Method for analyzing a sample to detect the presence of an active form of a biological species

Abstract

The invention relates to a method for analyzing a sample (ECH) to detect the presence of a biological species in a first form, called the active form (F2), in said sample, said sample containing said biological species in a second form, called the dormant form (F1), distinct from the active form (F2). The method consists of separating said sample (ECH) into two distinct parts (A, B) of identical volumes, called the first part (A) of the sample and the second part (B) of the sample, determining a first quantity (Q1) of biological species in the first part (A) of the sample and a second quantity (Q2) of biological species in the second part of the sample, and comparing the first quantity (Q1) obtained and the second quantity (Q2) obtained, in order to deduce the presence or absence of the active form (F2) of the biological species in the sample. Figure to be published with the abstract: Figure 1

Description

Description Title of the invention: Method for analyzing a sample to detect the presence of an active form of a biological species Technical field of the invention

[0001] — The present invention relates to a method for analyzing a sample in order to detect the presence of an active form of a biological species. State of the art

[0002] = In the field of agriculture, certain biological species may exist at the once in a so-called dormant and harmless form, that is to say resistant to any action external and inactive, non-virulent, as well as in an active form, susceptible to cause problems in crops. This situation can also to present in the field of the health of a living being. The two forms of the species biological cells possess the same genome and the same type of generic proteins but They do not have the same effect on crops or on the health of living beings.

[0003] — A particular known case is that of downy mildew, which is a common disease present in agriculture and particularly destructive. The pathogen responsible for this A disease can be both dormant and active. For example, it is likely to de- It develops on the leaves of plants and on bunches of grapes. It is currently treated by repeated and systematic applications of fungicide, which result Environmental pollution, toxicity, and the development of more resistant strains.

[0004] To adapt treatments and apply them only when necessary, it is necessary to to know if the pathogen is present in its active form or in its form dormant.

[0005] — For this, there are now various solutions, some more complex and others more or less effective:

[0006] - Installation of weather stations in fields and application of models predictive models designed to calculate the risks of the pathogen appearing in its active form; Analysis of samples taken on site and biomolecular detection; Optical detection by fluorescence type "Agrolase" (Registered Trademark);

[0007] — In the first proposed solution, the implementation of predictive models is lengthy and their execution can prove unreliable.

[0008] — In the second solution, biomolecular detection makes it possible to detect the presence of the biological species and possibly the quantity of biological species present, but not knowing if it is present in its dormant form or in its active form. In the third solution, the system is particularly cumbersome to implement and requires continuous monitoring. There is therefore a need for a solution that can detect the presence of an active form of a biological species, which is reliable and easy to implement and deploy, without using heavy and bulky equipment. The process of the invention makes it possible to meet this need. Description of the invention The invention thus consists of a method for analyzing a sample in order to detect the presence of a biological species in a first form, called the active form, in said sample, said sample containing said biological species in a second form, called the dormant form of said biological species, distinct from the active form, said method, starting from the same sample, comprises the following steps: Separation of said sample into two distinct parts of identical volumes, Please refer to the first part of the sample and the second part of the sample. Mild treatment or no treatment of the first part of the sample, First concentration of biological species contained in the first part of the sample, First quantitative detection of said biological species in the first part of the sample in order to determine an initial quantity of species biological in the first part of the sample, Active processing of the second part of the sample, Second concentration of biological species contained in the second part of the sample, Second quantitative detection of said biological species in the second part of the sample in order to determine a second quantity of species biologicals in the second part of the sample, Comparison between the first quantity obtained and the second quantity obtained, in order to deduce the presence or absence of the active form of the biological species in the sample. Depending on a specific characteristic, the active treatment for the second part of the sample is chosen from one or more of the following treatments, or a combination of at least two of these treatments: Chemical treatment, Biochemical treatment Thermal treatment, Mechanical treatment, Optical treatment. According to a particular embodiment, the active treatment is carried out by lysis of biological species against a rough surface. According to another particular embodiment, the active treatment is a heat treatment which consists of heating the second part of the sample by heating means to a given temperature and for a determined time. According to another particular realization, active treatment is a chemical or biochemical treatment which consists of adding a substance destructive of the active form of the biological species to the second part of the sample, in order to break its outer membrane and release its nucleic acids. According to another particular embodiment, active treatment is an optical treatment which consists of exposing the second part of the sample to a light signal emitted at a given wavelength and for a determined duration. According to another distinctive feature, the first and second concentrations are implemented using a microfluidic component that includes: A case with at least one opening, A first channel is provided in the said casing, A second channel is provided within the said casing, A chamber into which the first and second canals open, A filter separating said chamber into two distinct spaces so as to define a first space into which the said first injection channel opens and a second space into which said second channel, said filter, opens having a porosity adapted to retain the biological species present in the sample. According to another peculiarity, the first concentration and the second concentration are implemented simultaneously in two identical microfluidic components in parallel. According to another particularity, the gentle treatment and / or the said active treatment are implemented in the said micro-fluidic component. According to another peculiarity, the first quantitative detection and the second quantitative detection are carried out by biomolecular amplification. According to another particularity, the sample is obtained after a step of grinding and extraction of a sample to be analyzed. It should be noted that the method of the invention is suitable for detecting the presence of the active form of biological species, such as: Bacteria in a vegetative (active) or spore (dormant) form; Fungal pathogens with a sporulation cycle; This makes it possible to detect the onset of certain diseases such as downy mildew, Pseudoperonospora, powdery mildew, botrytis… Brief description of the figures Other features and advantages will appear in the detailed description that follows, in conjunction with the attached drawings, in which: Figure 1 represents a diagram illustrating the steps implemented in the process of the invention, according to a first embodiment variant; Figure 2 represents a diagram illustrating the steps implemented in the process of the invention, according to a second embodiment; Figure [Fig. 3] schematically represents the microfluidic component that can be used to implement the process of the invention, in the first variant and second variant of implementation; Detailed description of at least one embodiment For the purposes of this description, a target biological species may be present in the sample in a dormant form (hereafter F1) and an active form (hereafter F2). The dormant form F1 is the harmless and resistant form of the biological species, while its active form F2 is an active and destructive form. The terms "harmless" and "destructive" must, of course, be understood in relation to the environment in which the biological species is present; this environment could be a living organism, a plant, a food matrix, etc. The detection principle used in the invention is further based on the fact that the biological species is often more fragile in its active form F2 than in its dormant form F1, in the sense that, when an active treatment is carried out on the sample, the DNA molecules of the active form F2 tend to break more easily than those of the dormant form F1. The invention thus aims to provide a method for detecting the presence of an active F2 form of a biological species in an ECH sample. To achieve this, the process mainly consists of: Divide an initial ECH sample into two parts A, B of quantity identical, Perform a gentle T1 treatment or no treatment on the first part A of the sample and a so-called active T2 treatment on a second part B of the sample, Concentrate the biological species present in each part of the sample, Quantitatively detect the target biological species in each of the two parts of the sample, Perform a comparison of the results obtained for each part of the sample by performing differential treatment. Based on the ratio obtained between the quantity Q1 of biological species present in the first part A of the sample and the quantity Q2 of biological species present in the second part B of the sample, it can then be determined whether the biological species is present and whether it is present in its active form F2 and dormant form F1 or only in its dormant form F1. The method of the invention is particularly suitable for detecting the presence of the active F2 form of biological species, such as: Bacteria in a vegetative (active) or spore (dormant) form; Fungal pathogens with a sporulation cycle; This makes it possible to detect the onset of certain diseases such as downy mildew, Pseudoperonospora, powdery mildew, botrytis… To implement the process of the invention, a micro-fluidic component shown in [Fig.3] is advantageously used. This microfluidic component 1 comprises a housing including a lower wall 10, a side wall 11, and an upper wall 12. All the walls of the housing will preferably be made of the same material. This material will be particularly suitable for withstanding heating within a temperature range of 20°C to 100°C. Preferably, some of the housing walls will be made of a transparent material. Preferably, the material used will be a plastic, for example, PMMA (Polymethyl Methacrylate) or COC (Cyclic Olefin Copolymer). Device 1 includes a chamber 13 formed within the housing. This chamber represents the location where both the purification / concentration and the detection of the target biological species can be performed. Chamber 13 is closed at the bottom by the lower wall of the housing. The microfluidic component includes a first channel 14 formed in the housing and arranged for injecting fluids into the chamber or for expelling fluids from the chamber. The first channel 14 has a first end with an opening formed, for example, through the upper wall 12 of the housing, and a second end that opens into the chamber 13. The first end of the first channel 14 is, for example, arranged vertically, and its second end opens, for example, horizontally into the chamber 13. The first end of the first channel is, for example, flared to accommodate the tip of a pipette or is adapted to the type of device used to inject the fluid into the device. For example, it may be an opening with a Luer-type fitting for connecting a syringe or adapted for connecting a fluidic circuit. The device includes a second channel 15 housed within the casing. This second channel 15 also includes a first end which communicates with The exterior, forming an opening made, for example, through the upper wall of the housing, and a second end that communicates with the space formed by chamber 13. Fluids can also be injected into or discharged from this second channel 15 through this second channel. Its first end is, for example, arranged vertically and its second end horizontally. Chamber 13 is positioned between the first channel 14 and the second channel 15. Similarly, the first end of this second channel is, for example, flared to accommodate the tip of a pipette or adapted to the type of device used to inject the fluid into the device. For example, it could be an opening with a Luer-type fitting for connecting a syringe or adapted for connecting a fluid circuit. The chamber 13 can be closed at the top by an advantageously flexible and stretchable membrane 18, preferably transparent. The upper wall 12 of the device housing thus has an opening which is hermetically sealed by said membrane 18. Said membrane is anchored in the housing by any suitable fastening method, for example by adhesive. This membrane 18 will, for example, be composed of a film, for example a self-adhesive PET film, of a thickness, dimensions, and composition suitable for elastic deformation relative to its anchoring points, particularly to the bottom of the chamber 13. The term “transparent” means that the material used is at least partially transparent to visible light, fluorescence, or luminescence, allowing at least 80% of this light to pass through. This means it will be sufficiently transparent to see inside chamber 13, at least the second space located above filter 16 mentioned below. The microfluidic component | comprises a filter 16 arranged in said chamber 13 and dividing said chamber 13 into two spaces. The two spaces are, for example, superimposed and designated as such lower space 130 located below the filter and upper space 131 located above the filter and below the membrane 18. This filter 16 is preferably made, in whole or in part, in the form of a thin, flexible film, held in the space formed by the chamber so as to allow passage from one space to the other only through the pores of the filter 16. The film advantageously exhibits elastic deformability, enabling it to stretch when a support force is applied in a substantially vertical direction, this elastic deformability being sufficient to reach the lower surface of the chamber 13. The filter 16 has an average pore diameter of between 0.2 µm and 50 µm, for example, between 0.2 µm and 1 µm for the separation of microorganisms.The pore diameter is of course adapted to ensure separation between different biological species present in the sample. Filter 16 will, for example, be composed of a film of thickness, of di- . The dimensions and structure are adapted to allow the filter to deform to the bottom of chamber 13 relative to its anchor points. In a particular embodiment, the filter can also be made of a transparent material, for example, with the same transparency characteristics as the membrane. For bacteria, the filter can have a pore diameter ranging from 0.2 to 1 / 2000 mm to retain the bacteria. One of the active treatments that can be applied to a portion of the sample may consist of lysis of the biological species present in the sample. In the case of lysis, the microfluidic component may advantageously include a rough bearing surface 17 arranged on the bottom of the chamber 13. This rough bearing surface 17 extends over a major portion of the bottom of the chamber. It has an average surface roughness parameter between 0.01 µm and 10 µm, preferably between 0.2 µm and 3 µm. This rough bearing surface 17 is intended to allow mechanical lysis of the biological species present in the biological sample placed in the device. Preferably, the mechanical lysis is carried out by grinding said biological species through abrasion on said rough bearing surface.The grinding operation is carried out by a friction movement of the biological species against the rough support surface, using a suitable grinding element. This element will be, for example, a spatula or a rod, for example, made of plastic or metal. This element is applied from outside the chamber 13 and its end is applied against the external surface of the membrane 18 so as to stretch the membrane 18 and the filter towards the bottom of the chamber and thus rub the biological species present in a sample against the rough support surface 17. In the case of active thermal treatment, the enclosure can advantageously incorporate means for heating the internal space of the chamber, consisting, for example, of at least one heating element. The heating element is, for example, fixed under the lower wall of the enclosure. A power supply will be provided to power the heating element. The power supply will, for example, include one or more batteries, providing sufficient energy to heat the chamber to a temperature within the range defined above, i.e., from 20°C to 100°C. Of course, other heating methods could be used, including, for example, conductive ink applied by printing or screen printing under the lower wall of the enclosure. Thus, to summarize, the microfluidic component can advantageously include the following "multilayer" structure: Optionally, a rough lower support surface 17, A lower space 130 of room 13, located above the surface rough support 17, A filter 16, advantageously flexible and stretchable, located above the space less than 130, An upper space 131 of chamber 13 located above filter 16, A membrane 18, advantageously flexible and stretchable, located above the upper space 131, hermetically sealing the chamber and accessible from outside the device. The lower wall of the component and the membrane can be made of transparent materials, in particular to implement detection by biomolecular amplification. Without limitation, the microfluidic component may exhibit the following dimensional characteristics: A first channel 14 consisting of an inlet channel with a diameter of 1mm x 3mm height, then a rectangular channel of 1mm x 150um and 3mm long; A chamber 13 consisting of a lower space 130 for concentration / lysis which has a diameter of 8mm x 150µm in height and a greater space for elution of a diameter 8mm x 300mm height; Filter 16 with porosity adapted to the target to be retained (virus, yeast, mold…), for example with a porosity of 0.2 to 2 µm to retain bacteria or 10 to 100 nm to retain viruses; A second channel 15 consisting of a rectangular cross-section channel of Imm x 150µm and 3mm long, then a channel of 1mm diameter x3mm height ; Using this microfluidic component, the process of the invention can be implemented according to the two variants shown in Figures 1 and 2. It should be noted that the microfluidic component has the particular advantage of optimizing the quantity of target biological species obtained and concentrating them to the maximum in the lower space 130 of chamber 13 of component 1. For both embodiments, a sample P is first taken. This sample may consist, for example, of leaves of the targeted plant compound or any other sample for which the process of the invention may prove suitable. It may, in particular, consist of cells from a living organism or any other substance containing DNA molecules and capable of exhibiting biological species in both active and dormant forms. E0: This sample is initially processed to obtain an exploitable ECH sample that can be easily separated into two parts A, B of equal quantities. This initial treatment may consist of grinding the sample and then performing an extraction to obtain a liquid ready for analysis. This type of treatment is well known and not detailed in this application. The ECH sample obtained, in liquid form, can then be subjected to the process of the invention. The method of the invention can be implemented using the same microfluidic component 1 or using two identical microfluidic components in parallel. Advantageously, the two parts A and B of the ECH sample are injected into two identical components in parallel, and the process steps are performed simultaneously in both components to analyze both parts of the sample. This solution ensures reliable results and minimizes the risk of sample degradation over time. In the two variants described below, in a non-limiting manner, two identical microfluidic components are used in parallel, to analyze separately a first part of the sample and a second part of the sample. In the first variant, with reference to [Fig.1], the process follows the steps below. El: The ECH sample, containing the target biological species, is divided into two parts A, B of equal quantities. E2: The first part A of the sample undergoes no treatment or at most a mild treatment T1. This gentle T1 treatment may consist of a simple wash with a swab, for example, an aqueous swab with mild surfactants (0.01% Tween 20 swab - registered trademark). This gentle T1 treatment must not destroy the target biological species. F3: The second part B of the sample undergoes an active treatment T2. This active T2 treatment breaks down the DNA molecules of the active F2 form of the biological species. After purification (see below), these molecules will not be retained by the filter of the microfluidic component 1 and will be removed from chamber 13. This active T2 treatment can be chosen from the following treatments or a combination of at least two of these treatments: A chemical treatment, A biochemical treatment, A heat treatment, An optical treatment, A mechanical treatment. The active treatment T2 is chosen to be suitable for destroying the active form F2 of the targeted biological species in the ECH sample and for maintaining the form dormant F1 of the targeted biological species. Depending on the biological species and its active form, the most effective active treatment possible is therefore chosen. In the case of chemical or biochemical treatment, it is therefore a matter of choosing a substance that will break the DNA molecules of the active F2 form of the targeted biological species and adding it in sufficient quantity to the second part B of the sample to obtain this destruction, while keeping intact those of the biological species in its dormant F1 form. In the case of heat treatment, the second part B of the sample will be heated to a sufficient temperature and for a sufficient duration to break the DNA molecules of the active F2 form of the target biological species, while keeping intact those of the biological species in its dormant F1 form. In the case of optical treatment, the second part B of the sample is illuminated with a light signal at a given wavelength, at a given intensity and for a sufficient duration to break the DNA molecules of the active F2 form of the biological species, while keeping intact those of the biological species in its dormant F1 form. In the case of mechanical treatment, it will be a matter of applying sufficient mechanical pressure to the biological species of the second part B of the sample, for example by performing a lysis, to break the DNA molecules of the active form F2 of the biological species, while keeping intact those of the dormant form F1 of the biological species. E4: The first portion A of the treated sample is injected into the first microfluidic component through its first channel. The biological species present in this first portion of the sample remain trapped in the lower space 130 of the chamber 13 of the microfluidic component, thus allowing their concentration. They are then advantageously purified by injecting a buffer liquid through the first channel of the component; this liquid passes through chamber 13 and filter 16 and is then evacuated via the second channel 15. ES: The second part B of the treated sample is injected into the second microfluidic component through its first channel 14. As mentioned above, if the biological species is initially present in its active form, the F2 DNA molecules broken during the active processing step are not retained by the component's filter 16 during the purification step. They are therefore removed by elution, passing through filter 16 and then through the second channel 15 of the microfluidic component. The other biological species present in this second part B of the sample remain trapped in the lower space 130 of the chamber 13 of the microfluidic component, thus allowing their concentration. The remaining biological species are then advantageously purified by injecting a buffer liquid through the first channel of the component; this liquid passes through the chamber and filter 16 and is then discharged via the second channel 15. E6: This is a biomolecular amplification detection step to quantify the target biological species present in the first part A of the biological sample. This step is advantageously implemented directly in the microfluidic component. E7: This is a biomolecular amplification detection step to quantify the target biological species present in the second part B of the biological sample. This step is advantageously implemented directly in the microfluidic component. E8 / E9: This is the results processing step. It involves comparing the quantity Q1 of biological species obtained in the first part of the sample and the quantity Q2 of biological species obtained in the second part of the sample. If Q1 and Q2 are negative: we deduce that the biological species sought is absent from the initial sample. If Q1=Q2: We can deduce that the targeted biological species is indeed present, but only in its dormant form F1 in the initial sample. If the active form F2 had been present, the active treatment T2 performed on the second part of the sample should have eliminated it, and the two quantities Q1 and Q2 determined would then be distinct. If Q1>0 and Q2 <QI : Cela signifie que l'espèce biologique est présente sous sa forme active F2 dans l'échantillon ECH de départ. En effet, le traitement actif T2 a permis de casser les molécules d'ADN de la forme active de l'espèce biologique, celles-ci (référence F2") ont été évacuées du composant micro-fluidique 1 car non retenues par le filtre 16. La quantité Q2 d'espèces biologiques présentes dans la deuxième partie B de l'échantillon est donc plus faible que la quantité Q1 d'espèces biologiques présentes dans la première partie À de l'échantillon. In the second embodiment illustrated in [Fig. 2], two identical microfluidic components are also used in parallel. Furthermore, the mild treatment T1 and the active treatment T2, performed respectively on the first part A of the sample and the second part B of the sample, are implemented directly in each of the two microfluidic components used in parallel. The gentle T1 treatment is thus carried out in the first microfluidic component. The T2 active treatment is implemented in the second microfluidic component described above. This option is particularly advantageous when the active treatment involves the lysis of biological species and / or treatment thermal by heating, since the micro-fluid component 1 is directly capable of implementing these treatments. Apart from these specific features, steps E1 to E9 described above and implemented in this second embodiment are identical to those described for the first embodiment. The analysis of the results is identical to that described above. Example : The principle of the invention is demonstrated on samples of "Bacillus subtillis (hereinafter Bs)" bacteria which exist in the form of vegetative bacteria (active form) or more resistant spores (dormant form). It is known that spores and vegetative bacteria are not at all equally susceptible to heat treatment. This treatment is even used to eliminate all traces of bacteria in spores. To verify the principles of the invention, two samples were therefore used: A first sample containing only Bs spores; A second sample containing only the vegetative (active) form of Bs; For each of the two samples, the steps are then as follows: The sample is divided into two parts, first part A and second part B, of 0.5mL each. The first part A of the sample undergoes no treatment or at most one gentle treatment with no effect on the target biological species present. The second part B of the sample undergoes active treatment in the form of a heat treatment. The sample is heated to 80°C for 10 minutes. The two parts of the sample are injected into two micro-components distinct fluidics. In each of the two microfluidic components, a lysis of the biological species present in the lower space of the chamber. In each of the microfluidic components, elution using a qPCR mix (25uL) PCR detection of the two eluates to obtain the two quantities of target biological species, Q1 and Q2 for each of the two parts of the sample. Analysis of the results according to the principle already described above. The results obtained are summarized in the table below: Part B | Conclusion Sample #1 jo Estimate at 10000 Ct 25.2 qPCR* Quantity |10080 30.2 32.0 Sample #2 [Quantity [346 |102 100% active forms. Estimated at 10,000 Bs / 50Oul. 1 / 3 dormant forms and 2 / 3 active forms. Estimated at 350 Bs / 50Oul. #*Ct=qPCR threshold cycle. In sample #1, it was observed that the bacteria were broken down by the heat treatment; the DNA molecules were not retained in the microfluidic component, which explains the negative detection. We conclude that sample #1 of LO 000 Bs bacteria / 500uL consists solely of the biological species Bs in its vegetative (active) form. For sample #2, a total of approximately 350 bS / 500 yL was obtained, and after heat treatment, the quantity present was significantly reduced. Therefore, we conclude that 2 / 3 of the biological species Bs were present in their vegetative (active) form in this sample #2. In summary, the invention offers numerous advantages, including: The detection principle is simple to implement, requiring no bulky equipment; I use classic detection methods; It is particularly reliable and does not require the execution of mathematical models. complex matics; It is easily adaptable to different types of biological species;

Claims

Demands

1. Method for analyzing a sample (ECH) to detect the presence of a biological species in a first form, called the active form (F2), in said sample, said sample comprising said species biological in a second form, called dormant form (F1) of said biological species, distinct from the active form (F2), characterized in this that, starting from the same sample, it includes steps of: Separation of said sample (ECH) into two parts (A, B) distinct identical volumes, called first part (A) of the sample and second part (B) of the sample, Gentle treatment (T1) or no treatment of the first part (A) of the sample, First concentration of the biological species contained in the first part (A) of the sample, First quantitative detection of said biological species in the first part (A) of the sample in order to de- complete a first quantity (Q1) of biological species in the first part (A) of the sample, Active treatment (T2) of the second part (B) of the sample, Second concentration of biological species contained in the second part (B) of the sample, Second quantitative detection of the said biological species in the second part (B) of the sample in order to de- complete a second quantity (Q2) of biological species in the second part of the sample, Comparison between the first quantity (Q1) obtained and the second quantity (Q2) obtained, in order to deduce the presence or the absence of the active form (F2) of the biological species in the sample.

2. The method according to claim , characterized in that the active treatment of the second part (B) of the sample is chosen from one or several of the following treatments, or a combination of at least two of these treatments: Chemical treatment, Biochemical treatment Thermal treatment, Mechanical treatment, Optical treatment.

3. The method according to claim 2, characterized in that the active treatment is carried out by lysis of biological species against a rough surface.

4. A method according to claim 2 or 3, characterized in that the active treatment is a heat treatment and in that it consists of a heating by means of heating the second part of the sample at a given temperature and for a determined duration.

5. A method according to any one of claims 2 to 4, characterized in that the active treatment is a chemical or biochemical treatment and in that it consists of adding a substance that destroys the active form of the biological species in the second part of the sample, with a view to break its outer membrane and release its nucleic acids.

6. A method according to any one of claims 2 to 5, characterized in that the Active treatment is an optical treatment and in that it consists of exposing the second part of the sample to a light signal emitted at a given wavelength and for a determined duration.

7. A method according to any one of claims 1 to 6, characterized in that the the first concentration and the second concentration are put into works by employing a microfluidic component that includes: A case with at least one opening, A first channel (14) provided in the said box, A second channel (15) provided in said casing, A chamber (13) into which the first canal opens and the second channel, A filter (16) separating said chamber into two spaces distinct so as to define a first space (130) in which opens the said first injection channel and a second space (131) into which the said second canal opens, the said filter (16) having a porosity adapted to retain species biologicals present in the sample.

8. A method according to claim 7, characterized in that the first concentration and the second concentration are implemented simultaneously in two identical microfluidic components in parallel.

9. A method according to claim 7 or 8, characterized in that the treatment mild (T1) and / or said active treatment (T2) are implemented in said microfluidic component.

10. A method according to any one of claims 1 to 9, characterized in that the first quantitative detection and second quantitative detection are achieved through biomolecular amplification.

11. A method according to any one of claims 1 to 10, characterized in that the sample is obtained after a grinding and extraction step sample to be analyzed.