COMPOSITION AND METHOD OF AMPLIFICATION OF NUCLEIC ACID SEQUENCES

Abstract

The invention relates to a masterbatch composition for nucleic acid sequence amplification, intended to be mixed with a biological sample containing one or more target nucleic acid sequences and primers specific to the target nucleic acid sequence(s), as well as fluorescent probes to reveal amplification of the target nucleic acid sequence(s). The composition comprises nucleic acid amplification reagents and enzymes, characterized in that the composition further comprises a polymerizable reagent, which polymerizes under the influence of a stimulus selected from thermal and light stimulation, so as to form a hydrogel locally encapsulating each target nucleic acid sequence when said composition is exposed to said stimulation. The invention also relates to a method using this composition. (Short figure: Figure 1)

Description

Title of the invention: NUCLEIC ACID SEQUENCE amplification composition and method

[0001] The invention relates to a nucleic acid sequence amplification composition and a nucleic acid sequence amplification method that uses this composition. The invention also relates to a device specially designed for using the composition.

[0002] More generally, the field of the invention is that of the detection of nucleic acid sequences by amplification reaction, such as PCR or any other in vitro amplification process, in a controlled diffusion medium compatible with such processes.

[0003] In molecular biology, it is regularly necessary to identify, with a high level of precision and confidence, the presence of one or more nucleic acid sequences of interest (or target nucleic acid sequences) in a sample. It is thus possible to measure the relative or absolute concentration of these sequences of interest.

[0004] By way of example, target sequences can be DNA sequences such as gene fragments specifically present in the genome of human or animal pathogens. Target sequences therefore make it possible to identify the pathogen carrying them, or to characterize their pathogenicity or their resistance to one or more antibiotics or one or more antiviral molecules. Target sequences can also be DNA sequences located outside of genes, such as telomeres, transposons, or retrotransposons. Some target sequences can be distinguished from one another by one or more point mutations, that is, a change of one base by another (single nucleotide polymorphism), or by the addition or deletion of one or more bases.Target sequences can also be RNA, such as gene transcripts, microRNAs, untranslated RNAs, viral RNAs from RNA viruses, and retrotransposon transcripts. In some situations, target sequences are artificial DNA sequences used as barcodes or tags. This is notably the case for sequences covalently attached to antibodies used in immuno-PCR procedures, or for sequences integrated into padlock probes.

[0005] The target nucleic acid sequences may be in different states within the sample. Thus, the sequences may be free in the sample, possibly after a processing step of this sample, or included in Biological particles such as viruses, prokaryotic or eukaryotic cells, or organelles of the latter such as mitochondria. In this case, these nucleic acid sequences are generally chosen so that they allow the identification of the biological particles, or the characterization of a genotype or a particular property.

[0006] Regardless of the state of the sequences, when they are present in too small a quantity to be directly characterized, a commonly used method for detecting them consists of performing in vitro amplification of these sequences to obtain a sufficient quantity to allow their characterization. This amplification is carried out using a mixture of oligonucleotide primers located in the target sequences. The addition of one or more oligonucleotide probes conjugated to a fluorophore classically allows the presence or absence of the target sequences, as well as their quantity, to be characterized.

[0007] A well-known amplification method is the polymerase chain reaction, also known as PCR (Polymerase Chain Reaction). PCR is an in vitro exponential amplification method. It is the most commonly used amplification method currently. Besides PCR, there are other alternative methods such as LCR (Ligation Chain Reaction), NASBA (Nucleic Acid Sequence-Based Amplification), TMA (Transcription-Mediated Amplification), LAMP (Loop-Mediated Isothermal Amplification), SDA (Strand Displacement Amplification), and RCA (Rolling Circle Amplification).

[0008] PCR and other in vitro amplification reactions are usually carried out in a liquid reaction medium containing the various reagents. These typically include primers, fluorescent probes specific to the target sequences, enzymes for nucleic acid amplification, optionally additives, and the sample(s) to be analyzed containing the target sequence(s). During the amplification reaction, the reagents and the reaction products, called amplicons, diffuse freely into the liquid volume of the reaction medium. This free diffusion in the liquid reaction medium gives rise to several drawbacks that are prohibitive for certain applications.

[0009] Indeed, in some applications, the target sequences are present in small quantities in the samples (1 to 10 molecules, for example). It is therefore necessary to plan a procedure to ensure very high-sensitivity detection. This is particularly the case for the in vitro diagnosis of specific nucleic acid sequences in a person's blood sample, which may, for example, indicate the presence of septicemia, a tumor, or even a genetic disease in a fetus (detection in a (liquid biopsy). Even though amplification methods may theoretically be capable of detecting a single molecule of a target sequence, in practice, the diffusion of amplicons in the reaction mixture delays the point in the reaction where the signal intensity emitted by the amplicons and measured by the detector of the device used locally exceeds the detector's background noise level. This delay can be so significant that the amplicons are ultimately not detected during the reaction, leading to a false negative result.

[0010] Free diffusion also poses a problem in applications requiring the detection of multiple target sequences in the same reaction mixture. This is particularly true in applications related to the diagnosis of infectious diseases, where it is essential to simultaneously identify the pathogen(s) involved, as well as their resistance genotype to antibiotic or antiviral molecules. While one solution to meet this requirement might be to detect each sequence of interest in a monoplex test, this solution appears to be cumbersome, and it may be preferable to detect all the sequences of interest in a single reaction. The reasons for this may include urgency, small sample size, low concentration of the target sequences in the sample, or the cost of the test (reagents, consumables, analyzer utilization rate, etc.).However, the free diffusion of amplicons from the predominant target sequence in the sample interferes with the amplification of other, less abundant target sequences in that sample. This is primarily due to competition for the reagents required for the amplification reaction. Therefore, there is a constant risk that one or more less abundant target sequences will not be detected in the sample.

[0011] In other applications, it is necessary to measure the quantity of target sequence(s) present in a sample. There is thus a need for precise quantification or measurement of a distribution. More specifically, it is sometimes necessary to ensure that the presence of a target sequence in large quantities in a sample does not interfere with the measurement of the concentration of other target sequences present in smaller quantities (for example, 10, 100, or 1000 times less abundant). Such a measurement is impossible when the target sequences and their amplicons diffuse freely in the reaction mixture.

[0012] To overcome some of the problems mentioned above, the state of the art offers different approaches.

[0013] In particular, there is the approach of in situ PCR amplification. This approach is useful when the target sequences are contained within a cell. In situ PCR thus includes a local DNA or RNA amplification step to improve the sensitivity and specificity performance of the hybridization. In this technique Amplification can be performed in solution. This requires fixing the cell(s) and making them permeable to allow PCR reagents to enter. Amplification can also be performed on formalin-fixed histological sections (US5364790, US5538871, and US5804383). The main drawback is related to cell permeabilization, which allows reagents to penetrate the cells. This permeabilization enables amplicons to diffuse, rendering liquid-phase amplified product characterization methods inapplicable. Therefore, the sample must be manipulated after amplification to detect the amplicons fixed within the cells. This lengthens the reaction time and increases its complexity. Furthermore, it is a costly operation with a higher risk of cross-contamination.For these reasons, in situ PCR has notably failed to meet the needs of multiple genotyping (Uhlmann et al, 1998).

[0014] Another approach is a process called digital amplification or digital PCR. In this process, the reaction mixture is divided into a large number of distinct, small-volume compartments (for example, on the order of picoliters). The compartments are chosen so that each compartment contains at most one molecule of the target sequence. This physical compartmentalization can be achieved, for example, by producing an emulsion of droplets of the reaction mixture in an oily liquid using a microfluidic system. It can also be achieved by filling a microfluidic device consisting of a large number of small-volume cells. Regardless of the method used, this compartmentalization allows for the performance of a large number of completely separate amplification reactions, without the reaction mixture passing from one compartment to another.The compartments in which an amplification reaction occurs are detected and counted. This counting allows for a precise quantification of the number of target sequence molecules present in the sample. Digital PCR thus makes it possible to quantify the presence of several target sequences in a single reaction [Sensors (Basel) 2018, 18(4), 1271; J. Med. Virol. 2021, 93(7), 4182-4197]. However, performing a digital PCR reaction requires complex and expensive equipment. In particular, compartmentalizing the reaction mixture requires complex handling. Furthermore, digital PCR does not retain the advantages of real-time PCR (with the measurement of Ct, for example).

[0015] A promising approach is to perform PCR in a gel matrix and measure the PCR products generated at each cycle. The PCR can be carried out inside a polyacrylamide (PAM) gel (Anal. Biochem. 2006, 356, 300-302) or a PEG derivative. Thus, the reaction generates distinct molecular colonies. which are sometimes called "polonias" (P. Blainey et al. US 10487354; Huang et al. US2019 / 0202268; Huang et al. US2019 / 0202268; GM Church et al. US6485944; AB Chetverin et al. EP1999268; AB Chetverin et al. US6001568). The main advantages of these matrices are: i) with an appropriate mesh size, they delay the diffusion of linear molecules so that target molecules and their amplification products remain localized in the immediate vicinity; ii) the number of polonias formed is proportional to the number of target molecules initially present in the sample, which facilitates quantification in a manner similar to digital PCR; and iii) it is possible to attach at least one of the primers to the gel by covalent bonding. However, a significant drawback of state-of-the-art gels is their incompatibility with applications outside the laboratory.For example, state-of-the-art techniques are unsuitable for rapid diagnostic applications. This is primarily due to the gelation process, which is not spatially or temporally controllable. Furthermore, in the case of PAM gels, target molecules and amplification reagents must be incorporated into the already formed gel after it has been rinsed with buffer to remove unpolymerized acrylamide monomers, due to their strong inhibitory effect on the PCR amplification reaction.

[0016] The present invention improves the situation.

[0017] Thus, the invention relates to a masterbatch composition for the amplification of nucleic acid sequences, intended to be mixed with a biological sample containing one or more target nucleic acid sequences and with primers specific to the target nucleic acid sequence(s), as well as fluorescent probes to reveal an amplification of the target nucleic acid sequence(s), the composition comprising nucleic acid amplification reagents and enzymes, characterized in that the composition further comprises a polymerizable reagent, which polymerizes under the influence of a stimulation chosen from thermal stimulation and light stimulation, so as to form a hydrogel locally encapsulating each target nucleic acid sequence when said composition is exposed to said stimulation.

[0018] The invention thus makes it possible to carry out nucleic acid sequence amplification reactions in a medium in which the diffusion of the components is controlled. Therefore, the invention allows for the local encapsulation of the target nucleic acid sequence(s). This results in a number of advantages, particularly related to the detection of nucleic acids and / or the localized control of amplification reactions.

[0019] In one embodiment, the polymerizable reagent is a photosensitive reagent that polymerizes when exposed to light, preferably ultraviolet light. This allows precise control of the initiation of controlled polymerization of the composition of the invention.

[0020] The polymerizable reagent can also be a heat-sensitive reagent that polymerizes when exposed to a temperature above a threshold temperature. An advantage of this embodiment is that the temperature ranges used in nucleic acid amplification processes, whether isothermal or not, can be used directly.

[0021] Preferably, the polymerizable reagent comprises pre-polymerized chains, advantageously, of which only the terminal function is reactive to more easily control the pore size of the gel. These pre-polymerized chains can be chosen from water-soluble polymers that are non- or weakly inhibitory to PCR (for example, PEG, PAM, biopolymers, etc.).

[0022] In general, the polymerizable reagent can be a multi-arm poly(ethylene glycol) (or multi-arm PEG, or multi-arm PEG in English).

[0023] More generally, the polymerizable reagent is advantageously pre-polymerized and / or water-soluble and biologically inert (so as not to interfere with the amplification reaction) and / or capable of polymerizing (with an activatable function or by its radical nature for example).

[0024] Thus, in a particular embodiment, the polymerizable reagent is chosen from the class of multi-arm PEGs and their derivatives.

[0025] The polymerizable reagent can be selected from 4-Arm-PEG-SH, 4-Arm-PEG-norbornene, 2-Arm-PEG-SH, 8-Arm-PEG-norbornene (tripentaerythritol), nitrocinnamate-based 8-Arm-PEG, or a mixture thereof. These are photosensitive reagents that polymerize under light of a chosen wavelength and thus contribute to the precision of the controlled polymerization of the composition of the invention.

[0026] Advantageously, the photosensitive polymerizable reagent is a mixture of 2 Arm-PEG-Thiol and 8 Arm-PEG-Norbomene or 4 Arm-PEG-Thiol and 4 Arm-PEG-norbomene in the presence of the photoinitiator lithium phenyl-2,4,6-trimethyIbenzoyIpho sphinate (LAP).

[0027] The polymerizable reagent can be 4 Arm-PEG-acrylate. This is a reagent which polymerizes in the presence of a thermoinitiator when the latter is exposed to a given temperature.

[0028] Advantageously, the thermosensitive polymerizable reagent is a mixture of 4 Arm-PEG-Acrylate in the presence of the thermoinitiator 2,2'-azobis[2-(2-imidazolin-2-yl)propane]dihydrochloride.

[0029] More generally, the photosensitive polymerizable reagent can be chosen from water-soluble polymers bearing unsaturated (norbomene, nitrocinnamate, etc.) or non-saturated (thiols) functional groups, or a mixture thereof. These are reagents capable of forming reactive intermediate species, preferably radicals or radical ions, in the presence, where appropriate, of an activator (the polymer itself or a photoinitiator), by application of light with a wavelength between 200 and 800nm, preferably between 300 and 450nm.

[0030] Furthermore, the heat-sensitive polymerizable reagent can be chosen from water-soluble polymers bearing alkene functional groups (acrylate, acrylamide, etc.) or a mixture of activated unsaturated functional groups (maleic anhydride, maleimide, etc.) with conjugated 1,3-dienes (furan, thiophene, etc.). These reagents are capable of forming reactive intermediate species, preferably radicals or radical ions, or of participating in cycloaddition reactions by applying a threshold temperature between 30 and 95°C, preferably between 50 and 80°C.

[0031] In one embodiment, the polymerizable reagent is present in a content of 2% to 25%, preferably 5% to 15%, by weight per unit volume (w / V%).

[0032] In one embodiment, the composition further comprises a polymerization initiator. This may be a photoinitiator or a thermoinitiator. The initiator is present in a concentration of 0.02% to approximately 0.1% by weight per unit volume (w / v). Preferably, the photoinitiator is lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP), and the thermoinitiator is 2,2'-azobis[2-(2-imidazolin-2-yl)propane]dihydrochloride. The agent facilitates the initiation of polymerization and thus contributes to the controlled polymerization of the composition of the invention.

[0033] In addition, the composition may include, from the outset, a developer for the presence of nucleic acid sequences, preferably fluorescent. The developer may be a fluorescent nucleic acid intercalator or a sequence-specific fluorescent probe combined with a fluorescence quencher. The developer provides improved detection of amplified nucleic acids during, or at the end of, the amplification reaction.

[0034] The composition may further comprise ab initio primers specific to the target nucleic acid sequence(s). This makes it possible, in particular, to prepare master mixtures specifically designed to identify a known pathogen. In this embodiment, it is also advantageous to include directly in the composition the specific probe(s) to reveal the presence of the amplified target nucleic acid sequence(s).

[0035] In a preferred embodiment of the invention, the composition of the invention remains liquid prior to the application of said stimulation for a period of between several hours and several months, preferably between several days and several months, and said composition forms the hydrogel after the application of said stimulation in a time less than or equal to 10 minutes, preferably less than or equal to 1 minute.

[0036] Obviously, the composition is stable over time, that is, for as long as the composition remains liquid (or very low viscosity) and beyond, so as to function as a nucleic acid sequence amplification composition. Stability is understood to mean, in particular, that the reagents do not degrade (or hardly degrade) under normal storage conditions (for example, in a refrigerator, at room temperature, protected from light, etc.).

[0037] The invention also relates to a method for amplifying nucleic acid sequences comprising the following steps: (i) making available the composition of the invention; (ii) mixing said composition with a biological sample to be analyzed; (iii) applying a stimulation selected from thermal stimulation and light stimulation so as to polymerize the mixture of step (ii) to obtain a hydrogel; (iv) exposing the hydrogel to a nucleic acid sequence amplification reaction.

[0038] Step (ii) may include mixing the composition with primers specific to the target nucleic acid sequence(s) and / or with specific probes to reveal the presence of the amplified target nucleic acid sequences.

[0039] In one embodiment, the (i) making available step is maintained for a period of time between several hours (for example 1 to 5 hours) and several months (for example 1 to 18 months), preferably between several days (1 to 15 days) and several months (2 to 6 months).

[0040] A key point of the process of the invention is that the polymerization in step (ii) is initiated only after the application of a light or thermal stimulus. Thus, the light stimulus may consist of the application of light with a wavelength between 200 nm and 800 nm, preferably between 300 nm and 450 nm; and the thermal stimulus may consist of the application of a threshold temperature between 30°C and 95°C, preferably between 50°C and 80°C for a duration of less than 10 min.

[0041] Advantageously, the time between the provision of the composition and the application of the stimulus is greater than Ih, preferably greater than 1 month, which avoids preparing the composition just before its use.

[0042] In other words, the composition of the invention can be stored for a period of more than 1 month and which can reach up to six months and beyond.

[0043] Moreover, each step of the process can be characterized by a duration: (t0) of storage of the composition containing the polymer(s) and the amplification reagents, which can last up to several months depending on its composition and storage conditions; (ti) of handling the same composition once removed from storage and placed in the presence of the target without gelation under ambient conditions, which can can last up to several hours; and (t2) of gelation after the application of a stimulus (light or temperature), being less than 10 minutes.

[0044] In other words, the composition containing the polymerizable agent is stable for a very long period, up to several months. This is highly advantageous for storage conditions. The composition can then be mixed with the target sequence without the polymerization process directly transforming the solution into a gel. In practice, gelation progresses very slowly, and gel formation is only observed after several hours. Another major advantage is that there is no need to rush laboratory procedures. In particular, this allows for the parallel preparation of several samples. The polymerization of several samples can thus be initiated simultaneously in a single stimulus operation. The gelation of all the samples takes only a few minutes. Consequently, the amplification process of the target sequences can then be started for all the samples in parallel.Thus, in a particular embodiment, the hydrogel is obtained in a time less than or equal to 10 minutes, preferably less than or equal to 1 minute after the application of the stimulation of the process of the invention.

[0045] The hydrogel formed by the composition of the invention encapsulates the amplification reagents as well as the target nucleic acid sequence(s). Consequently, nucleic acid sequence amplification reactions are conducted locally at distinct locations within the gel. The amplification products remain encapsulated in the hydrogel. In other words, both the diffusion of the composition's constituents and the diffusion of the amplification reaction products are controlled. This results in numerous advantages, particularly related to the analysis and detection of nucleic acids.

[0046] In one embodiment, the amplification reaction is a polymerase chain reaction.

[0047] In one embodiment of the invention, polymerization is initiated by exposing the composition to ultraviolet light (when the polymerizable agent is photosensitive) or a threshold temperature (when the polymerizable agent is thermosensitive). This polymerization can be linked to a reaction selected from the group consisting of a bio-orthogonal thiol-ene reaction and a dimerization reaction.

[0048] Other advantages and features of the invention will become apparent from the detailed description below and the accompanying drawings, in which:

[0049] [Fig.1] shows a principle figure of the invention;

[0050] [Fig.2] shows photographs of hydrogels formed by the composition of the invention with a photosensitive agent having initial concentrations of sequences different targets, as well as a graph corresponding to the PCR data of a first example of embodiment of the invention;

[0051] [Fig.3] shows two photographs of a hydrogel formed by the composition of the invention of the first example of implementation; and

[0052] [Fig.4] shows photographs of hydrogels formed by embodiments different from the composition according to the invention having an initial concentration of identical target sequences (103 copies).

[0053] The figures, tables, and description below essentially contain elements of a certain nature. The figures and tables form an integral part of the description and may therefore not only serve to better understand the present invention but also contribute to its definition, if necessary.

[0054] In general, the present invention relates to compositions comprising a polymerizable agent. The compositions are biocompatible and exhibit low viscosity at room temperature. Thus, the compositions of the invention allow target molecules or target cells to be mixed with nucleotide sequence amplification reagents at room temperature. This has the advantage that manipulations by laboratory operators can be carried out routinely. The compositions of the invention are characterized by their ability to rapidly form a homogeneous network hydrogel by means of the polymerizable agent. The polymerizable agent triggers polymerization of the composition when exposed to a triggering factor, such as a light stimulus or a threshold temperature. The composition of the invention ensures sufficient PCR sensitivity, particularly through gel image analysis.When the composition of the invention is in a gel state, it can then be subjected to a nucleic acid amplification reaction.

[0055] The composition of the invention in the gel state has a matrix capable of withstanding the rapid temperature changes generally required during amplification reactions. Furthermore, the composition in gel form retains its structural integrity under the effect of the various physical and / or chemical stresses applied to it during nucleic acid amplification processes.

[0056] An objective of the invention is to produce gel matrices with a homogeneous structure. The gel has suitable meshes that drastically limit, or even prohibit, the diffusion of target molecules and amplification products, while allowing the diffusion of amplification reagents to carry out the nucleic acid amplification reaction(s). This objective is achieved by the main claim annexed to this description of the invention.

[0057] Classically, hydrogels are three-dimensional (3D) cross-linked polymer networks capable of absorbing and retaining large quantities of water. Due to their tunable properties and versatile manufacturing methods, hydrogels are used in a wide range of biomedical and technical applications. Examples include tissue engineering and regenerative medicine, wastewater treatment, and robotics. Hydrogels are generally formed from monomers or prepolymer chains through covalent and / or non-covalent bonds such as hydrogen bonds, electrostatic interactions, host-guest complexations, and combinations thereof (W. Wang, R. Narain, H. Zeng. Polymer Science and Nanotechnology, Fundamentals and Applications, 2020, Pages 203-244).

[0058] The prior art describes gel matrices used for nucleic acid amplification. These gel matrices are formed in polymerization reactions according to two basic mechanisms: chain polymerization and step-by-step polymerization.

[0059] Chain polymerization (or "chain growth polymerization") consists of forming a polymer from unsaturated monomer molecules (notably acrylamide, methacrylamide, acrylic acid, methacrylic acid, bis-acrylamide, styrene, etc.). This type of reaction requires initial activation of the unsaturated bond(s). In other words, it is necessary to first form a reactive species (radical, cation). For example, reactive radical particles can be formed after activation of specific compounds called initiators. Initiator activation can be achieved using photons, heating, redox potential, or enzymatic activity. The initiator(s) possess a radical center and thus the ability to easily transfer radicals to reactive molecules. The radicals are thus formed in a cascade.In summary, once the initiator is added and the stimulus applied if necessary, radicals begin to be generated and polymerization is initiated. During the polymer growth process, a chain containing repeating monomers is formed. Monomers are added sequentially to the ends of the polymer chains. The polymerization process can be interrupted, notably due to the achievement of a high conversion rate, the abrupt recombination of several different chains, or the presence of free radical scavenging compounds in the reaction medium. Oxygen is typically used as a free radical scavenging agent. Therefore, it is preferable to perform degassing before polymerization and / or to apply inert conditions during it.This requires complex manipulations and involves substantial laboratory equipment, such as desiccators, inert gas tanks or an inert gas line, pumps, etc. In addition, chain polymerization generates the formation of chains of different lengths, which leads to irregularities in the matrix. Gel matrices also contain heterogeneous, high-molecular-weight cross-links, resulting in a hydrogel composed of cells of varying sizes. The gel structure is thus often incompatible with nucleic acid amplification reactions. Furthermore, radical polymerizations are generally rapid and therefore difficult, if not impossible, to control.

[0060] Step-growth polymerization involves various types of reactions of bifunctional or multifunctional monomers (prepolymers). In particular, the thiol-based Michael conjugation reaction involving PEG derivatives is regularly used in this type of polymerization. The functional groups of the monomers or prepolymers are, by definition, reactive sites and can thus react directly with each other. Unlike chain polymerization, dimers, trimers, and tetramers are formed at the beginning of the polymerization. Subsequently, these oligomers combine with each other to form long polymer chains. The polymers formed by this type of polymerization generally do not possess heterogeneous cross-links [Biomacromolecules 2012, 13(8), 2410-2417; J. Appl. Polym. Sci. 2015, 132, 8]. This ensures a certain homogeneity of the lattice.For this reason, gel matrices of this type are preferred for PCR applications, such as the formation of polonia. However, the gel structure is very compact, which is often incompatible with nucleic acid amplification reactions. Furthermore, the step-by-step polymerization reaction can occur rapidly after mixing the reagents. Consequently, some of these reactions are also very difficult, if not impossible, to control.

[0061] Beyond the uncontrollable properties of the polymerization reactions and the unsuitable structure of the gels, there are other disadvantages, some of which are discussed below.

[0062] The reaction times of known gelation processes of the type described above (acrylamide-bis-acrylamide copolymers and multi-arm PEG-acrylate / PEG-SH copolymers) often require between 10 and 30 minutes at room temperature or at temperatures between 20 and 40°C to form a nearly complete polymer structure [P. Blainey et al. (US 10487354); Huang et al. (US2019 / 0202268); GM Church et al. (US 6,485,944); AB Chetverin et al. (EP1999268); AB Chetverin et al. (US6001568)]. This duration reduces the efficiency of amplification reagents, which are generally temperature-sensitive (enzymes, fluorophores, etc.). Furthermore, this duration is unsuitable for all applications where rapid testing is important, such as point-of-care testing. For such applications, a gelation time of approximately one minute would be preferable.

[0063] The onset of a polymerization reaction is uncontrollable in current techniques. Indeed, in the case of step-growth PEG-acrylate-PEG-SH hydrogels, the reaction is initiated as soon as the components are mixed, and in the case of chain-growth acrylamide-bis-acrylamide hydrogels, the reaction is initiated when the components are mixed with polymerization initiators (e.g., APS-TEMED). This complicates user handling (speed is particularly important) and affects the reproducibility of a protocol due to the high probability of gel matrix defects forming during the mixing and subsequent injection into the reaction chamber.

[0064] Furthermore, it is impossible to automate known polymerization processes, as handling replicates of 100-1000 reactions or more requires the separate preparation of batches of a maximum of 2-5 reactions. This also complicates the use of amplification chambers of various shapes (cassettes, narrow or elongated reaction chambers, i.e., rod or capillary type, specially folded or wound in a flat cassette to fit into a standard PCR cycler), thus limiting their number to those that can be filled quickly. More specifically, in known gel amplification processes, the viscosity of the reaction mixtures increases as soon as the polymers are introduced into the mixture. Consequently, the reaction mixture gels rapidly and must be introduced quickly into the amplification reaction chamber. Otherwise, the gelled mixture can clog the chamber inlet.The mixture then becomes unusable and the amplification fails.

[0065] The radical chain reaction, for example in the case of acrylamide-bis-acrylamide gels, is a reaction sensitive to the presence of oxygen in the air. Oxygen inhibits polymerization and contributes to the formation of networks containing heterogeneous, high-molecular-weight crosslinks. This results in hydrogels composed of meshes of varying sizes. Therefore, it is preferable to carry out a degassing process before the polymerization reaction and / or to apply inert conditions during it. This requires complex handling and the use of several pieces of laboratory equipment, such as desiccators, inert gas tanks or inert gas lines, pumps, etc.

[0066] Furthermore, a major drawback of these gels is that unreacted acrylate monomers are detrimental to the amplification reaction. Attempts have been made to overcome this problem by adding extra washing steps (AB Chetverin et al. in EP1999268 and US6001568). However, this increases the complexity of the processes by adding lengthy steps, often associated with yield losses. Indeed, the gel preparation process includes: a) treating the glass surface with a binder-silane-water-ethanol-acetic acid mixture to fix the gel (approximately 2 hours); b) degassing of the prepolymer solution (minimum approximately 15 minutes); c) incubation at 4°C for 1 hour, followed by the rapid addition of initiating agents and gelation at room temperature for approximately 40 minutes; d) washing of the gel for approximately 60 minutes, followed by overnight drying at room temperature; e) diffusion of the amplification reaction mixture into the gels. The entire process thus takes approximately 13 hours or more. Besides the time constraint, another major drawback of this type of process is that it makes it virtually impossible to use these gels for target molecules of varying sizes (DNA, RNA, bacteria, viruses). This is primarily due to diffusion problems within the gel, which result in an uneven distribution of the amplification products.

[0067] In the context of nucleic acid amplification reactions, the preparation of gels must take into account a large number of factors. These include the physical stability of the gel, its inertness, the toxicity of the components, the compatibility of the gel with the amplification reaction reagents, as well as the compatibility of the gel with the amplification conditions (i.e., on the one hand the influence of the gel components on the amplification reagents and on the other hand the influence of the amplification reagents on gelation).

[0068] Attempts have been made to produce PEG-based gels, in particular a copolymer of PEG 4-Arm acrylate and PEG 2-Arm thiol, and to apply a nucleic acid amplification reaction to them (P. Blainey et al. in US10487354 or Huang et al. in US2019 / 0202268). However, the excessively rapid onset of polymerization and its uncontrollable nature lead to difficulties that make industrialization impossible.

[0069] In conclusion, the problems encountered in the art are diverse and varied, and can be summarized as follows: a long preparation process; an initial polymerization that is too rapid and uncontrollable; a time that is too long for the formation of the gel; problems of reproducibility; the formation of non-homogeneous gels; complex manipulations; degassing or manipulations under an inert atmosphere; protocols with a large number of steps; the need to wash the gels to remove potential inhibitors of nucleic acid amplification; and the need for several pieces of laboratory equipment (desiccators, inert gas tanks or inert gas lines, pumps, washing baths, etc.).

[0070] All of this means that no nucleic acid amplification technique (PCR in particular) can be satisfactorily performed in gel form. Furthermore, no commercially available and / or industrially produced gel amplification technique currently exists.

[0071] The Applicant has developed a composition and a method for amplifying nucleic acids in hydrogel that solves the problems of the art. Thus, the invention It does not require complex manipulations, uses non-toxic reagents compatible with both nucleic acids and their enzymatic amplification. Polymerization with the composition of the invention is controllable by means of a polymerization agent that is sensitive to light and / or temperature. The polymerization agent of the invention is photosensitive and / or thermosensitive.

[0072] In particular, the invention offers drastically increased (i) ease of handling, (ii) speed, (iii) controllability, and (iv) storage compared to the prior art. Indeed, the invention makes it possible (il) to prepare the amplification reaction in a single step, without complex manipulations. Furthermore, (i.2) no degassing step is required (often linked to oxygen sensitivity). (ii.1) Preparation is rapid, i.e., notably without washing. For example, there is no need to wash the gels to remove potential inhibitors of amplification enzymes. (ii.2) Gelation is very rapid, and only after the application of a stimulus (light or heat). (iii.1) The stimulus is spatially and temporally controllable and is also perfectly (iii.2) reproducible. The (iii.3) size of the polonias is homogeneous, which in particular allows for greater control of the amplification process.The ingredients of the composition of the invention make it possible to (iv.1) mix them without risk of gelling before the application of the stimulus. (iv.2) Long-term storage, as well as carrying out a large number of (iv.3) parallel manipulations, become possible with the invention.

[0073] The invention relates in particular to a light-induced or temperature-induced polymerization of one or more monomers or prepolymers, with or without a photo- or thermo-initiation agent. The polymerization of the invention combines the advantages of the two polymerization mechanisms described above (chain-growth and step-growth).

[0074] Photo-polymerization and thermo-polymerization are among the chemical methods for forming hydrogels. It occurs by exposing a photosensitive or thermosensitive system composed of unsaturated components with / without a photo- or thermo-initiator (with / without other components bearing photo / thermosensitive functional groups) to ultraviolet (-200-400 nm) or visible (-400-800 nm) light or to a high temperature (above room temperature).

[0075] In the present description, and for the definition of the invention, "stimulus" or "stimuli" or "stimulus" means any chemical or physical element or factor that triggers polymerization.

[0076] The main advantage of photon-activated (photoactivation) or heat-activated (thermoactivation) polymerization is that hydrogels are formed in situ from aqueous solutions in a minimally invasive manner and with controlled activation. of the gelation process. This is made possible by the use of an external stimulus applied to the reagents. The stimuli used in the invention are, respectively, the targeted irradiation of the prepolymers with light of a chosen wavelength or the exposure of the monomers (which are also prepolymers) to a threshold temperature. The ability to adjust the intensity and duration of exposure to light or temperature via the PCR device provides additional control over the gelation and thus over the properties of the generated gel (control of the swelling rate, mechanical strength, degradability, etc.).

[0077] Regarding photoactivation: light can be directed to a precise location at a defined time. This allows for different technical designs of amplification chambers. The use of light energy enables polymerization at low doses of initiator radicals under simple reaction conditions. Typically, photopolymerization is defined by rapid polymerization rates (approximately a few minutes) and minimal heat production. This is particularly important for the stability of amplification systems (Biomaterials 2002, 23, 4307-4314). These advantages make this hydrogel formation method particularly well-suited for in vivo applications, the encapsulation of viable cells for tissue engineering and regenerative medicine strategies, as well as controlled drug release applications (B. Amsden).Chapter 8: Photocrosslinking methods for designing hydrogels. Gels Handbook, pp. 201-218 (2016).

[0078] Regarding thermoactivation: the temperature range used during PCR may be sufficient to initiate polymerization. Thus, gelation can occur simultaneously with the start of the nucleic acid amplification reaction. Unlike hydrogels formed by photopolymerization, this system may not require any additional time related to light irradiation. This makes this embodiment particularly suitable for rapid PCR applications. Furthermore, it does not require any additional equipment such as a UV lamp integrated into the amplification device (such as a PCR thermocycler, for example), since the stimulus for hydrogel formation is the same as that for nucleic acid amplification, namely temperature.

[0079] The invention provides for selecting one or more monomers or prepolymers for the construction of a hydrogel. The concentration of monomers or prepolymers in the composition of the invention is generally between 2% and 25%. More generally, the invention provides for a polymerizable reagent that polymerizes under the influence of an external factor. According to the invention, this factor is light or temperature. Thus, when the polymerizable reagent (i.e., the monomers or prepolymers) is exposed to light (when it is photosensitive) or to When exposed to temperature (when it is heat-sensitive), it polymerizes, forming a hydrogel.

[0080] Optionally, the composition of the invention may include a photoinitiator or a thermoinitiator. When a photoinitiator or a thermoinitiator is used, its quantity is generally chosen to be between 0.02% and 1%.

[0081] It should be noted that the present invention relates to a masterbatch composition intended to be mixed with a biological sample. This sample may, or may not, contain one or more target sequences that are to be identified by amplification, in particular to identify the presence or absence of a pathogen in the sample.

[0082] The masterbatch composition therefore includes ingredients necessary for the amplification of the target sequences. Thus, the composition includes in particular amplification reagents (for example dNTP nucleic bases, MgCl2, reaction buffer) and amplification enzymes (for example Taq polymerase, reverse transcriptase or reverse transcriptase).

[0083] The composition therefore does not initially contain the target sequence(s) that one seeks to amplify. Nor does the composition necessarily contain the primers specific to the target sequence(s), or the fluorescent probes to reveal amplification of the target nucleic acid sequence(s), since these two elements are dependent on the target sequence(s) being sought. Logically, when the target sequence is known (for example, a sequence specific to the coronavirus), the composition can contain the primers and probes specific to that sequence.

[0084] However, in the present description, reference is generally made to the composition of the invention in its state already mixed with the target sequence (including primers and probes). The technical effect related to the polymerization of the polymerizable reagent of the invention is observed under the operating conditions of a nucleic acid amplification process.

[0085] Indeed, classically the composition also includes enzymes for the amplification of nucleic acids, primers more or less specific to the target sequence(s), one or more fluorescent reporter molecules more or less specific to the target(s) of interest, a reaction buffer and other additives, if necessary.

[0086] The composition of the invention further comprises, in its usable state, a sample to be analyzed (DNA, RNA, plasmid, bacteria, virus, phages, fungi, etc.) containing target nucleic acid sequences. It should be noted that the sample may initially be in a second mixture, and / or undergo prior purification. Specifically, in this case, the sample is mixed with the gel components and PCR reagents. The resulting mixture is a composition according to the invention.

[0087] The composition can be injected into a reaction chamber compatible with a nucleic acid amplification instrument. The mixture is thus in a liquid state (more or less viscous) inside the reaction chamber and can therefore adapt to different shapes of reaction chambers.

[0088] According to the invention, the hydrogel formed by the polymerization of the polymerizable reagent has the effect of locally encapsulating each target nucleic acid sequence, while maintaining diffusion of the other constituents of the composition to allow efficient amplification of the target nucleic acid sequence(s).

[0089] When the composition of the invention is in a gel state in the reaction chamber(s), the amplification process of the target acid or nucleic acids can be carried out. The amplification process can be chosen from among those mentioned above in the description, in particular conventional or qPCR, real-time PCR, asymmetric PCR, RT-PCR, or other; NASBA; 3SR (self-sustained sequence replication); SDA; TMA; RCA; LAMP; or MDA (multiple displacement amplification).

[0090] Figure 1 shows a schematic diagram of the process of the invention. The amplification reaction mixture (PCR Mix) and a polymerizable reagent (polymers + activator(s)) are mixed to form the master mixture of the invention (MasterMix). The master mixture is thus ready for use and is mixed with the biological sample (target) containing the target sequence(s).

[0091] The composition of the invention, comprising the target sequences and primers in particular, can thus be distributed into reaction chambers of a nucleic acid amplification device (Amplification Chamber(s)). Next, a factor chosen from light or temperature is applied to initiate the polymerization of the polymerizable reagent. A hydrogel according to the invention is thus formed. According to the invention, the hydrogel locally encapsulates each target nucleic acid sequence while allowing the diffusion of the amplification reagents. The amplification process can then be carried out (Amplification).

[0092] The Applicant discovered, not without surprise, that the selection of polymers, and of activators where applicable, determines the state of the hydrogels formed. The hydrogels formed from the composition of the invention exhibit characteristics that overcome the prior art problems mentioned above in the description.

[0093] To select the polymers and activators of the invention and thus form a hydrogel from the composition of the invention, it is appropriate to proceed as follows.

[0094] The first step involves designing the hydrogel matrix according to the final requirements. The main considerations to be taken into account are: a) the chemical origin of the hydrogel components, their compatibility with the protocol; b) the use of functionalized monomers or prepolymers; c) the type of photocrosslinking or thermocrosslinking reaction; d) optionally the type of photoinitiator or thermoinitiator, as well as the source to be applied, i.e. respectively the light source or the heat source; e) the desired network structure and density (percentage of gel, ratio of reactants).

[0095] Various natural and / or synthetic materials can be used to form photo- or thermo-crosslinkable hydrogels. Natural substances, such as alginate, hyaluronic acid, chitosan, collagen, silk fibroin, and gelatin, can be used. However, it should be noted that batch-to-batch variation in naturally occurring hydrogels does not always reproduce their mechanical and biochemical properties and, consequently, can limit the possibility of obtaining matrices with well-defined properties. The material properties of synthetic hydrogels can be precisely controlled. Since these materials lack biologically relevant functionalities, some applications require the introduction of specific characteristics present in natural extracellular matrices (ECMs) in a highly controlled manner.Synthetic materials may include, but are not limited to, Pluronics derivatives, poly(vinyl alcohols) (PVA), poly(acrylic acids), polyethylene glycols (PEG) and polypeptides (Chinese Chem. Lett. 2020; ACS Macro Lett. 2013, 2, 5-9).

[0096] The use of monomers in polymerization is limited by the fact that most of them are cytotoxic and / or carcinogenic, and have inhibitory effects on the amplification process. Furthermore, this approach often results in inhomogeneous networks. An alternative solution is to use macromolecular hydrogel precursors (pre-polymers) that are functionalized with photo- or thermo-reactive groups to form photo- or thermo-polymerizable hydrogels, respectively. Two advantages of pre-polymers are their low toxicity and low inhibition rate, compared to the acrylamide / bis-acrylamide system, for example. In addition, pre-polymers are stable and water-soluble. As discussed above, these molecules possess at least one, and possibly several, reactive functional groups.For example, we can use PEG acrylate and methacrylate derivatives (linear and multi-arm configurations), PEG norbornene derivatives (linear and multi-arm configurations), PEG thiol derivatives (linear and multi-arm configurations), PEG acrylamide and methacrylamide derivatives (linear and multi-arm configurations), polyvinyl alcohol (PVA) derivatives, modified polysaccharides. such as hyaluronic acid derivatives, dextran methacrylate. Peptides can be incorporated into PEG-acrylate hydrogels by functionalizing the amino-terminal groups of the peptide with an acrylate portion (Biomaterials 2002, 23, 4307-4314).

[0097] There are several types of photocrosslinking or thermocrosslinking reactions. The choice of the appropriate reaction depends on the amplification reaction, the applied targets, and the temperature range, among other factors. Preferably, in the context of the present invention, a bio-orthogonal thiol-ene reaction and a dimerization reaction are used.

[0098] Regarding photosensitive polymerization, among bio-orthogonal reactions, the photoclic polymerization of thiol-norbornene (thiol-ene) is one of the preferred options here. This polymerization proceeds via a step-by-step growth mechanism, allowing the fabrication of structurally uniform hydrogels with virtually no network defects. It involves light-mediated orthogonal reactions between multifunctional macromers containing norbornene and thiol groups at their ends. Activated by UV or visible light, the thiol-ene reaction results in the rapid, step-by-step addition of thiols to the double bonds of the norbornene fragments, with the formation of thioether bonds, and is radical-mediated (it should be noted that a cationic ionic mechanism is also possible).This type of bio-orthogonal reaction is insensitive to water and oxygen, does not require the addition of a co-initiator or co-monomer, and can proceed under mild, selective, and efficient reaction conditions. This results in the formation of a homogeneous network and rapid kinetics compared to chain polymerization. The step-by-step polymerization mechanism allows for the formation of a homogeneous network with appropriate meshes that drastically limit the diffusion of target molecules / cells and amplification products, while allowing the diffusion of amplification reagents.

[0099] Another approach within the framework of photosensitive polymerization is the application of cycloaddition reactions, for example, photodimerization. Photodimerization occurs between two identical unsaturated molecules (after activation by light) and results in the formation of a dimer. Various structures containing double bonds, such as coumarins, cinnamates, thymine, anthracenes, stilbene, chalcones, and dimethylmaleimide (DMMI), can be used. They can be incorporated into natural or synthetic polymers, such as: conjugates of cinnamate, coumarin, and thymine with hyaluronic acid or chondroitin sulfate; conjugates of cinnamate with poly(N,N-dimethylacrylamide); PEGs containing terminal cinnamate groups and nitrocinnamate groups (Adv. Funct. Mater. 2001, 11, 1); modified gelatin with Nitrocinnamate; PVA with coumarin groups; poly(acrylamide) bearing DMMIs. (Chapter 8: Photo-Crosslinking Methods to Design Hydrogels. Gels Handbook, pp. 201-218 (2016)). The photoreactivity of these compounds could be adjusted by varying the substituents in the photosensitive reactive center. Among the additional advantages of this crosslinking strategy is that no initiator or catalyst is required. However, a high energy input must be applied to achieve efficient crosslinking. UV intensity and gelation time can be reduced in several ways. The first is to incorporate a greater number of photodimerizable groups onto the prepolymer core, which will also increase crosslinking (e.g., by incorporating photodimerizable fragments along the entire length of the prepolymer backbone, using multi-chain prepolymers).The second approach involves the use of water-soluble sensitizers, such as thioxanthone disulfate, to reduce energy intake (Polymer 2007, 48, 5599-5611).

[0100] Another group of photo-induced polymerizations includes reactions with the formation of reactive nitrene radicals as intermediates from UV-activated azides. The nitrene radical can rapidly undergo addition reactions with unsaturated bonds, insertion reactions in carbon-hydrogen or nitrogen-hydrogen bonds. Hydrogels can also be formed when the nitrene radical reacts with primary amines, for example, modified chitosan (B. Amsden. Chapter 8: Photo-Crosslinking Methods to Design Hydrogels. Gels Handbook, pp. 201-218 (2016)).

[0101] The choice of the appropriate photoinitiator (if necessary) is a crucial task in the design of photocrosslinkable matrices for nucleic acid amplification. A series of important characteristics must be taken into account: a photoinitiator must have an absorption spectrum that shows good overlap with the emission spectrum of the desired light source; a relatively high molar extinction coefficient; good water solubility; stability; the ability to produce free radicals (or other reactive particles); biocompatibility; compatibility with the amplification process, etc. (Biomaterials 2002, 23, 4307-4314; BioTechniques 2019, 66, 40-53).

[0102] Regarding temperature-sensitive polymerization, among bio-orthogonal reactions, the Diels-Alder reaction is one of the preferred options here. It proceeds via a stepwise growth mechanism, allowing the construction of structurally uniform hydrogels with virtually no defects. It involves temperature-mediated orthogonal reactions between multifunctional macromers containing diene and dienophile functionalities. Different structures can be used for the conjugated diene (furan, tetrazine, etc.) and for the substituted alkene. (maleimide, norbornene, etc.). These structures can be incorporated into natural or synthetic polymers such as poly(acrylates) (European Polymer Journal, 2013, 12, 3998-4007), poly(etheramines) (Polymers, 2019, 11, 930), hyaluronic acid (Polymer Chemistry, 2014, 5, 5116-5123), and alginate (Biomaterials, 2015, 50, 30-37). Activated by temperature, the Diels-Alder reaction is a radical-free reaction that results in a six-membered ring. This type of bio-orthogonal reaction is insensitive to water and oxygen, does not require the addition of a co-initiator or co-monomer, and can proceed under mild, selective, and efficient reaction conditions. This results in the formation of a homogeneous network and rapid kinetics compared to chain polymerization.The step-by-step polymerization mechanism allows for the creation of a homogeneous network with appropriate meshes that drastically limit the diffusion of target molecules / cells and amplification products, but allow the diffusion of amplification reagents.

[0103] Another approach within the framework of thermosensitive polymerization is the dimerization of a polymerizable group such as an acrylate. Dimerization occurs between two identical polymerizable functionalities in the presence of a thermoinitiator. Various structures containing double bonds, such as (meth)acrylates, (meth)acrylamides, or maleimides, can be used, and these structures can be incorporated into natural or synthetic polymers, such as PEGs or polysaccharides (hyaluronic acid, gelatin, etc.). The thermoreactivity of these compounds can be adjusted by varying the substituents of the thermosensitive core. The gelation temperature and time can be reduced in several ways.The first approach involves incorporating a greater number of thermodimerizable groups onto the prepolymer core, which will also increase cross-linking (for example, by incorporating thermodimerizable parts along the entire prepolymer backbone, using multi-armed prepolymers). The second approach involves using higher concentrations of thermoinitiator, while ensuring compatibility with PCR.

[0104] The choice of a suitable thermoinitiator (if necessary) is a crucial task in the design of thermocrosslinkable matrices for nucleic acid amplification. A number of important characteristics must be taken into account: a thermoinitiator must have a decomposition temperature that closely overlaps with the temperature used during the PCR process; a relatively high molar extinction coefficient; good water solubility; stability; the ability to produce free radicals (or other reactive particles); biocompatibility; and compatibility with the amplification process. Different types of thermoinitiators can be used to initiate thermal polymerization: - Persulfates, neutralized with a counterion (ammonium, potassium, sodium etc...), decompose into sulfate radicals which, in aqueous solution, form HS O4 ions and hydroxyl radicals, capable of initiating polymerization (Catalysis Today, 2015, 257,297-304); - Organic peroxides (methyl ethyl ketone peroxide, benzoyl peroxide, etc.) which undergo symmetrical fission (homolysis), forming two radicals capable of initiating polymerization; - Azo compounds (2,2'-azobis(isobutyronitrile), 2,2'-Azobis[2-(2-imidazolin-2-yl)propane dihydrochloride], etc.) which decompose with heat (or light) to form nitrogen gas and carbon radicals capable of initiating polymerization.

[0105] The polymerization process takes place through the formation and reaction of certain reactive species, i.e., radicals or ions. They can be formed as a result of high chemical reactivity of the participating components themselves (for example, cations in a Michael addition); as a result of special activation by one or more initiators (for example, radicals formed as a result of an oxidation-reduction reaction of the APS-TEMED system in a polymerization chain), by the application of temperature with or without initiators (for example, ammonium persulfate (APS), which can be decomposed into radicals by heating), or by the application of light with or without a photoinitiator.

[0106] In the present invention, this latter approach is used, namely the application of light or temperature, respectively, with or without a photoinitiator or thermoinitiator, for the generation of reactive radical species. In general, two main groups of initiators are used in the synthesis of hydrogels for biomedical applications: radical and cationic photoinitiators or thermoinitiators (i.e., those that induce the formation of reactive radicals or cations in the system, respectively). The use of cationic photoinitiators or thermoinitiators is possible but complicated because they lead to the formation of protonic acids, which are harmful to cells and enzymes, and can inhibit the amplification process.

[0107] In the context of photosensitive polymerization, cleavable (type I) or biomolecular (type II) photoinitiators can be used. Type I photoinitiators include, but are not limited to, benzoin and acetophenone derivatives, such as the Irgacure group of photoinitiators (Irgacure-2959; Irgacure-184; Irgacure-651; Irgacure-369; Irgacure-907; etc.), lithium 2,4,6-phenyltrimethylbenzoylphosphinate (LAP), etc. Under the influence of light, these compounds decompose into two parts bearing radical centers, capable of promoting subsequent polymerization. Typically, this type of compound has a maximum in the absorption spectrum around 300-400 nm and, therefore, can be used for UV polymerization. Conversely, type II photoinitiators, such as camphorquinone, thioxanthone, and benzophenone, have a maximum absorption spectrum in the visible range and can be used for visible light-mediated polymerization. Their mechanism of action is based on the extraction of hydrogen from the coinitiator (e.g., triethanolamine) with the generation of secondary radicals. Often, to induce efficient photogelation, an accelerator, such as N-vinylpyrrolidone, is required. However, compared to type I photoinitiators, type II is less cytotoxic and more water-soluble. One of the most widely used UV-sensitive photoinitiators is Irgacure-2959, due to its moderate water solubility, low cytotoxicity, and minimal immunogenicity.However, it is characterized by relatively low initiation efficiency and molar extinction coefficient in the UV-A spectral range. A good alternative is to use LAP, which is more efficient and biocompatible, and, being a lithium salt, is well soluble in water. In addition, LAP also absorbs in the blue light region (405 nm). V-50 (2,2'-azobis(2-methylpropionamidine) dihydrochloride) and V-086 (2,2'-azobis(N-(2-hydroxyethyl)-2-methylpropionamide)) can also be used (BioTechniques 2019, 66, 40-53; B. . Amsden. Chapter 8: Photo-Crosslinking Methods to Design Hydrogels. Gels Handbook, p. 201-218 (2016)).

[0108] The optimal concentration of photoinitiator or thermoinitiator is an important parameter for the polymerization of hydrogels used in the amplification process. High initiator concentrations allow for the generation of more free radicals, which normally leads to a higher conversion of monomers. However, too high a concentration of initiator can be detrimental to amplification because the radicals can damage cellular macromolecules, such as cell membranes, proteins, and nucleic acids, as well as fluorophores. This results in inhibition of amplification. Preferably, a concentration of 0.02%–1% is used, depending on the initiator applied, the light intensity or temperature, and the mechanokinetic aspects of the polymerization.

[0109] Finally, varying the percentage of gel and the ratio of components allows obtaining the desired network structure and density. The gel matrix must have a homogeneous structure with appropriate meshes that limit the diffusion of target molecules / cells and amplification products, while allowing the diffusion of amplification reagents. An average pore size ranging from 100 µm to 5 nm is capable of preventing diffusion and thus intermixing of target sequences, while allowing the diffusion of amplification reagents. Within this range, the gel concentration can be modified to influence two parameters: a) colony size molecular or polyunity; b) the restriction capacity of the gel matrix towards different types of targets (DNA, RNA, plasmids, bacteria, viruses, phages, etc.). As previously observed (GM Church et al. US6485944; AB Chetverin et al. EP1999268, AB Chetverin et al. US6001568), increasing the percentage of gel leads to a decrease in the size of molecular colonies. The concentration of the gel also has a considerable influence on the matrix's ability to trap different targets.

[0110] Various reaction chambers can be used. These may include, but are not limited to, flat chips, cassettes, narrow or elongated reaction chambers, i.e., rod- or capillary-type, specially folded or wound into a flat cassette to allow placement in a standard PCR thermocycler. The chamber may be of various suitable volumes confined within an enclosed space, excluding inlet / outlet holes for pouring the mixture. The reaction chamber consists of different parts. In a particular embodiment of the invention, the lower part of this chamber is in direct contact with a heating system on one side and with the mixture (liquid or gel) on the other. It is preferably flat to ensure optimal adhesion of the gel matrix. The upper part, the visible part, is also preferably flat and is closely bonded to the lower part.The upper part must be transparent to both the light wavelengths required to initiate polymerization and the light wavelengths used to detect the amplification reaction. The distance between the base and the apex can be between 1 µm and 1 mm, preferably the latter, which allows for the arrangement of a monolayer of the amplification products, i.e., molecular colonies. The other two dimensions (length and width) must be larger (ideally by a factor of at least 10) to provide sufficient surface area for the placement of different targets and their amplification products. To prevent gel dehydration, the inlet / outlet holes must be sealed in various ways: for example, mechanically, which is integrated into the chamber design; with the use of external adhesive films; and / or by applying pressure.

[0111] According to the first embodiment of the invention, the composition is exposed to light of a chosen wavelength and intensity for a predetermined duration. Light of different wavelengths (UV with 200-400 nm or visible light with 400-800 nm) and different intensities can be applied. The intensity and dose of irradiation must necessarily be taken into account, as they are required for the initiation of polymerization and the increase of photoinitiation. It should be noted that there is no linear proportionality between the intensity of UV light and photoinitiation and that excessively high intensity values ​​can lead to Polymer degradation results in an inhomogeneous gel. Furthermore, excessive intensity can destroy reagents necessary for amplification. The UV intensity range of 1 to 20 mW / cm² is suitable and allows gel formation in 1–5 minutes.

[0112] According to the second embodiment of the invention, the composition is exposed to a threshold temperature. In particular, it may be sufficient to start the PCR to initiate the polymerization of the composition of the invention.

[0113] Once the gel is formed, the nucleic acid sequence(s) are immobilized in the gel matrix. Amplification can then be performed on an amplification device. Examples include the MasterCycler Nexus flat panel thermocycler from Eppendorf; the ProFlex PCR system from Applied Biosystems; QuantStudio 3D from Applied Biosystems; SmartCycler from Cepheid; and Chronos from the Applicant.

[0114] EXAMPLES OF ACHIEVEMENTS

[0115] Examples 1 to 5 relate to hydrogels obtained from compositions comprising a photosensitive polymerizable reagent according to the invention, and examples 6 to 8 relate to hydrogels obtained from compositions comprising a thermosensitive polymerizable reagent according to the invention. Unless otherwise specified, the PCRs are performed on a commercially available Chronos machine of the Applicant.

[0116] EXAMPLE 1: Naked DNA in symmetric PCR.

[0117] A bio-orthogonal thiol-ene reaction is used for hydrogel formation. Equimolar amounts of 4 Arm-PEG-SH (MW 10,000 g / mol, Laysan Bio) and 4 Arm-PEG-norbomene (MW 10,000 g / mol, Sigma) are chosen for gel construction with w / v percentages in the range of 5–7%. LAP (lithium phenyl-2,4,6-trimethylbenzoylphosphinate, Sigma) is used as a photoinitiator at a concentration of 0.02% (w / v). The following amplification reagents are used: 1U of SsoAdvanced Universal SYBR Green Supermix (Bio-Rad), 8U of DNA polymerase Platinum™ Taq (ThermoFisher SCIENTIFIC), 0.4pM of primers targeting the 16S region of bacteria with a template length of 740 bp. Optionally, an additional intercalating dye (SybrGreen® fluorescent DNA dye (Jena Bioscience) or EvaGreen® dye (20X in water, Biotium)) can be added at concentrations in the range of 0.5-1.5 pM.

[0118] The mixture containing all the components with the target DNA from B. subtilis is adjusted with ddH2O to 25 pL and injected into the reaction chamber. Depending on the thermocycler used, the chamber is characterized by the following dimensions: Cepheid SmartCycler (5x5x1 mm plastic chamber, 25 pL); Eppendorf MasterCycler Nexus flat thermocycler (9x9x0.3 mm chamber, 25 pL (slide of Silane-treated glass microscope + frame-sealed chamber (Bio-Rad) + plastic coverslip); Chronos (10x10x0.25 mm, 25 pL chamber with an Al base and a cyclic olefin copolymer (COP) coverslip). The Cepheid SmartCycler is used here.

[0119] The gel resulting from the photosensitive composition formed at room temperature after 1 minute in a thiol-norbornene reaction activated by the application of UV light (365 nm, 3.4 mW / cm2) generated by a light source.

[0120] The PCR is carried out by 2 min at 95 °C for the initial denaturation, then 35 cycles of 5 s at 95 °C for denaturation and 30 s at 60 °C for elongation.

[0121] Molecular colonies of B. Subtilis DNA (104, 103, 102 copies and negative control) are visualized with the Axiovert 100 fluorescence microscope (ZEISS, Germany).

[0122] Figure 2 shows the respective photographs of the hydrogel of the samples containing 10⁴, 10³, 10² copies and a negative control after PCR. Figure 2 also shows a fluorescence signal curve as a function of the PCR cycles and the respective Ct values.

[0123] The photos were taken with a Samsung Galaxy S10 camera. The negative control contained 20-30 molecular colonies due to the amplification of E. coli impurities present in the SsoAdvanced Universal SYBR Green Supermix, amplifiable with highly sensitive primers targeting the 16S region.

[0124] EXAMPLE 2: Naked DNA in symmetric PCR.

[0125] In this example, a bio-orthogonal thiol-ene reaction is used for the formation of the polymer matrix. More specifically, equimolar amounts of 2 Arm-PEG-SH (MW of 3400 g / mol, Laysan Bio) and 8 Arm-PEG-norbornene (tripentaerythritol) (MW = 20000, Jenkem Technology) are taken for the construction of the gels with percentages (%) w / v in the range of 6-11%.

[0126] LAP (lithium phenyl-2,4,6-trimethylbenzoylphosphinate, Sigma) is used as a photoinitiator in an amount of 0.02% (w / v). The same amplification reagents as those in Example 1 are used.

[0127] The composition, including the target DNA sequence from B. subtilis (103 copies), is adjusted with ddH2O to 25 qL and injected into the reaction chamber.

[0128] The polymerization of the composition is initiated by the application of UV light (365 nm, 3.4 mW / cm2) from a UV torch. The gel is formed (at room temperature) after 1 minute by a stepwise thiol-norbornene polymerization reaction.

[0129] Fig. 3 shows that the number and size of the dots (which correspond to the polonies) were different depending on the percentage of frost: 80–100 sq m for a 10.8% gel (left image), 100-120 µm for an 8.8% gel (middle image), and 150-160 µm for a 6% gel (right image). Increasing the gel concentration results in fewer spots and smaller spots. The stronger signals for the 6% and 8.8% gels can be explained by greater PCR efficiency due to the larger mesh size of the matrix in these gels compared to the 10.8% gel. A larger mesh size allows for freer diffusion of amplification reagents, particularly relatively large molecules such as enzymes. Conversely, a larger mesh size also leads to increased diffusion of PCR products, resulting in larger spots.PCR is conducted on a Cepheid SmartCycler device according to the protocol of example 1 and photographs are taken with a Samsung Galaxy S10 camera on an Axiovert 100 fluorescence microscope (ZEISS, Germany).

[0130] Fig. 3 also shows a fluorescence signal curve as a function of PCR cycles and the respective Ct values.

[0131] Figure 3 also shows a curve of the derivative of the fluorescence signal with respect to temperature. The observed peak corresponds to the melting temperature of the product formed by amplification. Here, a value of approximately 88°C is obtained, the expected value for the amplified DNA.

[0132] EXAMPLE 3: Naked DNA in symmetric PCR.

[0133] In this example, a dimerization reaction is used for the formation of the polymer matrix: an 8-Arm-PEG based on nitrocinnamate (MW of 21,400 g / mol) is used to construct the gels with a w / v percentage of 10%. The following amplification reagents are used: 5U of SD Polymerase Hotstart (Bioron), an incomplete SD Polymerase Reaction Buffer (1Ox, Bioron), a 3 mM MgCl₂ buffer (Bioron), and 0.4 pM primers targeting the 16S region of the bacterium with a matrix length of 740 bp. EvaGreen® Dye, 20X in Water (Biotium), a DNA intercalating dye, is added at a concentration of 2X. The composition containing all components, including the naked DNA target from E. coli, is adjusted with ddH2O to 25 pL and injected into the Chronos reaction chamber.

[0134] The gel resulting from the photosensitive composition is formed at room temperature after 1 min by a dimerization reaction activated by the application of UV light (365 nm, 3.4 mW / cm²) generated by the UV torch. The PCR amplification process is carried out according to the following protocol: 2 min at 95 °C for initial denaturation, 35 cycles of 5 s at 98 °C for denaturation, and 30 s at 60 °C for elongation. The molecular colonies are visualized using the Axiovert 100 fluorescence microscope (ZEISS, Germany).

[0135] Fig. 4 shows nine photographs of hydrogels formed by different embodiments of the composition according to the invention having an initial concentration of target sequences identical (103 copies) or zero (see control shots with negative samples), corresponding to PCR data from example embodiments.

[0136] The first line of [Fig. 4] shows a first photograph taken with a Hamamatsu camera (103 copies) and a second photograph taken with the Apple iPhone 11 camera (negative). The negative control contains some molecular colonies due to the presence of impurities amplified by the highly sensitive primers targeting the 16S region.

[0137] EXAMPLE 4: Naked DNA in asymmetric PCR.

[0138] In this example, a bio-orthogonal thiol-ene reaction is used for the formation of the polymer matrix: equimolar amounts of 4 Arm-PEG-SH (MW of 10,000 g / mol, Laysan Bio) and 4 Arm-PEG-norbornene (MW of 10,000 g / mol, Sigma) are used for gel construction with a w / v percentage of 6.4%. LAP (lithium phenyl-2,4,6-trimethylbenzoylphosphinate, Sigma) is used as a photoinitiator in an amount of 0.02% (w / v). The following amplification reagents were used: 5U of SD Polymerase Hotstart (Bioron), a buffer (SD Polymerase Reaction Buffer incomplete (lOx), Bioron), 3 mM MgCl2 (Bioron), a pair of custom-designed primers targeting the E. coli FimH gene with a template length of 400 bp. An intercalation dye, EvaGreen® Dye, 20X in water (Biotium), was added to the IX concentration.

[0139] The composition comprising a target sequence (104 copies of naked DNA) of E. coli is adjusted with ddH2O to 25 qL and injected into the reaction chamber of Chronos.

[0140] The gel resulting from the photosensitive composition formed at room temperature after one minute in a step-growth thiol-norbornene click reaction activated by the application of UV light (365nm, 3.4 mW / cm2) generated by the UV torch.

[0141] The PCR amplification protocol is: 2 min at 92°C for initial denaturation, 40 cycles of 5 s at 92°C for denaturation, and 30 s at 64°C for elongation. Molecular colonies are visualized in real time with the Chronos camera.

[0142] EXAMPLE 5: Virus in RT PCR

[0143] The experimental protocol is that used in Example 1, except that the reverse transcriptase iScriptTM (Bio-Rad) is used for reverse transcription. The amplification protocol also includes a reverse transcription step for 60 s at 60°C. A pair of custom-designed primers targeting MS2 with a length 200 bp is used. The MS2 molecular colony networks (103 copies) are visualized on the Chronos machine.

[0144] The third line of [Fig.4] shows a photo taken with the camera of the Chronos machine (103 copies).

[0145] EXAMPLE 6: Naked DNA in PCR

[0146] This example implements an acrylate dimerization reaction for the formation of the polymer matrix: 4 Arm-PEG-acrylate (MW of 20000 g / mol, Laysan Bio) is taken for the construction of the gels at 10% w / v. VA-044 (2,2'-Azobis[2-(2-imidazolin-2-yl)propane]dihydrochloride, TCI Chemicals) is used as a thermoinitiator in an amount of 0.07% (w / v). The following amplification reagents are used: 6U SD Polymerase HotStart (Bioron), a 10X buffer containing 250 mM potassium acetate (KOAc), 83 mM ammonium sulfate ((NH4)2SO4), 30 mM magnesium chloride (MgCl2), 1.5 M Tris-HCl and 1 V% Tween-20, 0.2 pM dNTP mix (Sigma) and 0.4 pM primers targeting the 16S region of bacteria with a template length of 740 bp. Optionally, an additional intercalating dye (SybrGreen® fluorescent DNA dye (Jena Bioscience) or EvaGreen® dye (20X in water, Biotium)) can be added at concentrations in the range of 0.5–1.5 pM.

[0147] The mixture containing all the components with the target DNA from B. subtilis is adjusted with ddH2O to 25 pL and injected into the reaction chamber. Depending on the thermocycler used, the chamber is characterized by the following dimensions: Cepheid SmartCycler (5x5x1 mm plastic chamber, 25 pL); Eppendorf MasterCycler Nexus flat-plate thermocycler (9x9x0.3 mm chamber, 25 pL (silane-treated glass microscope slide + frame-sealed chamber (Bio-Rad) + plastic coverslip)); Chronos (10x10x0.25 mm chamber, 25 pL with an Al base and a cyclic olefin copolymer (COP) coverslip). The Chronos machine is used here.

[0148] The gel formed by the thermosensitive composition forms during PCR, thanks to the cycles between the denaturation temperature and the elongation temperature. Indeed, the threshold temperature allows the thermoinitiator to polymerize via the acrylic functionalities of 4-arm-PEG.

[0149] The PCR is carried out by 2 min at 95°C for the initial denaturation, then 35 cycles of 5 s at 95 °C for denaturation and 30s at 60°C for elongation.

[0150] Molecular colonies of B. Subtilis DNA are visualized with the Axiovert 100 fluorescence microscope (ZEISS, Germany) and photos taken with the camera of the Apple iPhone 11.

[0151] The fourth line of [Fig. 4] shows two photos taken with the Apple iPhone 11 (103 copies and negative). The negative control contains some molecular colonies due to the presence of impurities amplified by the highly sensitive primers targeting the 16S region.

[0152] EXAMPLE 7: Naked RNA in RT PCR

[0153] The experimental protocol is that used in Example 6, except that WarmStart® LAMP reverse transcriptase (New England Biolabs) is used for reverse transcription, and the amplification protocol additionally includes the reverse transcription step for 60 seconds at 60°C. A pair of custom-designed primers targeting MS2 with a template length of 200 bp is used instead of the one previously used to target the 16S gene of B. subtilis. Molecular colonies of naked MS2 RNA are visualized with the Chronos machine camera.

[0154] The fifth line of [Fig. 4] shows two photographs taken with the Chronos machine camera (103 copies and negative). The negative control contains some molecular colonies due to the presence of impurities amplified by the highly sensitive primers targeting the 16S region.

[0155] EXAMPLE 8: Virus in RT-PCR

[0156] The protocol is that used in Example 6, except that WarmStart® LAMP reverse transcriptase (New England Biolabs) is used for reverse transcription, and the amplification protocol additionally includes the reverse transcription step for 60 seconds at 60°C. A pair of custom primers targeting MS2 with a template length of 200 bp is used. Molecular colonies of MS2 bacteriophage are visualized with the Chronos instrument camera.

[0157] The sixth line of [Fig.4] shows a photo taken with the camera of the Chronos machine (103 copies).

[0158] In view of the embodiments related to photosensitive polymerization, the Applicant has developed a device specifically adapted for use with the composition of the invention. Thus, the present description also discloses an invention relating to a device for amplifying nucleic acid sequences. This device can be defined as follows:

[0159] A nucleic acid sequence amplification device comprising a thermocycler arranged to perform a series of thermal cycles with a nucleic acid amplification reaction mixture comprising one or more target nucleic acid sequences, and amplification reagents comprising primers specific to the target nucleic acid sequence(s), fluorescent probes for revealing the amplification of said target sequences, and nucleic acid amplification enzymes, the reaction mixture further comprising a photosensitive reagent, polymerizable when exposed to light from the light source so as to form a gel; the device further comprising a reaction chamber disposed in the thermocycler and arranged to receive the nucleic acid amplification reaction mixture, a light source directed towards said reaction chamber, and a control of the light source so as to turn it on or off and control the wavelength and intensity of the light.

[0160] The device is therefore arranged to generate a stimulus to the photosensitive reagent such that said reagent polymerizes when exposed to light from the light source. This results in the formation of the gel.

[0161] In other words, when the light source illuminates the reaction mixture, a hydrogel forms locally encapsulating each target nucleic acid sequence and the amplification reagents.

[0162] The invention can further be marketed in the form of a kit comprising the composition as described above and a nucleic acid sequence amplification device. Thus, the invention also relates to a: Nucleic acid sequence amplification kit comprising (i) the composition according to the invention and (ii) a nucleic acid sequence amplification device including a thermocycler arranged to perform a series of thermal cycles with said composition, a reaction chamber disposed in the thermocycler and arranged to receive the nucleic acid amplification reaction mixture, and further comprising a light source directed towards said reaction chamber as well as a control for said light source so as to turn it on or off and control the wavelength of the light.

Claims

Demands

1. Liquid masterbatch composition for nucleic acid sequence amplification, intended to be mixed with a biological sample containing one or more target nucleic acid sequences and with primers specific to the target nucleic acid sequence(s), as well as fluorescent probes to reveal amplification of the target nucleic acid sequence(s), the composition comprising nucleic acid amplification reagents and enzymes, characterized in that the composition further comprises a polymerizable reagent comprising one or more prepolymers, which polymerizes in a time less than or equal to 10 minutes, preferably less than or equal to 1 minute, under the influence of a stimulus selected from thermal stimulation between 50 and 80°C and light stimulation by application of light with a wavelength between 200 nm and 800 nm,so as to form a hydrogel locally encapsulating each target nucleic acid sequence when said composition is exposed to said stimulation.

2. Composition according to claim 1, wherein the polymerizable reagent is selected from a photosensitive reagent which polymerizes when exposed to light, preferably ultraviolet, and a thermosensitive reagent which polymerizes when exposed to a threshold temperature.

3. Composition according to any one of the preceding claims, wherein the polymerizable reagent is selected from the class of PEGs and other water-soluble polymers compatible with PCR.

4. Composition according to any one of the preceding claims, wherein the polymerizable reagent is present in a content of 2% to 25%, preferably 5% to 15%, by weight per unit volume (w /

5. * )■ Composition according to any one of the preceding claims, further comprising an initiating agent selected from a photoinitiator, preferably lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) and a thermoinitiator, preferably 2,2'-azobis[2-(2-imidazolin-2-yl)propane]dihydrochloride, in a content of 0.02% to 1% by weight per unit volume (w / V).

6. Composition according to any one of the preceding claims, comprising primers specific to the target nucleic acid sequence(s), and fluorescent probes to reveal amplification of the target nucleic acid sequence(s).

7. A method for amplifying nucleic acid sequences comprising the following steps: (i) making available a composition according to claim 1 to 6; (ii) mixing said composition with a biological sample to be analyzed; (iii) applying a stimulation selected from thermal stimulation and light stimulation so as to polymerize the mixture of step (ii) to obtain a hydrogel; (iv) exposing the hydrogel to an acid sequence amplification reaction, wherein the hydrogel is obtained in a time less than or equal to 10 minutes, preferably less than or equal to 1 minute after the application of said stimulation.

8. A method according to claim 7, wherein the step (i) of making available is maintained for a period of time ranging from several hours to several months, preferably ranging from several days to several months.

9. A method according to any one of claims 7 and 8, wherein said reaction is a polymerase chain reaction.

10. Amplification method according to any one of claims 7 to 9, wherein step (iii) of applying a stimulus involves exposing said composition to UV light or a threshold temperature.

11. Amplification process according to any one of claims 7 to 10, wherein step (iii) comprises a reaction selected from the group consisting of a bio-orthogonal thiol-ene reaction and a dimerization reaction.

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