Digital biomolecular detection and / or quantification using isothermal amplification

Abstract

The present invention relates to a digital method for detecting and / or quantifying at least one target biomolecule in a sample, wherein said biomolecule is selected from DNA, RNA, and protein, based on isothermal amplification. The present invention further relates to various applications and kits of the digital method.

Description

[Technical field]

[0001] The present invention relates to a digital method for detecting and / or quantifying biomolecules such as DNA, RNA, and proteins in a sample, based on isothermal amplification with specific molecular design. The present invention further relates to a method for diagnosing diseases selected from the group including cancer, neurological diseases, cardiovascular diseases, inflammatory diseases, autoimmune diseases, diseases due to viral or bacterial infections, skin diseases, skeletal muscle diseases, dental diseases, and prenatal diseases, as well as a method for agricultural diagnosis. The present invention also relates to a method for detecting biomarkers (biomolecules) in the food and agriculture food sector and in the environment. All such diagnostic and detection methods include using the digital method of the present invention. The present invention also relates to a kit for detecting and / or quantifying at least one target biomolecule, comprising an enzyme, an oligonucleotide, and a partitioning agent. [Background technology]

[0002] Several biomolecules, such as DNA, RNA, or proteins, are used as attractive diagnostic, prognostic, or predictive biomarkers. There is accumulating clinical evidence that such molecules, and especially nucleic acids, are closely related to various diseases (cancer, neurological or cardiovascular diseases, diabetes, etc.). Moreover, such molecules, and especially nucleic acids, are present in body fluids and therefore accessible via minimally invasive liquid biopsies (serum, plasma, urine). Circulating biomarkers can be repeatedly evaluated, allowing regular follow-up of treatment and recurrence, large population screening, or early diagnosis. Detection of these biomolecules is often a challenging task as the results must be accurate for clinical use, therefore, accurate measurement of such biomarkers has become a major bottleneck, prompting the development of sensitive and specific quantitative detection techniques.

[0003] The most commonly used techniques for sensitive detection of proteins are the enzyme-linked immunosorbent assay (ELISA) and, for nucleic acids, the polymerase chain reaction (PCR) for detecting DNA and reverse transcription quantitative polymerase chain reaction (RT-qPCR) for detecting RNA. All these techniques allow specific and sensitive detection, but they have some limitations and need improvement. For example, currently detection of DNA by PCR and related methods requires amplification of the target nucleic acid, which can be prone to non-specific or poor signal amplification, which reduces the specificity and sensitivity of the detection method. Despite the high sensitivity of RT-qPCR with regard to RNA detection, the technique has several drawbacks: the RT step is known to introduce significant bias in quantification (Bustin et al., 2015), primers and probes must be designed for each target, the short length of the targets requires relying on sophisticated designs, thermocycling protocols should be optimized for each assay, the target itself is amplified and thus a deleterious source of carryover contamination (Aslanzadeh et al., 2004), and PCR reactions are known to be inhibited in biological samples (Opel et al. 2010). In addition, the procedure relies on real-time tracking of amplification and requires standard calibration, thus providing only a qualitative estimate of the amount of biomolecules.

[0004] The above mentioned drawbacks can be partially overcome by using digital procedures based on isolating and analysing single nucleic acid molecules in small compartments. Digital techniques, in particular, offer several advantages: i) Compatible with endpoint assays and do not require continuous monitoring of the response; ii) providing absolute quantification without the use of calibration standards; iii) providing ultimate sensitivity; iv) It also improves the sensitivity and specificity of the assay, especially for rare targets in a complex background.

[0005] However, an assay can only be transferred to a digital format if its bulk sensitivity is already higher than the concentration corresponding to a single target per compartment. For example, if the internal volume of the compartments used for the digital procedure is about 1 nanoliter, the concentration of a single molecule in such a compartment is on the order of femtomolar, and the digital assay will require an amplification technique with a detection limit lower than femtomolar. Amplification methods with detection limits higher than femtomolar will not be usable in this situation. Although PCR generally meets the requirement of high sensitivity and provides a powerful method for precise and absolute quantification of biomolecules, especially nucleic acids, the weaknesses of RT-qPCR remain to be evaluated in a digital format (Campomenosi et al., 2016).

[0006] Isothermal alternatives have been proposed (Zhao et al., 2015) that rely on simpler one-step protocols, do not require thermocycling, and are free from reverse transcription (EXPAR, LAMP, RCA, HCR, etc.). Despite their reasonable sensitivity, these techniques suffer from non-specific amplification reactions and are therefore not suitable for digital readout. For example, Zhang et al. (Zhang et al., 2015) clearly demonstrated this limitation for EXPAR (Exponential Amplification), a method well known to be prone to fast non-specific amplification. The authors showed that when adapted to a digital format, blank droplets are amplified only minutes after target-containing droplets. Thus, isothermal nucleic acid amplification methods, although showing good sensitivity in bulk, cannot be transferred to robust digital formats. Thus, robust methods for the digital measurement of enzymes and nucleic acids, particularly microRNAs, are not currently available.

[0007] The object of the present invention is to provide a method that allows absolute quantification and higher sensitivity in detecting biomolecules used as biomarkers, such as biopolymers, in particular nucleic acids and enzymes, which is based on the digitalization of a specific isothermal amplification method, but does not have the drawbacks listed above. Furthermore, the present invention aims to provide such a method that can be easily implemented in a system. Summary of the Invention

[0008] The inventors of the present invention have previously developed a method for eliminating background amplification for detecting nucleic acids (WO2017140815). The inventors of the present invention have surprisingly found that they can successfully digitize this analog method to obtain a digital method that increases the detection sensitivity of biomolecules used as biomarkers and allows one of them to be accurately quantified in bulk measurements without the need for continuous monitoring of the amplification reaction or the use of a calibration curve. Thus, according to a first aspect, the present invention provides a digital method for detecting and / or quantifying at least one biomolecule in a sample, comprising: a) mixing the sample with a mixture comprising a buffer, an enzyme, a first oligonucleotide that is an amplification oligonucleotide, a second oligonucleotide that is a leak absorption oligonucleotide, and a third oligonucleotide that is a target-specific conversion oligonucleotide; b) distributing the mixture obtained in step a) into several compartments, some of the compartments not containing the target biomolecule; c) converting the target biomolecule into a signal, said signal being preferably a single strand of DNA; d) amplifying the signal; and e) detecting and / or measuring the amplified signal in each compartment. The present invention relates to a digital method comprising:

[0009] The main advantage of this method is that it allows rapid and selective detection of biomolecules such as DNA, RNA and proteins, as well as absolute quantification of these biomolecules. In particular, when detecting and / or quantifying RNA, advantages of the method of the present invention compared to RT-PCR analogue methods are that 1) it does not require a reverse transcription step that can interfere with the quantitative nature of the assay, 2) it does not require the high temperatures and temperature cycles required for the PCR step, and 3) isothermal amplification is robust against many chemicals that may be found in crude samples and have been shown to inhibit PCR reactions (Huggett et al. 2008), avoiding cross-contamination issues since the target biomolecules are not amplified.

[0010] The inventors have also demonstrated that the methods of the present invention can be used to directly detect biomolecules from any type of sample that contains biomolecules, such as blood samples and other biological fluids. The specificity, simplicity and rapidity of the method allow it to be used in medical diagnostic procedures, in particular in the diagnosis of diseases such as cancer, neurological diseases, cardiovascular diseases, inflammatory diseases, autoimmune diseases, diseases caused by viral or bacterial infections, skin diseases, musculoskeletal diseases, dental diseases and prenatal diseases. Also, according to a second aspect, the present invention relates to an in vitro method for diagnosing a disease selected from the group comprising cancer, a neurological disease, a cardiovascular disease, an inflammatory disease, an autoimmune disease, a disease caused by a viral or bacterial infection, a skin disease, a musculoskeletal disease, a dental disease and a prenatal disease, comprising using the digital method.

[0011] The specificity, sensitivity, simplicity and rapidity of the digital methods of the invention allow them to be used in agronomic diagnostic methods, in particular for diagnosing diseases caused by biotic stresses, such as infectious and parasitic diseases, or diseases caused by abiotic stresses, such as nutritional deficiencies or adverse environments. According to a third aspect, the present invention also provides a method for producing ... composition comprising the steps of: Diseases caused by biotic stress, preferably of infectious and / or parasitic origin, or Diseases caused by abiotic stress, preferably by nutritional deficiency and / or adverse environment An in vitro method for the agricultural diagnosis of a disease selected from the group comprising: In vitro methods involving the use of the digital methods of the invention are also provided. Kits for carrying out the methods of the invention are also provided.

[0012] Thus, according to a fourth aspect, the present invention provides a kit for detecting and / or quantifying at least one target biomolecule, comprising: a) a mixture of enzymes, preferably selected from the group comprising polymerases, nicking or restriction enzymes, and exonucleases, b) a mixture of oligonucleotides comprising a first oligonucleotide which is an amplification oligonucleotide, a second oligonucleotide which is a leak-absorbing oligonucleotide, and a third oligonucleotide which is a target-specific conversion oligonucleotide, and optionally a fourth oligonucleotide which is a reporting probe; and c) Partitioning agents, preferably water-in-oil emulsions The present invention relates to a kit comprising: DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] As indicated above, the present invention relates to adapting modern background-free amplification chemistry to a digital readout, thus providing absolute quantification of biomolecular targets. In particular, an adaptive background-free amplification chemistry involving the elimination of background amplification in isothermal amplification of biomolecular targets, particularly nucleic acid targets, has been implemented by some of the inventors of the present invention and is disclosed in WO2017140815. Thus, according to a first aspect, the present invention provides a digital method for detecting and / or quantifying at least one biomolecule in a sample, comprising: a) mixing said sample with a mixture comprising a buffer, an enzyme, a first oligonucleotide that is an amplification oligonucleotide, a second oligonucleotide that is a leak-absorbing oligonucleotide, and a third oligonucleotide that is a target-specific conversion oligonucleotide; b) distributing the mixture obtained in step a) into several compartments, some of the compartments not containing the target biomolecule; c) converting the target biomolecule into a signal, said signal being preferably a single strand of DNA; d) amplifying the signal; and e) detecting and / or measuring the amplified signal in each compartment. The present invention relates to a digital method comprising:

[0014] The digital method according to the invention allows the simultaneous detection and / or quantification of one or several biomolecules, said biomolecules having the same or different structures and functions. In the context of the present invention, the term "biomolecule" relates to large macromolecules or biopolymers such as proteins, carbohydrates, lipids, and nucleic acids, as well as small molecules such as primary metabolites, secondary metabolites, and natural products. Biomolecules in the present invention are usually endogenous, but may also be exogenous (e.g., biopharmaceutical drugs). According to one preferred embodiment of the invention, the biomolecule is selected from the group comprising proteins, preferably enzymes, or from nucleic acids.

[0015] The inventors have surprisingly found that coupling enzyme activity with a versatile molecular circuit in which a threshold exponential amplification of DNA signals is performed in a digital bioassay allows enzyme detection and / or quantification with a very high sensitivity compared to other digital enzyme tests in the art (allowing the detection of several enzymes). The circuit includes a module that links target activity to the generation of a short DNA trigger, and a DNA amplification system that produces a detectable readout, in particular a fluorescent readout. Since the enzyme catalytic rate is decoupled from the signal generation, this versatile framework allows digital detection, in particular droplet digital detection of a wide range of DNA processing enzymes using standard microcompartments, in particular microfluidic droplets with internal volumes in the picoliter range. Thus, the method of the present invention allows the conversion of weak catalytic activity associated with a single enzyme into a detectable signal, in particular a fluorescent signal of sufficient intensity for easy detection in microcompartments, in particular microdroplets.

[0016] In the context of the present invention, the term "enzyme" refers to a protein that has a catalytic function and is capable of catalyzing the conversion of a molecular substrate. The group of enzymes that can be detected and / or quantified by the digital method of the present invention is selected from DNA-related enzymes with a wide range of activities, such as nucleases, DNA N-glycosylases, polymerases, ligases, and kinases, or non-DNA-related enzymes. In particular, the enzymes are selected from the group comprising nicking enzymes, restriction enzymes, endonucleases, DNA N-glycosylases, AP-endonucleases, exonucleases RNA / DNA polymerases, ligases, kinases, and methylases. More specifically, the method of the present invention can be carried out to identify and / or quantify nicking enzymes and restriction endonucleases. Specific examples of enzymes that can be detected and / or quantified by the methods of the present invention include, but are not limited to, Nt.BstNBI, RNAseH2. APE-endonuclease 1 (APE-1), uracil DNA glycosylase (UDG), alkyladenine glycosylase (AAG), BsmAI restriction enzyme, poly(A) polymerase (PAP), T4 DNA ligase, and T4 polynucleotide kinase (T4 PNK).

[0017] In the context of the present invention, the term "module" relates to an oligonucleotide or a group of oligonucleotides that perform a specific function, such as signal transduction, amplification, reporting, etc. If a nucleic acid is detected, the module corresponds to an oligonucleotide (template). If an enzyme is detected, the module corresponds to a group of oligonucleotides. In another embodiment, the biomolecular target of the present invention is a nucleic acid, such as DNA, cDNA, RNA, mRNA, microRNA, etc. Even more preferably, the biomolecular target of the present invention is a ribonucleic acid (RNA). In the context of the present invention, the term "nucleic acid" relates to a biopolymer or small biomolecule composed of nucleotides, which are monomers made of three components: a five-carbon sugar, a phosphate group, and a nitrogenous base. If the sugar is the compound ribose, the polymer is RNA (ribonucleic acid), and if the sugar is deoxyribose, which is derived from ribose, the polymer is DNA (deoxyribonucleic acid).

[0018] As mentioned above, the digital methods of the present invention can be used to detect and / or quantify DNA molecules or complementary DNA (cDNA) encoding molecules. Even more preferably, the method of the present invention is used to detect and / or quantify a ribonucleic acid (RNA) molecule selected from messenger RNA (mRNA), small interfering RNA (siRNA), and microRNA (miRNA). According to a most preferred embodiment, the method of the present invention can be used to detect and / or quantify microRNA molecules.

[0019] In the context of the present invention, the term "microRNA" refers to endogenous short non-coding RNA strands having a length of about 22 nucleotides that are involved in the post-transcriptional regulation of gene expression. To carry out the method of the invention, in a first step (step a)), it is necessary to mix a molecular program that allows avoiding background amplification with a biological sample containing the target biomolecule. As used herein, the term "target biomolecule" or "biomolecular target" relates to a biomolecule as defined above which is detected and / or quantified by the method of the present invention.

[0020] These target biomolecules are present in a sample. The sample may be of any type. For example, the sample may be obtained from a subject to be tested, the subject being an animal, preferably a mammal, more preferably a human. Such a sample may also be called a biological sample. In the context of the present invention, the term "biological sample" relates to solid or fluid biological material obtained from a living organism. A solid biological sample may be a cell, a part of a tissue (biopsy), a whole tissue, or an organ, etc. Preferably, the sample used in the method of the present invention is a fluid sample. In the context of the present invention, the term "sample of a biological fluid" or "fluid sample" relates to any sample obtained from a bio-organic fluid produced by a living organism. The biological fluid is selected from the group comprising extracellular fluid, intravascular fluid, interstitial fluid, lymphatic fluid and transcellular fluid.

[0021] In particular, the sample of biological fluid is selected from the group comprising blood and blood components, urine, saliva, etc. In a more preferred embodiment, the sample of biological fluid is a blood sample or a blood component sample. By "blood sample" or "blood component sample" is meant whole blood or one of its components, in particular selected from the red blood cell fraction, the white blood cell fraction, platelets, plasma, or serum. In another embodiment of the invention, the sample may be obtained from a non-living organism. For example, the sample may be obtained from air, water, soil, digestive products, etc. The type of sample depends on the application of the digital method of the invention. According to the invention, said sample contains or is likely to contain biomolecules. As indicated above, a sample containing a target biomolecule is mixed with a molecular program in step a) of the digital method of the invention.

[0022] The molecular program used in the method of the present invention is disclosed in detail in International Publication No. WO2017140815, the contents of which are incorporated herein by reference. In the context of the present invention, the term "molecular programming" relates to the design of biomolecular circuits to perform information processing tasks in vitro. Molecular programming is based on predictable Watson and Crick base pairing that endows DNA with inherent programmability that allows the rational design of molecular circuits. The molecular program used in the method of the invention is based on a versatile molecular programming language called PEN-DNA toolbox (Polymerase Exonuclease Nickase-Dynamic Network Assembly, Montagne et al., 2011), developed by some of the inventors. The topology of the network is defined by a set of short oligonucleotides (templates). The network is interpreted by a mixture of enzymes (polymerases, exonucleases and nickases, or restriction enzymes), which process the information flux by producing and degrading DNA strands, which in turn activate or inhibit other nodes of the network.

[0023] According to one embodiment of the digital method of the invention, the enzymes in the mixture of step a) are selected from the group comprising a polymerase, a nicking enzyme, a restriction enzyme and an exonuclease, the polymerase, the nicking enzyme and the restriction enzyme driving the isothermal amplification and the exonuclease being able to avoid saturation of the system. In particular, the polymerases used in the digital method of the present invention are Bst2.0 DNA polymerase, Bst large fragment DNA polymerase, Klenow fragment (3'->5' exon - )(Klenow DNA polymerase (Klenow(exo - ) or Klenow polymerase), Phi29 DNA polymerase, Vent (exo - ) DNA polymerase, more particularly the polymerase is selected from the group consisting of Vent(exo) - ) DNA polymerase (purchased from New England Biolabs (NEB)). According to one embodiment of the method of the invention, one or more polymerases can be used simultaneously. According to a preferred embodiment, the polymerase Vent(exo - ) DNA polymerase and Klenow fragment (3'->5' exo -) are used together. Klenow polymerase and Vent(exo - The simultaneous use of Vent(exo) DNA polymerase allows for an increase in the rate of the amplification reaction, especially when the biomolecule to be detected is RNA. The increase in rate is essentially due to the Klenow polymerase, but in order to avoid the appearance of non-specific amplification products, the Vent(exo) - ) DNA polymerase is still required.

[0024] In order to obtain an increased rate of the amplification reaction, the concentration of Klenow polymerase is comprised between 1 and 50 u / mL, in particular between 8 and 25 u / mL, and even more particularly, this concentration is limited to 16 u / mL. The nicking enzyme is selected from the group including Nb.BbvCI, Nb.BstI, Nb.BssSI, Nb.BsrDI, in particular the nicking enzyme is Nb.BsmI and / or Nt.BstNBI (purchased from New England Biolabs (NEB)). More than one nickase can be used simultaneously. According to one embodiment of the method of the present invention, the nicking enzyme may be replaced by a restriction enzyme. In contrast to the nicking enzyme that cuts a single strand, the restriction enzyme cuts a double strand. Therefore, when a restriction enzyme is used instead of the nicking enzyme, the template used in the method of the present invention needs to be protected. This protection can be obtained, for example, by chemically modifying the template. Such modifications include backbone modifications such as phosphorothioate linkages.

[0025] The exonuclease used in the digital method of the present invention is, for example, RecJ f , exonuclease I, exonuclease VII, in particular the exonuclease is ttRecJ exonuclease obtained according to the protocol described by Yamagata (Yamagata et al., 2001). More than one exonuclease can be used simultaneously. The buffer used for the mixture of enzyme and oligonucleotide is adapted to the selected oligonucleotide template. Those skilled in the art will be able to adapt conventional buffers to specific molecular designs. Examples of such buffers are also provided in the experimental section of this application. For example, in a preferred embodiment, the reaction buffer comprises 20 mM Tris HCl pH 7.9, 10 mM (NH4)2SO4, 40 mM KCl, 10 mM MgSO4, 50 μM each dNTP, 0.1% (weight / solution) Synperonic F104, 2 μM netropsin, 200 μg / mL BSA.

[0026] According to one embodiment, in particular when an enzyme is to be detected and / or quantified, the mixture of step a) comprises using at least one module. Each oligonucleotide or module of the mixture of step a) of the digital method of the invention ultimately has a specific function that allows the conversion of the target biomolecule and the amplification of the signal obtained without simultaneously inducing background amplification, i.e. the amplification reaction of the signal that occurs in the absence of the target biomolecule. Thus, the mixture comprises a first oligonucleotide or a first module which is an amplification oligonucleotide, a second oligonucleotide or a second module which is a leak-absorbing oligonucleotide, and a third oligonucleotide or a third module which is a target-specific conversion oligonucleotide. In particular, the mixture comprises a third module for detecting and / or quantifying an enzyme.

[0027] In the context of the present invention, the term "amplification oligonucleotide" or "autocatalytic template" or "aT" refers to an oligonucleotide that can exponentially amplify a trigger sequence. Examples of oligonucleotides that can function as amplification oligonucleotides are shown in Table 1 below. In the context of the present invention, the term "leak-absorbing oligonucleotide" or "pseudo-template" or "pT" refers to an oligonucleotide that binds to the amplified sequence more strongly than the autocatalytic template, but only adds a few nucleotides to its 3' end, thus inactivating the amplified sequence with respect to further priming to the autocatalytic template (since its 3' end is now mismatched to the autocatalytic template). The leak-absorbing oligonucleotide allows the avoidance of non-specific amplification, also referred to herein as background amplification (i.e. amplification occurring in the absence of an amplified signal sequence). The leak-absorbing oligonucleotide drives the inactivation of the trigger synthesized from the leaky reaction. The pseudo-template, just like the autocatalytic template, needs to be protected from degradation. This is done using a few phosphorothioate modifications (or other exonuclease blocking capabilities such as biotin-streptavidin modifications, inversions or modified nucleotides) at the 5' end. Examples of leak-absorbing oligonucleotides are shown in Table 1 below.

[0028] In the context of the present invention, the term "target-specific conversion oligonucleotide" or "conversion template" or "cT" refers to an oligonucleotide that converts a target biomolecule into a universal trigger sequence (also referred to herein as "signal" or "signal sequence"). The conversion template, like the autocatalytic template, may or may not be protected from degradation. The term "conversion module" refers to a group of conversion oligonucleotides as defined herein. According to one embodiment of the digital method of the present invention, a first oligonucleotide comprises a partially repeated structure that includes a nicking enzyme recognition site, and a second oligonucleotide is capable of binding to the products of polymerization along the first oligonucleotide, extending them, inactivating them and slowly releasing them, thereby inducing a threshold.

[0029] According to another embodiment of the digital method of the present invention, particularly when the target is a DNA or RNA sequence, the 3' side of the third oligonucleotide is capable of binding to the target sequence, and upon polymerization and nicking, the third oligonucleotide outputs a sequence capable of activating the first oligonucleotide above a threshold that is adjusted by controlling the concentration of the second oligonucleotide. According to one embodiment of the digital method of the present invention, the 3' end of the first oligonucleotide exhibits a reduced affinity for the amplified sequence. In yet another embodiment, the 3' end of the second oligonucleotide is complementary to the sequence amplified by the first oligonucleotide, and the 5' end of the second oligonucleotide serves as a template for adding an inactivated tail to the amplified sequence.

[0030] In one embodiment for carrying out the digital method, the concentrations of the first and second oligonucleotides are selected such that the reaction of the first oligonucleotide is faster than the reaction of the second oligonucleotide with a high concentration of the amplified sequence, but the reaction to the second oligonucleotide is faster than the reaction of the first oligonucleotide with a low concentration of the amplified sequence, thereby effectively eliminating amplification unless the stimulation threshold is exceeded. In another embodiment, the third oligonucleotide (conversion template or module) can be designed in different formats depending on the nature of the biomolecule to be detected and / or quantified. In particular, when detecting an enzyme, the conversion template (module) can be designed in different formats depending on the nature of the enzyme to be detected. For example, RNASe H and AP-endonuclease (APE-1) can be detected by introducing ribonucleotides and abasic sites (AP), respectively, into the stem structure of the sensing template. Uracil DNA glycosylase (UDG) can be detected, for example, by replacing the AP site with a deoxyribouridine moiety. Detection of restriction enzymes can be achieved by adding recognition sites to the 5' portion of the sensing template. The conversion template can be designed to serve as a substrate for the target enzyme and includes one or more chemical modifications, such as specific sequences or structural features such as phosphate modifications, modified nucleobases, sugar or backbone moieties, wobble, mismatch, blunt or overhanging ends, flap sequences, etc.

[0031] In yet another embodiment, more than one conversion oligonucleotide can be used, especially for detecting and / or identifying the enzyme. Such templates can be designed as described above. The use of modules composed of two or more oligonucleotides is possible, for example, when the target enzyme is specific for a double-stranded DNA substrate or when the target enzyme uses multiple nucleic acid strands as substrates. Such enzymes include some DNA N-glycosylases (e.g., alkyladenine glycosylases) or DNA or RNA ligases or kinases, transposases, DNA or RNA nucleases. In addition, it is possible to design conversion modules such that the target enzyme induces a cascade of modifications that ultimately lead to the production of a trigger, initiating amplification. As an example, the enzyme poly(A) polymerase (PAP) can be detected and quantified using two oligonucleotides. The first oligonucleotide serves as a substrate for polyadenylation, and the second oligonucleotide serves as a template to produce a trigger output using the newly polymerized poly(A) tail as input.

[0032] According to one embodiment of the digital method of the present invention, a fourth oligonucleotide is added which is a reporting probe. In the context of the present invention, the term "report probe" or "report template" or "rT" refers to an oligonucleotide that converts the presence of a trigger sequence into a detectable signal. Such detectable signals refer, for example, to particle agglutination, medium gelation, electrochemical signals, or chemiluminescent signals, preferably fluorescent signals. Thus, the report probe is preferably a fluorescent probe. In one embodiment, the report probe detects a signal chain amplified by an amplification oligonucleotide.

[0033] In yet another embodiment, the report probe is a self-complementary DNA strand modified at both ends with a fluorophore and / or a quencher. As used herein, the term "self-complementary" means that two different parts of the same molecule can hybridize with each other due to base complementarity (AT and GC). In the present case, the two ends of the single-stranded probe (a few nucleotides at the 3' and 5' parts) can hybridize with each other and induce the quenching of the fluorophore by the quencher. The amplified sequence can bind to a portion of the report template, leading to the opening of the stem-loop structure, thus enhancing the fluorescent signal. The reporting probe may also include a loop that contains a nicking recognition site.

[0034] Compared to PCR probes (used for detection) used in the prior art that must be designed (sequence) and optimized (length) for each target, the reporting probes used in the methods of the present invention are modular, meaning that the reporting probes used in the methods of the present invention can be used for any target with signal transduction by the appropriate bistable module (autocatalytic template + pseudotemplate). Examples of different oligonucleotides (templates) used in step a) of the digital method of the invention are given in Table 1 below and may also be given in the Examples section of this application.

[0035] [Table 1-1] [Table 1-2] [Table 1-3]

[0036] In Table 1 above, biotin and bioteg refer to biotinylated synthons using the aminoethoxy-ethoxyethanol linker and the longer triethylene glycol linker, respectively. * " indicates phosphorothioate backbone modification, and "p" indicates 3' phosphate modification. In SEQ ID NOs: 16-23 and 30, thymidine (T) is replaced with deoxyuridine (U) to avoid nicking of the polymerization product of the trigger on the template. Atto633, FAM, Cy5, Hex, Cy3.5, BMN3 are fluorophores, and BHQ1, BHQ2, BMNQ530 are quenchers.

[0037] Based on the sequences listed above and the examples described herein, various combinations of templates can be used to carry out the digital method of the invention. Starting from this description and general knowledge of nucleotide design, the skilled artisan will be able to determine other templates and other combinations to carry out the method of the invention on any target of interest.

[0038] In particular, the choice of the template sequence depends on the experimental parameters such as the reaction temperature, the rate and specificity of the enzyme used, in particular the nickase. Preferably, amplification templates containing Nb.BsmI nickase sites and templates reacting with Nt.BstNBI can be used. According to one embodiment of the method of the present invention, the templates, preferably four templates (autocatalytic template, conversion template, pseudotemplate, and reporting template), are linked by the sequence of an amplified sequence, the "universal trigger." FIG. 3 shows one embodiment of the circuit connectivity in the molecular program preferably used in the digital method of the present invention. The universal signal amplification part generates a bistable node, i.e. an amplification system that allows two states: a non-productive state in the absence of a trigger and a productive state where amplification is initiated when the concentration of the trigger exceeds a given threshold. This bistable node is composed of two types of templates: an autocatalytic template (aT), which is preferably composed of a double repeat sequence that catalyzes the exponential replication of a 12-mer oligonucleotide; a pseudotemplate (pT), which absorbs the leakage products resulting from non-specific reactions in the autocatalytic template, thereby avoiding background amplification. A conversion template (cT) is connected upstream of aT, and when a target binds to the input part of cT, the conversion template catalyzes the production of multiple output strands, which in turn trigger the autocatalytic reaction in aT. Downstream of aT, a report template (rT) captures the amplified signal strand to generate a fluorescent signal. A detailed reaction network according to a preferred embodiment of the method of the present invention is shown in FIG. 2.

[0039] According to another embodiment, the readout of the amplified signal can be performed not by using a reporting template, but for example by DNA double-stranded inserts (Evagreen or SYBR green), by aggregating nanoparticles, or by forming specific DNA structures, DNAzymes, etc. Step b) of the digital method of the invention is carried out by partitioning the mixture obtained in step a) into several compartments such that some of the compartments do not contain the target biomolecule. Sample partitioning is the basis of the digital quantification method. Digital quantification relies on partitioning the sample containing the amplification mixture such that the target biomolecules are randomly distributed such that only a small number of target biomolecules, preferably 0-10, more preferably 0-5, even more preferably 0-2, even more preferably 1 molecule, are present in most of the compartments. The random partitioning process generates a distribution of the number of target biomolecules per compartment that follows Poisson's law. Digital quantification can be applied when at least some of the compartments do not contain any target biomolecules. This proportion of empty compartments should be higher than 1%, preferably higher than 10%. Thus, according to the invention, most of the compartments contain at least one target biomolecule, but not all of these compartments do.

[0040] In other words, the distribution of the mixture obtained in step a) into several compartments is carried out such that the distribution of the target biomolecules makes the number of compartments that have received at least one biomolecule less than 100% of the total number of compartments. As used herein, the term "digital method" or "digital amplification method" refers to a detection method in which a test sample containing target biomolecules is distributed into several microcompartments and targets are randomly isolated within the compartments according to a Poisson distribution. Reactions are then carried out in each individual compartment, allowing direct counting of individual events and absolute target quantification. Sample distribution can be performed by several means, including microfabricated chambers (such as Thermofisher Scientific's SlipChip or QuantStudio 3D) or microdroplets (such as Biorad's QX200 system or Stilla Technologies' Naica system). According to a preferred embodiment of the digital method of the present invention, the test sample is distributed into millions of water-in-oil droplets fabricated by combining two immiscible liquids: a sample aqueous phase; and a continuous hydrophobic phase such as undecan-1-ol, silicone oil, mineral oil, and more preferably perfluorinated oils because they are highly immiscible with water, biocompatible, low viscosity, transparent, and generally compatible with microfluidic devices.

[0041] The average size of the droplets is very important because it determines the feasibility of the assay, the time required to perform the assay (corresponding to the time required to amplify a droplet containing a single target), and the dynamic range of the assay (which is the range of concentrations constituted between the lower limit of the sensitivity of the assay and the highest concentration at which a significant fraction of droplets do not turn on). In fact, in digital methods, the size of the droplets must be adjusted so that the concentration corresponding to a single copy of the target encapsulated in one droplet reaches a value above the detection limit in bulk. If the droplets are too large, the target will not be detected with the typical sensitivity of the assay because it will simply be too diluted to induce a signal distinguishable from the background. Also, the size of the droplets must be small enough (and therefore the concentration of single molecules in the droplets large enough) to ensure that amplification is induced in a reasonable time (typically a few hours) for target-positive droplets. The smaller the droplets, the faster the assay will be. Finally, once the appropriate droplet size for the above two conditions is determined, the dynamic range of the digital assay is comprised between the lower bulk target concentration at which the percentage of droplets that turn on is significantly higher than the negative control, and the concentration at which a significant percentage of droplets do not turn on. Smaller droplets shift the dynamic range to higher concentrations, and larger droplets, if compatible with the assay, shift the dynamic range to lower concentrations.

[0042] In addition, some technical constraints apply to the size of the compartments or droplets: smaller monodisperse droplets can be more difficult to generate, but are typically more stable during high temperature incubation or cycling. Most PCR-based digital assays in the art use droplets with a diameter of 20-100 micrometers (i.e., 4-500 pL). This is due to the high sensitivity of the PCR amplification method, which makes it compatible with very low initial target concentrations. Isothermal techniques are less sensitive and therefore better suited to smaller droplet sizes. Depending on the target of interest, the assays described herein achieve sensitivities without droplet dispensing that are generally better than 1 pM and in some cases, such as enzyme detection, can achieve 1 fM. Therefore, it is preferable to use droplets that are relatively small for nucleic acid detection, especially microRNA detection, and may be larger for enzyme detection. It is further possible to vary the size of the droplets in order to shift the dynamic range of the assay. In one embodiment, the size of the droplet is adjusted so that the single copy of the target enclosed in the volume of one compartment reaches a value of the order of picomole.This also ensures that the target positive droplets will trigger amplification in a reasonable time (typically several hours).If the compartment is too large, the target will simply be too diluted and will not allow amplification at the typical sensitivity of the assay.

[0043] Miniaturization of reaction compartments (from microliter scale in tubes to picoliter scale in droplets) introduces stochastic effects in the amplification reaction (due to template and enzyme distribution within the droplet, surface effects, biomolecule-surfactant interactions, temperature heterogeneity). These phenomena imply a dispersion in the droplet amplification onset time (i.e., droplets containing a single target will not all amplify at exactly the same time). This dispersion is less evident in PCR-based reactions, since the thermocycling process synchronizes the replication cycle among all compartments. Since isothermal reactions have a larger on-time dispersion, it is important that empty droplets remain off until all target-containing droplets are on. Classical isothermal methods (EXPAR, RCA, HCR, etc.), although they exhibit good sensitivity in bulk, cannot address this constraint. Isothermal digital methods require the use of compartments of precisely selected size together with methods that provide a sufficient time window for the target concentration, so that a single copy of the target biomolecule present in one compartment of this size reaches the mentioned target concentration.

[0044] According to one embodiment, the droplets used in the digital method of the invention have a size comprised between 0.001 pL and 100 pL, preferably between 0.1 pL and 10 pL, more preferably between 0.5 and 5 pL, or between 0.5 and 8 pL. Preferably, when the method of the invention is used to determine and / or quantify an enzyme, the size of the droplets is comprised between 5 and 8 pL, preferably the size of the droplets in this case is 7.2 pL. According to another embodiment, when the method of the invention is used for the detection and / or quantification of an enzyme, the size of the droplets may be comprised between 0.5 and 100 pL, in particular between 5 and 50 pL, more particularly between 6 and 10 pL. As shown above, the main advantage of the method of the present invention is that the target biomolecule is not directly detected but converted into a signal sequence, which makes it possible to avoid the problems of sensitivity and specificity caused by the direct detection of the target biomolecule, and also limits the risk of carryover contamination.

[0045] Thus, in the method of the invention, after partitioning the mixture in step b), the sequence of the target biomolecule is converted into a signal in step c), preferably by means of a conversion template. As used herein, the term "signal" or "signal sequence" relates to a nucleic acid sequence, preferably a single-stranded DNA, obtained by converting the sequence of a target biomolecule, preferably a nucleic acid molecule, more preferably a microRNA, into a sequence that can be amplified. The converted signal sequence is then amplified in step d) of the method of the invention. According to one embodiment, the amplification of the signal sequence in step d) is carried out at a constant working temperature in the range of 35-60°C, more preferably 37-55°C, even more preferably 45-50°C.

[0046] According to the invention, the chemical reactions of steps c) and d) occur simultaneously. Steps c) and d) can also be designed as incubation steps. In order to detect and / or measure the amplified signal sequence, said molecules need to be labeled. Preferably, labeling is performed by using fluorescent probes labeled with organic or inorganic dyes such as quantum dots, particle aggregates, etc. A person skilled in the art will be able to adapt conventional labeling means to the digital method of the present invention. According to a preferred embodiment of the digital method of the present invention, the label used is a fluorescent signal. During the incubation for amplifying the signal sequence in step d), the target biomolecule induces signal amplification in its encapsulated droplet. Thus, after incubation, the droplet exhibits a positive fluorescent signal. Thus, according to one embodiment of the digital method of the invention, step e) of detecting and / or measuring said amplified signal comprises detecting and / or counting areas that emit strong fluorescence.

[0047] In one embodiment, when the digital method of the present invention is used to measure the absolute concentration of a target biomolecule in a test biological sample, the number of compartments that receive the target biomolecule and that exhibit strong fluorescence, and the number of non-fluorescent compartments are counted and the ratio between them is calculated. Preferably, imaging by fluorescence microscopy allows counting of positive / negative droplets, thus allowing calculation of the exact target biomolecule concentration in the initial sample. According to another embodiment, other conventional methods, such as flow cytometry, may be used to count positive / negative droplets. According to a preferred embodiment, the digital method of the invention is implemented as follows: a mixture containing the molecular program (buffer, the enzymes mentioned above, polymerase, nicking enzyme, and exonuclease, as well as the four oligonucleotide templates mentioned above) and the target biomolecule-containing sample is dispensed into picoliter-sized (approximately 0.1-5 pL) water-in-oil droplets using standard microfluidic techniques. The targets are randomly distributed in the droplets according to Poisson's law. The emulsion is then incubated at a constant working temperature (in the range of 42-55 °C) to allow the conversion of the target biomolecule into a signal and to amplify the signal. The signal is amplified and fluorescence is turned on in droplets that received at least one target biomolecule during the dispensing step. At the same time, empty droplets remain at a low fluorescence level. The droplets are finally imaged using a fluorescent microscope (other readouts such as fluorescence-activated droplet sorting (FADS) or flow cytometry may be used). The readout therefore relies on end-point measurements, as opposed to the real-time monitoring required for test-tube experiments. The ratio of positive / negative droplets (also called the "on / off" ratio) allows calculation of the exact target concentration in the initial sample without reference to a calibration standard.

[0048] The above-mentioned biomolecules may be present in all types of samples. For example, the above-mentioned biomolecules may be present in samples obtained from non-living organisms (e.g., soil samples, water samples, air samples, food samples, etc.) or samples obtained from living organisms, such as cells, body fluids, or tissues. According to one embodiment of the method of the present invention, the target biomolecules detected and / or measured by the digital method of the present invention are used as biomarkers. In the context of the present invention, the term "biomarker" relates to a naturally occurring molecule, preferably a biomolecule, gene or trait by which a particular pathological or physiological process, disease or the like can be identified.

[0049] Such biomarkers can be used to detect disease in organisms such as plants, animals, preferably mammals, and more preferably humans. Furthermore, such biomarkers can be used for the detection of one or more abnormalities, as well as for the food and agri-food industries or in the environment. Biomarkers, for example, can be present in all bodily fluids and therefore accessible via minimally invasive liquid biopsies (serum, plasma, urine, tears, saliva, sweat, etc.). Preferably, they are used as biomarkers for detecting diseases selected from the group including cancer, neurological diseases, cardiovascular diseases, inflammatory diseases, autoimmune diseases, diseases caused by viral or bacterial infections, skin diseases, musculoskeletal diseases, dental diseases, and prenatal diseases.

[0050] In the context of the present invention, the term "detection" is used in a general manner to define the detection of a target biomolecule in a sample as defined above. Also, as used herein, the term "detection" refers to the diagnosis or prognosis of one or several of the above-listed diseases or their symptoms.Detection also includes predicting one or several of the above diseases or predicting the risk that a subject will develop one or several of these diseases. Moreover, the term "detection" further relates to agronomic diagnosis, i.e. the diagnosis of plant pathologies, in particular plant pathologies having a biotic or abiotic origin as defined in the present invention. In the context of the present invention, the term "cancer" refers to a malignant neoplasm characterized by deregulated or uncontrolled cell proliferation. In particular, a "cancer cell" refers to a cell that exhibits deregulated or uncontrolled cell proliferation.

[0051] The term "cancer" includes primary malignant tumors (e.g., those whose cells have not migrated to sites in the subject's body other than the site of the original tumor) and secondary malignant tumors (e.g., metastases, resulting from the migration of tumor cells to secondary sites different from the site of the original tumor). Such cancers may be selected, in particular, from the group of solid tumors and / or the group of hematopoietic cancers.

[0052] In one embodiment of the invention, the cancer is selected from the following: osteolysis, sarcoma of bone (osteosarcoma, Ewing's sarcoma, giant cell tumor of bone), bone metastasis, glioblastoma and brain cancer, lung cancer, acoustic neuroma, acute leukemia, acute lymphocytic leukemia, acute myeloid leukemia (monocytic, myeloblastic, adenocarcinoma, angiosarcoma, astrocytoma, myelomonocytic, and promyelocytic), acute T-cell leukemia, basal cell carcinoma, cholangiocarcinoma, bladder cancer, breast cancer, bronchogenic carcinoma, cervical cancer, chondrosarcoma, chordoma, choriocarcinoma, chronic leukemia, chronic lymphocytic ... Myeloblastic leukemia, chronic myeloid (granulocytic) leukemia, chronic myelogenous leukemia, colon cancer, colorectal cancer, craniopharyngeal carcinoma, cystadenocarcinoma, diffuse large B-cell lymphoma, proliferative abnormal changes (dysplasia and metaplasia), embryonal carcinoma, endometrial cancer, endotheliosarcoma, ependymoma, epithelial carcinoma, erythroleukemia, esophageal cancer, estrogen receptor positive breast cancer, essential thrombocythemia, fibrosarcoma, follicular lymphoma, germ cell testicular cancer, glioma, heavy chain disease, hemangioblastoma, hepatocellular carcinoma, hominis Cancer of the prostate, leiomyosarcoma, liposarcoma, lung cancer, lymphangioendotheliosarcoma, lymphangiosarcoma, lymphoblastic leukemia, lymphoma (Hodgkin and non-Hodgkin), malignant tumors and hyperproliferative disorders of the bladder, breast, colon, lung, ovary, pancreas, prostate, skin, and uterus, lymphoid malignancies of T-cell or B-cell origin, leukemia, lymphoma, medullary carcinoma, medulloblastoma, melanoma, meningioma, mesothelioma, multiple myeloma, myeloid leukemia, Myeloma, myxosarcoma, neuroblastoma, non-small cell lung cancer, oligodendroglioma, oral cancer, osteogenic sarcoma, ovarian cancer, pancreatic cancer, papillary adenocarcinoma, papillary carcinoma, pinealoma, polycythemia vera, prostate cancer, rectal cancer, renal cell carcinoma, retinoblastoma, rhabdomyosarcoma, sarcoma, sebaceous gland carcinoma, seminoma, skin cancer, small cell lung cancer, solid tumors (carcinoma and sarcoma), small cell lung cancer, gastric cancer, squamous cell carcinoma, synovium, sweat gland carcinoma, thyroid cancer, Waldenstrom's macroglobulinemia, testicular tumor, uterine cancer, and Wilms' tumor.

[0053] In the context of the present invention, the term "neurological disease" refers to diseases of the central and peripheral nervous system, including the brain, spinal cord, cranial nerves, peripheral nerves, nerve roots, autonomic nervous system, neuromuscular junction, and muscles. Neurological diseases are selected from neurodevelopmental diseases, neurodegenerative diseases, or psychiatric diseases. Neurological diseases include epilepsy, Alzheimer's disease and other dementias, cerebrovascular diseases including stroke, migraine, and other headache disorders, multiple sclerosis, Parkinson's disease, neuroinfections, brain tumors, traumatic disorders of the nervous system due to head trauma, and the like. As used herein, "cardiovascular disease" refers to heart or vascular failure including coronary heart disease: disease of the blood vessels that supply the heart muscle; cerebrovascular disease: disease of the blood vessels that supply the brain; peripheral artery disease: disease of the blood vessels that supply the arms and legs; rheumatic heart disease: damage to the heart muscle and heart valves due to rheumatic fever caused by streptococcus bacteria; congenital heart disease: malformations of the heart structure present at birth, and deep vein thrombosis, and pulmonary embolism: blood clots in the leg veins that can break off and travel to the heart and lungs.

[0054] As used herein, "inflammatory disease" preferably refers to acute pancreatitis; ALS; Alzheimer's disease; cachexia / anorexia; asthma; atherosclerosis; chronic fatigue syndrome, fever; diabetes (e.g., insulin-dependent diabetes); glomerulonephritis; graft-versus-host rejection; hemorrhagic shock; hyperalgesia, inflammatory bowel disease; inflammatory conditions of the joints, including osteoarthritis, psoriatic arthritis, and rheumatoid arthritis; ischemic injury, including cerebral ischemia (e.g., brain injury as a result of trauma, epilepsy, hemorrhage, or stroke, these each of which may be linked to neurodegeneration); pulmonary disease (e.g., ARDS); multiple myeloma; multiple sclerosis; myeloid (e.g., AML and CML) and other leukemias; myopathy (e.g., muscle protein metabolism, especially in sepsis); osteoporosis; Parkinson's disease; pain; preterm labor; psoriasis; reperfusion injury; septic shock; side effects of radiation therapy, temporomandibular joint disease, tumor metastasis; or inflammatory conditions resulting from strains, sprains, cartilage injuries, trauma, orthopedic surgery, infection, or other disease processes.

[0055] In the context of the present invention, an "autoimmune disease" is defined as a condition in which a subject's body produces antibodies against the subject's own tissues and cells. Examples of autoimmune diseases are type I diabetes, Graves' disease, inflammatory bowel disease, multiple sclerosis, psoriasis, rheumatoid arthritis, systemic lupus erythematosus, etc.

[0056] Viral or bacterial diseases are caused by pathogenic viruses or bacterial strains. Examples of such diseases are AIDS, ascariasis, athlete's foot, dysentery, chickenpox, cholera, cold, dengue fever, diarrhea, diphtheria, filariasis, gonorrhea, herpes, hookworm, influenza, leprosy, measles, mumps, oriental cyst, pinworm disease, plague, pneumonia, poliomyelitis, rabies, ringworm, septicemic sore throat, sleeping sickness, smallpox, syphilis, tetanus, typhoid, vaginitis, viral encephalitis, whooping cough, etc. "Skin disease" is a condition affecting the skin, such as, for example: acne, alopecia areata, basal cell carcinoma, Bowen's disease, congenital erythropoietic porphyria, contact dermatitis, Darier's disease, disseminated superficial actinic porokeratosis, dystrophic epidermolysis bullosa, eczema (atopic eczema), extramammary Paget's disease, epidermolysis bullosa simplex, erythropoietic protoporphyria, fungal infections of the nails, Hay fever, and the like. Leahayley disease, herpes simplex, hidradenitis suppurativa, hypertrichosis, hyperhidrosis, ichthyosis, impetigo, keloids, keratosis pilaris, lichen planus, lichen sclerosus, melanoma, melanosis, mucous membrane pemphigoid, pemphigoid, pemphigus vulgaris, pityriasis lichenoides, pityriasis rubra pilaris, plantar warts (verrucae), polymorphous photoeruption, psoriatic pyoderma gangrenosum, rosacea, scabies, shingles, squamous cell carcinoma, Sweet's syndrome, urticaria, and angioedema, vitiligo.

[0057] As used herein, "muscle skeletal disease" refers to diseases of bone, muscle (myopathy) and musculoskeletal junction. For example, such diseases are selected from back pain, bursitis, fibromyalgia, fibrous dysplasia, growth plate injury, hereditary connective tissue disorder, Marfan syndrome, osteogenesis imperfecta, osteonecrosis, osteoporosis, Paget's disease of bone, scoliosis, spinal stenosis, tendonitis, etc. As used herein, "dental disease" refers to problems with the teeth and mouth. Examples of such diseases are dental cavities, periodontal (gum) diseases, oral cancer, oral infectious diseases, traumatic injuries, and genetic disorders. As used herein, "prenatal disease" relates to diseases that may affect pregnancy or fetal development, such as: AIDS, amniotic fluid, bleeding during pregnancy, cervical disorders, gestational diabetes, disseminated intravascular coagulation (DIC), ectopic pregnancy, erythroblastosis fetalis, fetal growth problems, high blood pressure during pregnancy, HELLP syndrome, hydatidiform mole, hyperemesis gravidarum, intrauterine growth retardation, large for gestational age (LGA), miscarriage, placental abruption, placenta previa, placental insufficiency, polyhydramnios, prenatal testing, pregnancy loss, preterm labor and delivery, rubella, small for gestational age (SGA), systemic lupus erythematosus, toxoplasmosis, twin-to-twin transfusion syndrome, twins, triplets, multiple births, vaginal bleeding during pregnancy.

[0058] Thus, the digital method of the present invention can be used in an in vitro diagnostic method for diagnosing a disease selected from the group including cancer, neurological diseases, cardiovascular diseases, inflammatory diseases, autoimmune diseases, diseases caused by viral or bacterial infections, skin diseases, musculoskeletal diseases, dental diseases, and prenatal diseases. Thus, in a second aspect, the present invention relates to an in vitro method for diagnosing a disease selected from the group comprising cancer, a neurological disease, a cardiovascular disease, an inflammatory disease, an autoimmune disease, a disease due to a viral or bacterial infection, a skin disease, a musculoskeletal disease, a dental disease and a prenatal disease, comprising using a digital method according to the present invention.

[0059] According to one embodiment, the diagnostic method comprises: Providing a sample from a subject; and Detecting the presence or absence of one or more of the above diseases by using the digital method of the present invention. Includes. The specificity, sensitivity, simplicity and rapidity of the digital methods of the invention allow them to be used in agronomic diagnostic methods, in particular for the diagnosis of diseases caused by biotic stresses, such as infectious and parasitic diseases, or by abiotic stresses, such as nutritional deficiencies or adverse environments.

[0060] According to a third aspect, the present invention also provides a method for producing ... composition comprising the steps of: Biotic stress, preferably a disease caused by infectious and / or parasitic causes, or Diseases caused by abiotic stress, preferably nutritional deficiencies and / or adverse environments An in vitro method for the agricultural diagnosis of a disease selected from the group comprising: It also relates to in vitro methods involving the use of the digital methods of the present invention. According to one embodiment, the agricultural diagnostic method comprises: Providing a sample from any one of the plant parts; and Detecting the presence or absence of one or more of the above diseases by using the digital method of the present invention. Includes.

[0061] In the context of the present invention, the term "agronomic diagnosis" relates to the diagnosis of plant pathologies, said diagnosis comprising carrying out an analysis of a plant sample to identify fungi, viruses, bacteria, nematodes and any other organisms causing biotic stress and / or to identify biomolecules whose presence is due to abiotic stress. In the context of the present invention, the term "plant pathology" or "plant disease" relates to a plant abnormality manifested by alterations in the morphological, physiological characteristics or behavior of the plant due to biotic or abiotic stress. As used herein, the term "biotic stress" also relates to stress or disease caused by a living organism. The biotic stress may be caused by any living organism, but preferably by organisms of infectious and / or parasitic origin and selected from fungi, viruses, bacteria and nematodes.

[0062] According to one embodiment, the agronomic diagnostic method of the present invention can be used to diagnose diseases caused by fungi, said diseases being selected from anthracnose, black knot disease, blight, chestnut blight (such as leaf blight), canker, clubroot, wilt, elm wilt, ergot, Fusarium wilt, Panama disease, leaf blister, mildew (such as downy mildew and powdery mildew), oak wilt, rot (such as base rot, gray mold rot, heart rot), rust (such as blister rust, cedar apple rust, and coffee rust), scab (such as apple scab), ear smut, wheat smut, corn smut, snow rot, sooty mildew, and verticillium wilt. According to another embodiment, the agronomic diagnostic method of the present invention can be used to diagnose a disease caused by a virus, said disease being selected from dwarf disease, mosaic disease, sorosis, and spotted wilt disease.

[0063] According to yet another embodiment, the agronomic diagnostic method of the present invention may be used to diagnose a disease caused by bacteria, said disease being selected from aster yellows, bacterial wilt, blight (such as fire blight and rice bacterial spot), canker, crown gall, rot, base rot, and scab. The agricultural diagnostic method of the present invention can also be used to detect root-knot nematodes (Meloidogyne spp., etc.), cyst nematodes (Heterodera spp. and Globodera spp., etc.), root lesion nematodes (Pratylenchus spp., etc.), burrowing nematodes (Radopholus similis, etc.), Ditylenchus dipsaci, pinewood nematodes (Bursaphelenchus xylophilus, etc.), reniform nematodes (Rotylenchulus reniformis, etc.), grape spur nematode (Xiphinema index), false root-knot nematode (Nacobbus The present invention can be used for the diagnosis of diseases caused by nematodes selected from the group consisting of Aphelenchoides besseyi, Aphelenchoides aberrans, and Aphelenchoides besseyi.

[0064] According to one embodiment, the agronomic diagnostic method of the present invention is used to diagnose diseases caused by "abiotic stress", the term "abiotic stress" defines diseases that are not caused by living organisms. Such diseases are preferably caused by nutritional deficiencies and / or adverse environments. For example, abiotic stress can be caused by inappropriate pH, water availability (drought stress), temperature (heat stress and cold stress), oxygen and / or gas availability, mineral deficiencies (salt stress), and toxic compounds (such as pollutants). The specificity, sensitivity, simplicity and rapidity of the digital method of the invention also make it possible to use it in methods for detecting biomolecules in the field of food, agri-food industry and in the environment. In particular, the digital method of the invention is used to detect abnormalities in food, agri-food and the environment. This detection is carried out by using the digital method of the invention to detect biomolecules that can be considered as biomarkers for said abnormalities.

[0065] Such biomolecules may, for example, be part of a living organism, or may be produced by the activity of a living organism, or may be artificial biomolecules. Such biomolecules (or biomarkers) are selected from the group comprising biopolymers, in particular DNA, RNA, proteins, and enzymes. Said biomolecules are present in the original and / or transformed products of agri-foods and foods. Biomolecules may also be present in the environment, for example, in the air, water, and / or soil. Thus, according to one aspect, the present invention also relates to an in vitro method for detecting biological molecules (biomarkers) in agri-food, the food industry and / or the environment, comprising using the digital method of the present invention.

[0066] In the context of the present invention, the term "food" relates to any food product, basic or transformed, produced without the use of industrial processes or by using such processes. In the context of the present invention, the term "agri-food" relates to the agri-food industry, i.e. the commercial production of food by agricultural enterprises. The term "environment" or "environmental" relates to the natural environment, i.e. an ecological unit functioning as a natural system without large-scale civilized human intervention, including all plants, microorganisms, soil, rocks, atmosphere, and natural phenomena occurring within their boundaries and properties. Such terms also relate to non-natural or artificial environments that humans may create. According to one aspect, the present invention also relates to an in vitro method for detecting anomalies in the food and agri-food industry and / or the environment, using the digital method of the present invention.

[0067] Preferably, the method comprises the steps of: Preparing samples obtained from food, agri-food or from the environment; Detecting a test biomolecule (biomarker) in said sample by the digital method of the present invention. Includes.

[0068] The inventors have also demonstrated that the method of the present invention can be used to simultaneously or in parallel detect and / or quantify different target biomolecules from different samples. To this end, in one embodiment, the samples may be barcoded, for example, with a fluorescent dye, so that the barcode fluorescence intensity of each droplet can be assigned to each of the initial samples. Barcode protocols are disclosed, for example, in Brouzes et al. (2009) and Genot et al. (2016). Thanks to this barcode procedure, multiple samples can be emulsified, collected, incubated, and analyzed simultaneously, thus reducing detection time and costs. Thus, according to a fourth aspect, the digital method of the invention can also be used to assay more than one sample, or more than one target biomolecule in a sample, in parallel.

[0069] According to a preferred embodiment, in order to directly detect and / or quantify more than one biomolecule in one or more than one different samples, even if the signal provided is weak, the inventors have surprisingly found that the use of a fifth oligonucleotide, which is a crosstalk-inhibiting oligonucleotide (herein referred to as a "killer oligonucleotide" or "killer template"), allows to significantly improve the specificity of the method. This embodiment relies on a target-specific DNA circuit (comprising aT, cT, pT, and / or rT) interconnected with a DNA-encoded inhibitor that suppresses non-specific signal amplification due to the fact that molecular crosstalk generates undesired communication between multiplexed detection channels. Thus, in the context of the present invention, a "killer oligonucleotide" or "killer template" or "kT" refers to an oligonucleotide that can generate a pseudotemplate (pT) of the reverse switch, thereby acting as a cross-inhibitor of amplification and reducing non-specific crosstalk. Examples of sequences of killer templates are shown in Table 1A below.

[0070] [Table 2]

[0071] In order to control the strength of the inhibitory reaction, 1 pM and 100 nM, preferably 10 pM to 20 nM, even more preferably 100 pM to 10 nM. Thus, according to this embodiment, the digital method for detecting and / or identifying biomolecules of the present invention further comprises adding a fifth oligonucleotide, which is a cross-inhibiting oligonucleotide, to detect and / or quantify two or more biomolecules. The present invention also relates to kits that can be used to detect and / or quantify target biomolecules according to the methods of the present invention.

[0072] According to one embodiment, the present invention provides a kit for detecting and / or quantifying at least one target biomolecule, comprising: a) a mixture of enzymes, preferably selected from the group comprising polymerases, nicking enzymes, and exonucleases; b) a mixture of oligonucleotides, comprising a first oligonucleotide that is an amplification oligonucleotide, a second oligonucleotide that is a leak-absorbing oligonucleotide, and a third oligonucleotide that is a target-specific conversion oligonucleotide, and optionally a fourth oligonucleotide that is a report probe; and c) Partitioning agents, preferably water-in-oil emulsions The present invention relates to a kit comprising:

[0073] The components of the kit, ie the enzyme, the oligonucleotide and the dispensing agent, are as defined above. The kit of the present invention may also include instructions for use. Specific embodiments of the present invention will become apparent from the following examples and figures. [Brief description of the drawings]

[0074] [Figure 1] Microfluidic chip design (scale bar represents 100 μm). [Diagram 2] Droplet analysis. a) Water-in-oil droplets were sandwiched between two hydrophobic slide glasses and imaged with an epi-fluorescence microscope. b) Atto633 fluorescence channel. c) Bright-field channel, d) Bright-field channel with a 10-μm offset blurred focus. This setting emphasizes the droplet contour and facilitates droplet differentiation. e) Binarize the bright-field image (d), f) Use the morphological component command to differentiate all droplets. g) Filter the droplets according to their size and circularity. h) Extract the fluorescence of each droplet from a disk with radius r (3 < r < 6 pixels) and center xy (xy corresponds to the centroid of the selected component). i) Positive droplets correspond to droplets with fluorescence exceeding the set threshold (here, threshold = 7). If the droplet volume is known, the concentration is calculated from Poisson's law. [Diagram 3] Molecular program specialized for microRNA detection. a) The 4-template DNA circuit encodes the connectivity of the molecular program, and its reaction is catalyzed by a set of enzymes (polymerase, exonuclease, endonuclease): the conversion template (cT) converts the target microRNA into a universal trigger sequence; the autocatalytic template (aT) exponentially amplifies the trigger sequence; the pseudo-template (pT) drives the inactivation of the trigger synthesized from the leaky reaction to avoid non-specific amplification (in the absence of microRNA); the reporter template (rT) generates a fluorescence signal using the trigger sequence. b) Real-time monitoring of the amplification reaction in the presence of increasing concentrations of Let-7a. c) Correlation between the amplification time (Cq) and the Let-7a concentration. Error bars were calculated from three independent replicate experiments. [Figure 4] Detailed chemical reaction network of the molecular program for detecting the microRNA shown in Figure 3. [Diagram 5]Bulk detection of Let-7a. a) Full molecular program, b) molecular program without converter template, or c) molecular program without pseudotemplate (pT). Amplification reactions are monitored in real time and amplification time (Cq) is plotted as a function of Let-7a concentration. [Figure 6] Droplet digital detection of microRNA. a) Samples are mixed with a molecular program and distributed into millions of monodisperse droplets, resulting in a random distribution of microRNA targets throughout the compartment. After incubation, the droplets are imaged by fluorescence microscopy. Droplets that received at least one target exhibit a positive fluorescent signal (1), while other droplets remain negative (0). b) Fluorescence snapshots of emulsified samples spiked with increasing concentrations of Let-7a after amplification. c) Analysis of 30,000 droplets. d) Plot of the linear relationship between expected target concentration (theoretical concentration) and experimentally measured target concentration (measured concentration). e) Other microRNA assays by adapting the converter template. f) Specificity of the method of the present invention evaluated from the cross-reactivity of Let7a against Let7c (one mismatch) and Let7b (two mismatches). [Figure 7] Effect of pseudotemplate concentration and Nb.BsmI concentration on the detection of Let-7a. Samples containing defined concentrations of pT (0-15 nM) and Nb.BsmI (0.1-0.4 u / μL) were spiked with 0 or 1 pM of Let-7a and the amplification reaction was monitored in real time. a) Cq is plotted as a function of pT concentration and Nb.BsmI concentration. MDS is plotted as a function of b) pseudotemplate concentration and c) Nb.BsmI concentration. [Figure 8] Optimization of Nt.BstNBI concentration. Samples spiked with 0 or 1 pM Let7a are incubated in the presence of various concentrations of Nt.BstNBI. Cq is plotted as a function of Nt.BstNBI concentration. [Figure 9] A molecular program to reduce the false positive droplet rate. Target-free samples with various concentrations of pseudotemplate (pT) are dispensed into the droplets. The fluorescence of the emulsion is monitored in real time. [Figure 10] Elimination of non-specific amplification reactions. Molecular programs with pT of 0 or 15 nM were spiked with 0 or 1 pM synthetic Let-7a before dispensing. Droplets are incubated at 50 °C and the bulk fluorescence (emulsion average) is continuously monitored (solid line). At different time points the incubation is stopped and the droplets are imaged under a fluorescence microscope to extract the percentage of positive compartments (diamonds). [Figure 11] MicroRNA detection from biological samples. a) Let7a detection from H1975 cell line. b) Let7a and mir-39ce detection from human colon total RNA. [Figure 12] Effect of Klenow (exo-) on the detection of Let7a microRNA. a) Real-time amplification reactions in the presence of 0 or 10 pM Let7a and various concentrations of Klenow (exo-). b) Amplification time (Cq) as a function of Klenow (exo-) concentration. c) Experimental conditions. [Figure 13] Digital detection of Let7a using a mixture of Klenow (exo-) and Vent (exo-). a) Measured concentrations as a function of spike-in expected concentration. b) Experimental conditions. Mix A (containing the enzyme) and Mix B (containing the template) are mixed on-chip using a 3-inlet flow focusing device. Droplets are incubated at 50° C. for 200 min. [Figure 14] Detection of cel-miR39 in plasma samples. Human blood samples were collected from healthy donors and plasma was obtained by centrifugation. 0 or 1 pM of cel-miR39 is spiked into 5% plasma (vol / vol) and the amplification mixture supplemented with RNAse inhibitors. The measured concentrations reported in the plots indicate complete recovery of exogenous microRNA in 5% plasma. [Figure 15]Conversion module design and bulk detection of enzyme activities (standard deviation calculated from at least three independent data points). a) Nt.BstNBI, b) RNAseH2. c) APE-endonuclease 1 (APE-1). d) Uracil DNA glycosylase (UDG). e) Alkyl adenine glycosylase (AAG). f) BsmAI restriction enzyme. g) Poly(A) polymerase (PAP). h) T4 DNA ligase. i) T4 polynucleotide kinase (T4 PNK). [Figure 16] Digital detection of enzymes. a) Microscopic snapshots of 2D droplet arrays of Nt.BstNBI enzyme at six different concentrations. b) Measured concentrations (fM) as a function of spike-in concentrations (u / mL) of various enzymes. The linear relationship indicates that the digital readout provides absolute quantification. Error bars correspond to the 95% confidence interval of the measurement. [Figure 17] Comparison of digital assay quantification of Nt.BstNBI in small (0.95 pL) and large (7.2 pL) droplets. Concentrations calculated in both experiments were consistent. Error bars correspond to the 95% confidence interval of the measurements. [Figure 18]A tetrastable system constructed with two cross-inhibitory bistable switches. (a) Schematic of the tetrastable DNA circuit. Two microRNA sensing circuits (cT, aT, pT, rT) are interconnected by the killer templates αkβ and βkα, which suppress unwanted cross-activation. (b) Detailed mechanism of the five types of templates (pol.=Vent(exo-), nick1=Nt.BstNBI, nick2=Nb.BsmI, RE=BsmI, exo=ttRecJ). The conversion template (cT) converts the complementary microRNA target into a signal strand (α or β). The autocatalytic template (aT) exponentially amplifies the signal strand. The pseudotemplate suppresses background amplification caused by biochemical noise by inactivating a part of the signal strand. The report template (rT) converts the molecular signal (α or β) into a detectable fluorescent signal (green = Oregon green fluorophore, red = Atto633 fluorophore). The killer template (kT) produces the reverse-switch pT from either the α or β strand, reducing non-specific crosstalk. All produced strands are continuously degraded by exonucleases, maintaining the dynamics of the system. Only half of the tetrastable circuit is shown here; the other half can be obtained by replacing α with β and vice versa. [Figure 19] Killer template efficiency. (a) An α-switch, triggered at 10 pM in the case of mir39, is connected to the killer template αkβ to produce pTβ of various lengths (with inactivating tails ranging from 0 to 5 adenylate moieties). (b) Fluorescence of the β-switch as a function of the concentration of kT (t = 1000 min). [Figure 20] Extended data from Figure 19. (a) Amplification curves of various concentrations of αkβ producing pTβ of various lengths. (b) Cq plotted as a function of αkβ concentration. [Figure 21]Determination of kT concentrations to suppress cross-activation between the α- and β-switches. (a) The A and β-circuits are induced with 0 or 10 pM mir92a and let7a, respectively, in the presence of increasing concentrations of αkβ and βkα. (b) Amplification times (Cq) of the α- and β-switches. (c) Color-coded representation of Cq as a function of kT concentration. The dashed blue box represents the concentration of kT at which the system reaches tetrastability. (d) Amplification curves of mir39 / mir7 duplex assays (0 or 10 pM of each target). (e) Cq measured in seven different duplex assays. The left inset represents the average Cq and standard deviation of the seven assays. [Figure 22] Principle of the digital duplex assay. (a) Droplets were generated using a microfluidic chip from four samples (0 pM mir39 / 0 pM let7a, 3 pM mir39 / 0 pM let7a, 0 pM mir39 / 3 pM let7a, 3 pM mir39 / 3 pM let7a) and the emulsions were analyzed by microscope. (b) 2D histograms of the fluorescence of the probes (α switch = green fluorescence-mir39, β switch = red fluorescence-let7a). The vertical and horizontal dashed lines indicate the positive thresholds for the α switch and β switch, respectively. (c) Histograms of measured vs. expected target concentration. (d) Digital duplex assays of samples with different compositions (microRNA targets and concentrations). [Diagram 23] Singleplex versus duplex assays in solution. (a) Amplification curves of α (top) and β (bottom) bistable circuits incubated separately with amplification mix and 0 or 3 pM let7a and mir39 targets (singleplex assays). (b) Amplification curves of α- and β-switches embedded in intact tetrastable circuits (duplex assays). (c) Amplification times measured in duplicate experiments. From these data, we conclude that the killer template has little effect on amplification times under these conditions. [Figure 24]Limitations of blank singleplex vs duplex assay. Four samples are assembled as follows: sample A = α circuit only, 0 pM target, sample B = β circuit only, 0 pM target, sample C = α and β circuits, 0 pM target, sample D = α and β circuits, 1 pM target mir39 and let7e. (a) Amplification curves of the four samples in solution. (b) Composite image of a portion of the microfluidic chamber (bright field, green fluorescence, and red fluorescence). (c) 2D histogram of droplet fluorescence (α switch = green fluorescence, β switch = red fluorescence). (d) Percentage of positive droplets. The percentage of false positive droplets is qualitatively similar whether the assay is performed in singleplex or duplex (false positive α = 1.1% for singleplex and 1.1% for duplex; false positive α = 0.25% for singleplex and 0.28% for duplex). EXAMPLES

[0075] The purpose of the following examples is to demonstrate that the present invention enables highly sensitive molecular detection by digitizing analog methods of isothermal nucleic acid amplification.

[0076] Example 1 Detection of microRNAs by the digital method of the present invention Methods and Materials Chemicals: Oligonucleotides (templates and synthetic microRNAs) were purchased from Biomers (Germany). Sequences were purified by HPLC and confirmed by matrix-assisted laser desorption / ionization mass spectrometry. Templates were designed according to the rules described by Montagne et al. (Montagne et al, 2011 and Montagne et al., 2016). The autocatalytic template (aT), pseudotemplate (pT), and report template (rT) are protected from exonuclease degradation by 5' phosphorothioate backbone modifications. To avoid non-specific polymerization, a 3' blocking moiety (phosphate group for aT, pT, cT and quencher for rT) is used. Table 2 below summarizes all sequences used throughout this invention.

[0077] [Table 3]

[0078] Nb.BsmI and Nt.BstNBI nicking enzymes, Vent(exo - ) DNA polymerase, and BSA were purchased from New England Biolabs (NEB). Ten-fold dilutions of Nt.BstNBI were prepared by dissolving stock enzyme in diluent A (NEB) supplemented with 0.1% Triton X-100. The exonuclease ttRecJ was expressed in in-house culture and purified by chromatography according to the protocol published by Yamagata (Yamagata et al., 2001). The enzyme was stored at 1.53 μM in diluent A + 0.1% Triton X-100. All proteins were stored at -20°C. Cell culture. Human non-small cell lung cancer cell line H1975 cells and colorectal cancer cell line HCT116 cells were used for miRNA extraction. HCT116 cells were cultured in DMEM / F12 medium supplemented with 10% FCS, 100 units / mL penicillin G, and 100 μg / mL streptomycin. H1975 cells were cultured in RPMI1640 medium supplemented with 10% (vol / vol) FBS, 100 units / mL penicillin, and 100 μg / mL streptomycin. Cells were grown in a 5% CO2 incubator at 37 °C.

[0079] MicroRNA extraction: Human colon total RNA (Thermofisher Scientific) was aliquoted to 13 μg / mL and stored at -20°C until use. For cell extraction, TaqMan® miRNA ABC Purification Kit (Applied Biosystems) was used, and approximately 1 × 10 6MicroRNAs were extracted from cells. Briefly, cells were resuspended in 50 μL of 1×PBS and mixed with 150 μL of lysis buffer. After the cell lysis step, 2 μL of 1 nM external control cel-miR-39-3p oligonucleotide (Biomers) was spiked into the prepared sample and vortexed to evaluate the extraction efficiency. Target microRNAs were captured using magnetic Human Panel beads and eluted with 100 μL of elution buffer. Reaction mixture assembly: All reaction mixtures were assembled in 200 μL PCR tubes at 4 °C. Templates were mixed with reaction buffer (20 mM Tris HCl pH 7.9, 10 mM (NH4)2SO4, 40 mM KCl, 10 mM MgSO4, 50 μM each dNTP, 0.1% (wt / vol) Synperonic F104, 2 μM netropsin, all purchased from Sigma Aldrich) and BSA (200 μg / mL). After homogenization, enzymes were added (300 u / mL Nb.BsmI, 10 u / mL Nt.BstNBI, 80 u / mL Vent(exo - ), 23 nM ttRecJ). Each sample was spiked with a microRNA target and serially diluted in 1x Tris-EDTA buffer (Sigma Aldrich) using a low-binding DNA chip (Eppendorf). Samples (bulk or emulsion) were incubated in a qPCR thermocycler (CFX96 Touch, Biorad) at 50 °C and fluorescence was recorded in real time. For bulk experiments, time traces were normalized and Cq (time of amplification onset) was determined as 10% of the maximum fluorescent signal.

[0080] Droplet generation: A two-inlet (one for oil and one for aqueous samples) flow focusing microfluidic mold was prepared by patterning a 4-inch silicon wafer by standard soft lithography techniques using SU-8 photoresist (MicroChem Corp., MA, USA). A 10:1 mixture of Sylgard184 PDMS resin (40 g) / crosslinker (4 g) (Dow Corning, MI, USA) was poured into the mold, degassed under vacuum, and baked at 70 °C for 2 h. After curing, the PDMS was peeled off from the wafer and 1.5 mm diameter inlet and outlet holes were punched with a biopsy punch (Integra Miltex, PA, USA). Immediately after oxygen plasma treatment, the PDMS layer was fixed onto a 1 mm thick glass slide (Paul Marienfeld GmbH&Co.KG, Germany). Finally, the chip was subjected to a second baking at 200 °C for 5 h to render the channels hydrophobic (Kaneda et al., 2012). Details of the microfluidic chip are shown in Figure 1. The aqueous sample phase and the continuous phase, consisting of fluorinated oil (Novec-7500, 3M) containing 1% (wt / w) fluorosurfactant (RAN Biotechnologies, MA, USA), were mixed on-chip using a pressure pump controller MFCS-EZ (Fluigent, France) and 200 μm diameter PTFE tubing (CIL, France) to generate 0.5 pL droplets by hydrodynamic flow focusing.

[0081] Droplet imaging: After incubation, the droplets were imaged by fluorescence microscopy. The bottom slide (76 × 52 × 1 mm glass slide) was spin-coated with 200 μL of Cytop CTL-809M (Asahi Glass) and dried at 180 °C for 2 h. The emulsion was sandwiched between 0.17 mm thick coverslips treated with Aquapel. 10 μm polystyrene particles (Polysciences, Inc., PA, USA) were used as spacers to support the top slide and avoid emulsion compression. The imaging chamber was finally sealed with epoxy glue (Sader) and images were acquired using an epifluorescence microscope Nikon Eclipse Ti equipped with a motorized XY stage (Nikon), a camera Nikon DS-Qi2, an Apochromat 10× (NA 0.45) (Nikon), and a CoolLed pE-4000 illumination source. Composite images were generated using the open source ImageJ software. Quantitative data were extracted from microscopic images using Mathematica software (Wolfram) following the procedure detailed in Figure 2 .

[0082] result Analog amplification method according to International Publication No. 2017140815 The inventors of the present invention have previously developed a versatile molecular programming language called PEN-DNA Toolbox (Polymerase Exonuclease Nickase Dynamic Network Assembly) (Montagne et al., 2011 and Baccouche et al., 2014). The topology of the network is defined by a set of short oligonucleotides (templates). The network is interpreted by a mixture of enzymes (polymerases, exonucleases, and nickases) that process the information flux by producing and degrading DNA strands, which in turn activate or inhibit other nodes of the network.

[0083] Using this set of reaction modules, we have previously designed a universal molecular program dedicated to the detection of microRNA. Figure 3 shows the connectivity of the circuit. The universal signal amplification part corresponds to a bistable node composed of two types of templates. The autocatalytic template (aT) is composed of a double repeat sequence that catalyzes the exponential replication of 12-mer oligonucleotides, and the pseudotemplate (pT) absorbs the leakage products resulting from non-specific reactions in the autocatalytic template, thus avoiding background amplification. The conversion template (cT) is connected upstream of aT, and when the target binds to the input part of cT, the latter catalyzes the production of the output strand, which in turn triggers the autocatalytic reaction in aT. Downstream of aT, the report template (rT) captures the amplified signal strand to generate a fluorescent signal. The detailed reaction network is shown in Figure 4.

[0084] Figures 5b-5c show an evaluation of the sensitivity of this approach for bulk detection of Let-7a. The four templates are mixed together with the enzyme processor and spiked with synthetic target Let-7a at concentrations ranging from 0 to 1 nM. The fluorescence of rT is monitored in real time in a PCR thermocycler set at a constant temperature of 50 °C. The negative control (no target) does not generate a positive signal for longer than 20 h. The sensitivity of the assay is about 1 fM and the dynamic range in bulk ranges from 1 fM to 100 pM, i.e., 6 orders of magnitude. In the absence of pT (Figure 5b), the sensitivity is negatively affected, with a detection limit of 1 pM (estimated from the mean amplification time of the negative control, which is 3 standard deviations). These results demonstrate the importance of this active leak absorption mechanism to control the amplification threshold and thus eliminate background amplification. In the absence of cT (Figure 5c), no amplification reaction is observed, demonstrating the specificity of the molecular program for the target microRNA.

[0085] Digitalization of analog amplification methods To convert the analog signal into a digital readout, we move on to assess the possibility of detecting single molecules compartmentalized in droplets. Molecular programs spiked with known concentrations of Let-7a were dispensed into picoliter-sized water-in-oil droplets using a flow-focusing microfluidic junction. The monodisperse emulsions were incubated at 50°C, amplifying the target-containing droplets, after which the reaction was stopped. Finally, the droplets were imaged by fluorescence microscopy and the concentrations were recalculated from Poisson's law. Figure 6 shows microscopic snapshots of the emulsion after 200 min of incubation. A linear correlation is observed between the spiked miRNA concentration and the measured concentration according to Poisson's law. The calculated detection limit is 2.1 fM.

[0086] The modular approach of the system allows the molecular program to be reused by redesigning only the converter template (Figure 6d) to hybridize to any target of interest, without affecting quantification that relies on a universal signal amplification mechanism. A major concern for microRNA quantification is the high sequence homology between microRNA targets. Therefore, we evaluated the specificity of our detection method for the Let-7 family. Figure 4e shows that the Let-7a sequence is very well discriminated from analogs containing single (Let-7c) or double (Let-7b) mismatched bases. Effect of pseudotemplate (pT) and nuclease concentration We also assayed the effect of pseudotemplate (pT) and nuclease concentration (Nb.Bsml). To that end, samples containing defined concentrations of pT (0–15 nM) and Nb.BsmI (0.1–0.4 u / µL) were spiked with 0 or 1 pM of Let-7a and the amplification reaction was monitored in real time. Cq is plotted as a function of pT and Nb.BsmI concentrations (Figure 7a). The microRNA detection score (MDS) is calculated for each set of concentrations according to the following formula:

[0087]

number

[0088] We also investigated the optimal concentration of the nuclease Nt.BstNBI. To do so, samples spiked with 0 or 1 pM Let7a are incubated in the presence of various concentrations of Nt.BstNBI. Cq is plotted as a function of Nt.BstNBI concentration. Figure 8 shows that the optimal concentration of the endonuclease Nt.BstNBI is around 0.01 u / μL.

[0089] (Comparative Example) Most isothermal nucleic acid amplification techniques cannot be transferred to digital formats. This is generally due to non-specific reactions that eventually trigger amplification in all compartments regardless of the presence of target, as described by Zhang et al. (Zang et al., 2015). Due to the reliance on end-point analysis, it becomes important to have a sufficient time window to distinguish between target-containing droplets (exhibiting a positive signal) and target-free droplets. Figure 9 shows that in the absence of pT and target, all droplets turn on in less than an hour. This result has a considerable impact on the time frame required to separate the two populations and is consistent with the previously described EXPAR system described by Zhang et al. (Zhang et al., 2015). By increasing the pT concentration, and thus raising the amplification threshold, spontaneous initiation is delayed until it is completely abolished at 15 nM pT. Figure 10 is a comparison of the time window for detecting 1 pM Let-7a with 0 or 15 nM pT. In the absence of pT, it is nearly impossible to distinguish between target-containing samples and negative controls. However, by absorbing the leakage that causes false-positive droplets, the stabilization of the off state is guaranteed for longer than 16 hours without interfering with the amplification of target-containing droplets. Thanks to the complete elimination of the background, the method of the present invention shows unparalleled robustness with respect to incubation times and a theoretically infinite time window.

[0090] Furthermore, compared with other previously implemented digital amplification methods (Zhang et al., 2015, Cohen et al., 2016, and Tian et al., 2016), the selectivity of the present method is estimated to be better than 97%. Detection of endogenous microRNAs from human cells by the methods of the present invention The success of microRNA-based diagnostics as a routine biomedical procedure depends on the robustness and reproducibility of microRNA quantification. Therefore, we evaluated the feasibility of detecting endogenous microRNAs from human cells. MicroRNAs were extracted from the cell line H1975 (adenocarcinoma) and Let-7a was quantified by the method of the present invention at varying concentrations of RNA extract. The measured Let-7a concentrations for samples containing 1% and 10% RNA extract are 90 fM and 1 pM, respectively (Figure 11a).

[0091] In addition, we quantified Let-7a from human colon total RNA. Figure 11b shows the linear relationship between total RNA concentration (range 0-4 μg / mL) and measured Let-7a concentration. As a negative control experiment, mir-39ce, which is not present in the human genome, was not detected in these samples. Overall, this demonstrates the accuracy of the method of the present invention and its robustness in highly complex background samples.

[0092] Example 2 Klenow (3'->5' exon) for accelerating microRNA-induced amplification - ) Use of DNA polymerase To accelerate microRNA-targeted amplification, we used Klenow (exo - We investigated the effect of adding another DNA polymerase, Vent(exo - ) polymerase) at various concentrations of Klenow (exo - ) is used to complete the exoplasmic reticulum. - In the absence of exon 16, specific amplification occurs in approximately 100 minutes, whereas the negative control shows no amplification within 1000 minutes. - Using exocycline, the specific amplification is shortened to 20 minutes, while the negative control is unaffected. Above this concentration, we observed undesirable self-amplification of the negative control sample in less than 40 minutes. Taken together, these results support the conclusion that the Klenow (exocycline) - ) efficiently initiates RNA primer elongation, so Vent(exo - ) plus optimal concentration of Klenow (exo -) is beneficial in accelerating the amplification reaction.

[0093] We then investigated the use of a mixture of DNA polymerases for droplet digital detection of microRNAs. - Since the polymerases have non-negligible activity at room temperature, the oligonucleotides (template) and enzymes are assembled separately (Mix A and Mix B) and mixed on-chip using a three-inlet microfluidic device (one inlet is for the continuous phase and two inlets are for parts of both amplification mixes A and B) just before dispensing the droplets. This prevents the reaction from starting before target encapsulation. The measured concentrations are consistent with the spike-in concentrations of each sample, demonstrating accurate digital quantification of the target microRNAs using this polymerase mixture (Figure 13).

[0094] Example 3 MicroRNA detection directly from plasma samples The inventors also evaluated the detection of microRNAs in plasma samples using the digital detection method of the present invention. Human blood samples were collected from healthy donors (HIV, HBV, and HCV negative) into 10 mL collection tubes (Streck tubes supplied by Biopredic International). Plasma was obtained by centrifugation at 2000 x g for 10 min at 4 °C followed by centrifugation at 2000 x g for 15 min at 4 °C. Plasma was aliquoted into clean polypropylene tubes using a Pasteur pipette and stored at -80 °C until use. 0 or 1 pM of cel-miR39 (a microRNA derived from C. elegans) is spiked into the amplification mixture supplemented with 5% plasma (vol / vol) and 1 u / μL mouse RNAse inhibitor. The measured concentrations reported in the plot indicate complete recovery of exogenous microRNA in 5% plasma. The results are shown in Figure 14, thus demonstrating the quantitative measurement of microRNA concentration in crude plasma samples.

[0095] Example 4 Detection of enzymes by using the digital method of the present invention Oligonucleotides were obtained from Biomers or Eurofins (Table 3). Nicking enzymes nt.BstNBI (R0607), Nb.BsmI (R0706), DNA polymerase Vent (exo - )(M0257), Klenow(exo - ) polymerase, restriction enzyme BsmAI (R0529), AP-endonuclease APE-1 (M0282), uracil DNA glycosylase UDG (M0280), alkyladenine glycosylase hAAG (M0313), poly(A) polymerase (M0276), T4 DNA ligase (M0202), and T4 polynucleotide kinase PNK (M0201) were purchased from New England Biolabs. RNAse H2 enzyme (11-03-02-02) was purchased from Integrated DNA Technologies. Exonuclease ttRecJ was purified in-house according to a procedure previously reported (8). All oligonucleotides and proteins were stored at -20 °C.

[0096] [Table 4]

[0097] Reaction Assembly: All reactions were assembled in 200 μL PCR tubes at 4° C. Templates and enzymes (Table 4) were mixed with reaction buffer (20 mM Tris HCl pH 7.9, 10 mM (NH4)2SO4, 40 mM KCl, 10 mM MgSO4, 50 μM each dNTP, 0.1% (wt / vol) Synperonic F104, 2 μM Netropsin, all purchased from Sigma Aldrich) and BSA (200 μg / mL). Samples were spiked with 10% volume / volume of various concentrations of target enzyme serially diluted in 200 μL PCR tubes with 1× reaction buffer supplemented with BSA (200 μg / mL). Following an optional pre-incubation step, samples are incubated at 48° C. in a CFX96 Touch thermocycler instrument. Detailed experimental conditions are shown in Table 4.

[0098] [Table 5]

[0099] Digital Assay: For the digital assay, enzyme (Mix A) and template (Mix B) were mixed in 1x reaction buffer in two separate tubes to prevent the reaction from initiating before encapsulation of single enzymes into water-in-oil droplets. To increase the throughput of the assay, we used a sequential emulsification strategy previously reported (Menezes et al., 2019). Mix A, containing various concentrations of the target enzyme, is barcoded with various combinations of three fluorescently labeled dextrans (Dextran Texas Red 70,000 MW, Dextran Alexa Fluor 488 3,000 MW, and Dextran Cascade Blue 10,000 MW Lysine-tetherable (ThermoFisher Scientific)). Mix A (loaded into a pressurized sample exchanger) and Mix B were combined and sequentially emulsified on the chip using a three-inlet flow-focusing microfluidic PDMS chip. The continuous phase consists of fluorinated oil (Novec-7500, 3M) containing 1% (wt / wt) fluorosurfactant (RAN Biotechnologies, MA, USA). A microfluidic mold patterned on a 4-inch silicon wafer was prepared by standard soft lithography techniques using SU-8 photoresist (MicroChem Corp. MA, USA) and manually aligned using an MJB4 mask aligner (SUSS Microtec). A 10:1 mixture of Sylgard 184 PDMS resin (40 g) / crosslinker (4 g) (Dow Corning, MI, USA) was poured into the mold, degassed under vacuum, and baked at 70 °C for 2 h. After curing, the PDMS was peeled off the wafer and 1.5 mm diameter inlet and outlet holes were punched with a biopsy punch (Integra Miltex, PA, USA). Immediately after oxygen plasma treatment, the PDMS layer was fixed onto a 1 mm thick glass slide (Paul Marienfeld GmbH&Co.KG, Germany). Finally, the chip was baked a second time at 200°C for 5 h to make the channels hydrophobic.

[0100] Droplet imaging and analysis: Droplets were analyzed by transmission and epifluorescence microscopy. 70x50x1 mm glass slides (Paul Marienfeld, GmbH & Co. KG, Germany) were made hydrophobic by pouring 3 mL of Novec 1720 (3M) and baking at 100°C for 1 min on a heating plate. 10 μm polystyrene beads (Polysciences, Inc., PA, USA), used as hard sphere spacers, were spotted onto the glass slide and left at 100°C for evaporation. The emulsion was deposited onto the glass slide and covered with a 22x22 mm cover slip (VWR) treated with Novec 1720. The chamber was sealed with epoxy glue (Sader) and images were acquired using an epifluorescence microscope Nikon Eclipse Ti equipped with a motorized XY stage (Nikon), a Nikon DS-Qi2 camera, and a CoolLed pE-4000 illumination source, and an apochromatic 20× (NA 0.75, WD 1.0) objective. Pseudocolor images were generated with the open source ImageJ software. Mathematica software (Wolfram) was used to analyze the images and classify the different sample populations using the fluorescent barcodes. The number of negative and positive droplets for each sample allows the calculation of the target enzyme concentration, as determined by Poisson statistics.

[0101] result Proof of principle with the nicking enzyme Nt.BstNBI The isothermal signal amplification system used here is based on three coding deoxyribonucleotides. The first one is based on an autocatalytic template (sequence Cbo12-2PS3 SEQ ID NO: 73), a DNA polymerase (Vent(exo -)) and a nicking enzyme (Nb.BsmI) to catalyze the exponential replication of the trigger strand. The second pseudotemplate module (sequence pTBoT5PS3 SEQ ID NO: 74) inactivates a portion of the trigger strand and acts as a catalytic drain to avoid non-specific target-independent amplification caused by leak reactions. The third report module (sequence: rTBo-2BsmICy5 SEQ ID NO: 75) is a pre-fluorescent hairpin-shaped probe that hybridizes to the trigger to generate a fluorescent signal upon polymerization. Together, these enzyme and nucleic acid components create a bistable molecular circuit that can be used in a variety of ultrasensitive biosensing applications.

[0102] As a proof of principle, we designed a first sensing module (sequence: nbitoBo-2+2 SEQ ID NO: 76) to connect the activity of the nicking enzyme Nt.BstNBI to a bistable amplification switch (Figure 15a). The hairpin template contains a 5' output site, complementary to the trigger strand, immediately upstream of the nick recognition and cleavage site. The 3' end is self-complementary and primes extension along the template by the polymerase. In its double-stranded form, the duplex can be nicked by Nt.BstNBI to release the trigger. The catalytic cycle of polymerization / nicking is coupled to the linear production of the trigger strand, which initiates amplification after crossing a concentration threshold set by the pseudotemplate. We monitored the reaction in real time in the presence of increasing concentrations of Nt.BstNBI. As expected, the higher the concentration, the faster the production of the trigger and therefore the faster the amplification. The sensitivity of this technique in bulk is approximately 5 mu / ml (milliunits per milliliter), which is three orders of magnitude lower than when using traditional cleavage assays of pre-fluorescent probes.

[0103] Demonstrated versatility for 9 enzymes Based on these results, we designed various sensing strategies for ultrasensitive detection of other DNA-related enzymes. We designed a reaction cascade that links the enzymatic activity of interest to the generation of a specific trigger strand. The detection of nucleases was based on blocking the trigger production in the constitutive presence of Nt.BstNBI. RNAseH (RNAseH2) and AP-endonuclease (APE-1) were detected by introducing ribonucleotides and abasic sites (AP), respectively, into the stem structure of the sensing template (Figures 15a and 15b, sequences: nbitoBo-2+2(rG) SEQ ID NO: 77, and nbitoBo-2+2AP SEQ ID NO: 78). In these designs, polymerization of unprocessed substrates with protruding 3' polythimidylate extensions is blocked. Processing of these substrates by the corresponding enzymes induces endonuclease cleavage of the stem, restoring the production of the trigger by the polymerization / nicking cycle. Uracil DNA glycosylase (UDG) was detected by replacing the AP site with a deoxyribouridine moiety (sequence: nbitoBo-2+2UDG(2) SEQ ID NO:79), adding another step to the enzymatic cascade (Figure 15c). Excision of the uracil base by the glycosylase introduces an abasic site that is further incised by APE-1, ultimately reactivating the production of the trigger.

[0104] We tested a different strategy to detect another monofunctional DNA N-glycosylase, alkyladenine glycosylase (AAG, FIG. 15d). An inosine residue (hypoxanthine nucleobase, Hx) is incorporated into a short double-stranded oligonucleotide (sequence: Aagtorna-top / Aagtorna-bot, SEQ ID NO: 80 / SEQ ID NO: 81). Upon excision by AAG, the AP site is incised by APE-1. The 5' portion of the nicked strand is dissociated and can bind to the input portion of a second NBI-dependent template, the output of which is the trigger strand (sequence: dnatoBo-2+2P SEQ ID NO: 82). Detection of the restriction enzyme was achieved by appending a recognition site to the 5' portion of the sensing template (Figure 15e). In the absence of the target enzyme, futile cycles of polymerization / nicking generate a non-productive trigger with a 3' extension (containing the restriction site). In the presence of the target enzyme, the double-stranded restriction site is cleaved, releasing the extension from the sensing template, which in turn produces the trigger.

[0105] Polymerases with specific activity, such as poly(A) polymerase (PAP), which catalyzes the addition of a polyadenine tail to the 3' end of an RNA strand, can also be detected using this technique (Figure 15f). After polyadenylation of the RNA strand (sequence: rna SEQ ID NO:84), the poly(A) tail binds to the poly(T) input site of the sensing template (sequence: polyAtoBo-2+2P SEQ ID NO:85), which in turn triggers the output. We also adapted this strategy to detect DNA ligase (Figure 15d). The sensing module is composed of three templates: a hairpin template with a modified 5' phosphate moiety (sequence: lig2P SEQ ID NO: 87); a linear template with a 5' complementary activator sequence (sequence: lig1toBo SEQ ID NO: 86); and a splint strand (sequence: lig3P SEQ ID NO: 88) that partially hybridizes to both of the other strands. The resulting triplex contains a nick that can be sealed by T4 DNA ligase using ATP as the energy source. As a result, the splint strand is strand-displaced by DNA polymerase, restoring a functional source of the activator strand. Detection of polynucleotide kinase (T4 PNK) was made possible by using the unphosphorylated form of the hairpin template (seq: lig2noP SEQ ID NO: 89). After its phosphorylation, which is necessary for ligation, T4 DNA ligase seals the nick and rescues the production of the activator strand.

[0106] We performed detection of each enzyme in bulk solution separately using a cognate design. Figure 15 shows the amplification times (Cq) extracted from the fluorescence time traces recorded in real time. For each enzyme, we observed an inverse relationship between Cq and the concentration of the target enzyme down to the low concentration range of μU to mU / mL. This demonstrates the specific and sensitive detection of enzyme activity using a versatile DNA amplification mechanism.

[0107] Digital counting of single enzymes To further demonstrate the sensitivity of this technique, we performed digital counting of single enzymes isolated in microfluidic droplets. As for the bulk assay, a proof of principle was realized with a detection circuit of Nt.BstNBI. Two series of samples spiked with various concentrations of NBI were prepared and emulsified individually into picoliter-sized droplets (~0.95 pL) using a flow-focusing microfluidic chip. The droplets were incubated at 48 °C for 3 h and analyzed by fluorescence microscopy (Figure 16a). Figure 16b (top, left panel) shows the linear correlation between the spiked NBI concentration and the measured concentration calculated from Poisson's law (R 2 >0.99). Figure 16b shows the results of digital droplet assays of other enzymes, including RNAseH2, APE-1, UDG, BsmAI, PAP, T4 DNA ligase, and T4 PNK. Similarly, the linear relationship between spike-in and measured concentrations demonstrates that this method is successful for digital enumeration of these enzymes.

[0108] Digital detection of Nt.BstNBI was performed using larger droplets (7.2 pL) (Figure 17). Very similar concentrations were calculated compared to smaller droplets. The proportional relationship between spike concentration and measured concentration of target, coupled with the consistency of results obtained with different droplet sizes, clearly demonstrates a direct absolute quantification of active enzyme.

[0109] Example 5 Multiplexed detection of microRNAs Materials: HPLC-purified oligonucleotides were purchased from Biomers or Eurofins and resuspended at 100 μM in 1× Tris-EDTA pH 7.5 for long-term storage. Templates were designed according to the protocol described in Example 1 above. Template sequences aT, pT, rT, and kT were protected from the 5′->3′ exonuclease activity of ttRecJ by adding three 5′ phosphorothioate backbone modifications. Templates aT, pT, cT, and kT were blocked from undesired polymerization by adding a 3′ phosphate moiety. aT was designed to hybridize only to the last 10 bases of the corresponding input (α or β) to facilitate deactivation by pT of the signal strand produced by the leaky reaction. kT presents the same truncated input binding site to reduce competitive binding of the signal strand. This prevents kT from being nonspecifically activated prior to target-induced amplification. Table 5 summarizes all sequences used throughout this assay (SEQ ID NOs: 104, 106, 108, and 109 are also referenced as SEQ ID NOs: 54, 55, 56, and 57, respectively).

[0110] [Table 6]

[0111] The nicking enzymes Nb.BsmI and Nt.bstNBI, the restriction enzyme BsmI, and the DNA polymerase Vent(exo - ), BSA, and dNTPs were obtained from New England Biolabs (NEB). Thermus thermophilus RecJ exonuclease was produced in-house according to a previously published protocol (Yamagata et al. Nucleic Acids Res. 2001, 29 (22), 4617-4624). Sodium chloride, potassium chloride, magnesium sulfate, ammonium sulfate, Trizma hydrochloride, netropsin, and synperonic F104 were purchased from Merck (Sigma-Aldrich).

[0112] Reaction mixture assembly: All reaction mixtures were assembled in 200 μL PCR tubes at 4 °C. Templates and enzymes were first mixed with reaction buffer (20 mM Tris-HCl, pH 8.9, 10 mM (NH4)2SO4, 40 mM KCl, 10 mM MgSO4, 50 μM each dNTP, 0.1% (wt / vol) synperonic F104, 2 μM netropsin, and 200 mg / mL BSA). Optimized template concentrations were as follows: aTα=50 nM, aTβ, 50 nM, pTα=15 nM, pTβ=11 nM, rTα=40 nM, rTβ=40 nM, cT (each)=0.5 nM, αkβ=1 nM, and βkα=2.5 nM. The enzyme concentrations were Nb.BsmI = 300u / mL, Nt.BstNBI = 10u / mL, Vent(exo - ) = 60u / mL, BsmI = 60u / mL, and ttRecJ = 23nM. After homogenization, samples were spiked with microRNA solution and serially diluted in 1x Tris-EDTA buffer using low-binding DNA chips (Eppendorf). Samples (bulk or emulsion) were incubated at 50°C in a qPCR machine CFX96 touch (Bio-Rad).

[0113] Microfluidic droplet generation: A two-inlet flow focusing device was prepared using standard soft lithography techniques. Briefly, a microfluidic mold was obtained by coating a 4-inch silicon wafer with SU-8 photoresist (Micro-Chem Corp.) and reticulating upon UV exposure. After careful cleaning of the mold with isopropanol, a 10:1 mixture of Sylgard184 PDMS resin (40 g) / hardener (4 g) (Dow Corning) was poured into the mold, degassed under vacuum, and baked at 70 °C for 2 h. The PDMS slabs were removed from the mold, stacked, and punched with a 1.5 mm diameter biopsy puncher (Integra Miltex). The PDMS slabs were fixed onto a 1 mm thick glass slide (Paul Marienfeld GmbH&Co) immediately after oxygen plasma activation. The chip was baked at 200 °C for 5 h to render the channels hydrophobic. -Monodisperse water-in-oil droplets were generated by mixing aqueous samples and continuous phase (fluorinated oil Novec7500, 3M+1% (w / w) fluosurf, Emulseo) on-chip using a pressure pump controller MFCS-EZ (Fluigent) and 200 μm inner diameter PTFE tubing (CIL).

[0114] Droplet imaging and analysis: After incubation, the emulsions were imaged by microscope. A monolayer of droplets was sandwiched between two glass slides (1 mm thick bottom slide, Paul Marienfeld GmbH&Co, 0.17 mm thick top slide, VWR) spaced with 10 μm polystyrene particles (Polysciences, Inc.) to avoid droplet compression. The chamber was sealed with epoxy glue (Sader). Images were acquired with an epifluorescence microscope Eclipse Ti equipped with a motorized XY stage (Nikon), a camera Nikon DS-Qi2, an apochromatic 10× objective (NA 0.45, Nikon), and a CoolLed pE-4000 illumination source. Composite images were generated using the open source software ImageJ. Droplets were analyzed using Mathematica software (Wolfram) according to the procedure described in the examples above. The concentration of microRNAs is calculated by the following formula:

[0115]

number

[0116] result Killer templates counter switch cross activation We converted two parallel bistable switches into a tetrastable biochemical circuit. The rationale is that each of the four alternative states can result from four possible chemical "states" (0:0, 0:1, 1:0, and 1:1) associated with the presence / absence of each target, allowing appropriate classification in each case. To that end, we designed a cross-inhibitory template (killer template, kT) that connects the two switches bidirectionally (Figure 18). When activated by a cognate input (α or β), kT produces a pseudotemplate of the inverse switch, thereby acting as a cross-inhibitor of amplification. For the system to allow four states, the inhibitor needs to be strong enough to stabilize states 1:0 and 0:1 (only one of the two switches is on), but not too strong to allow the existence of state 1:1 (both switches are on). We therefore assessed the effect of endogenous pT length, as determined by the length of the inactivated 5' tail, on the strength of the killer template. Figure 19 shows the amplification reaction of a simple β-switch in the presence of 5 nM cel-mir-39 and increasing concentrations of αkβ-induced α-switches producing different pTβ. The system is set up so that in the absence of αkβ, the β-switch spontaneously turns on in about 100 min (Figure 20).

[0117] We observed that increasing the concentration of kT enhanced the preamplification delay, as expected. In addition, it is clear that αkβ, which produces shorter pT, is a more potent inhibitor: less than 100 pM kT αkβA1 (meaning that the resulting pTβ will add only one thymidine nucleotide to the 3' end of the α chain) is needed to completely prevent α-switch amplification, whereas more than 100-fold more is needed to observe the same effect with αkβA4 (Figure 18c). Interestingly, no inhibition was observed with αkβA5 in the concentration range tested. Similarly, αkβA0 (producing a complementary strand without catalytic elongation activity from α) had no effect on β-switch amplification, confirming a pseudotemplate catalytic mechanism.

[0118] Following these measurements, we selected kT to produce a pT with a 4-nucleotide extension whose inhibitory strength could be easily regulated by adjusting the concentration. Next, we evaluated the ability of kT to suppress cross-reactivity between α- and β-switches while retaining sensitivity to their cognate targets. Two microRNA sensing circuits are spiked with 0 or 10 pM mir92a (α-switch) and let7a (β-switch) in the presence of various concentrations of both αkβ and βkα (Figure 21). Figures 21b and 21c show the amplification times of both switches. In these experimental conditions, tetrastability is achieved at 2.5-10 nM βkα and 0.63-1.3 nM αkβ. In this concentration range of kT, the absence of target results in the absence of amplification (Cq>1000 min, state 0:0); when only one microRNA target is present, only the corresponding switch amplifies the fluorescent signal (Cq ~200 min, states 1:0 and 0:1); and finally, when both microRNAs are injected, the two switches are turned on (state 1:1).

[0119] We tested the generalization of this strategy to detect other microRNAs. The modular design of this programmable DNA circuit allows, in principle, the detection of any nucleic acid strand (RNA or DNA) with a known 3'-hydroxyl end by adapting only the converter template input domain. The rest of the duplex circuit (i.e., both aT, pT, rT, and kT) sequence and concentration remain unchanged. In these experiments, we used five microRNAs: has-mir-92a-5p, cel-mir-39, hsa-mir-7-5p, hsa-let-7a-5p, and hsa-let-7e-5p (abbreviated as mir92a, mir39, mir7, let7a, and let7e, respectively). Figure 21e shows the amplification times (Cq) of seven different duplex experiments in solution to detect 0 or 10 pM of the microRNA target. As expected, the system behaves as a tetrastable biochemical circuit in each case. Importantly, the amplification time of each switch is independent of the target sequence (Cqα=178±44 min, Cqβ=149±19 min). This therefore confirms that the cross-inhibition circuit suppresses undesired cross-reactivity and allows for programmable target detection.

[0120] Duplex digital detection of microRNAs. We finally transferred this multiplex assay to a digital readout using droplet microfluidics (Figure 22). The sample mixture is distributed into thousands of picoliter-sized droplets using a flow-focusing microfluidic device. As a result, the target microRNAs are randomly distributed within the water-in-oil droplets and occupy them according to a Poisson distribution. After incubation, which allows the droplet fluorescence to turn either green, red, or orange depending on the initial content, the droplets are imaged with an epifluorescence microscope. By knowing the droplet size and the percentage of positive droplets of each color, the concentration of the two microRNAs in the original sample was calculated. We demonstrated that the tetrastable circuit does not affect the detection (Figure 23) and limit of blanks compared to the singleplex assay (Figure 24). To better demonstrate, we prepared four samples spiked with 0 or 3 pM of microRNAs mir39 (α-switch) and let7a (β-switch). Each sample is barcoded with a combination of two fluorescent dextran barcodes and continuously emulsified using a home-made sample exchanger. After incubation, the droplets are imaged by fluorescence microscopy (Fig. 22b-22d). Although a small number of false positive events were recorded, accurate quantification of the two microRNAs was achieved within 12% ± 6% error (which may be partly due to concentration uncertainties due to serial dilution of the targets). To evaluate the reproducibility of the technique, we repeated this experiment for samples of different composition (different concentrations of the different microRNAs). For 15 samples, we observed a good correlation between the expected and measured concentrations of the spike-in microRNAs (Fig. 22d). Finally, we confirmed that the proportion of double-positive droplets (both green and red droplets) corresponds to the proportion expected from a Poisson distribution of the two targets (F o =F g .F r , in the formula F o , F g , and F r is the percentage of orange, green, and red droplets).

[0121] conclusion We have previously demonstrated that MP-based isothermal amplification strategy has the ability to completely eliminate background amplification. In the present invention, we take advantage of this feature to convert analog readout (real-time fluorescence monitoring) into digital (endpoint compartment analysis). Thus, the method of the present invention allows for highly sensitive and specific quantitative measurement of target biomolecules, particularly enzymes and nucleic acids, more specifically microRNAs. Based on a one-step procedure, the method of the present invention reduces sample manipulation and therefore reduces the risk of carryover contamination. The fact that the system relies on signal amplification mechanisms rather than target sequence replication further reduces the problem of contamination. The versatile DNA-based circuit can be adapted to any biomolecule of interest by designing the corresponding conversion template. Note that for microRNAs, all other circuit parts are common to all microRNAs, eliminating the need for primer and probe design, reducing assay costs.

[0122] Furthermore, the above results demonstrate that the DNA circuit architecture can be adapted for the detection of enzymes, especially DNA-associated enzymes with a broad range of activities (nucleases, DNA N-glycosylases, polymerases, ligases, and kinases). The sensitivity of the present invention allows for direct digital counting of individual enzymes isolated in picoliter-sized compartments. Also, the above method can be used for quantification of active enzymes after purification processes and to determine the effect of physical (temperature) or chemical treatments on enzyme activity at the single enzyme level. The above examples also demonstrate that the methods of the invention can be adapted to multiplex detection in a more sensitive manner. Preferred aspects of the present invention are as follows. [1] A digital method for detecting and / or quantifying at least one target biomolecule in a sample, comprising: a) mixing said sample with a mixture comprising a buffer, an enzyme, a first oligonucleotide that is an amplification oligonucleotide, a second oligonucleotide that is a leak-absorbing oligonucleotide, and a third oligonucleotide that is a target-specific conversion oligonucleotide; b) distributing the mixture obtained in step a) into several compartments, some of the compartments not containing the target biomolecule; c) converting said target biomolecule into a signal, said signal being preferably a single strand of DNA; d) amplifying said signal; and e) detecting and / or measuring said signal in each compartment. Digital methods including. [2] The digital method described in [1], wherein the target biomolecule is a nucleic acid or a protein, and the protein is preferably an enzyme. [3] The method according to [2], wherein the target biomolecule is a nucleic acid, preferably selected from the group including DNA, cDNA, RNA, mRNA, and microRNA, and more preferably, the nucleic acid is microRNA. [4] The method according to any one of [1] to [3] above, wherein the enzyme used in step a) is selected from the group consisting of a polymerase, a nicking enzyme or a restriction enzyme, and an exonuclease. [5] The method according to any one of [1] to [4] above, wherein the first oligonucleotide comprises a partially repeated structure including a nicking enzyme recognition site, and the second oligonucleotide can bind to the products of polymerization along the first oligonucleotide, extend them, inactivate them, and slowly release them, thereby inducing a threshold effect. [6] The method according to any one of [1] to [5] above, further comprising adding a fourth oligonucleotide which is a report probe, preferably the report probe being a fluorescent probe. [7] The method according to any one of [1] to [6] above, further comprising adding a fifth oligonucleotide which is a cross-inhibitory oligonucleotide for detecting and / or quantifying two or more types of biomolecules. [8] The method according to any one of [1] to [7] above, wherein the mixture obtained in step a) is partitioned into droplets, preferably water-in-oil emulsion droplets, in step b). [9] The method according to [8] above, wherein the size of the droplets is between 0.001 and 100 pL, preferably between 0.01 and 10 pL, more preferably between 0.1 and 5 pL, or between 0.1 and 8 pL.

[10] The method according to any one of [1] to [9] above, wherein the signal is labeled, preferably fluorescently labeled.

[11] The method according to any one of [1] to

[10] above, wherein the step d) of detecting and / or measuring the signal comprises detecting and / or counting the compartments that emit fluorescence.

[12] The method according to

[11] , wherein the number of compartments receiving a fluorescent signal and the number of non-fluorescent compartments are counted and the ratio between them is calculated to measure the absolute concentration of the target biomolecule in the tested biological sample.

[13] The method according to any one of [1] to

[12] above, wherein the target biomolecule is used as a biomarker.

[14] An in vitro method for diagnosing a disease selected from the group consisting of cancer, neurological diseases, cardiovascular diseases, inflammatory diseases, autoimmune diseases, diseases caused by viral or bacterial infections, skin diseases, musculoskeletal diseases, dental diseases, and prenatal diseases, comprising using the method according to any one of [1] to

[13] above.

[15] Diseases caused by biotic stress, preferably infectious and / or parasitic causes, or Diseases caused by abiotic stress, preferably nutritional deficiencies and / or adverse environments An in vitro method for the agricultural diagnosis of a disease selected from the group comprising: An in vitro method comprising using the digital method described in any one of [1] to

[13] above.

[16] A kit for detecting and / or quantifying at least one target biomolecule, comprising: a) a mixture of enzymes, preferably selected from the group comprising polymerases, nicking or restriction enzymes, and exonucleases, b) a mixture of oligonucleotides comprising a first oligonucleotide which is an amplification oligonucleotide, a second oligonucleotide which is a leak-absorbing oligonucleotide, and a third oligonucleotide which is a target-specific conversion oligonucleotide, and optionally a fourth oligonucleotide which is a report probe; and c) Partitioning agents, preferably water-in-oil emulsions Kit including:

[0123] References TIFF0007675022000011.tif235170 TIFF0007675022000012.tif100169

Claims

1. 1. A digital method for detecting and / or quantifying at least one target biomolecule selected from the group consisting of nucleic acids and enzymes in a sample, comprising: a) mixing said sample with a mixture comprising a buffer, an enzyme selected from the group comprising a polymerase, a nicking or restriction enzyme, and an exonuclease, a first oligonucleotide which is an amplification oligonucleotide that exponentially amplifies a signal sequence, said first oligonucleotide comprising a partially repeated structure containing a nicking enzyme recognition site, a second oligonucleotide which is a leaky absorbing oligonucleotide that drives the inactivation of a signal sequence synthesized by a leaky reaction, thereby enabling the avoidance of non-specific amplification, said second oligonucleotide having a 3' end complementary to the sequence amplified by the first oligonucleotide and a 5' end that serves as a template for adding an inactivation tail to the amplified sequence, and a third oligonucleotide which is a target-specific conversion oligonucleotide that converts said target biomolecule into a signal sequence, said third oligonucleotide being capable of binding to a target sequence at its 3' end and outputting, upon polymerization and nicking, a sequence capable of activating the first oligonucleotide above a threshold value that is adjusted by controlling the concentration of the second oligonucleotide; b) distributing the mixture obtained in step a) into several compartments, the target biomolecules being randomly distributed according to Poisson's law such that a percentage of the compartments higher than 1% does not contain the target biomolecule, between 0 and 10 biomolecules are present in the compartments, the size of the compartments being comprised between 0.01 and 10 pL, c) converting the target biomolecule into a signal; d) amplifying said signal; and e) detecting and / or measuring said signal in each compartment. thereby allowing for the direct counting of individual events for each target biomolecule population, thereby allowing for absolute quantification of said target biomolecules in the initial sample.

2. The digital method of claim 1 , wherein the target biomolecule is a DNA processing enzyme.

3. 2. The digital method of claim 1, wherein the target biomolecule is a nucleic acid selected from the group comprising DNA, cDNA, RNA, mRNA, and microRNA.

4. A digital method described in any one of claims 1 to 3, wherein the second oligonucleotide is capable of binding to, extending, inactivating and releasing products of polymerization along the first oligonucleotide, thereby inducing a threshold effect.

5. The digital method of any one of claims 1 to 4, further comprising adding a fourth oligonucleotide that is a report probe.

6. The digital method according to any one of claims 1 to 5, further comprising adding a fifth oligonucleotide which is a cross-inhibitory oligonucleotide for detecting and / or quantifying two or more biomolecules, said fifth oligonucleotide being capable of generating a reverse switch leak-absorbing oligonucleotide, thereby acting as a cross-inhibitor of amplification and reducing non-specific crosstalk.

7. The digital method according to any one of claims 1 to 6, wherein the mixture obtained in step a) is dispensed into droplets in step b).

8. The digital method according to claim 7, wherein the size of the droplets is comprised between 0.1 and 5 pL.

9. The digital method according to any one of claims 1 to 8, wherein the signal is labeled.

10. The digital method according to any one of claims 1 to 9, wherein the step e) of detecting and / or measuring the signal comprises detecting and / or counting the compartments that fluoresce.

11. The digital method according to claim 10, wherein the compartments receiving a fluorescent signal and the non-fluorescent compartments are counted and their ratio is calculated to determine the absolute concentration of the target biomolecule in the tested biological sample.

12. The digital method according to any one of claims 1 to 11, wherein the target biomolecule is used as a biomarker.

13. The digital method of claim 1, wherein the first oligonucleotide comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-3, 5, 12-15, 23, 24, 26, 28, 29, 31, 33, 35, 36, 40, 43, 44, 46, 52, 54, 61, 73, 103 and 104; the second oligonucleotide comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 4, 6-11, 16-22, 25, 27, 30, 37, 41, 49, 55, 62, 74, 105 and 106; and the third oligonucleotide comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 32, 34, 38, 39, 50, 51, 57-59, 64-66, 76-90 and 109-113.

14. An in vitro method for detecting the presence or absence of a disease selected from the group comprising cancer, a neurological disease, a cardiovascular disease, an inflammatory disease, an autoimmune disease, a disease caused by a viral or bacterial infection, a skin disease, a musculoskeletal disease, a dental disease and a prenatal disease, comprising using a digital method according to any one of claims 1 to 13, wherein the result of the detection and / or quantification of said at least one target biomolecule is indicative of the presence or absence of said disease.

15. A kit for detecting and / or quantifying at least one target biomolecule selected from the group consisting of nucleic acids and enzymes, comprising: a) a mixture of enzymes selected from the group comprising polymerases, nicking or restriction enzymes, and exonucleases; b) a mixture of oligonucleotides comprising a first oligonucleotide, which is an amplification oligonucleotide that exponentially amplifies a signal sequence, the first oligonucleotide comprising a partially repeated structure that includes a nicking enzyme recognition site; a second oligonucleotide, which is a leaky absorbing oligonucleotide that drives the inactivation of a signal sequence synthesized by a leaky reaction, thereby enabling the avoidance of non-specific amplification, the second oligonucleotide having a 3' end that is complementary to the sequence amplified by the first oligonucleotide and a 5' end that serves as a template for adding an inactivation tail to the amplified sequence; and a third oligonucleotide, which is a target-specific conversion oligonucleotide that converts the target biomolecule into a signal sequence, the 3' side of which is capable of binding to the target sequence and which, upon polymerization and nicking, outputs a sequence that can activate the first oligonucleotide above a threshold that is adjusted by controlling the concentration of the second oligonucleotide; c) A partitioning agent capable of forming compartments, in which a percentage of the compartments greater than 1% does not contain the target biomolecule, the target biomolecule is randomly distributed according to Poisson's law such that between 0 and 10 biomolecules are present in the compartment, and the size of the compartments is comprised between 0.01 and 10 pL. Kit including:

Images