EP2269738B1 - Device for thermo-dependent chain reaction amplification of target nucleic acid sequences - Google Patents

Abstract

A heating plate(s) for a polymerase chain amplification (PCA) system comprising three distinct zones operating at different temperatures corresponding to the three cycles of the PCA, is new. Independent claims are included for the following: (1) a polymerase chain amplification (PCA) system has plate(s) with reaction chambers and heating plate(s) as above.; (2) PCA using the above system where the plate has reservoir(s) for filling with a fluid including DNA samples and supplying this fluid to the chambers in the plate that have already been given markers for specific DNA sequences, where the plate is moved relative to the heating plate according to the cycle of the PCA.

Description

The present invention relates to the field of genetics.

More specifically, the present invention relates to a device for the amplification of target nucleic sequences, and modes of use of this device.

The object of the present invention is in particular to enable the detection and, where appropriate, the quantification in real time of target nucleic acid sequences in one or more samples.

Detection of target nucleic acid sequences is an increasingly popular technique in many fields, and the range of applications of this technique is expected to expand as it becomes more reliable, cost-effective and more reliable. fast. Thus, in human health, the detection of certain nucleic acid sequences in certain cases allows a reliable and rapid diagnosis of viral or bacterial infections. Similarly, the detection of certain genetic peculiarities can make it possible to identify susceptibilities to certain diseases, or to establish an early diagnosis of genetic or neoplastic diseases. The detection of target nucleic sequences is also used in the food industry, in particular to ensure the traceability of products, to detect the presence of genetically modified organisms and to identify them, or to carry out a health control of food.

Nucleic acid-based detection methods almost invariably involve a molecular hybridization reaction between a target nucleic acid sequence and one or more sequences nucleic acid complementary to said target sequence. These methods have numerous variants such as the techniques known to those skilled in the art under the terms "transfer techniques" ( blot, dot block, Southern blot, Restriction Fragment Length Polymorphism, etc.), or else the miniaturized systems on which the complementary sequences of the target sequences ("biochips") are prefixed. In the context of these techniques, the complementary nucleic sequences are generally called probes. Another variant, which may constitute in itself the basis of a diagnostic method or be only an additional step in one of the techniques mentioned above (in particular to increase the concentration of the target sequence and therefore the sensitivity of the diagnosis), consists in amplifying the targeted nucleic acid sequence. Several techniques for the specific amplification of a nucleic acid sequence have been described, the most commonly used of which is Polymerase Chain Reaction (PCR) or Polymerase Chain Rection (PCR). In the context of this latter technique, complementary nucleic sequences of the target sequences, called primers, are used to amplify said target sequences.

The PCR reactions involve a repetition of cycles, the number of which generally ranges from 20 to 50, and which are each composed of three successive phases, namely: denaturation, hybridization, elongation. Respectively, the first phase corresponds to the transformation of the double-stranded nucleic acids into single-stranded nucleic acids, the second phase to the molecular hybridization between the target sequence and the complementary primers of said sequence, and the third phase to the elongation of the hybridized complementary primers. to the target sequence, by a DNA polymerase. These phases are carried out at specific temperatures: generally 95 ° C for denaturation, 72 ° C for elongation, and between 30 ° C and 65 ° C for hybridization, depending on the hybridization temperature (Tm) of the primers used. It is also possible to perform the hybridization and elongation steps at the same temperature (generally 60 ° C).

où
X_n-1 est la quantité de produit obtenu au cycle précédent, et r_n le rendement de la PCR au cycle n (0 < r_n ≤ 1).A PCR reaction therefore consists of a series of repetitive thermal cycles in which the number of target DNA molecules serving as template is theoretically doubled at each cycle. In fact, the yield of the PCR is less than 100%, so that the amount of product X _n obtained after n cycles is: $${\mathrm{X}}_{\mathrm{not}}\mathrm{=}{\mathrm{X}}_{\mathrm{not}\mathrm{-}\mathrm{1}}\left(\mathrm{1}\mathrm{+}{\mathrm{r}}_{\mathrm{not}}\right)\mathrm{,}$$

or
X _n-1 is the amount of product obtained in the previous cycle, and _n the yield of the PCR at cycle n (0 <r _n ≤ 1).

Considering the constant yield, that is to say the same for each cycle, the quantity of product X _n obtained after n cycles from an initial quantity X ₀ is then: $${\mathrm{X}}_{\mathrm{not}}\mathrm{=}{\mathrm{X}}_0{\left(\mathrm{1}\mathrm{+}\mathrm{r}\right)}^{\mathrm{not}}$$

In reality, the yield r decreases during the PCR reaction, because of several factors, such as an amount mimicking at least one of the reagents necessary for the amplification, the inactivation of the polymerase by repeated passages. at 95 ° C, or its inhibition by the pyrophosphates produced by the reaction.

Because of this decrease in yield, the kinetics of a PCR reaction first exhibit an exponential phase (as long as r is constant), which then progresses to a plateau phase as r decreases.

During the exponential phase, equation (A) above is valid, and can also be written: $$\log \left({\mathrm{X}}_{\mathrm{not}}\right)\mathrm{=}\log \left({\mathrm{X}}_{\mathrm{0}}\right)\mathrm{+}\mathrm{n\; log}\left(\mathrm{1}\mathrm{+}\mathrm{r}\right)$$

Thus, in the exponential phase of the PCR, the curve presenting the quantity of product, in logarithmic scale, as a function of the number of cycles, and a slope line (1 + r) and which intersects the y-axis with a value equal to the logarithm of the initial concentration.

The real-time measurement of the quantity of product obtained can therefore make it possible to know the initial matrix concentration, which is particularly useful in a large number of applications, for example for measuring the viral load of a patient, or again to know the variability of a transcriptome.

Generally, the PCRs involve reaction volumes ranging from 2 to 50 μl and are carried out in tubes, microtubes, capillaries or systems known to those skilled in the art under the term "microplates" (in fact sets of micro tubes secured). Each batch of tubes or equivalent containers must be successively brought to the three temperatures corresponding to the different phases of the PCR, and as many times as desired cycles.

The use of tubes or systems approaching it requires the user to perform multiple manipulations to prepare as many tubes and solutions (known to those skilled in the art as "PCR mix") that target sequences that it wishes to amplify even from a single sample of nucleic acids, with the exception of "multiplex" amplification methods, which allow the amplification of several target sequences simultaneously in the same container, either by the use of so-called unspecific primers which can hybridize with several target sequences, for example the RAPD- Random Amplified Polymorphism DNA technique , or by the use of specific primers but in larger numbers, each pair of primers used to amplify a target sequence. These multiplex amplifications correspond to particular cases and are not the norm. Of moreover, they do not guarantee the absence of interactions of one amplification reaction on another, and for reasons including possible hybridizations between the primers, can only be very limited in the number of target sequences amplified by container.

These different manipulations involve many disadvantages.

In the first place, they are time consuming. Second, they are not without risk from the point of view of possible contamination from one tube to another or from the external environment (dust, bacteria, aerosol or any other contaminant likely to contain nucleic acid molecules or molecules likely to influence the efficiency of the amplification reaction). In addition, they do not ensure homogeneity of volume and concentration of reagents from one tube to another. Finally, they require the use of manually manipulable volumes, generally greater than 1 .mu., which has an impact on the costs associated with carrying out the PCRs, the reagents used being expensive.

The use of devices designed to automate at least partially such manipulations overcomes some of these disadvantages. However, such automata are relatively expensive and their use is therefore generally economically justified only in the case of large-scale PCRs, for example for the sequencing of genomes.

There are also some automata for performing kinetic PCR reactions. As has been seen above, carrying out a kinetic PCR requires quantifying in real time, and specifically, the amplified target sequence. The use of a fluorescent intercalant in the reaction mixture makes it possible to measure the increase in the total amount of double-stranded DNA in said mixture. However, this method does not discriminate amplification of the target sequence with respect to background noise or a possible non-specific amplification. Several probe systems have recently been described to specifically measure the amplification of a specific target sequence. They are based on oligonucleotides complementary to said sequence, and linked to couples of fluorophore or fluorophore / quencher moieties, such that hybridization of the probe to its target and subsequent amplification cycles result, depending on the case an increase or decrease in the total fluorescence of the mixture, in proportion to the amplification of the target sequence.

Examples of useful probes for performing kinetic PCR include the TaqMan ^™ system (ABI ^®), the AmpliSensor ^™ system (InGen) and the Sunrise ^™ system (Oncor ^{®, ®} Appligene).

The most used system today is the Taq Man ^{™ system} .

This method combines the DNA polymerase and 5 '→ 3' nuclease activities of Taq polymerase during PCR. Its principle is as follows: in addition to the two primers of sequence complementary to that of the target to be quantified, a probe, called probe reporter, is added in the reaction medium. It has the ability to hybridize to the target in the body of the amplified sequence, but can not be amplified itself. Indeed, a phosphoryl group added to the 3 'end of the probe prevents its extension by Taq polymerase. A fluorescein derivative and a rhodamine derivative are incorporated into the probe at the 5 'and 3' ends, respectively. The probe is small, so the rhodamine derivative, located near the fluorescein, absorbs the energy emitted by the fluorescein subjected to a source of excitation ( quenching phenomenon).

Once the primers hybridized to the target, during the extension reaction, the Taq DNA polymerase attacks the probe by its 5 'activity. nuclease, releasing the quencher group and thereby restoring the fluorescence emission. The intensity of the fluorescence emitted is then proportional to the quantity of PCR products formed, which makes it possible to obtain a quantitative result. The fluorescence emitted is proportional to the number of starting target molecules. The kinetics of fluorescence development can be monitored in real time during the amplification reaction.

This technique has the advantage of being easily automatable. An apparatus for carrying out this technique, the ABI Prism 7700 ^™ , is marketed by Perkin-Elmer. This device combines a thermocycler and a fluorimeter. It is able to detect the increase in fluorescence generated during a quantification test according to the TaqMan ^™ process, thanks to optical fibers located below each tube and connected to a CCD camera that detects, in real time , the signal emitted by the fluorescent groups released during the PCR. The quantitative data are derived from the determination of the cycle at which the signal of the amplification product reaches a certain threshold determined by the user. Several studies have shown that this number of cycles is proportional to the amount of initial material (Gibson, Heid et al., 1996, Heid, Stevens et al., 1996, Williams, Giles et al., 1998).

The number of potential applications of such a device is considerable, both in human health, agri-food and quality control. Unfortunately, the ABI Prism 7700 ^™ and the few other competing devices currently on the market are extremely expensive, and they can only be used by a skilled manipulator. In practice, such devices are therefore used only in some very specialized structures.

There is therefore today a real need for a nucleic acid amplification system, if necessary measured in real time, and which does not have the drawbacks mentioned above of the state of the art.

The objective of the present invention is to propose such a system which makes it possible to considerably reduce the number of manipulations required to implement an amplification method on a plurality of target sequences and, consequently, to reduce the time required. necessary for this operation.

Another object of the present invention is to provide such a system which minimizes the risk of contamination from one container to another.

Another objective of the present invention is to propose such a system which reduces the volumes of reagents involved and therefore the costs.

Another objective of the present invention is to propose such a system which optimizes a homogeneous distribution in volume and in concentration of the reagents necessary for the PCR in the containers.

Another objective is to provide all potential users, including hospitals, medical laboratories, agribusiness and health control laboratories, an easy-to-use and easy-to-use device to routine quantified nucleic acid amplifications in real time.

• Une "réaction d'amplification d'acides nucléiques" fait référence à n'importe quelle méthode d'amplification d'acides nucléiques connue de l'homme du métier. On peut citer, à titre d'exemples et de façon non restrictive, l'Amplification en Chaîne par Polymérase (ACP), plus souvent désignée par l'homme du métier sous son acronyme anglophone, PCR (Polymerase Chain Reaction), la TMA (transcription mediated amplification), la NASBA (nucleic acid sequence base amplification), la 3SR (self sustained sequence replication), l'amplification par déplacement de brin, ou SDA (strand displacement amplifîcation) et la LCR (ligase chain reaction).
  La matrice initiale de l'amplification peut être n'importe quel type d'acide nucléique, ADN ou ARN, génomique, plasmidique, recombinant, ADNc, ARNm, ARN ribosomal, ARN viral ou autre. Lorsque la matrice initiale est un ARN, une première étape de transcription réverse est en général réalisée pour obtenir une matrice ADN. Cette étape ne sera en général pas mentionnée dans ce texte, car l'homme du métier sait exactement quand et comment la réaliser. Il est bien entendu que les dispositifs de l'invention sont utilisables pour amplifier et éventuellement quantifier spécifiquement des séquences d'ARN aussi bien que d'ADN. Dans la suite du texte, le terme "PCR" sera donc le terme générique utilisé pour désigner aussi bien la PCR proprement dite que la RT-PCR (Reverse Ttranscription - Polymerase Chain Réaction).
• Parmi les réactions d'amplification citées ci-dessus, certaines sont isothermes. D'autres, notamment la PCR et la LCR, impliquent de porter le mélange réactionnel à différentes températures au cours du temps, de façon cyclique. De telles réactions sont appelées ici "réactions d'amplification d'acides nucléiques thermo-dépendantes". Dans la suite de ce texte, le dispositif de l'invention sera principalement décrit dans son application à la PCR. Toutefois, il est bien évident que ce dispositif n'est pas limité à cette technique, et qu'il peut être utilisé aussi pour n'importe quelle réaction d'amplification d'acides nucléiques, voire pour d'autres réactions enzymatiques et/ou de biologie moléculaire. Ce dispositif est particulièrement adapté aux réactions qui nécessitent des volumes faibles et le passage cyclique du mélange réactionnel à plusieurs températures, comme cela apparaîtra clairement dans la suite.
• Un des objectifs de la présente invention est de fournir un nouveau dispositif pour effectuer des réactions d'amplification dites "quantitatives", c'est-à-dire permettant de déterminer la concentration de séquence cible initialement présente dans le mélange réactionnel. Plusieurs types de réactions d'amplification quantitatives ont été décrits. On peut distinguer les amplifications quantitatives basées sur l'emploi d'un standard externe, les amplifications compétitives, utilisant un standard interne, et enfin les amplifications cinétiques, dont le principe a été évoqué plus haut, et qui consistent à mesurer en temps réel, l'augmentation de la quantité de la séquence cible. Ce type d'amplification sera désigné ici indifféremment sous les termes "amplification cinétique (d'acides nucléiques)" "PCR cinétique", "amplification (d'acides nucléiques) quantifiée en temps réel", ou encore "PCR en temps réel". Les termes entre parenthèses sont parfois omis.
• Dans cette demande, le terme "réactif' doit être compris au sens large, comme désignant n'importe quel élément nécessaire soit à la réaction d'amplification proprement dite, soit à sa détection. Suivant cette définition, les sels, les dNTP, les amorces ou encore la polymérase, sont des réactifs nécessaires à la PCR. De même, un intercalant fluorescent, ou une sonde, sont ici considérés comme des réactifs participant à la détection des produits amplifiés, bien qu'ils ne réagissent pas au sens littéral.

In this application, several terms are used, the meaning of which is as follows:

• A "nucleic acid amplification reaction" refers to any method of nucleic acid amplification known to those skilled in the art. Nonlimiting examples include Polymerase Chain Amplification (PCR), plus frequently designated by those skilled in the art under its acronym, PCR ( Polymerase Chain Reaction ), TMA ( transcription mediated amplification), NASBA ( nucleic acid sequence base amplification ), 3SR ( self-sustained sequence replication ), amplification by strand displacement, or SDA ( strand displacement amplification ) and LCR ( ligase chain reaction ).
  The initial template of the amplification can be any type of nucleic acid, DNA or RNA, genomic, plasmidic, recombinant, cDNA, mRNA, ribosomal RNA, viral RNA or other. When the initial template is an RNA, a first reverse transcription step is generally performed to obtain a DNA template. This step will generally not be mentioned in this text, because the skilled person knows exactly when and how to achieve it. It is understood that the devices of the invention are useful for amplifying and optionally quantifying specifically RNA sequences as well as DNA. In the remainder of the text, the term "PCR" will therefore be the generic term used to designate both the actual PCR and the RT-PCR ( Reverse Transcription - Polymerase Chain Reaction ).
• Among the amplification reactions mentioned above, some are isothermal. Others, including PCR and LCR, involve carrying the reaction mixture at different temperatures over time, cyclically. Such reactions are referred to herein as "thermo-dependent nucleic acid amplification reactions". In the rest of this text, the device of the invention will be mainly described in its application to PCR. However, it is obvious that this device is not limited to this technique, and that it can also be used for any nucleic acid amplification reaction, or even for other enzymatic reactions and / or molecular biology. This device is particularly suitable for reactions that require low volumes and cycling the reaction mixture at several temperatures, as will become clear later.
• One of the objectives of the present invention is to provide a new device for performing so-called "quantitative" amplification reactions, that is to say, for determining the target sequence concentration initially present in the reaction mixture. Several types of quantitative amplification reactions have been described. We can distinguish the quantitative amplifications based on the use of an external standard, the competitive amplifications, using an internal standard, and finally the kinetic amplifications, the principle of which has been mentioned above, and which consist in measuring in real time, increasing the amount of the target sequence. This type of amplification will be referred to herein as "kinetic amplification (of nucleic acids)""kineticPCR","amplification (of nucleic acids) quantized in real time", or "real-time PCR". The terms in parentheses are sometimes omitted.
• In this application, the term "reagent" should be understood in a broad sense to mean any element necessary for either the amplification reaction itself or for its detection. The primers or the polymerase are necessary reagents for PCR, and a fluorescent intercalator, or a probe, are considered as reagents involved in the detection of the amplified products, although they do not react in the literal sense.

Other terms designating certain elements of the device of the invention will be defined later in the detailed description of invention.

• la figure 1 représente une vue latérale d'une réalisation simplifiée du dispositif selon la présente invention ;
• la figure 2 représente une vue supérieure de la platine chauffante, dans le cas où les blocs (21 à 23) sont des secteurs de disque (figure 2A), et dans le cas où ils sont constitués de secteurs de couronne (figure 2B);
• la figure 3 représente une vue en perspective d'un premier exemple de réalisation de la cartouche (1), pourvue des chambres réactionnelles et d'une partie des moyens de déplacement ;
• la figure 4 représente une vue en coupe de cette cartouche en accord partiel avec l'invention selon la ligne AA ;
• la figure 5 représente une vue supérieure de la partie inférieure (socle) d'un deuxième mode de réalisation particulier de la cartouche selon l'invention. Les cotes sont données à titre purement indicatif, et ne sont en aucun cas limitatives;
• la figure 6 représente une vue en coupe de cette cartouche inférieure, selon la ligne AA de la figure 5 ;
• la figure 7 représente une vue supérieure de la partie supérieure (couverte) de la cartouche représentée aux figures 5 et 6 ;
• la figure 8 représente une vue en coupe de cette cartouche supérieure, suivant la ligne BB de la figure 7 ;
• la figure 9 représente une cartouche complète, constituée du socle représenté aux figures 5 et 6 (traits pleins), et du couvercle représenté aux figures 7 et 8 (traits discontinus) ;
• la figure 10 montre trois modélisations de la cartouche de la figure 9, au-dessus de laquelle se trouvent les moyens d'excitation/mesure de la fluorescence (5) ;
• la figure 11 montre une coupe schématique d'un canal (12) possédant un dispositif de "perte de charge".

Some elements of the device are numbered with reference to the drawings, which illustrate some non-limiting embodiments and variants of the invention, and in which:

• the figure 1 is a side view of a simplified embodiment of the device according to the present invention;
• the figure 2 represents an upper view of the heating plate, in the case where the blocks (21 to 23) are disk sectors ( Figure 2A ), and in the case where they consist of crown sectors ( Figure 2B );
• the figure 3 is a perspective view of a first embodiment of the cartridge (1), provided with the reaction chambers and a portion of the displacement means;
• the figure 4 represents a sectional view of this cartridge in partial agreement with the invention along line AA;
• the figure 5 represents an upper view of the lower part (base) of a second particular embodiment of the cartridge according to the invention. The ratings are given for information only, and are in no way limiting;
• the figure 6 represents a sectional view of this lower cartridge, according to the line AA of the figure 5 ;
• the figure 7 represents an upper view of the upper (covered) part of the cartridge shown in Figures 5 and 6 ;
• the figure 8 represents a sectional view of this upper cartridge, along the line BB of the figure 7 ;
• the figure 9 represents a complete cartridge, consisting of the base shown in Figures 5 and 6 (solid lines), and the lid shown in Figures 7 and 8 (discontinuous lines);
• the figure 10 shows three modelizations of the cartridge of the figure 9 above which are the means for exciting / measuring the fluorescence (5);
• the figure 11 shows a schematic section of a channel (12) having a "pressure drop" device.

The invention relates firstly to any device according to one of claims 1 to 16.

The temperature of each zone of the platen can be homogeneous or, where appropriate, this temperature can vary according to a gradient.

Several types of molecular biology reactions require placing the reaction mixture at different temperatures as a function of time. This is the case, for example, when it is desired to inactivate an enzyme after using it (for example, a restriction nuclease), or to test the stability of a complex. In the latter case, it is conceivable to place a complex (for example, an antigen / antibody complex, or receptor / ligand), one of which is coupled to a fluorophore and the other to a quencher of fluorescence, one of the reaction chambers of the device. The stage is then programmed to present several temperatures in increasing order, possibly in the form of a gradient. The stability of the complex is then tested by moving the cartridge on the plate, so that the temperature of the reaction chamber rises gradually, and observing the increase in fluorescence, using excitation means measurement of the fluorescence placed opposite the reaction chamber. The increase in fluorescence then reflects the dissociation of the complex.

The device of the invention is particularly suitable for reactions requiring a cyclic variation in the temperature of the reaction chambers, which is the case for certain nucleic acid amplification reactions, for example for polymerase chain reaction ( PCR ), or for the ligase chain reaction (LCR).

The invention therefore relates in particular to a device for the thermo-dependent chain amplification of target nucleic acid sequences according to one of claims 1 to 16.

Such a system according to the invention is less complex than the systems of the prior art, insofar as the temperatures required for the cycles of the chain amplification are provided by distinct zones of constant temperatures and not by a plate of which one must vary the temperature.

It is important to note that thermo-dependent chain amplification reactions require the passage of samples at at least two temperatures. For example, the CSF requires at each cycle a phase at about 95 ° C to denature the target DNA, then a phase between 55 and 65 ° C (depending on the Tm probes) to give rise to hybridization / ligation . With regard to PCR, each cycle generally breaks down into three phases, namely denaturation at about 95 ° C, hybridization whose temperature depends on the Tm of the probes, and elongation, usually performed at 72 ° C. . However, it is possible to perform PCRs with simplified cycles, in which the hybridization and elongation are at the same temperature, so that each cycle requires only two different temperatures.

• des amorces spécifiques de séquences cibles à amplifier sont pré-réparties dans les chambres réactionnelles (13),
• le réservoir (11) est destiné à recevoir un fluide composé notamment d'un échantillon d'acides nucléiques à analyser et des réactifs nécessaires à une réaction d'amplification en chaîne par polymérase, à l'exception des amorces,
• la platine de chauffage (2) présente trois zones distinctes pouvant être portées à trois températures différentes, correspondant aux trois phases des cycles d'amplification en chaîne par polymérase.

It may be considered different variants of the device described above. According to a preferred variant of the invention, the system comprises the following characteristics:

• primers specific for target sequences to be amplified are pre-distributed in the reaction chambers (13),
• the reservoir (11) is intended to receive a fluid composed in particular of a sample of nucleic acids to be analyzed and reagents necessary for a polymerase chain reaction reaction, with the exception of the primers,
• the heating stage (2) has three distinct zones which can be raised to three different temperatures, corresponding to the three phases of the polymerase chain amplification cycles.

According to this preferred embodiment, it is possible to dispense, from a reservoir, a fluid containing a sample of nucleic acids to be analyzed and the reagents necessary for the PCR in a plurality of reaction chambers containing specific primers of target sequences. nucleic acids to be amplified, and to allow the amplification process by subjecting the contents of the chambers successively to different temperatures (namely those necessary for denaturation, hybridization and elongation) a multitude of times through a movement relative between the cartridge including said reaction chambers and said heating platen having two or three distinct zones that can be brought to different temperatures.

If desired, the reaction chambers (13) may contain reagents necessary for a real-time PCR reaction other than the primers mentioned above. In a preferred embodiment of the device of the invention, the reaction chambers also comprise, in addition to the primers, one or more specific probe (s) of the sequence to be amplified. The distribution of the probes in the reaction chambers may also be such that some chambers contain probes specific for the sequences to be amplified and other chambers comprise control probes, not recognizing a priori the sequence to be amplified. These probes may be labeled and, if several probes are present in the same reaction chamber (for example a probe specific for the sequence to be amplified and a control probe), these probes will preferably be labeled with different fluorophores.

In another variant of the device, additional reagents, such as dNTPs or salts, are initially deposited in the reaction chambers. These reagents will then be absent, or present in a smaller amount, in the fluid deposited in the reservoir (11). In the extreme case, all the reagents necessary for the PCR reaction, with the exception of the matrix, are deposited in the reaction chambers (13), and the fluid deposited in the reservoir (11) will then comprise only the sample. DNA (or RNA) to amplify.

The variants described above assume that several reactions are carried out in parallel, with different primers and / or probes, on the same sample. It is therefore the characterization of a single sample (or a few samples if the reservoir is divided into a few sub-reservoirs) according to several criteria. In some applications, on the contrary, it is desired to characterize a multitude of samples according to a single criterion or a small number of criteria. This is the case for example in research, when it is desired to screen a library of phages or bacteria for the presence of a given gene. In this case, it is necessary to carry out a PCR on a large number of samples, from a given pair of primers. The device of the invention is also suitable for this type of manipulation. For this, the samples are deposited in the reaction chambers (13). The primers can be introduced into the fluid deposited in the reservoir (11), along with the other reagents necessary for the PCR. Of course, this configuration does not exclude that certain reagents other than the sample to be analyzed are pre-deposited in the reaction chambers (13).

Whatever the variant of the device chosen, and whatever the reagents deposited in the reaction chambers (13), they can be advantageously deposited there by a simple liquid deposit, followed by drying. The arrival of the fluid from the reservoir (11) then allows the re-solution of these reagents. The amount of each reagent deposited is calculated as a function of the volume of fluid that will penetrate into each reaction chamber (13), so that the reactivation of the reagents will result in the desired final concentration for each of them. Cartridges as described above, in which at least a portion of the reaction chambers (13) comprise reagents which have been loaded therein by a liquid deposit, followed by drying, so that these reagents are returned to solution by the arrival of a fluid in these reaction chambers, are also an integral part of the invention.

The device described above has the advantage of allowing a concomitant filling of all the reaction chambers, which reduces the preparation time and the risks of contamination from one chamber to another. This device also has the advantage of being able to be miniaturized and to involve the use of smaller volumes of reagents than in the state of the art.

Finally, it will also be noted that, thanks to the specific heating platinum that it recommends, the invention makes it possible to accelerate PCR cycles, since it is not necessary to perform the various phases (denaturation, hybridization, elongation). to vary the temperature of the heating stage or of the atmosphere as in the state of the art, the relative movement between the cartridge and the plate making it possible to quickly and successively submit the contents of each of the chambers reaction at three distinct temperatures dedicated to each of these phases. The use of small reaction volumes, and a thin floor for the cartridge (1), also limit the thermal inertia at the reaction chambers, and therefore contribute to the speed of the reaction.

The invention also relates to a device for thermo-dependent chain amplification of target nucleic acid sequences, measured in real time, characterized in that it comprises the same elements as any of the devices described above. above, and further comprising optical means (5) for excitation / measurement of fluorescence, arranged to excite and measure at each cycle the fluorescence of the contents of the reaction chambers.

• chaque chambre réactionnelle est reliée au réservoir par un canal (12) ayant une section droite comprise dans un cercle de diamètre inférieur à 3 mm,
• la capacité du réservoir est inférieure à 10 ml,
• la disposition des chambres réactionnelles et des canaux par rapport au réservoir permet de répartir un fluide de façon homogène dans les chambres réactionnelles, à partir du réservoir.

One of the particularly original elements of the devices described above is the element indifferently called plate or reaction cartridge (1). This element may be recyclable or, preferably, consumable, and constitutes in itself an aspect of the present invention. Thus, the invention preferably uses a reaction cartridge comprising a plurality of reaction chambers (13) and at least one reservoir (11) and having the following characteristics:

• each reaction chamber is connected to the reservoir by a channel (12) having a cross-section included in a circle of diameter less than 3 mm,
• the capacity of the tank is less than 10 ml,
• the arrangement of the reaction chambers and the channels with respect to the reservoir makes it possible to distribute a fluid homogeneously in the reaction chambers from the reservoir.

The diameter of the channels will preferably be chosen small enough not to allow a gravity distribution of the fluid present in the tank in the reaction chambers, so as to avoid non-reproducible filling of these chambers. This diameter will thus preferably be less than or equal to approximately 0.2 mm. With regard to this diameter, it will be noted that the section of the channels will preferably be circular but that it may also be of any other shape and in particular polygonal, the "diameter" of the channels then aiming at their greatest width in section.

The reservoir intended to receive the sample of nucleic acids and the reagents necessary for the PCR may have a variable capacity, for example between about 0.1 ml and about 1 ml.

The cartridge preferably comprises between about 20 and about 500 reaction chambers and, more preferably, between 60 and 100 reaction chambers.

The volume of these rooms may also vary according to the embodiments. Advantageously, these chambers have a volume of between approximately 0.2 and 50 μl, preferably between 1 μl and 10 μl.

In the cartridges, the junction between the channels (12) and the reservoir (11) is at the periphery of the reservoir, and the bottom of said reservoir is inclined and / or convex, so as to ensure the distribution of a fluid contained in the tank at the entrance of the channels.

It will be noted that a cartridge used according to the invention may have multiple forms. However, according to a preferred variant of the invention, this cartridge has a circular shape. The reservoir is provided substantially in the center of the cartridge, the reaction chambers being distributed in a circle around the reservoir, and the channels connecting the reservoir to the chambers being provided essentially radially. Such an architecture allows to optimize the filling of the reaction chambers from the central tank.

In a particular embodiment of the circular cartridges of the invention, the bottom of the tank (11) is conical.

Also preferably, said reaction chambers are provided relative to the periphery of said cartridge. Thus, it is possible to optimize the number of reaction chambers that can be provided on the cartridge and filled from the central tank.

According to one variant of the invention, such a cartridge comprises as many channels as there are reaction chambers. However, in some embodiments, it will be possible to provide channel sections common to several reaction chambers.

One of the advantages of the present invention is to allow a miniaturization of the device that it proposes. Thus, advantageously, the cartridge preferably has a diameter of between about 1 and 10 cm.

A variant of the cartridges of the invention described above, whatever their geometry, consists in dividing the reservoir (11) into 2 to 20, preferably 2 to 8 sub-reservoirs, making it possible to simultaneously analyze several samples on a same cartridge. In this case, each of the reaction chambers (13) is connected to only one of these sub-tanks by a channel (12).

The depth of the reaction chambers (with respect to the channels) may also vary depending on the embodiments of the invention. According to a preferred variant, these chambers have a depth of between approximately 0.5 mm and 1.5 mm.

Note also that the thickness of the cartridge depends on several factors including the material constituting it. In practice, this cartridge is preferably made of plastic material, preferably polycarbonate, whose physical, optical and thermal properties are suitable for carrying out the present invention. The thickness of the cartridges of the invention is preferably between 0.5 and 5 mm.

In order to facilitate the heat exchange between the contents of the reaction chambers and the platinum, the thickness of the "floor" thereof will preferably be as low as possible. This thickness depends on the material used to make the cartridge. Preferably, it is between 0.05 and 0.5 mm, for example about 0.25 mm.

The reaction chambers of the cartridges of the invention are preferably closed by a transparent upper wall (17), for example of transparent plastic, in order to allow the excitation and the measurement of the fluorescence of the reaction fluid, in good conditions.

In a particular embodiment of the invention, the chambers are provided with vents (open system), allowing the air they contain to escape during their filling by the fluid from the tank.

In the above case, where the chambers (13) are provided with vents (14), the channels (12) preferably consist of at least two parts of different diameters (121 and 122), the diameter of the second part (122) being smaller than that of the first part (121), so as to create a pressure drop in the channel (12). Thus, if one channel fills faster than another under the effect of pressure, the pressure drop phenomenon stops the progression of the fluid in the channel or channels whose first portion (121) is filled, until all the channels are filled in the same way. This allows to "pre-calibrate" the volumes for each channel, to ensure a homogeneous filling of the different reaction chambers. The second portion of the channel (122) may consist for example of a glass capillary, much smaller in diameter than the first portion (121), said capillary being included in a plastic cartridge.

It is also possible to provide enclosures (15) into which open the vents (14) of the reaction chambers. These enclosures have an opening (16) towards the outside of the cartridge (open system), and have the advantage, on the one hand, of recovering, without pollution, any excess fluid that would come out of the reaction chambers through the vents (14). ) and, on the other hand, to be able to close after the filling of the reaction chambers. This closure can be done for example in using an adhesive tape, and allows to go into closed system to perform the amplification itself. This makes it possible to avoid or at least limit the evaporation of the fluid contained in the cartridge (1). This embodiment of the invention is illustrated in figure 11 .

Alternatively, it is possible to work in a closed system as soon as the reaction chambers are filled, causing the cartridge to depressurise followed by a reestablishment of the pressure, as will be detailed below. Cartridges in which the reaction chambers have no other opening than the arrival of the channel (12) (so-called "closed" reaction chambers) are also part of the invention.

The cartridges described above, provided for use in an open system or for use in a closed system, preferably comprise an opening that can be adapted to means (4) for modulating the pressure in the reservoir (11), making it possible to moving the fluid present in the reservoir to the reaction chambers.

• emplir au moins partiellement le réservoir (11) avec un fluide,
• connecter la cartouche (1) aux moyens (4) de modulation de la pression,
• appliquer une dépression à l'intérieur de la cartouche, puis rétablir la pression.

The invention also relates to a closed system filling process of the reaction chambers (13) of a cartridge (1) as described in the previous paragraph, in the variant where the reaction chambers are closed, which method comprises the following steps:

• at least partially filling the reservoir (11) with a fluid,
• connect the cartridge (1) to the means (4) for modulating the pressure,
• apply a vacuum inside the cartridge, then restore the pressure.

In a variant of the cartridges of the invention, each channel (12) is equipped with an anti-reflux cavity (123) at its junction with the reservoir (11), said anti-reflux cavity consisting of a substantially vertical channel portion of a diameter greater than or equal to that of the channel (12). This variant has two main advantages. On the one hand, these anti-reflux cavities make it possible to prevent cross-contamination in the event of an inadvertent return of fluid to the reservoir (11), or in the case where all the fluid has not been engaged in the channels. On the other hand, these cavities make it possible to provide, in the devices of the invention, a plug whose serrations come to marry these vertical inlets, in order to plug the channels after the addressing of the editorial fluid but before the amplification reaction. This allows to work in perfectly closed system, and thus to avoid any risk of contamination and evaporation. However, it is important to note that the anti-reflux cavities, and the use of a plug at the reservoir to plug the entrance of the channels on the tank side, can be used also in the case of open systems such as described above, where the reaction chambers are provided with vents.

In a preferred embodiment of the cartridges of the invention, at least a portion of the reaction chambers (13) comprise oligonucleotides. More preferably, each of the reaction chambers (13) comprises two primers specific for a nucleic acid sequence to be amplified and, optionally, one or more labeled probe (s) specific for said sequence. Such a probe may be labeled so that its signal is increased when it hybridizes to its target sequence (Sunrise ^™ system), or so that elongation from a strand on which it is hybridized causes a decrease or increase in the signal (AmpliSensor ^™ system or TaqMan ^™ system, respectively). The presence of such probes in the reaction chambers makes it possible to carry out quantized amplifications in real time, with a device of the invention having means (5) for excitation / measurement of the fluorescence, as described above. Control probes, nonspecific of the sequence to be amplified, and labeled in a different way from the specific probes, can also be used to detect possible contaminations.

In the embodiment of the invention described above, where the reaction chambers comprise primers and, optionally one or more probe (s), these different probes and primers will preferably be chosen such that their melting temperatures (Tm) respective ones are close. In particular, the Tm of the different primers will preferably be in the same range of about 5 ° C. Similarly, the different probes will preferably have a Tm within the same range of 5 ° C, which may be different from the range of Tm primers. In this case, the probes will be chosen so that their Tm is greater than that of the primers, the difference between Tm of the different categories of oligonucleotides then being preferably of the order of 5 ° C. The hybridization temperature chosen to carry out the amplification then corresponds to the lowest of the melting temperatures of the primers.

The reaction chambers (13) of the cartridges of the invention may also comprise, in addition to the primers and any probes, one or more other reagents necessary for the PCR reaction or measurement of the amplification. It may be, for example, salts, dNTPs, or a fluorescent interlayer of double-stranded DNA, SybrGreen type (trademark). As mentioned above, all these reagents are advantageously deposited at the level of the reaction chambers (13) by the deposition of a liquid solution, followed by drying.

According to an alternative embodiment of the cartridges of the invention, the cartridges are intended for screening a large number of samples according to a small number of criteria. This implies that the user of these cartridges can easily deposit his samples in each of the reaction chambers (13). For this, the cartridge can for example have a removable cover which, when removed, gives direct access to the reaction chambers. Such cartridges may also be pre-charged and comprise, in the reaction chambers (13), one or more reagents necessary for amplification and / or detection.

Of course, the devices of the invention mentioned above may comprise one or more cartridges corresponding to any of the cartridges described above.

In the particular embodiment of the device of the invention, where the cartridge is circular, the distinct heating zones of the heating plate (2) are preferentially distributed according to disk portions ( Figure 2A ) or crown ( Figure 2B ). Each portion may be heated to a different temperature to successively bring the contents of the reaction chambers to the desired different temperatures, by means of relative displacement means (3) between the cartridge (1) and the heating stage (2). In order to limit the problems of evaporation and condensation in the cartridge (1), the thermoblocks are preferably sufficiently wide to heat also part of the channels, as represented for example in FIG. figure 11 , as part of a rectangular cartridge.

It is important to note that the number of separate heating zones can be two, three, or more. For example, in the case of two-temperature PCR, platinum may have a 95 ° C zone for denaturation of double-stranded nucleic acids, and a 60 ° C region for primer hybridization and elongation. . In the case of a three-temperature PCR, the platinum will have an area at 95 ° C (denaturation), a region between 40 and 70 ° C (primer hybridization), and an area at 72 ° C (elongation). Finally, the plate may have a number of zones greater than three, for example to temporarily block the reaction at a given moment in each cycle. The platen can also have a number of zones that is a multiple of two or three, so that one revolution of the cartridge corresponds to several PCR cycles. Finally, it is important to note that the relative size of the different heating zones is advantageously chosen proportionally to the desired incubation time for the reaction fluid at the temperature of said zone. So, in the platen represented at a Figure 2B , the thermoblock 21, dedicated to the denaturation step, has a surface twice as small as that of the thermoblocks for the hybridization and elongation steps (blocks 22 and 23, respectively). By choosing a relative speed of rotation of the cartridge on the plate such that a 360 ° rotation is carried out in 150 seconds, cycles are thus obtained in which the denaturation lasts 30 seconds, the hybridization 1 minute and the elongation 1 minute.

With regard to the displacement means, it will be noted that, according to a preferred embodiment of the invention, the plate (2) is fixed and the cartridge (1) is moved by means of displacement (3).

However, in other embodiments, it will also be possible to provide a fixed cartridge and a heating plate set in motion by means of displacement means.

In the particularly preferred embodiment of the invention, wherein the cartridge is circular, the displacement means (3) allow the rotation of said cartridge and / or said platen.

A conductive element can be provided between the cartridge and the heating plate. However, according to a preferred embodiment of the invention, said cartridge is in direct contact with said heating stage. In this case, said plate is advantageously provided with a coating promoting displacement between said cartridge and said plate. Such a coating may for example be made of Teflon (registered trademark).

As indicated above, the system heating plate may have at least two or three zones that can be raised to different temperatures. Preferably, this plate consists of two or three independent thermal blocks ("thermoblocks") connected to separate means for programming their temperature. In the case where the plate comprises three thermoblocks (21 to 23), the first of these thermoblocks (21) is heated to the denaturation temperature, the second (22) to the hybridization temperature, the third (23) to the elongation temperature. The use of such thermoblocks constant temperature simple realization of the heating stage.

The relative displacement means of the cartridge relative to the platen can be realized in multiple forms. According to a preferred embodiment, illustrated in FIG. figure 10 , the cartridge (1) has on the underside a central projection (181) having a notch (182), so that the projecting portion (181) fits into the heating stage (2) and connects the cartridge (1). ) with the displacement means (3) at a cleat or shaft (32) set in motion by a micromotor (31). The protruding part (181) thus makes it possible, on the one hand, to position the cartridge with respect to a plate (2) such as that represented in FIG. Figure 2B and, secondly, to ensure its connection with the moving means (3).

According to an alternative embodiment, shown in figures 1 and 3 , the cartridge has at least one lug (183) and the displacement means (3) include at least one axis (32) cooperating with said lug to instill in said cartridge a rotary movement.

The relative mode of movement between the plate and the cartridge may vary according to the embodiments. It may be a movement at continuous speed or in jerks. The speed of movement may be constant or vary over time.

Advantageously, the system according to the invention also comprises optical means of excitation / fluorescence measurement, provided for example above or on the side of said cartridge. According to a preferred variant of the invention, these means will constitute a single and fixed system. An advantage of a preferred variant of the invention according to which the cartridge is circular and moved in a rotary displacement is to be able to successively bring each reaction chamber under said optical system, thus reducing its complexity. A tracking system, located for example on the cartridge (1), makes it possible to determine at each instant which reaction chamber is located opposite the optical system.

The means for supplying the fluid present in said reservoir to said reaction chambers can be made in different forms. As has been described above, two categories of modes of addressing the fluid towards the reaction chambers can be distinguished: open system addressing, which assumes an increase in pressure at the reservoir and the presence of vents (14). ) at the level of the reaction chambers, and the addressing in closed system, which begins on the contrary by the establishment of a vacuum in the cartridge (1), followed by a recovery of this pressure.

The means (4) for supplying the fluid into the reaction chambers differ according to the embodiment chosen. Thus, in an open system, the fluid contained in the reservoir is distributed under pressure in the reaction chambers so as to allow uniform filling of these chambers. In this case, the feed means (4) preferably include a piston device (41) whose penetration rate in the tank will be calculated-to promote the proper filling of the reaction chambers, alternatively, these supply means include a pump connected to increase the pressure in the tank (11).

As has been seen above, another preferred variant of the invention involves working in a closed system. The fluid contained in the reservoir is then distributed in the reaction chambers as follows: in a first step, a vacuum is created inside the cartridge, where appropriate by a piston device or a pump (42), connected this time to reduce the pressure in the cartridge (1). The pressure is then restored, allowing the fluid to engage the channels and fill the peripheral reaction chambers.

• remplir au moins partiellement le réservoir (11) avec un fluide contenant un échantillon d'acides nucléiques à analyser ainsi que tout le nécessaire à une réaction d'amplification, à l'exception des amorces, et facultativement, un intercalant fluorescent des acides nucléiques ;
• répartir ledit fluide dans les chambres réactionnelles (13) prévues dans la cartouche (1), dans lesquelles sont pré-réparties des amorces et, facultativement, une ou plusieurs sondes marquées spécifiques de la séquence nucléique cible;
• mettre en oeuvre les moyens de déplacement relatif entre la cartouche et la platine chauffante pour amener successivement, et autant de fois que désiré, le contenu de chaque chambre aux températures définies par les deux, trois ou davantage zones de ladite platine de chauffage.

The invention also relates to any method of nucleic acid amplification using a system as described above, characterized in that it comprises the steps of:

• at least partially filling the reservoir (11) with a fluid containing a nucleic acid sample to be analyzed as well as all the necessary for an amplification reaction, with the exception of the primers, and optionally, a fluorescent intercalator of the nucleic acids;
• distributing said fluid in the reaction chambers (13) provided in the cartridge (1), in which primers and, optionally, one or more labeled probes specific for the target nucleic sequence are pre-distributed;
• implementing the relative displacement means between the cartridge and the heating plate to successively bring, as many times as desired, the contents of each chamber to the temperatures defined by the two, three or more zones of said heating plate.

In a variant of the above process, reagents necessary for the amplification reaction and / or the detection of the products of the amplification, and distinct from the primers and probes, are pre-distributed in the reaction chambers (13). of the cartridge (1). The fluid introduced into the reservoir (11) does not then contain these reagents.

The fluid distribution step in the reaction chambers (13) is carried out either by applying a vacuum inside the cartridge, then by restoring the pressure (closed system), or by increasing the pressure at the reservoir (11). ), provided that the reaction chambers are provided with vents (open system).

The invention, as well as the various advantages that it presents, will be better understood thanks to the following description of some non-limiting embodiments thereof, illustrated in the figures.

Example 1 : Simplified embodiment of the device of the invention.

The system for detecting and quantifying target nucleic sequences represented in figure 1 comprises a circular plastic cartridge 2 mm thick having a diameter of 5 cm. This cartridge (1) is provided with a central reservoir (11) and will be described in more detail with reference hereinafter to Figures 3 and 4 . The capacity of the reservoir is, in the context of the present embodiment, 400 .mu.l. Its floor is flat but it will be noted that in other embodiments it may be curved to facilitate the passage of fluid to the chambers without the formation of air bubbles, especially at the end of addressing when the reservoir is almost empty.

The system further comprises a heating plate (2) in direct contact with the underside of the cartridge (1) and means (3) for moving the cartridge (1) relative to the heating plate (2). These displacement means include a micromotor (31) connected to two axes (32) which cooperate with two lugs (183) of the cartridge (1) to inculcate in it a rotary movement on the heating plate (2), the latter while remaining fixed.

The described system also comprises a piston (41) intended to cooperate with said reservoir (11) as well as an optical device (5) for excitation / fluorescence measurement (emitting source allowing excitation at a given, programmable wavelength). and fluorescence receiver emitted) fixed and placed above the cartridge (1) and the heating plate (2).

As can be seen on the Figure 2A , the heating plate (2) consists of three metal blocks (21, 22 and 23) (hereinafter referred to as thermoblocks) in the form of disk portions. Note that in this embodiment, these thermoblocks have substantially the same size but that, in other embodiments, they may have a different size, the size being understood as the occupied angular surface in top view. Each thermoblock (21, 22 and 23) is designed to be brought to a constant and programmable temperature, corresponding to one of the phases (denaturation, hybridization or elongation) amplification cycles (PCR), generally respectively 94 ° C for denaturation, 72 ° C for elongation, and between 30-40 and 65-70 ° C for hybridization according to the Tm (hybridization temperature) of the primers used, the temperatures of the thermoblocks can be controlled by all means known to those skilled in the art.

With reference to the figure 3 , the cartridge (1) is provided with a central tank (11) of capacity 400 μl connected to 36 reaction chambers (13) by as many channels (12), distributed uniformly over the entire periphery of the cartridge (on the figure 3 , we did not represent all the channels and rooms but only some of them). These reaction chambers (13) are further provided with vents (14) abutting on the edge of the cartridge (1). In the present embodiment, the channels have a diameter of 0.2 mm and the volume of the reaction chambers is 2.5 microliters. In other embodiments, this diameter and this volume may of course be different.

As already stated, this cartridge (1) is also provided with two lugs (183) each pierced with an orifice for passing an axis (32) connected to the micromotor (31).

According to figure 4 the reaction chambers have a depth of 1 mm. Their floor has a thickness of about 0.2 mm. This thickness is sufficiently small to facilitate good heat exchange between the chambers (13) and the thermoblocks (21, 22 and 23). The reaction chambers (13) are closed in their upper part by a wall (17) transparent, also forming the wall of the tank (11).

• Le réservoir central (11) est destiné à recevoir l'échantillon d'acides nucléiques à analyser ainsi que tout le nécessaire à une réaction d'amplification, et facultativement un intercalant fluorescent des acides nucléiques (l'ensemble est ci-après dénommé fluide), à l'exception des amorces préréparties dans chaque chambre réactionnelle périphérique 10.

The use of the device shown is as follows:

• The central reservoir (11) is intended to receive the sample of nucleic acids to be analyzed as well as all the necessary for an amplification reaction, and optionally a fluorescent intercalant of the nucleic acids (the assembly is hereinafter referred to as a fluid). with the exception of pre-distributed primers in each peripheral reaction chamber 10.

• dNTP : 200 µM
• Tampon Taq:1x
• MgCl₂ : 1,5 mM
• Taq : 4U
• SybrGreen (marque déposée) : 1 x
• H₂O : qsp

In the context of the present embodiment, the user places in the central reservoir 90 μl (that is to say 36 times 2.5 μl) of fluid, including 75 ng of nucleic acids. The reagent concentrations of said fluid are as follows:

• dNTP: 200 μM
• Taq buffer: 1x
• MgCl ₂ : 1.5 mM
• Taq: 4U
• SybrGreen (registered trademark): 1 x
• H ₂ O: qs

Each chamber 10, except a few for negative control purposes, contains two primers specific for a target sequence to be amplified, and optionally one or more labeled probes, allowing a subsequent specific measurement of fluorescence. In the present embodiment, 10 ng of each primer were distributed in each chamber except for those serving as a negative control.

After partially filling the reservoir (11) with the fluid whose volume is equal to the sum of the volumes of the chambers (the volume of a chamber is defined as being the product of its "floor" surface by its depth), the piston (41) is actuated to dispense this fluid into the plurality of reaction chambers (13). This piston increases the pressure within the reservoir (11) and allows the passage of fluid in the channels to the chambers. The speed of movement of the piston in the reservoir is about 1 mm per second and said displacement is stopped at a level that depends on the volume of fluid to be addressed in the chambers.

The small diameter of the channels (12) makes it possible to prevent the diffusion of the fluid from the reservoir (11) to the channels (12) and the chambers (13) under the effect of gravity (at this scale, the processes are usually negligible as the capillary forces become pregnant, and in this case are enough to keep the fluid in the tank). Thanks to the vents (14), the air present in the chambers (13) is evacuated, which ensures the filling thereof.

The thermoblocks (21, 22, 23) are brought to the three temperatures corresponding to the three temperatures of the PCR phases (or at slightly higher temperatures given the possible heat losses between the heating stage (2) and the cartridge 1) and the displacement means (3) are implemented so as to animate the cartridge (1) by a gyratory movement in order to pass each reaction chamber successively and as many times as desired over the three thermoblocks.

More precisely, the block (21) is brought to the temperature corresponding to the denaturation phase (94 ° C.), the thermoblock (22) is brought to the temperature corresponding to the hybridization phase (36 ° C.) and the thermoblock (23) is brought to the temperature corresponding to the phase of elongation (72 ° C).

In the present embodiment, the micromotor (31) of the displacement means (3) is adapted to inculcate a rotation of 10 degrees every 2.5 seconds to the cartridge (1) (ie a 1.5 PCR cycle). min). However, in other embodiments, this movement may have a different speed and be continuous instead of jerky.

It will be noted that the optical device (5) is provided above the corresponding block 23 raised to a temperature corresponding to the elongation temperature, and more particularly to a location corresponding to the end of the elongation phase. Of course, the optical device (5) can be placed at a different location, chosen in particular according to the chemistry used. For example, using TaqMan ™ chemistry or nonspecific fluorescence, it makes sense to perform the measurement at the end of the elongation phase, as described above. On the other hand, the use of a Molecular Beacons ™ type chemistry implies that the measurement is done rather at the time of the hybridization.

The system presented makes it possible to rapidly and reproducibly fill a large quantity of reaction chambers and to carry out PCR and fluorescence measurements on each PCR cycle.

The embodiment described here is not intended to reduce the scope of the invention. It can therefore be made many changes without departing from the scope thereof.

Example 2 : Improved Circular Cartridge

The Figures 5 to 10 represent an example of a circular cartridge exhibiting certain modifications with respect to the cartridge of example 1.

This cartridge is intended for use in a closed system, that is to say that the reaction chambers (13) have no other opening than the arrival of the channel (12). The cartridge consists of two elements that fit into each other: the lower part, or base, is represented in Figures 5 and 6 , and the upper part, or cover, is represented in Figures 7 and 8 . The assembly of the two is illustrated in figures 9 and 10 .

This cartridge is loaded as follows:

The user places in the central reservoir the nucleic acid extract to be analyzed. He places the consumable in the automaton. The latter creates a vacuum inside the cartridge (P = 0.05 bar approximately), for example by the use of a pump (42). The pressure is then reestablished, allowing the fluids to engage the channels and fill the peripheral reaction chambers. Thus, compared to the device of Example 1, the fluid is no longer addressed by an increase in pressure but by depression, which presents the advantage of not requiring a vent and therefore working in a closed system.

If necessary, several sub-tanks can be provided and no longer a single tank, which has the advantage of simultaneously processing several samples.

The bottom of the tank has a conical shape allowing the fluid to be distributed at its periphery, that is to say near the entrance of the channels.

At the junction between the channels and the reservoir, there is an anti-reflux system consisting of a vertical channel portion (123), which, on the one hand, prevents cross contamination in the event of inadvertent return of fluid to the central part or in the case where all the fluid would not be engaged in the channel and, on the other hand, allows once the addressing done but before the PCR, to come to plug the channels by means of a plug whose serrations come to marry these vertical entries, to work in closed system (no contamination, no evaporation).

The cartridge is made of plastic, preferably polycarbonate because this polymer has interesting physical, optical and thermal behavior characteristics.

The size of the channels is for example 0.4 x 0.2 mm (half-moon) in section.

The size of the consumable is for example 100 mm (diameter), the number of chambers is 80, the number of sub-tanks is between 1 and 8.

As illustrated in figure 10 , the cartridge (1) has on the underside a central projection (181) having a notch (182), so that the projecting portion (181) fits into the heating stage (2) and connects the cartridge (1). ) with the moving means (3) at a cleat or shaft (32) moved by a micromotor (31). The protruding part (181) thus makes it possible, on the one hand, to position the cartridge with respect to a plate (2) such as that represented in FIG. Figure 2B and, secondly, to ensure its connection with the moving means (3).

The reaction chambers are loaded with primers specific for target sequences and, where appropriate, with TaqMan ^™ or other probes specific for said targets. Depending on the applications, the targets will be viral or bacterial genes, junctions between a transgene and the genome of a plant to detect and / or identify certain GMOs, etc.

• DUTP 400 µM
• dNTP : 200 µM
• Tampon Taq : 1 x
• MgCl₂ : 3 mM
• Taq: 15 U
• TWEEN (marque déposée) : 0,007 %
• SybrGreen (marque déposée) : 0,1 x
• ADN génomique de Salmonella enteritidis : 1 ng
• H₂O: qsp

A variant of the cartridge described above, comprising 36 reaction chambers with a volume of 8 .mu.l and channels with a diameter of 0.3 mm, was used to perform a test for detection of Salmonella bacteria, 288 .mu.l ( 36 times 8 μl) of the following solution were placed in the central tank:

• DUTP 400 μM
• dNTP: 200 μM
• Taq buffer: 1 x
• MgCl ₂ : 3 mM
• Taq: 15 U
• TWEEN (registered trademark): 0.007%
• SybrGreen (registered trademark): 0.1 x
• Genomic DNA of Salmonella enteritidis: 1 ng
• H ₂ O: qs

1.6 picomole FinA1 and FinA2 primers described in Cohen, Mechanda et al. 1996 were deposited in the reaction chambers.

This experiment gave positive results, as expected.

BIBLIOGRAPHY

• Cohen, HJ, SM Mechanda, et al. (1996). "PCR amplification of the fimA gene sequence of Salmonella typhimurium, a specific method for detection of Salmonella spp." Appl Environ Microbiol 62 (12): 4303-8 .
• Gibson, UE, CA Heid, et al. (1996). "A novel method for real time quantitative RT-PCR." Genome Res 6 (10): 995-1001 .
• Heid, CA, J. Stevens, et al. (1996). "Real time quantitative PCR." Genome Res 6 (10): 986-94 .
• Williams, P. M., T. Giles, et al. (1998). "Development and application of real-time quantitative PCR." In F; Ferré (Ed.), Gene Quantification. Birkhäuser, Bost we .

Claims (18)

1. Device for carrying out enzymatic and/or molecular biological reactions requiring at least two different incubation temperatures, comprising at least one cartridge (1) of circular shape comprising a plurality of reaction chambers (13), in which primers are pre-disposed, a reservoir (11) and channels (12), characterized by:

   - this cartridge (1) having a geometry of revolution, in which the reservoir (11) is positioned substantially at the centre, the reaction chambers (13) being distributed in a circle around said reservoir, and the channels (12) connecting said reservoir (11) to said chambers (13), the channels being provided, in particular, essentially radially;

   - at least one heating plate (2) having at least two distinct zones that can be heated to at least two different temperatures,
   in particular, the heating plate having three distinct zones that can be heated to three different temperatures;

   - means (3) for relative displacement between said cartridge and said plate, allowing a cyclic variation of the temperature of the reaction chambers.
2. Device according to claim 1, in which said reaction chambers (13) are placed at the periphery of said cartridge.
3. Device according to claim 1 or 2, in which the enzymatic reaction is a thermodependent chain amplification of nucleic acid sequences, and in which the zones of the heating plate (2) can be heated to at least two different temperatures, corresponding to the phases of the nucleic acid amplification cycles.
4. Device according to claim 3, characterized in that:

   - primers specific for target sequences to be amplified are pre-distributed in the reaction chambers (13),

   - the reservoir (11) is intended to receive a fluid composed, in particular, of a sample of nucleic acids to be analysed and reagents required for a polymerase chain amplification reaction, with the exception of the primers,

   - the heating plate (2) has three distinct zones that can be heated to three different temperatures corresponding to the three phases of the polymerase chain amplification cycles.
5. Device according to claim 3 or 4, for thermodependent chain amplification of nucleic acid sequences, measured in real-time, characterized in that it comprises optical means (5) for fluorescence excitation/measurement, disposed so as to excite and measure the fluorescence of the contents of the reaction chambers for each cycle.
6. Device according to any one of claims 1 to 5, in which the distinct heating zones of the plate (2) are distributed corresponding to at least two or three sections of a disk.
7. Device according to any one of claims 1 to 5, in which the distinct heating zones of the plate (2) are distributed corresponding to sections of a ring.
8. Device according to any one of claims 1 to 7, in which said displacement means (3) allow rotation of said cartridge (1) and/or said heating plate (2).
9. Device according to any one of claims 1 to 8, in which the cartridge (1) is in direct contact with the heating plate (2).
10. Device according to any one of claims 1 to 9, in which the plate (2) is provided with a coating promoting relative displacement between said cartridge (1) and said plate (2).
11. Device according to any one of claims 1 to 10, in which the heating plate (2) comprises two or three distinct thermoblocks (21, 22 and, optionally, 23) connected to means for programming their temperature.
12. Device according to any one of claims 1 to 11, comprising optical means (5) for fluorescence excitation/measurement disposed above or to the side of the cartridge.
13. Device according to any one of claims 1 to 12, further comprising means (4) for supplying the fluid present in the reservoir (11) to the reaction chambers (13).
14. Device according to claim 13, in which said supply means (4) include a piston device (41), and the fluid is supplied to the reaction chambers by increasing the pressure.
15. Device according to claim 13, in which said supply means (4) include a pump (41), and the fluid is supplied to the reaction chambers by reestablishing the pressure after establishing an underpressure.
16. Device according to claim 5, in which the reaction chambers (13) of the cartridge (1) are closed.
17. Method for nucleic acid amplification using a device according to any one of claims 1 to 16, comprising the following steps:

    - at least partially filling the reservoir (11) with a fluid containing a sample of nucleic acids to be analysed and all that is required for an amplification reaction, with the exception of the primers, and, optionally, a fluorescent nucleic acid reporter;

    - distributing said fluid to the reaction chambers (13) of the cartridge (1), in which are pre-disposed primers and, optionally, one or more labelled probes;

    - employing the means (3) for relative displacement between the cartridge and the heating plate to successively bring the contents of each reaction chamber to the two, three or more temperatures defined by the two, three or more zones of said heating plate (2) as many times as is desired.
18. Amplification method according to claim 17, in which the step for distributing the fluid to the reaction chambers (13) is carried out by applying an underpressure to the interior of the cartridge, then reestablishing the pressure.

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