybridization using a nucleic probe, does not have good specificity. It is indeed not uncommon to obtain false positive results which significantly reduce the overall specificity of the test. This phenomenon is due to accidental molecular contamination of negative samples with at least one DNA or RNA molecule that one seeks to detect (Lo et al., 1988). These contaminations which alter the overall specificity of the test take place between the taking of the biological sample and the end of the amplification of the DNA sequences by PCR. The false positive results are ultimately a direct consequence of the very high sensitivity of the test.

In other words, the use of an amplification technique as powerful as PCR, has made it possible to discover the existence of molecular contaminations hitherto unsuspected.

 The extreme sensitivity reached by diagnostic tests based on PCR is thus at the origin of a new type of false positive results linked to molecular contamination. The magnitude of the problem is such that it is now necessary to carry out the different stages of the test in different premises in an attempt to minimize this phenomenon.

This partitioning makes it possible to physically separate the pre-PCR manipulations, carried out on samples poor in specific DNA, from the post-PCR manipulations, carried out on samples very rich in
Specific DNA. A minimum of three independent rooms (but preferably four) with their own security airlock is beginning to be installed in a large number of research laboratories around the world. These parts are under overpressure or under depression depending on whether the step taking place there is pre-PCR or post-PCR respectively.

 This partitioning makes it possible to significantly reduce the number of false positive results but not to eliminate them completely. This approach remains costly in any event, and makes a test which was basically simple to be very complex.

 There are currently in the commercial circuit a certain number of devices which can only execute one of the 3 or 4 steps which make up a diagnostic test using PCR and molecular hybridization using a nucleic probe. The only steps thus automated are the step of extracting nucleic acids from the biological sample and the step of amplifying DNA sequences by PCR.

 To carry out the step of extracting nucleic acids from the biological sample, a single device is today available on the world market. It is an expensive device, being able to process only a very small number of samples (8 at most) in about 6 hours.

All of its characteristics mean that this device can only be used in a very specialized research laboratory.

 Between 12 and 15 different devices make it possible to automatically perform the step of amplifying DNA sequences by PCR.

All of these machines subject disposable and independent plastic tubes at different temperatures, with a capacity varying from 0.75 ml to 1.50 ml. Each tube contains the biological sample to be treated. Two devices are also available with an adapter that can receive a microtiter plate.

 In general, existing devices have been designed to automatically carry out a precise technological stage. None of them makes it possible to carry out the entire diagnostic test by molecular hybridization. Thus the consecutive use of a nucleic acid extraction apparatus and a PCR apparatus obliges to carry out, between these two automated steps, manipulations on each sample. Such manual intervention is compulsory between these two stages and during the stages carried out after the CPR.

 These manipulations mainly consist in opening and closing disposable tubes with removable stopper, and in precision pipetting. When handling the tubes, the experimenter wears latex or vinyl gloves for the sake of greater cleanliness and above all to avoid contaminating the sample himself with nucleases present on the skin. Nucleases are enzymes that degrade DNA and RN. Unfortunately, the gloves are coated with talc, or an equivalent product, which is a great potential source of contamination of the sample during the opening and closing of the tube. In addition, when a tube is opened, the significant danger of contamination is further accentuated by the presence of neighboring tubes potentially containing contaminating molecules. During the pipetting of a determined quantity of liquid in a donor tube and the delivery of this liquid into a recipient tube, the pipetted liquid can be the sample itself, a buffer solution, a preparation containing at least one active principle ( enzymes, oligonucleotides, nucleotide triphosphates, etc.).

 For each biological sample processed, 12 to 15 pipettings should be performed using a precision pipetting device for the only pre-PCR step consisting of extraction of nucleic acids. The precision pipetting device is very often the source of molecular contamination between two different reaction mixtures. Faced with this major problem, experimenters are increasingly using positive displacement pipetting systems which seem to slightly decrease the number of contaminations, but which are high in cost.

 In any event, All of these manipulations are a constant danger in terms of molecular contamination which may generate false positive test results. This situation is a major obstacle to the use of molecular hybridization technologies for biological diagnosis. For human health in particular, it cannot be envisaged to develop a reliable test, because of the large number of false positive results.

 The current situation is therefore paradoxical today, since there are very powerful technologies, PCR in particular, but that they cannot be used due to unsuitable general handling conditions. These handling conditions, however deemed very strict in a molecular biology laboratory, are not sufficient when the sensitivity of the tests developed allows the detection of a single DNA or RNA molecule.

 The impact of the use of molecular hybridization diagnostic tests in human health would be considerable since various estimates report that approximately 10 to 20% of individuals tested in transfusion centers, hospitals and specialized laboratories are contaminated with a pathogen but are not detected (false negatives) by conventional immunoassays.

 In the present description and in the claims, the term “sample” will be understood to mean a set of molecules at a given time during the overall processing of the biological sample, the said set of molecules being initially derived from the biological sample taken during sampling. When there is physical separation into at least two families of molecules, the sample is that which contains the greatest number of specific DNA or RNA sequences sought in the state of purification or of association with other molecules the most advanced compared to the global treatment composed of several successive stages. In the same way, the term "active ingredient" will be understood to mean a molecule or a family of molecules intervening during the treatment of the sample. An active ingredient can, in particular, be an enzyme, an ion, a salt, a nucleotide triphosphate or an oligonucleotide. By the term "target sequence" is meant the DNA or RNA sequence which will be specifically amplified during the amplification step. Once amplified, the target sequence can possibly be demonstrated by molecular hybridization using at least one probe sequence. The term "sampling" is understood to mean the taking of biological material from an individual, an animal, a plant, or a product from the food industry. The biological material thus taken will be called "initial sample".

 The main objective of the present invention is to completely eliminate the problems of molecular contamination during the automatic processing of a biological sample which comprises a step of in vitro amplification of the number of a defined sequence of DNA or RNA. Such a result can be achieved by the use of a method for amplifying the number of at least one defined sequence of nucleic acid or of its complementary sequence, hereinafter called target sequences, in a biological sample, characterized by on the one hand, it comprises an addition of active principles to the said sample and at least one preliminary step, intended to increase the accessibility of the nucleic acids present in the sample, followed by an amplification step, on the other hand that the said sample as well as the active ingredients are confined within a hermetically closed space during the entire duration of carrying out the process. In order to more effectively overcome these problems of molecular contamination, the method can advantageously be characterized by the fact that the sampling is carried out directly in said hermetically closed space.

 Such a method preferably makes it possible to carry out a complete biological diagnostic test comprising, on the one hand, a preliminary step making it possible to increase the accessibility of DNA and RNA to molecules contained in the initial sample and at least one active principle, such as DNA and RNA polymerization enzymes and partially or fully complementary DNA and RNA sequences, on the other hand at least one additional step subsequent to step d amplification intended to demonstrate the possible presence in the sample of all or part of at least one target sequence of deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), the number of which has increased during the amplification stage.

 The aforementioned amplification step preferably uses, on the one hand, the activity of a DNA polymerase or of an RNA polymerase responsible for the synthesis of the amplified DNA or RNA sequences, on the other hand, either at least one DNA or RNA sequence recognized as a primer by the abovementioned enzyme and partially or totally complementary to a part of the target sequence to be amplified specifically, ie a perfectly defined promoter sequence recognized by the enzyme and belonging to the target sequence to be amplified.

 The process in question is advantageously characterized in that at least one additional step, situated before or after the amplification step, uses at least one DNA or RNA probe sequence, modified or not, which in precise physico-chemical conditions hybridizes specifically by complementarity between the bases with at least one target sequence, and which is present in the sample in free or supported form.

 In the form of a complete diagnostic test, the method described above can advantageously be characterized by the fact that the amplification step uses the polymerase chain reaction (PCR) technique to specifically increase the number of molecules of a target sequence defined with a modified oligonucleotide primer which carries a molecule capable of emitting a measurable signal, on the other hand that after the amplification the sample is brought into contact with a supported DNA probe, at least partially complementary to the amplified target sequence, and that the presence of said amplified target sequence specifically retained on said support is evaluated using means external to the bar making it possible to quantitatively detect the molecule emitting a signal carried by the amplified sequences.

 The implementation of the method having any of the above-mentioned characteristics is the fact of a device characterized in that it comprises, on the one hand at least one enclosure containing the active ingredients necessary for the preliminary step intended for increase the accessibility of the nucleic acids, on the other hand at least one enclosure containing the active principles necessary for the amplification step, and that the enclosures are hermetically connected to each other so as to constitute an assembly, hereinafter called a bar, delimiting an enclosed space, means being provided for circulating and for thermally regulating the sample and the active ingredients. Such a device preferably uses a single-use bar, that is to say disposable after the treatment of a single biological sample.

 Advantageously, the above-mentioned device is characterized by the fact that, on the one hand, the means providing partial or total movement of the sample and of the active ingredients in the strip consist of a peristaltic pump acting on the flexible wall a capillary belonging to the bar, on the other hand that the connections between the different enclosures are capillaries equipped with means intended to allow or prevent the passage of a fluid at a given time in at least one precise capillary in order to '' ensure the smooth running of the treatment and that the said means consist of a rigid element having at least two positions in space, such that a so-called closed position allows the flexible capillary to be compressed in this place in order to prevent the circulation of a fluid, and such that the other so-called open position does not have this compression effect and allows the passage of a fluid in this place of the capil laire.

 Advantageously, several bars can be grouped together to form a cartridge making it possible to simultaneously process several samples with common means for ensuring thermal regulation, displacement and selective circulation of the samples and of the active ingredients.

 The device for implementing the process described in this invention consists, on the one hand of a bar containing the active ingredients necessary for the proper conduct of the process, on the other hand of means for ensuring displacement, thermal regulation and the selective circulation of the sample and of the active ingredients inside the said strip.

The present invention will be better understood with the aid of the additional description which follows, which refers to the appended drawings in which:
- Figure 1 shows a schematic perspective view and partially cut of a bar fitted to a preferred embodiment of the device according to the invention.

 - Figures 2, 3 and 4 show sectional views of certain components of the bar shown in Figure 1. It is the base plate comprising a network of grooves (Figure 2), capillaries formed by the association of the base plate and of a perforated plate (FIG. 3), of the same capillaries represented in FIG. 3 but cut at a site for connection of the perforated plate with a treatment enclosure (FIG. 4).

 - Figure 5 is a schematic illustration of the network of capillaries allowing the connections between the treatment chambers belonging to the bar shown in Figure 1, the said network being equipped with valves.

 - Figures 6, 7, 8 and 9 show different functional loops of the network of Figure 5, each loop being associated with a configuration of opening and closing of the valves.

 - Figure 10 shows a schematic view of a cartridge fitted to a preferred embodiment of the device according to the invention.

 The bar, fully or partially shown in Figures 1, 2, 3 and 4, as well as the cartridge shown in Figure 10, are for single use. This approach makes it possible to have, inside said strip and cartridge, a hermetically closed space containing active principles which have never been in contact with a potential source of contaminations harmful to the smooth running of the process.

The re-use of at least part of the strip increases the risk of molecular contamination of the sample at a given point in the processing, even if the re-used part is not in the path of the sample or a liquid having been in contact with the sample. This is the case, for example, of a device made up of two connected parts, on the one hand of a single-use part inside which takes place all the movements of the sample and of the active ingredients, d other part of a reusable part consisting of a cartridge containing a gas which due to its pressure is used for moving the sample and the active ingredients. Although the disposable part can carry, near its connectable end, a system filtration preventing the passage of previously defined contaminating molecules, the risks of contamination are greater than those inherent in the use of a single-use bar.

 The aforementioned bar (FIGS. 1 to 4) and the cartridge (FIG. 10) are designed to carry out a complete diagnostic test defined as a biological process making it possible to carry out all the stages preliminary to amplification, the amplification itself and the stages subsequent to the amplification resulting in a final qualitative, semi-quantitative or quantitative analysis. The abovementioned final analysis is intended to demonstrate the possible presence in the sample of at least one defined sequence of DNA or RNA whose number increased during the amplification step.

 Advantageously, the introduction of the initial sample into the bar or the cartridge will take place as soon as it is sampled from an individual, an animal or a product from the food industry.

The aforementioned introduction can however be carried out from a storage compartment serving as an intermediary between the sampling and the actual processing of the sample in the bar or cartridge.

 The aforementioned bar (FIGS. 1 to 4) and the cartridge (FIG. 10) are characterized by the fact that the hermetically closed space is surrounded by a sealed physical barrier, advantageously made of plastic material which may have hermetic junction zones between two separate components. These junction zones can result from a bonding, a heat-sealing or a "strength" assembly of two aforementioned elements. Due to the construction materials used, the bar does not allow the passage of contaminating molecules from the outside to the inside, nor from the inside to the outside. The passage of such molecules would be likely to disrupt the processing of the sample, in particular if it is a DNA or RNA sequence of a length greater than or equal to two nucleotides. It is mainly a question of preventing the passage of DNA or RNA sequences, the number of which could be amplified during the treatment of the sample.

 The bar (Figure 1) consists of a cover (5) fixed on a base plate (1), which itself rests on a flexible perforated plate (2). At the end of the plate (1), two outlets allow each to receive a flexible hose (E7.4) with an internal diameter of 1.2 mm. The cover (5) protects 6 elements, (El), (E2), (E3), (E4), (E5) and (E6) fixed on the plate (1). The elements (E7) and (4) are connected to an enclosure (E8) carrying a window (3).

 The biological process which takes place inside this bar comprises at least one preliminary step intended to increase the accessibility of the nucleic acids contained in the initial sample.

These nucleic acids will thus be more accessible to at least one molecule which is either present in the sample at the time of this step and resulting from the initial sample or from the addition of an active principle, or added in the form of 'an active ingredient in a subsequent step. The increase in accessibility of nucleic acids can be obtained either by modification of the molecules and biological structures contained in the sample, or by partial purification of these nucleic acids. This step to increase the accessibility of nucleic acids is necessary because any living cell, such as a virus, a bacteria, or a eukaryotic cell, contains more or less advanced biological structures intended to compartmentalize the cell and allowing to protect essential molecules such as nucleic acids. Accessibility to nucleic acids therefore requires the partial or total destruction of such structures which are generally of protein and lipid nature. The most commonly used technique consists in destroying or sufficiently modifying the molecules making up this biological structure. The destruction or profound modification of structural proteins, either by a proteolytic enzyme such as proteinase K, or by the action of heat (treatment at 110 "C) is an effective technique and perfectly compatible with the process and the device. described in this invention.

 Partial purification of nucleic acids can consist of the partial elimination of lipid molecules and proteins contained in the sample. This method therefore indirectly alters the above-mentioned biological structures. The first technique consists in passing the sample through a column composed of a resin having a high affinity for proteins at neutral pH and a very low affinity for nucleic acids. A second technique consists in passing the sample at least once through an ion exchange column which will retain the nucleic acids by ionic bonds between the negatively charged phosphates of DNA or RNA and the cations attached to the column. .In a second step, a strong ionic elution buffer will be used to unhook the DNA or the RNA by creating a competition between the anion of the column and the anion of the salt (Na + preferably) for the fixation on the negative charges of phosphates. This simple technique is entirely compatible with the method and the device described in this invention. A third technique consists in extracting molecules like lipids and proteins using organic solvents like phenol and chloroform. A fourth technique consists in retaining defined DNA or RNA sequences by hybridization with at least partially complementary supported sequences present on a column.

 The preliminary step intended to increase the accessibility of the nucleic acids present in the sample can be a simple heating of the latter to a temperature capable of denaturing certain proteins, that is to say at a temperature preferably between 900C and 1 1 00C. By following such a protocol, the aforementioned preliminary step can be confused with the first heat treatment used to denature the DNA and RNA sequences during the amplification step.

 Advantageously, two successive preliminary steps are used during the process which takes place in the bar shown in FIG. 1. The first preliminary step consists in a proteolytic treatment of the sample with proteinase K. To do this, I ' sample is mixed with this enzyme in the enclosure (E6) then it moves in the enclosure (E7) to be incubated there at 70 "C for 20 minutes. The second preliminary step consists in passing the sample through low ionic strength conditions, on an anion exchange column located in (E2), the nucleic acids retained on the column are then removed using a high ionic strength buffer located in (ex).

 In the bar shown in FIG. 1, it is advantageous, after the two successive preliminary steps intended to increase the accessibility of the nucleic acids, to use the polymerase chain reaction (PCR) with at least one oligonucleotide primer carrying the fluorescein signal molecule at its 5 'end. Thus, at least part of the neosynthesized sequences during the amplification step will carry the said signal molecule and can be recognized selectively during a subsequent step. All of the active ingredients necessary for such an amplification step are contained in (E5).

After adding the said active principles, the sample is brought to (E7) where it is subjected cyclically to the different temperatures enabling the PCR to be carried out. 15 to 40 cycles of PCR amplification are generally sufficient to obtain a high sensitivity of the diagnostic test. However, for the detection of certain pathogens such as the HIV viruses (type 1 and 2) and the hepatitis C virus (Garson et al., 1990) it is necessary to carry out a second PCR using oligonucleotide primers internal to the specifically amplified sequence. This approach achieves significant sensitivity even when the initial number of pathogens is close to unity.

 According to other versions of the method described in the present invention, the amplification step can use the activity of a DNA polymerase, or of an RNA polymerase, responsible for the synthesis of the amplified sequences, and in which two families enzymes can be distinguished. The first family is composed of enzymes which require the presence of a primer sequence of DNA or RNA. Such enzymes in fact use as substrate a molecular duplex constituted by the hybridization between a target sequence, or template sequence, and a primer sequence carrying a free 3 'hydroxyl (OH) end. The enzyme then extends the primer sequence from its 3 ′ end by incorporating, for a given position on the target sequence, the complementary nucleotide by using free nucleotide triphosphates present in the medium. This results in the synthesis of a sequence complementary to the target sequence. The second family consists of enzymes which require the presence of a perfectly defined promoter sequence which they recognize and from which it synthesizes the sequence complementary to the sequence located immediately 3 'to the said promoter sequence using nucleotides free triphosphates present in the environment. The RNA polymerase of bacteriophages T7 and SP6 are enzymes which behave in the latter way (Stoflet et al., 1988).

 Advantageously, a signal molecule other than fluorescein can be used to modify the oligonucleotide primer used during the process amplification step described in the present invention. Indeed, there are two main families of signal molecules. The first family is composed of molecules which, under precise physicochemical conditions1 can emit a signal of a particulate nature which can be a radioactive, fluorescence, luminescence, bio-luminescence and phosphorescence emission. The second family is composed of molecules of which at least one of their parameters is easily measurable. It can be the soluble or precipitable product resulting from the degradation of a substrate in particular by an enzyme such as alkaline phosphatase or peroxidase. The presence of the soluble degradation product can be detected by colorimetry. There are degradation products of substrates belonging to the first family of aforementioned signal molecules due to the nature of their emission.

 According to still other versions of the method described in the present invention, at least one oligonucleotide primer used during the amplification step can be modified in different ways, other than that resulting in the attachment of a signal molecule. Indeed, a nucleic acid sequence can be modified by the addition of a radioactive isotope (32P, 355, 3H), a chemical group, a hapten, a molecule with a molecular weight greater than 300 daltons or of an enzyme.

 The method which takes place in the bar represented in FIG. 1 preferably comprises a final step of analysis of the specifically amplified sequences. To do this, the amplified sample is brought into contact with a supported DNA probe, at least partially complementary to the amplified target sequence, then the presence of said amplified target sequence, specifically retained on said support. , is evaluated using means making it possible to quantitatively detect the fluorescein emitting a signal carried by the amplified sequences. The probe sequence is preferably an oligonucleotide of 20 to 35 nucleotides covalently linked to a solid support consisting of silica.

 The above-mentioned final analysis step consists, first of all, of at least one passage of the sample at a precise temperature in a column included in (E8) and containing a solid support on which is immobilized at least one aforementioned probe sequence, then by stalling and re-solution at an elution temperature of at least one target sequence amplified using an elution buffer stored in (E3), followed by detection in the eluate of the signal molecule. Said elution temperature is calculated as a function of the melting temperature (Tm) of the molecular duplex formed by the hybridization of the supported probe sequence with at least one amplified target sequence. Due to the genetic variability of certain target sequences and therefore, from the approximate knowledge of the target sequence actually contained in the samples, several elution temperatures may successively be advantageously used in the direction of increasing temperatures. At least one eluate can then be subjected to the detection of the signal molecule carried by the amplified target sequence using a detector external to the strip.

 According to a simplified version of the method described in the present invention, the amplification step represents the end of said method. Still other variants of the process described in the present invention can be used depending on the nature of an additional step. Said additional step may be before or after the amplification step, and use at least one DNA or RNA probe sequence, modified or not, which under specific physico-chemical conditions hybridizes specifically by complementarity between bases with at least one target sequence. The aforementioned probe sequence can be present in the sample in free or supported form. The purpose of such an additional step is to lead at the end of treatment to a qualitative, semi-quantitative or quantitative analysis of the presence of at least a target sequence in the initial sample and therefore to have a complete diagnostic test carried out automatically and under the previously defined isolation conditions.

 During the said additional step, the probe sequence can advantageously be added before the amplification step and serve as a specific inhibitor during the amplification. Indeed, a DNA polymerase or an RNA polymerase is stopped or strongly slowed down if during its relative progression on the matrix sequence, and while it synthesizes the complementary sequence, it collides with the 5 'end of a sequence DNA or RNA, called blocking, hybridized to said matrix sequence. A probe sequence, complementary to at least one target sequence to be amplified, added before the amplification step behaves like a blocking sequence and therefore has an inhibiting effect on the specific amplification of at least one target sequence. given sample, if the amplification step is applied independently, on the one hand on said sample containing a probe sequence or blocking sequence, on the other hand on said sample containing no probe sequence, the comparison of the analyzes of amplified sequences in these two cases makes it possible to diagnose with certainty the presence or absence of the target sequence in the initial sample. To be effective, such a probe sequence can be modified at its 3 ′ end, preferably by incorporating a dideoxynucleotide, so that it cannot be used as a primer by DNA or RNA polymerase during amplification.

 During the said additional step, the probe sequence can preferably be added after the amplification step and specifically hybridize to at least one amplified target sequence. According to this approach, very many different hybridization models can be used depending on whether the target sequence is immobilized on a solid or free support, depending on whether the probe sequence is immobilized on a solid or free support, depending on the number of different probe sequences used and depending on the type of possible modification of at least one target sequence or at least one probe sequence. The hybridization model used conditions the following stages of sample processing, the final stage of which is an analysis of the amplified sequences.

 Advantageously, when the abovementioned hybridization model and the final analysis are intended to differentiate at least one amplified target sequence from all the other sequences present in the sample, it is necessary to use either means making it possible to differentiate these molecules as a function of at least one of their physico-chemical parameters, ie a hybridization complex between the said amplified target sequence and at least one complementary probe sequence. The most used physico-chemical parameter is the molecular weight of a DNA or RNA sequence, or its length in number of nucleotides, but its evaluation requires the use of a separation technique such as electrophoresis or gel-filtration.

 Preferably, the method described in this invention can make it possible to carry out a complete diagnostic test, the last step then being a qualitative, semi-quantitative or quantitative analysis of the presence of a precise signal molecule making it possible to estimate the presence at least one specifically amplified target sequence and since there is a correlation, depending on the nature of the analysis, between the presence of the amplified target sequence and the presence of all or part of this target sequence in the initial sample. The aforementioned signal molecule can be carried either by an amplified target sequence or a part thereof, or by a probe sequence or a part thereof.

The signal molecule is characterized by the fact that its presence can be directly measured using a detector device.

 For the smooth running of the process implemented in the bar of FIG. 1, certain enclosures (FIG. 1) will advantageously be provided with external means of heating and cooling. Thus, (E7) is equipped with three independent heating elements which allow, on the one hand to submit the sample to a constant temperature between 37 "C and 700C during the proteolytic treatment with proteinase K, on the other hand to successively submit the sample to the three temperatures associated with an amplification cycle by PCR and making it possible to carry out the denaturation of the double stranded DNA, the hybridization of the primers and the extension by a DNA polymerase. (E8) is also equipped with a heating element which allows during the previously described analysis step to unhook an amplified target sequence. The plurality of steps composing the overall treatment of the sample requires system dynamics.

This dynamic consists of a displacement of the sample between different enclosures and a displacement towards the sample of an active principle necessary for the treatment. The dynamic in question is ensured by the operation of a peristaltic pump acting on the flexible wall of (E7).

 According to still other versions of the device for implementing the method described in the present invention, the speed of movement of the sample or of the active principle can be continuous or discontinuous. The motive force necessary for such displacements can be of different nature: fluid under pressure, displacement of a magnetic system, passive capillarity, thermal gradient, peristaltic pump acting on the wall of a flexible capillary, difference in pressure between the fluid ( gas for example) upstream of the sample and the fluid downstream of the sample (this pressure difference being created by a mechanical system acting locally), or a combination of the various preceding possibilities.

 Some of the capillaries (FIG. 5) of the bar in question (FIG. 1) are equipped with means intended to allow or prevent the passage of a fluid from one enclosure to another. These means are intended to allow or prevent the passage of the sample, at least one gas or at least one active principle at a given time in at least one precise capillary in order to ensure the proper functioning of the treatment. To do this (Figure 2 to 4), the plate (2) can advantageously be flexible and carry grooves (7) on one of its faces (Figure 2). The path layout was established to meet a precise functional diagram. The intimate contact, by heat sealing or by gluing (Figure 3), of the plates (1) and (2) makes it possible to define capillaries coming from the grooves (7). The flexible structure of the plate (2) allows a set of cams (9) outside the bar to intermittently crush the protrusions (6) of the grooves (7). This has the function of preventing the passage of a fluid in the capillary concerned). The set of three cams (9) integral with a single support (8) shown in FIG. 3, makes it possible to crush the three capillaries simultaneously, and therefore to prevent the circulation of a fluid in the area of crushing. The set of plates (1) and (2) forms a well-defined network of capillaries, to which the speakers (El), (E2), (E3), (E4), (E5), (E6) and (E7), and the flexible pipe (4). The connections (figure 4) with the enclosure elements (E) are ensured by a perforation (10) in the plate (1), on which is fitted a sleeve (11 ) carrying a pipe (12). The connection with the flexible hose (4) is ensured by a perfectly similar system. A valve (X) (FIG. 5), corresponding to an external cam of the type of those mentioned above, can be either in the open position (X = 1) and allow the free circulation of a fluid at its level, or in the closed position (X = 0) and prevent the circulation of a fluid at its level.

The raw biological sample, in liquid form, is introduced into the enclosure (E4) where it is mixed with active principles which will make the viral DNA accessible. After mixing, the concentrations are as follows: 25 mM sodium acetate pH 6.5; 2.5 mM EDTA; 0.5% SDS; 2.5 mg / ml proteinase K. All the valves (figure 5) being in the configuration [X1 = 0, X2 = 0, X3 = 1, X4 = 0, X5 = 0, X6 = 0] (figure 6 ),
The sample moves to (E7) through the connections (a16) and (a18). Inside the enclosure (E7), the sample is brought to 700C for 15 minutes. All the valves being in the configuration [Xi = 0, X2 = 0, X3 = 1, X4 = 0, X5 = 0, X6 = 0], the sample returns to (E6) passing through (au8) and (a16). All the valves being in the configuration [X1 = 0, X2 = 0, X3 = 1, X4 = 0, X5 = 0, X6 = 0], the sample flows from (E6) to (E2 ) through (a15) and (a5). In (E2), the sample crosses an ion exchange column which specifically retains nucleic acids. The eluate not retained on the column circulates from (E2) to (E4) passing through (a3) and (a9). Due to the spatial configuration of connections (a9) and (a10), the eluate is blocked in (E4). All the valves being in the configuration [X1 = 1, X2 = 1, X3 = 0, X4 = 0, X5 = 1, X6 = 0] (Figure 7), the elution buffer contained in (El) is brought into (E2) where it crosses the ion exchange column and detaches the nucleic acids. The liquid sample thus formed by this elution is diluted then it moves from (E2) to (E5) via (a4) and (at 12). Due to the spatial configuration of the connections (ail) and (a12), the sample is blocked in (E5) where it is mixed with the active ingredients necessary for the amplification reaction by PCR. After mixing the concentrations are as follows : 200ru deoxyadenosine5'triphosphate; 200M deoxythymidine-S'triphosphate; 200, ut deoxyguanosine-5'triphosphate; 200C1M deoxycytidine-5'triphosphate; 250 nM of each oligonucleotide primer; 10mM Tris-HCl, pH 8.4; 1.5 mM magnesium chloride; 100, ug / ml gelatin and unit for 50ffi1 dtDNA heat-resistant polymerase (Taq polymerase for example).

All the valves being in the configuration [X1 = 0, X2 = 0, X3 = 0,
X4 = 0, X5 = 1, X6 = 1] (figure 8), the sample flows from (E5) to (E7), passing through (a14) and (a18), where it undergoes the heat treatment necessary for the amplification step by PCR. Heating and cooling means external to the bar allow the sample contained in (E7) to be subjected to 30 successive thermal cycles defined as follows: 1 cycle = 1 minute at 95 "C, then 1 minute at 50" C, then 1 minute at 72 "C. All the valves being in the configuration [X1 = 0, X2 = 0, X3 = 0, X4 = 0, X5 = 1, X6 = 1], the sample moves from (E7 ) to (E8) passing through (a19) and (a21). Inside the enclosure (E8) equipped with external heating means, the sample is brought into contact with an oligonucleotide probe fixed on a solid support and the specific amplified sequences are selectively retained by subjecting (E8) to 60 "C. The eluate containing non-specific sequences continues its migration to (E4), passing through (a20), (a17) and (a10), where it is blocked due to the spatial configuration of the connections (a10) and (ail) . All the valves being in the configuration [X1 = 0,
X2 = 0, X3 = 0, X4 = 1, X5 = 0, X6 = I] (figure 9), the buffer contained in (E3) is brought into (E8) where it is brought into contact with the support retaining the sequences specific DNA samples to be detected. The enclosure (E8) is then subjected to a temperature allowing dehybridization between the supported probe sequence and the amplified sequence to be detected. The latter is therefore redissolved and its presence directly detected inside (E8) thanks to a detector centered on the axis of the window (3). In a configuration of the valves identical to that previously cited and after its analysis by the detector, the sample continues its migration towards (E4) where it is blocked due to the configuration of (a10) and (a8). (E4) is a trap enclosure intended to accumulate the products extracted from the sample and the active ingredients already used. (E4) has 4 connections, (a8), (a9), (a10) and (ail), allowing the free circulation of a gas between any two of these connections. Its compartmentalized structure, however, allows the amplified and analyzed sample to be collected in an element not soiled by other liquids from the treatment. The amplified sample can therefore be used later by simple sampling in (E4).

 Several bars can be grouped together to form a cartridge (Figure 10) which is structured in 4 distinct parts: (C1), (C2), (C3) and (C4). Part (Cl) contains the speakers (ex), (E2), (E3), (E4), (ES) and (E6). The part (C2) is a flexible junction structure between (C1) and (C3). The part (C3) contains the enclosure (E7) and the part (C4) contains the enclosure (E8).

REFERENCES CITED

Claims (10)

 1 / Method for amplifying the number of at least one defined sequence of nucleic acid or of its complementary sequence, hereinafter called target sequences, in a biological sample, characterized in that, on the one hand, it comprises a contribution of active principles to the said sample and at least one preliminary step, intended to increase the accessibility of the nucleic acids present in the sample, followed by an amplification step, on the other hand than the said sample as well as the active ingredients are confined within a hermetically sealed space during the entire process.  2 / A method according to claim 1, characterized in that the sampling is done directly in said hermetically closed space.  3 / Method according to one of claims 1 and 2, characterized in that it makes it possible to carry out a complete test of biological diagnosis comprising, on the one hand a preliminary step making it possible to increase the accessibility of the DNA and RNA to molecules contained in the initial sample and to at least one active principle, such as DNA and ART polymerization enzymes and partially or fully complementary DNA and RNA sequences , on the other hand at least one additional step subsequent to the amplification step intended to demonstrate the possible presence in the sample of all or part of at least one target sequence of deoxyribonucleic acid (DNA) or d ribonucleic acid (RNA) whose number increased during the amplification step.  4 / Method according to one of claims 1 to 3, characterized in that the amplification step uses, on the one hand the activity of a DNA polymerase or an RNA polymerase responsible for the synthesis of the sequences amplified DNA or RNA, on the other hand, either at least one DNA or RN sequence recognized as a primer by the abovementioned enzyme and partially or completely complementary to a part of the target sequence to be amplified specifically , or a perfectly defined promoter sequence recognized by the enzyme and belonging to the target sequence to be amplified.  5 / Method according to one of claims 1 to 4, characterized in that at least one additional step, located before or after the amplification step, uses at least one DNA or RNA probe sequence, modified or not, which under specific physico-chemical conditions hybridizes specifically by complementarity between the bases with at least one target sequence, and which is present in the sample in free or supported form.  6 / Method according to one of claims 1 to 5, characterized in that, on the one hand that the amplification step uses the polymerase chain reaction technique (PCR) to specifically increase the number of molecules d '' a target sequence defined with a modified oligonucleotide primer which carries a molecule capable of emitting a measurable signal, on the other hand that after the amplification the sample is brought into contact with a DNA probe supported at least partially complementary to the sequence amplified target and that the presence of said amplified target sequence specifically retained on said support is evaluated using means external to the bar making it possible to quantitatively detect the molecule emitting a signal carried by the amplified sequences.  7 / Device for implementing the method according to one of claims 1 to 6, characterized in that it comprises, on the one hand at least one enclosure containing active ingredients necessary for the preliminary step intended to increase the accessibility of the nucleic acids, on the other hand at least one enclosure containing the active principles necessary for the amplification step, and that the enclosures are hermetically connected to each other so as to constitute an assembly, ci -after called a bar, delimiting an enclosed space, means being provided for circulating and for thermally regulating the sample and the active ingredients.  8 / Device according to claim 7, characterized in that the assembly defined by the hermetically closed space and the active ingredients it contains is for single use, that is to say disposable after the treatment of a single sample organic.  9 / Device according to one of claims 7 and 8, characterized in that, on the one hand that the means ensuring partially or completely the movement of the sample and the active ingredients in the strip consist of a peristaltic pump acting on the flexible wall of a capillary belonging to the bar, on the other hand that the connections between the various enclosures are capillaries equipped with means intended to allow or prevent the passage of a fluid at a given time in at least one precise capillary in order to ensure the smooth running of the treatment and that the said means consist of a rigid element having at least two positions in space, such that a so-called closed position makes it possible to compress the flexible capillary in this place in order to 'prevent the circulation of a fluid, and such that the other so-called open position does not have this compression effect and allows the passage of a fluid at this location of the capillary.  10 / Device according to one of claims 7 to 9, characterized in that several bars are grouped together to form a cartridge for simultaneously processing several samples with common means to ensure thermal regulation, movement and selective circulation of samples and active ingredients.