Microfluidic device including an amplification reaction chamber

Abstract

To provide a microfluidic system equipped with an integrated solution to control an amplification reaction or to identify multiple targets during one analysis.SOLUTION: A microfluidic system is designed for analysis of a biological sample containing biological species. A device comprises a support configured such that a so-called amplification chamber 20 allows an amplification reaction to be carried out therein, and an input channel opening into the amplification chamber. The amplification chamber includes at least one recess A1 to receive a compound for internal control of the amplification reaction.SELECTED DRAWING: Figure 4A

Description

[Technical field]

[0001] The present invention relates to microfluidic systems and more particularly to the structure of chambers located in the devices of the system and intended for carrying out amplification reactions. [Background technology]

[0002] The qPCR reaction consists of the amplification of a target DNA or RNA sequence (representative of a particular organism) bound to an intercalator, or probe, which produces a fluorescence that can be detected by optical equipment if the sequence is amplified. An increase in fluorescence therefore means that the amplification reaction has taken place and that the DNA or RNA of the target organism was indeed present. However, if there is no reaction, it must be made clear that this is due to the absence of the target organism and not due to an inhibition of the amplification reaction resulting in a false negative. In practice, the enzymes involved in the amplification reaction are sensitive to many inhibitors provided by the sample being tested.

[0003] To ensure that the absence of amplification means the absence of target, internal reaction controls are used. These are almost always another DNA target intentionally added in the test that is amplified simultaneously with the sample of interest. It is therefore necessary to be able to distinguish between the two reactions. Several strategies are used in this field: - One is to carry out a control reaction in parallel with a portion of the sample as an independent reaction. This requires splitting the initial sample, resulting in a loss of sensitivity / representativeness of the test. The advantage is that it is possible to use any amplification detection technique for this control (e.g. fluorescent DNA intercalation). - Another strategy to avoid splitting the sample is to perform a control reaction in the same reaction as the target sequence. This solution allows testing of the entire sample, but is not available for all detection techniques, especially for non-specific intercalating agents, non-specific sequences or any other non-sequence specific detection methods (colorimetry, pH measurement...). EP 0 586 112 and US Pat. No. 6,312,930 each describe detection methods that eliminate false negatives by adding a control target. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] European Patent Application Publication No. 0586112 [Patent Document 2] U.S. Patent No. 6,312,930 Summary of the Invention [Problem to be solved by the invention]

[0005] In view of the above, it is an object of the present invention to provide a microfluidic system with an integrated solution for controlling amplification reactions and for identifying multiple targets during one assay. [Means for solving the problem]

[0006] This object is achieved by a microfluidic system designed for the analysis of a biological sample containing a biological species, said system being configured as follows: - an optical detection device comprising a light source configured to emit an optical signal and at least one sensor having a capture surface defining an area for reading the optical signal; - A microfluidic device, including: A support in which a so-called amplification chamber is formed, in which an amplification reaction can be carried out an inlet channel opening into said amplification chamber; Equipped with - the amplification chamber comprises at least one first area located within the area of ​​sensor reading and at least one protrusion designed to receive a compound for an internal control of the amplification reaction and arranged to be located outside the area of ​​sensor reading or forming a recess that is opaque to the optical signal.

[0007] According to one feature, the amplification chamber comprises a first mass having a first compartment and a second mass having a second compartment that is narrowed relative to the first compartment to form a protrusion, the protrusion forming the recess.

[0008] According to another feature, the support comprises a plurality of superimposed layers, the amplification chamber being formed by at least two of the superimposed layers, referred to as an upper layer and a lower layer, and the recess being formed in only one of the two layers.

[0009] According to another feature, the protrusions forming the recesses are formed in a lower layer.

[0010] According to another feature, the amplification chamber comprises a main cavity formed in the upper layer and one or more sub-cavities formed in the lower layer, each forming a separate recess of the chamber.

[0011] According to another feature, the internal reaction control compound comprises a set of DNA primers targeting a known DNA sequence or a predefined DNA target, allowing its amplification according to the amplification method used.

[0012] According to another feature, the first section of the chamber is transparent to pass an optical signal provided by a light source of the detection device, and the second section has at least one opaque portion configured to block the passage of the optical signal.

[0013] The invention also relates to a method for analyzing a biological sample containing a biological species, said method being carried out by a system as defined above and consisting of: - disposing an internal reaction control compound in a recess of the amplification chamber; - Injecting a fluid sample into an amplification chamber of a microfluidic device; - detecting the presence of a target compound in the fluid sample and located in a first area of ​​the amplification chamber with a sensor; - A time lag is provided for detecting the presence of an internal reaction control compound in the first zone of the amplification chamber. Other features and advantages will become apparent from the following detailed description which refers to the accompanying drawings. [Brief description of the drawings]

[0014] [Figure 1] FIG. 1 shows the overall steps of a method for the preparation and analysis of biological samples by biomolecule amplification. [Diagram 2] FIG. 1 shows a two-unit microfluidic device for carrying out the steps of the preparative and analytical method defined in FIG. [Diagram 3] 3 illustrates an example of an embodiment of the device of FIG. 2 in an exploded view. [Figure 4A] 3 shows an example of an embodiment of an amplification chamber that can be used in the apparatus of FIG. 2. [Figure 4B] 3 shows an example of an embodiment of an amplification chamber that can be used in the apparatus of FIG. 2. [Figure 5A] 13A-13C show different alternative embodiments of the amplification chamber embodiment. [Figure 5B] 13A-13C show different alternative embodiments of the amplification chamber embodiment. [Figure 5C] 13A-13C show different alternative embodiments of the amplification chamber embodiment. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0015] The microfluidic device of the present invention is for the analysis of biological samples, for example in the form of a liquid containing a biological species that contains the biological material to be investigated.

[0016] Biological species refers in particular to microorganisms, cells, spores, viruses.... Biological substances investigated mean for example nucleic acid molecules (RNA, DNA) of cellular origin, proteins, lipopolysaccharides (LPS), lipoteichoic acid (LTA)....

[0017] By fluid is meant liquid, gas.... Liquids may have different viscosities and may for example be paste-like or gel-like.

[0018] In the following description, the terms "lower", "upper", "top" and "bottom" should be understood with reference to the vertical major axis (X).

[0019] In the following description, the terms "external", "outer", "internal" and "inside" should be understood with reference to the chamber of the device described below.

[0020] With reference to FIG. 1, the complete analysis of a biological sample as defined above may involve carrying out the following steps in order: - E1: Concentration of biological species present in a biological sample; - E2: Purification wash, removal of culture interferences, - E3: Addition of medium, - E4: Cultivation of biological species, - E5: Optical growth monitoring during incubation and colony counting - E6: Washing, removal of PCR inhibitors, - E7: Mechanical lysis of biological species present in a sample in order to extract the biological material under investigation. - E8: Separation of the biological material under investigation from any contaminants present; - E9: Detection of the presence of pathogens in biological material by qPCR, LAMP, RPA-based biomolecular amplification and optical detection such as fluorescence, colorimetry, holographic imaging, turbidimetry, pH measurement associated with the amplification reaction.

[0021] Of course, the method may be limited to certain steps, not all of which are necessarily performed within the apparatus.

[0022] In the concentration step, a biological sample, for example in liquid form, containing the biological species is injected into the chamber and passed through the filter. The liquid portion of the sample and any particles / molecules that pass through the filter are collected through the outlet and discarded from the analysis. The biological species are then concentrated in the space within the chamber.

[0023] The biological species present within the chamber can be washed by injecting a cleaning / rinsing solution.

[0024] Inject medium for culturing biological species.

[0025] The proliferation monitoring step allows for monitoring of cell proliferation during the culturing step by optical reading using sensor C.

[0026] Mechanical lysis of biological species is used to disrupt the biological species present in a sample using a rough bearing surface, making the biological material available for investigation, consisting of, for example, DNA molecules and pollutants.

[0027] In order to separate the biological material of interest from the contaminants, a solution containing an amplification reagent is injected to elute the biological material of interest, with a portion of the liquid solution thus injected retaining and isolating those biological material of interest, e.g. DNA molecules, which pass through the filter.

[0028] Once the separation between the contaminant and the biological material under investigation is completed, the presence of the pathogen in the separated biological material is detected by an amplification reaction of the biological material. The amplification reaction is carried out by adding an amplification mixture and heating the chamber in which the sample is placed. The temperature to which the chamber is heated depends on the type of amplification reaction to be carried out. The type of amplification reaction can be any, for example, loop-mediated isothermal amplification (LAMP), polymerase chain reaction (PCR), nucleic acid sequence-based amplification (NASBA), recombinase polymerase amplification (RPA)... For LAMP, heating at a temperature between 60°C and 65°C is preferred. This reaction makes it possible to amplify the molecules of the biological material to be detected, for example DNA molecules. During the amplification reaction of the biological material, the aim is to detect whether the pathogen is present or not. Various methods can be used for this, for example colorimetry, fluorescence, electrochemical methods, pH measurement, turbidimetry, etc. Other detection methods may also be considered. For detection methods such as pH measurement, a pH detection electrode can be integrated into the device.

[0029] A microfluidic device for carrying out the above steps is shown in Figure 2. The device consists of a single rigid support S. Figure 3, described below, shows an example of an embodiment of this device.

[0030] This rigid support S integrates a microfluidic network adapted for the implementation of the steps of an analytical method. The microfluidic network can have different configurations depending on the configuration of the analytical method to be implemented.

[0031] The support S preferably comprises a flat bottom wall and a multi-layer structure stacked on the bottom wall along said major axis.

[0032] The microfluidic network of the device is composed of at least two units U1, U2.

[0033] The first unit U1 comprises a first chamber 10 formed in said support. This chamber 10 has a non-zero volume and is bounded by the walls of the support S.

[0034] The first unit U1 has a first flow path 11 formed in the support, which injects a fluid into the first chamber 10 or discharges a fluid from the first chamber 10. The first flow path 11 has a first end having an opening formed, for example, through a top wall of the support S, and a second end opening into the first chamber 10. The first end of the first flow path is disposed, for example, vertically, and the second end opens, for example, horizontally into the first chamber 10.

[0035] The first unit U1 has a second flow path 12. This second flow path 12 also has a first end communicating with the outside, for example forming an opening formed to penetrate the top plate of the support S, and a second end communicating with the space formed by the first chamber 10. Through this second flow path 12, a fluid is injected into the first chamber and a fluid is discharged from the first chamber. The first end is, for example, disposed vertically, and the second end is disposed horizontally. The first chamber 10 is disposed between the first flow path 11 and the second flow path 12.

[0036] The first chamber 10 can be closed at the top by a flexible and stretchable membrane 13. In the first chamber layer, the opening in the top plate of the support is therefore covered and sealed by the membrane 13. For this purpose, the membrane 13 is fixed in the support by a suitable fixing solution, for example an adhesive. This membrane 13 is, for example, a membrane of the MicroAmp type from 3M (registered trademark), and has a thickness, dimensions and structure such that it deforms superelastically at least from its fixing point towards the bottom of the first chamber.

[0037] The membrane 13 can reversibly deform and assume several shapes, that is, it can be stretched by superelastic deformation towards the outside of the support S and can be compressed or relaxed and retracted inside the first chamber 10. By superelastic material is meant here a material capable of having a surface that can be changed from a first surface area to a second surface area, the second surface area being at least 5 times the first surface area, for example 10 times the first surface area or even equal to 50 times the first surface area.

[0038] The first unit U1 also comprises a lateral filter 14 arranged in the first chamber 10 and separating the first chamber 10 into two spaces 100, 101. The two spaces are superimposed, for example, such that the lower space 100 is located below the filter 14 and the upper space 101 is located above the filter 14. The filter 14 is preferably formed in whole or in part in the form of a thin flexible film, which is drawn into the space formed by the chamber and can pass from one space to the other only through the pores of the filter 14. The film has elastic deformation such that it expands and contracts when a supporting force acts in a substantially vertical direction, and the level of this elastic deformation is sufficient to reach the bottom of the chamber 10. The filter 14 has an average pore size of 0.2 μm to 50 μm, for example, 0.2 μm to 1 μm for the separation of microorganisms. The pore size is designed to ensure separation between different biological species present in the sample. This filter 14 is, for example, made of a film having a thickness, dimensions and structure such that it deforms from its fixed point towards the bottom of the chamber 10. It may also be a superelastic membrane having similar properties to the membranes described above.

[0039] In one embodiment, the first flow channel 11 opens into the upper space 101 of the first chamber 10 and the second flow channel 12 opens into the lower space 100 of the first chamber 10. The mouths of the two flow channels are therefore separated by a filter 14 located within the chamber.

[0040] As shown in FIG. 2, the first unit U1 preferably has a rough grinding surface 15 arranged at the bottom of the first chamber 10. This rough grinding surface 15 extends over most of the bottom of the first chamber. It has an average surface roughness parameter between 0.1 μm and 10 μm, preferably in the range of 0.2 μm to 3 μm. This rough grinding surface 15 is designed to perform a mechanical lysis of the biological species present in the biological sample placed in the device. The mechanical lysis preferably involves grinding and pulverizing the biological species on the rough grinding surface. This grinding operation is performed by frictional movement of the biological species against the rough grinding surface with a suitable grinding member. This grinding member may be, for example, a spatula or a rod made of plastic or metal. The grinding member is inserted from the outside of the chamber 10 and its end is pressed against the outer surface of the membrane 13 so that the membrane 13 and filter 14 extend toward the bottom of the first chamber 10, thereby rubbing the biological species present in the sample against the rough grinding surface 15.

[0041] Concerning the rest of the device, the second unit U2 consists of a second chamber 20 of non-zero volume bounded by the wall of the support S. The second unit U2 also constitutes a third channel 21 formed in the support. This third channel 21 consists of a first end consisting of an opening formed, for example, through the upper wall of the support, and a second end opening only into the second chamber 20. The first end of this third channel 21 is arranged, for example, vertically, and its second end opens, for example, horizontally into said second chamber 20. The first end of this third channel is sealed, for example, by a hydrophobic membrane 210, i.e. a membrane that is impermeable to liquids but permeable to gases such as air. This hydrophobic membrane 210 can be made of a material such as polytetrafluoroethylene (PTFE).

[0042] The two transverse walls of the support, preferably the parallel upper wall 200 and lower wall 201 that partially delimit the second chamber 20, are made of a transparent material, thus allowing optical reading through the bulk of the second chamber. The term "transparent" means that the material used is at least partially transparent to visible light, allowing at least 80% of that light to pass through. This means that it has sufficient transparency to allow viewing of the inside of the chamber. The lower plate may be made of glass and the upper plate may be a peelable adhesive that may stick to the top side of the second chamber, thus closing it.

[0043] According to one feature of the invention, the device also comprises a first transfer channel 22 provided in the support. This first transfer channel 22 is designed to connect the first chamber 10, or more precisely its lower space 100, to the second chamber 20.

[0044] The first transport channel 22 preferably has a first end that opens directly into the second channel 12, thereby forming a bypass node on the second channel 12, and has a second end that opens directly into the second chamber.

[0045] The device further comprises a switching means, eg arranged on the second flow path 12, for selecting the connection of the first chamber. - only via the second flow path to the outside, or - only via the first transfer channel to the second chamber.

[0046] These switching means may consist of a hollow, removable cone 120 in the shape of a funnel. When its tip is inserted into the second channel 12, it establishes communication between the outside and the first chamber and its walls block the inlet to the first transfer channel 22 made at the diversion point. When it is removed, the first end of the second channel 12 is closed, for example by means of an adhesive 121 applied to the surface of the support, and the connection between the first transfer channel 22 and the second channel 12 is opened, so that the fluid can flow between the first chamber 10 and the second chamber 20.

[0047] Of course, the switching means can also be implemented in other alternative embodiments. Its general principle is to close the transfer channel to allow access to the first chamber or to allow connection between the first and second chamber. It can therefore be a simple valve: In the first position, the bypass node closes the mouth of the transport channel 22, thereby making the second channel accessible. In the second position, the connection between the second channel 12 and the transport channel 22 is open.

[0048] Without being limited thereto, the device can be fabricated according to the structure shown in FIG.

[0049] In this FIG. 3, the support S has the following characteristics: the support comprises a slide 50, for example made of glass or of plastic, such as PMMA or COC; the slide 50 is covered in a first area with a grinding surface, so that a rough grinding surface 15 for melting is formed on part of its upper surface; the slide 50 is transparent in at least one second area, so that the bottom plate 201 of the transparent second chamber 20 is made transparent and an optical reading can be made; - a first layer 51 with an imprint of a first microchannel is laminated on the upper surface of the slide 50, the first imprint consisting of a first cavity defining the lower space 100 of the first chamber 10, a second cavity defining the lower part of the second chamber 20 and the lower part of the third channel 21; the edge of the first cavity is around the first grinding area and the edge of the second cavity is around the second reading area; - thus, the filter 14 is applied to the first layer so as to cover the lower space 100 of the first chamber, and the hydrophobic membrane 210 is applied to the lower part of the third flow channel 21; - a second layer 52 with a second microchannel cutout is laminated on top of the first layer 51 and covers the filter 14 and the hydrophobic membrane 210. This second microchannel cutout consists of a cavity forming the headspace 101 of the first chamber 10, a second cavity forming the central part of the second chamber 20 and the top of the third channel 21; - a third layer 53 with a third microchannel cutout is laminated on top of the second layer 52, this third microchannel cutout consisting of the top of the first chamber 10 and the top of the second chamber 20; - a glass or plastic strip is dimensioned to cover the top side of the third layer 53 in the second chamber, forming the upper transparent plate 200 of the carrier; a cap 54, for example made of PMMA, which is placed above the first chamber 10 and which is provided on its underside with a membrane 13 designed to close the first chamber from above; - the cover is provided with an upper axial opening to allow access to the membrane 13 provided for dissolution; The cap 54 has two inlets / outlets for fluids on its upper surface: a first inlet / outlet is connected to a first axial through-channel formed through the membrane 13 and the three layers and opening into the first chamber 10, forming the first channel 11 of the support; a second inlet / outlet is connected to a second axial through-channel formed through the membrane 13 and the second and third layers and opening above the hydrophobic membrane 210, forming the third channel 21 of the support. Finally, the support has two further through channels forming a first transfer channel 22 connecting the two chambers 10,20. The other two layers 55, 56 are responsible for the inlet to the second channel 12 and for the connection of this channel with the transport channel 22.

[0050] The present invention is more particularly directed to the second chamber 20, known as the amplification chamber, which has a structure suitable for carrying out the detection steps described above.

[0051] The amplification chamber has an internal volume of suitable shape to perform reliable control experiments in the amplification reaction or to identify several targets simultaneously. In the latter case, the chamber may be shaped to allow the simultaneous detection of several targets. These tests, known as multiplex tests, are used, for example, to detect a group of pathogens that correspond to similar clinical symptoms, and to detect not only bacteria but also potential antibiotic resistance genes.

[0052] The principle is to form at least one recess Ax (x ranging from 1 to N, N corresponding to the number of recesses, one or more) in the amplification chamber to store internal reaction control compounds (e.g. amplification primers targeting a selected DNA sequence or a predefined DNA) that are compatible with the amplification technique used (PCR, LAMP, RPA...). According to this principle, the DNA is dried in the recess and then the primers are carried by a liquid introduced into the chamber, or the primers are dried in the recess and the DNA is carried by a liquid introduced into the chamber.

[0053] This recess Ax is formed outside a section of the chamber dedicated to optical reading, which corresponds to an optical reading zone Z, which is the only zone of the chamber visible by the sensor C. The chamber may have areas outside this optical reading zone Z and may be located outside the reading range of the sensor C.

[0054] For reaction controls, an internal control compound can be deposited in recess Ax at a known concentration and then dried directly in the chamber, thus remaining permanently in the device and ready for use.

[0055] The advantage of depositing compounds outside the optical read zone Z, rather than directly within it, is that it allows for optimal spatial gas exchange and also makes it possible to distinguish between control and target amplifications. Indeed, the control amplification can be shifted both in time and in space.

[0056] A multi-layer device structure allows the amplification chamber to be constructed with layers of different designs: a lower layer may actually contain one or more recesses, and an upper layer of the chamber may define the entire optical read cross-section of the chamber.

[0057] This multi-layering principle is illustrated in Figures 4A and 4B. These figures show a lower layer S1 with a recess A1 (here exemplarily represented as a rectangle) and an upper layer S2 that defines a cross section of an optical readout zone Z. The recess A1 is here formed in the shape of a half moon. Figure 4A represents a three-dimensional view of the amplification chamber, showing the position of a sensor C relative to the chamber, whose surface defines the optical readout zone Z of the chamber.

[0058] As shown in Fig. 4A, the recess A1 can be located outside the optical readout zone Z of the sensor C. In Fig. 4B, the amplification chamber is formed by superposing two layers S1, S2. This figure also shows the inlet to the chamber, which is formed in the lower layer S1 and corresponds to the first transport channel 22.

[0059] 5A-5C show different configurations of the lower layer of the amplification chamber, in which the optical readout zone Z is shown in grey.

[0060] In Figure 5A, the bottom layer S1 has a bulge forming a recess A1 which in cross section extends beyond the section of the optical reading zone Z of the chamber, which is located opposite the entry point of the liquid into the chamber (corresponding to the design of Figures 4A and 4B). This offset zone makes it possible to shift the amplification reaction both in time and in space.

[0061] In Fig. 5B, the bottom layer S1 has three different recesses A1, A2, A3 formed by three cavities, and thus can accommodate three different primers. These three areas are located in the optical readout area Z, making the multiplexing method feasible.

[0062] FIG. 5C shows a lower concept, in which five distinct zones are defined, four recesses A1-A4 each in the form of a cavity, the main compartment being a protrusion and a fifth recess A5. The upper layer of the immediate upper chamber defines an optical read zone. The optical read zone Z is designed to cover the four recesses A1-A4, such that the masses are in fluid communication. The fifth recess may be outside the optical read zone Z, as shown in FIG. 5A.

[0063] As mentioned above, each primer may be placed in a separate recess of the chamber in dry form. To facilitate drying, it is preferable to dissolve the primers in an acidic buffer (about pH 6) to facilitate binding to the glass slide. Sugars (e.g., trehalose) may be used to limit the spreading of the droplets, potentially increasing the stability of the dried DNA sequences.

[0064] It is further noted that, whatever its configuration, the chamber 20 has rounded corners and contours to optimize liquid flow within the chamber and prevent the formation of air bubbles.

[0065] From the above it can be seen that the device of the invention has many advantages. The amplification chamber of the device of the invention makes it possible to achieve several goals. - Fluid optimization (no air bubbles); - Push air out of each section but retain liquid; - Do not pollute the surrounding environment; - Enabling multiplexing; - Including an internal response control; - Reducing dead volume; - Do not split the sample;

Claims

1. 1. A microfluidic system for the analysis of a biological sample containing a biological species, comprising: an optical detection device comprising a light source configured to emit an optical signal and at least one sensor having a capture surface defining an area for reading the optical signal; a microfluidic device comprising: a support in which a so-called amplification chamber (20) is formed in which an amplification reaction can be carried out, an inlet channel opening into said amplification chamber; Equipped with the amplification chamber comprises at least one first zone located within the zone of sensor readout and at least one protrusion forming a recess (Ax) designed to receive an internal reaction control compound for an internal control of the amplification reaction and arranged so as to be located outside the zone of sensor readout; the amplification chamber comprises a first mass having a first compartment and a second mass having a second compartment narrowed relative to the first compartment to form a protrusion, the protrusion forming the recess (Ax); system.

2. the support comprises a plurality of superposed layers, the amplification chamber being formed by at least two of the superposed layers (S1, S2), called upper layer (S1) and lower layer (S2), the recess being formed in only one of the two layers, The system of claim 1 .

3. The protrusion forming the recess (A1) is formed on the lower layer. The system of claim 2.

4. The amplification chamber (20) comprises a main cavity formed in the upper layer and one or more sub-cavities formed in the lower layer, each forming a separate recess of the chamber.

4. A system according to claim 2 or 3.

5. The internal reaction control compound comprises a set of DNA primers that target a known DNA sequence or a predefined DNA target and allow its amplification according to the amplification method used. A system according to any one of claims 1 to 4.

6. a first area of ​​the chamber being transparent to allow a light signal provided by a light source of the detection device to pass therethrough, and a second area having at least one opaque portion configured to prevent the light signal from passing therethrough; A system according to any one of claims 1 to 5.

7. A method for analyzing a biological sample containing a biological species, carried out in a system as defined in any one of claims 1 to 6, comprising: - disposing said internal reaction control compound within said recess of said amplification chamber; - injecting a fluid sample into the amplification chamber of the microfluidic device; - detecting the presence of a target compound in the fluid sample and located in the first area of ​​the amplification chamber with the sensor; - A time lag is applied to detect the presence of the internal reaction control compound in the first area of ​​the amplification chamber. method.

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