Method for detecting an allergenic, contaminating, or fraudulent species in an agri-food sample

Abstract

The invention relates to a method for detecting a target species, such as an allergen, contaminant, or fraudulent substance, in an agri-food sample (ECH). The method comprises: a chemical attack step (E2) by mixing the agri-food sample (ECH) with a corrosive solution (S_corr) to obtain an extract (EX) in solution; a first analysis (A1) comprising a detection step (E41) of the DNA of the target species present in the extract by sequencing or biomolecular analysis; and / or a second analysis (A2) comprising a detection step (E51) of a protein from the target species in the extract by immunological or spectrometric analysis. Figure to be published with the abstract: Figure 1

Description

Title of the invention: Method for detecting an allergenic, contaminating or fraudulent species in an agri-food sample Technical field of the invention

[0001] The present invention relates to a method for detecting an allergenic, contaminating or fraudulent species in an agri-food sample. State of the art

[0002] The presence of allergenic, contaminating or fraudulent species in an agri-food sample is a sensitive subject.

[0003] For example, allergenic food groups include gluten, soy, sesame, almonds, hazelnuts, etc.

[0004] For example, the contaminating species can be mycotoxins, bacteria, viruses, cells, pesticides, antibiotics, endocrine disruptors.

[0005] For example, species of fraudulent nature are compounds supposedly not present in the food such as almond powder in flour, horse meat mixed with beef, curry spices in saffron.

[0006] The agri-food sector today needs reliable, robust, automated, rapid, easy-to-implement tests that can be carried out directly on production sites. Similarly, to enable them to detect the presence of allergenic or contaminating species in their food, users must have access to high-performance portable devices that implement a reliable and rapid test.

[0007] Allergenic or contaminating agri-food species: - Are mainly proteins or small molecules sometimes degraded by manufacturing / transformation processes; - Are inserted into complex matrices; - Change over the life of the product and its possible transformation (cooking, freezing, etc.); - Can be introduced accidentally in small quantities;

[0008] As a reminder, schematically, a DNA molecule will be transcribed into RNA which will then be translated into protein. In theory, the same type of information is found on both the initial DNA and the protein. However, for various reasons, this is not always the case: - There are a multitude of possible transcripts from the same DNA; - There are a multitude of possible translations into protein from RNA; - Manufacturing processes (heating, roasting, freezing, exposure, mixing with other substances, etc.) are likely to modify proteins, these processes being able to eliminate an allergenic power or, on the contrary, reveal and make accessible an allergenic zone of the protein;

[0009] The implementation of a reliable and rapid detection method can therefore prove to be more complex than one might think.

[0010] Many methods and devices have already been proposed in the state of the art. This is the case in patent application WO2017 / 059103A1, WO2008 / 110655A1, US2016 / 266083A1 and patent US7585529B2.

[0011] Some of these prior documents propose solutions for detecting allergenic species by biomolecular analysis, others solutions for detecting allergenic species by immunological analysis.

[0012] Detection methods by biomolecular analysis generally have the following disadvantages: - They are solely dedicated to biomolecular analysis; - They can be toxic; - They must be implemented at a higher temperature than the room temperature; - They require a centrifuge and specific thermocycling, heating and fluorescence detection equipment, which makes them difficult to implement in the field; - They require a grinding step of the sample taken, resulting in a potential loss of sensitivity to detection, by dilution of the traces in a large ground material;

[0013] Detection methods by immunological analysis often have the following disadvantages: - They are only dedicated to immunological analysis; - They can be toxic, particularly due to the classic use of 2[3-mercaptoethanol known for its CMR effects (carcinogenic, mutagenic and toxic for reproduction); - They can be long; - They may require handling under a hood and the use of a centrifuge; - They can cause inactivation of the antibody added for detection;

[0014] The aim of the invention is to propose a method for detecting an allergenic, contaminating or fraudulent species in an agri-food sample which can resolve one or more of the drawbacks listed above and observed in prior methods.

[0015] The method of the invention will in particular make it possible to carry out a detection of an allergenic, contaminating or fraudulent species in an agri-food sample which is reliable, simple and quick to implement, in particular directly in the field. Statement of the invention

[0016] This aim is achieved by a method for detecting a target species, of the allergenic, contaminating or fraudulent type in an agri-food sample, said method comprising: - A chemical attack step by mixing said agri-food sample with a corrosive solution in order to obtain an extract in solution; - A first analysis comprising a step of detecting the DNA of the target species present in said extract by sequencing or biomolecular analysis, and / or - A second analysis comprising a step of detecting a protein from the target species in said extract by immunological or spectrometric analysis.

[0017] According to a particular feature, a first part of the extract is used for the step of detecting the DNA of the target species and a second part of the extract is used for the step of detecting the protein from the target species.

[0018] According to another feature, the method may comprise a step of filtration of said extract present in solution to recover a lysate.

[0019] According to another feature, the method comprises a step of taking said agri-food sample and the chemical attack step is carried out on said agri-food sample without an intermediate step of mechanical grinding of said agri-food sample.

[0020] According to another feature, the first analysis includes a DNA purification / extraction step.

[0021] According to another particularity, the step of detecting the DNA of the target species comprises the implementation of a q-PCR type method, or by isothermal amplification of the LAMP or RPA type.

[0022] According to another feature, the second analysis comprises a step of solubilization and / or denaturation and / or reduction of the proteins present in the extract obtained after the chemical attack step.

[0023] According to another feature, the step of detecting a protein from the target species is carried out by ELISA or EIA type test.

[0024] According to another feature, the agri-food sample is mixed with the corrosive solution in a ratio of a volume of sample to a volume of corrosive solution which is between 0.1 and 1000.

[0025] According to another feature, the agri-food sample is mixed with the corrosive solution in a ratio of a volume of sample to a volume of corrosive solution which is between 1 and 100.

[0026] According to another feature, the agri-food sample is mixed with the corrosive solution in a ratio of a volume of sample to a volume of corrosive solution which is between 1 and 10.

[0027] According to another feature, the corrosive solution comprises chaotropic agents, acidic agents, basic agents, oxidizing and / or detergent solutions, and / or salts.

[0028] According to another feature, the corrosive solution comprises one or more of the components from the following list: - Urea, - Guanidine salts, - Sodium perchlorate or hypochlorite, - Potash, - Soda, - Sulfuric acid, hydrochloric acid, nitric acid, - Hydrogen Peroxide, - Detergent or Surfactant (Tween, Triton, SDS), - Sodium or Potassium Chloride.

[0029] The invention also relates to a device for detecting a target species, of the allergenic, contaminating or fraudulent type in an agri-food sample, said device being capable of implementing the method as defined above and comprising a housing housing a receptacle intended to receive the agri-food sample taken, a hermetic container intended to receive the corrosive solution, a human-machine interface and a central unit equipped with software modules configured to: - Order a mixture of the corrosive solution with the food sample taken; - Order an execution of the first analysis by detection of the DNA of the selected target species; and / or - Order an execution of the second analysis by detection of the protein of the selected target species; - Recover and analyze the first measurements taken during the first analysis; - Recover and analyze second measurements taken during the second analysis; - Determine the presence of the target species based on the first and second measurements collected and analyzed; - Control the human-machine interface to display an analysis result;

[0030] It should be noted that it may be particularly relevant to implement the two types of analysis in parallel and that it is even more interesting when this is carried out from the same starting sample.

[0031] It should be remembered that: - The presence of DNA from the allergenic species definitely indicates the presence of the species. However, proteins derived from this DNA may potentially not be allergenic, or may not exist at all (no translation and / or no transcription). - Detecting the presence of proteins from an allergenic species only allows targeting a few specific areas that correspond to the epitopes of the protein recognized by the antibodies that allow the assay. In other words, a protein may very well have an allergenic character but this will not necessarily be detected if no antibodies targeting these allergenic areas are available.

[0032] For example, for the allergenic species of gluten type: - A PCR analysis allows the quantification of gluten DNA, or more precisely the DNA of wheat, barley, rye and oats. - An ELISA-type immunological test can measure prolamins, proteins that make up gluten (gliadin for wheat, secalin for rye, hordein for barley) containing the peptide sequences likely to trigger an allergic reaction in people.

[0033] The two approaches therefore make it possible to correlate the data obtained after the PCR analysis and the ELISA test and to compare the potential differences in quantification between these two approaches.

[0034] Even if in certain cases, a single analysis may prove sufficient, the implementation of the two analyses in parallel may therefore prove particularly relevant. Brief description of the figures

[0035] Other characteristics and advantages will appear in the detailed description which follows given with reference to the appended drawings in which:

[0036] [Fig.l] - [Fig.l] schematically represents the principle of the process of the invention;

[0037] [Fig.2] - [Fig.2] illustrates the principle of chemical attack carried out on a heterogeneous agri-food sample such as seeds;

[0038] [Fig.3] - [Fig.3] schematically represents the architecture of a device capable of implementing the method of the invention;

[0039] Detailed description of at least one embodiment

[0040] The method for detecting an allergenic species, a contaminating species or a fraudulent species is implemented on an ECH agri-food sample.

[0041] In the remainder of the description, we will refer to the target species to refer to the allergenic species, the contaminating species or the fraudulent species sought.

[0042] This agri-food sample ECH can be of any type, for example seeds, flour, a paste (for example spread) or be formed from any sample of an agri-food matrix produced on a production line or directly by the final consumer during a meal (e.g.: finished product such as Bolognese sauce, cassoulet, etc.). It will be seen that the method of the invention is of particular interest when the agri-food matrix is ​​heterogeneous (seeds, pieces, etc.).

[0043] The method of the invention consists in particular in implementing a step of obtaining an extract in solution which can be used both for: - A first analysis by detection of the DNA of the target species present in said extract, by sequencing or biomolecular analysis, and / or - A second analysis by detection of a protein from the target species in said extract, by immunological or spectrometric analysis.

[0044] With reference to [Fig.l], after a step E1 of sampling the agri-food sample ECH (for example on a FAB production line), the method thus comprises a preparation step E2 common to the two types of analyses A1, A2 which can be carried out subsequently, whether by detecting the presence of the DNA of the target species or by detecting the presence of a protein from the target species.

[0045] The preparation step E2 consists of a chemical attack on the agri-food sample ECH taken, by mixing said agri-food sample with a corrosive solution S_corr in order to obtain an extract EX in solution.

[0046] By way of example, the corrosive solution S_corr may comprise chaotropic agents, acidic agents, basic agents, oxidizing and / or detergent solutions, and / or salts.

[0047] The corrosive solution may comprise one or more of the following components: Urea, Guanidine salts, Sodium perchlorate or hypochlorite, Potash, Soda, Sulfuric acid, hydrochloric acid, nitric acid, Hydrogen Peroxide, Detergent or Surfactant (Tween, Triton, SDS), Sodium or Potassium Chloride.

[0048] According to a particular feature, the agri-food sample ECH is mixed with the corrosive solution S_corr in a ratio of a volume of sample to a volume of corrosive solution which is between 0.1 and 1000, preferably between 1 and 100 and preferably between 1 and 10, ideally equal to 4.

[0049] Ideally, a volume of sample is therefore diluted in four volumes of corrosive solution.

[0050] In some cases, the quantities to be detected are very low in the ECH agri-food sample taken and may be present only in trace amounts. Carrying out a chemical attack on the agri-food sample makes it possible to recover the dust and over-represent the traces of the target species present in the agri-food sample.

[0051] This is particularly effective when the ECH agri-food sample is of a heterogeneous type, for example composed of seeds or pieces. In this particular case, the chemical attack will make it possible to attack the seeds or pieces on the surface to detach the traces of the target species and thus make them more easily detectable. This principle is notably illustrated by [Fig.2].

[0052] On this [Fig.2]

[0053] At T0: the seeds G are taken from a receptacle 1;

[0054] ATI: The corrosive solution is added. The arrows F illustrate the principle of attack chemical on seeds G.

[0055] At T2: Thanks to the chemical attack, the traces 10 and dust are detached from the surface of the seeds G allowing an over-representation.

[0056] According to a particular aspect of the invention, when the agri-food sample ECH taken is in a heterogeneous state, there is therefore no need to grind it before the preparation step E2 by chemical attack, which makes it possible to save time over the entire analysis process while improving the detection efficiency.

[0057] After preparation step E2, an extract in solution (EX - E3) is obtained.

[0058] This extract in EX solution can then be used both for the first analysis A1 by detection of the DNA of the target species and for the second analysis A2 by detection of a protein of the target species.

[0059] According to a principle of the invention, it is in fact possible to implement the two types of analysis in parallel from the same extract obtained after the chemical attack step. Of course, it will also be possible to implement one or the other of the two types of analysis.

[0060] The analysis of the presence of the DNA of the target species can then be carried out by any known solution. It can include a step of extraction / purification E40 of the DNA molecules present in the solution extract obtained. This extraction / purification step can for example consist of a precipitation of the DNA present in the solution extract. This precipitation step can be carried out by any known method, for example alcoholic precipitation, in the cold or in the presence of salts.

[0061] Once the DNA has been extracted, the step E41 of detecting the DNA of the target species can be carried out by sequencing or by biomolecular analysis. In a non-limiting manner, the biomolecular analysis can be carried out according to conventional techniques of the qPCR ("Quantitive Polymerase Chain Reaction") type or by isothermal amplification of the LAMP (Loop mediated isothermal Amplification) or RPA ("Recombinase Polymerase Amplification") type.

[0062] The analysis of the presence of protein of the target species can also be carried out by any known solution. In a non-limiting manner, it can include a prior step E50 of solubilization and / or denaturation and / or reduction of the proteins present in the extract EX obtained after the chemical attack step. This step E50 can be carried out by any known method, by adding an alcohol (ethanol), by adding a chaotropic agent (for example Guanidine), by adding a reducing agent (for example Dithiothreitol DTT).

[0063] Subsequently, after extraction of the proteins from the target species, the detection step E51 can be carried out by immunological or spectrometric analysis. The immunological analysis can consist of a conventional immunological test of the ELISA or EIA type.

[0064] Optionally, the extract in solution EX which is obtained in step E3 can be filtered during a filtration step E30 through a filter FT to retain only a lysate LY which can then be used for a first part for the first Al analysis by detection of the DNA of the target species and for a second part for the second analysis by detection of the protein of the target species according to the two principles mentioned above.

[0065] To validate the relevance of the method of the invention, a first experiment was conducted. A guaranteed gluten-free agri-food sample is spiked with gluten in two different ways: - Addition of a grain of wheat in a 3g sample of seeds which is guaranteed gluten-free; - Adding a wheat grain to a 3g sample of seeds that is guaranteed gluten-free, stirring manually and removing the wheat grain with forceps;

[0066] The two samples are then mixed with the corrosive solution S_corr according to the method of the invention. By then performing a gluten DNA detection by q-PCR, the presence of gluten is detected in both samples, even in the one where the wheat grain has been removed. The traces are therefore detectable, thanks to the chemical action carried out to over-represent the traces.

[0067] It is understood from the above that the method of the invention has the following advantages: - Increased level of sensitivity, obtained in particular by the absence of grinding of the ECH agri-food sample taken and thanks to the implementation of chemical attack which makes it possible to detach the dust and therefore to obtain an over-representation of the traces present; - Quick solution to implement: The preparation of the EX extract to be analyzed is common to both subsequent analyses Al, A2, detection of the presence of DNA or detection of DNA proteins. In addition, there is no need to allow a delay between the ECH sample collection step and the E2 preparation step, which avoids any degradation of the samples. - Easy to implement solution, which can be carried out in the field (production line) or by a user using a suitable device. - Process compatible with all types of agri-food samples (seeds, dough, flour, etc.), even if more favorable to heterogeneous matrices compared to existing conventional methods. - Solution compatible with the dosage of any allergenic species (gluten, soy, sesame, almond, hazelnut, etc.), contaminating species (for example: mycotoxin, bacteria, viruses, cells, pesticides, antibiotics, endocrine disruptors) or fraudulent nature (almond powder with flour or other seeds, horse meat in beef, curry spice mix in saffron, etc.) - Solution compatible with DNA detection methods, such as q-PCR, isothermal amplifications (LAMP, RPA, etc.) and sequencing, and with detection methods by immunological analysis (EIA, ELISA). - Solution allowing both types of analysis (molecular biology and immunology) to be carried out from a single sample, so there is no problem of representativeness, since the two measurements will be obtained from the same sample.

[0068] The method can in particular be implemented in a suitable device, advantageously portable and easy to use whether in the field or by a user who wishes to carry out tests for personal use.

[0069] With reference to [Fig.3], this device 2 can have the following simplified architecture: - An external 20 box; - A receptacle 21 intended to receive the agri-food sample taken; - A container 22 hermetically storing the corrosive solution S_corr; - A human-machine interface (HMI) of the button and / or screen type to control the device 2, arranged on the housing; The human-machine interface may in particular allow the target species sought to be selected. - A central processing unit UC comprising a microcontroller responsible for executing software modules configured to implement the method of the invention;

[0070] The central unit is responsible for: - Receive commands from the human-machine interface to start the detection process; - Order the mixing of the corrosive solution with the ECH agri-food sample taken; - Order the execution of the first Al analysis by detection of the DNA of the selected target species; and / or - Order the execution of the second A2 analysis by detection of the protein of the selected target species; - Recover and analyze the Ml measurements carried out during the first Al analysis; - Recover and analyze the M2 measurements carried out during the second A2 analysis; - Determine the presence of the target species based on the Ml, M2 measurements recovered and analyzed; - Control the human-machine interface (HMI) to communicate an analysis result, for example via a display on a screen;

[0071] Mechanical and pneumatic means are advantageously integrated into the device to allow material transfers during the process.

[0072] Optical means may also be present to carry out q-PCR type analyses for example.

Claims

Claims

1. Method for detecting a target species, of allergenic, contaminating or fraudulent nature in an agri-food sample (ECH), characterized in that it comprises: • A chemical attack step (E2) by mixing said agri-food sample (ECH) with a corrosive solution (S_corr) in order to obtain an extract (EX) in solution; • A first analysis (A1) comprising a detection step (E41) of the DNA of the target species present in said extract by sequencing or biomolecular analysis, and • A second analysis (A2) comprising a detection step (E51) of a protein from the target species in said extract by immunological or spectrometric analysis.

2. Method according to claim 1, characterized in that a first part of the extract (EX) is used for the step of detection (E41) of the DNA of the target species and in that a second part of the extract (EX) is used for the step of detection (E51) of the protein from the target species.

3. Method according to claim 1 or 2, characterized in that it comprises a step of filtration (E30) of said extract present in solution to recover a lysate (LY).

4. Method according to one of claims 1 to 3, characterized in that it comprises a step of sampling (El) of said agri-food sample and in that the chemical attack step (E2) is carried out on said agri-food sample (ECH) without an intermediate step of mechanical grinding of said agri-food sample.

5. Method according to one of claims 1 to 4, characterized in that the first analysis (A1) comprises a step of purification / extraction (E40) of the DNA.

6. Method according to one of claims 1 to 5, characterized in that the step of detecting (E41) the DNA of the target species comprises the implementation of a q-PCR type method, or by isothermal amplification of the LAMP or RPA type.

7. Method according to one of claims 1 to 6, characterized in that the second analysis (A2) comprises a step (E50) of solubilization and / or denaturation and / or reduction of the proteins present in the extract (EX) obtained after the chemical attack step (E2).

8. Method according to one of claims 1 to 7, characterized in that the step of detecting (E51) a protein from the target species is carried out by ELISA or EIA type test.

9. Method according to one of claims 1 to 8, characterized in that the agri-food sample (ECH) is mixed with the corrosive solution in a ratio of a volume of sample to a volume of corrosive solution which is between 0.1 and 1000.

10. Method according to one of claims 1 to 8, characterized in that the agri-food sample (ECH) is mixed with the corrosive solution in a ratio of a volume of sample to a volume of corrosive solution which is between 1 and 100.

11. Method according to one of claims 1 to 8, characterized in that the agri-food sample (ECH) is mixed with the corrosive solution in a ratio of a volume of sample to a volume of corrosive solution which is between 1 and 10.

12. Method according to one of claims 1 to 9, characterized in that the corrosive solution (S_corr) comprises chaotropic agents, acidic agents, basic agents, oxidizing and / or detergent solutions, and / or salts.

13. Method according to one of claims 1 to 9, characterized in that the corrosive solution (S_corr) comprises one or more of the components from the following list: • Urea, • Guanidine salts, • Sodium perchlorate or hypochlorite, • Potash, • Soda, • Sulphuric acid, hydrochloric acid, nitric acid, • Hydrogen peroxide, • Detergent or surfactant (Tween, Triton, SDS), • Sodium or potassium chloride.

14. Device for detecting a target species, of an allergenic, contaminant or fraudulent nature in an agricultural sample. food (ECH), characterized in that it is capable of implementing the method as defined in one of claims 1 to 13 and in that it comprises a housing (20) housing a receptacle (21) intended to receive the agri-food sample (ECH) taken, a hermetic container intended to receive the corrosive solution (S_corr), a human-machine interface (HMI) and a central unit (UC) equipped with software modules configured to: • Order a mixture of the corrosive solution with the agri-food sample (ECH) taken; • Order an execution of the first analysis (AI) by detection of the DNA of the selected target species; and / or • Order an execution of the second analysis (A2) by detection of the protein of the selected target species; • Recover and analyze the first measurements (Ml) taken during the first analysis (Al); • Recover and analyze second measurements (M2) carried out during the second analysis (A2); • Determine the presence of the target species based on the first measurements (Ml) and second measurements (M2) collected and analyzed; • Control the human-machine interface (HMI) to display an analysis result;

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