Kits and methods for detecting targeted nucleic acids

Abstract

The subject of this invention is a kit for highly sensitive and robust detection of a target nucleic acid in a sample, and a sandwich method using the kit. This invention also relates to the use of the kit or method according to the present invention for target nucleic acids for the diagnosis of a disease or pathological condition. The kit and method according to the present invention are particularly useful for detecting point mutations in target nucleic acids.

Description

[Technical Field]

[0001] The subject of this invention is a kit for highly sensitive and robust detection of target nucleic acids in a sample, and a sandwich method using the kit. The invention also relates to the use of the kit or method according to the present invention, for example, for molecular diagnosis of a disease or pathological condition. The invention relates to the field of molecular biological testing methods. [Background technology]

[0002] Nucleic acids are considered important markers for diseases such as cancer and infectious diseases, but their detection can be difficult because they exist at very low concentrations.

[0003] Because disease-related nucleic acid biomarkers often exist in extremely low abundances, accurate and robust detection of analytes, primarily in large volumes of biological fluids, is crucial for biological research, precision medicine, and early diagnosis. The COVID-19 pandemic highlights the need for rapid, highly sensitive, and specific detection methods to control rapidly evolving pandemics. of This highlights a critical need. In clinical diagnosis, direct nucleic acid testing for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is not applied due to the difficulty in detecting ultra-low concentrations of RNA molecules in suspension. Direct detection is limited by strong background noise from nonspecific proteins, nucleic acids, or other biomolecules.

[0004] In molecular diagnostics, there are two types of testing methods, each with its own unique advantages: (1) targeted amplification tests and (2) signal amplification tests. Targeted amplification tests, such as PCR, are highly sensitive, but require target extraction and purification, as well as enzymatic reactions, and are prone to false positives. In contrast, signal amplification tests tend to be simpler, as they often omit nucleic acid purification and enzymatic amplification. Therefore, there is less material loss and a lower rate of false positives. However, signal amplification tests are less sensitive than PCR-based targeted amplification tests. Much effort has been made to develop ultra-sensitive signal amplification tests to detect unamplified nucleic acids. Portable microfluidic devices have shown specific and highly sensitive detection of Ebola nucleic acids compared to RT-PCR (Cai, H. et al., 2015). Interestingly, some of these studies were able to directly detect target nucleic acids from whole blood lysates (Ngo, HT et al., 2018) (Zheng, Z. et al., 2006).

[0005] To date, the recommended method for diagnosing COVID-19 is based on quantitative reverse transcription polymerase chain reaction (qRT-PCR), which requires nucleic acid extraction from pharyngeal swabs and targeted amplification procedures. Such procedures require skilled individuals, specialized equipment, and long processing times (over 2 hours), making the implementation of SARS-CoV-2 nucleic acid testing in the field difficult. Furthermore, due to the high demand for commercially available RNA extraction kits and the resulting shortage of these reagents, several diagnostic workflows have been developed to bypass the lengthy intermediate RNA extraction step. Over the past decade, the performance of magnetic beads in nucleic acid extraction from biological fluids before molecular detection has been demonstrated in several studies. Magnetic beads consist of iron oxide nanoparticles embedded in a polymer matrix, and nucleic acids can be isolated separately by functionalizing the surface. Functionalization affects binding rate and compatibility with molecular detection strategies. For example, silica-coated beads bind non-selectively to all nucleic acids via electrostatic interactions and are therefore primarily used in detection methods resistant to high concentrations of non-targeted background nucleic acids, such as RT-PCR. Oligonucleotide-conjugated beads, such as oligo(dT) beads, or beads surface-functionalized with specific sequences, are used for mRNA target extraction and exhibit high recovery rates. This purification can be performed manually, in a microfluidic chip, or using automated robotic equipment. In addition to nucleic acid isolation and elution, several specific nucleic acid sequences have been directly detected on the bead surface by hybridization assays using various materials (biotin-avidin, protein-enzyme, fluorescent dyes, quantum dots, etc.). However, most of these methods have limitations, such as low signal intensity or rapid photodecolorization (Lim et al., 2009).

[0006] EP3536806, "Oligonucleotide-functionalized hydrophobic polymer nanoparticles," describes nanoparticles comprising a hydrophobic polymer and a luminescent component as an energy donor element, which can be used to detect target nucleic acids. These nanoparticles can be conjugated to oligonucleotides complementary to a specific target or a non-specific sequence of the target. The target-specific oligonucleotides conjugate to the energy donor element, and their nucleotide sequences are complementary to another nucleotide sequence labeled with an excitation energy acceptor compound. A FRET (fluorescence resonance energy transfer) effect occurs between the acceptor and donor compounds of the nanoparticles. In the described method, the lower limit of detection using these nanoparticles is a target nucleotide concentration of 5 pM.

[0007] WO2017 / 220453 describes the detection of mutations using magnetic beads. A magnetic bead-bound probe hybridizes with one end of the target nucleic acid, and a surface-bound probe hybridizes with the other end of the target nucleic acid. Stringency is applied to the hybridization complex by magnetic force and / or temperature.

[0008] There is still a need for simple, rapid, highly sensitive, and robust methods for detecting ultra-low abundance nucleic acids. [Overview of the project]

[0009] In this study, the inventors aimed to develop a sandwich hybridization method for detecting low-abundance nucleic acid molecules from a sample. The method according to the present invention includes hybridization to detect the target nucleic acid by dual direct hybridization using different complementary oligonucleotides that specifically bind to different regions of the target nucleic acid.

[0010] The method according to the present invention combines the following: • Capture of target nucleic acid molecules using solid surface-based hybridization of sequence-specific probes to targets in solution (the capture probe is immobilized on a solid substrate, allowing direct isolation and concentration of target molecules from raw samples with minimal processing), and • At least a second sequence-specific capture method that enables direct detection on the solid surface by binding to ultra-high-brightness light-emitting particles.

[0011] Capture of target nucleic acid molecules using solid surface-based hybridization involves a solid surface, which may be either an immobilized surface or a bead, such as a magnetic bead or a glass bead. Isolation of complex beads / targets can be achieved, depending on the properties of the solid surface, for example, by applying magnetic force or gravity, centrifugation, or filtration. This isolation is important for removing excess unbound DNA-modified nanoprobes and / or ultraluminescent particles. It may also be useful to isolate the target nucleic acid from cell lysates without requiring RNA extraction. Detection of the fluorescence signal is performed directly on the solid surface by fluorescence quantification, as the fluorescent particles bind to the target through complementary hybridization.

[0012] The kit and method according to the present invention are useful for detecting target nucleic acids in samples such as environmental or biological samples. The kit and method according to the present invention are also useful for diagnostic purposes.

[0013] The advantages of the kits and methods according to the present invention are their extremely high sensitivity, robustness, and ease of use, as well as the fact that they are enzyme-free amplification detection methods.

[0014] In fact, the kit and method according to the present invention enable accurate and robust detection of nucleic acids with a detection limit of approximately 1 fM. The extremely high brightness of the luminescent particles is important for achieving a high signal-to-noise ratio for rapid detection of targets using a simple technique.

[0015] Furthermore, unlike conventional detection kits and methods, it is possible to detect and isolate low-abundance target nucleic acid molecules without requiring a supplemental nucleic acid extraction step.

[0016] The target nucleic acid molecules may include double-stranded and single-stranded DNA molecules, double-stranded and single-stranded RNA molecules, and may include short-sized and long-sized nucleic acid molecules. Point mutations can be detected using the kit or method of the present invention.

[0017] The brightness of the ultra-high-luminosity particles and the high concentration of complementary oligonucleotides bound to the beads and ultra-high-luminosity luminescent particles are major advantages of the kits and tests of the present invention for improving the detection limit of nucleic acids. In fact, the detection limit of the test method according to the present invention is about 1 fM when using a fluorescence plate reader. This represents a dramatic improvement in the detection limit compared to prior art tests. The kits and tests according to the present invention enable the detection of RNA and DNA of different lengths and can be used for the detection of nucleic acids in a sample. For example, the kits and tests according to the present invention can be used for the diagnosis of diseases or the detection of pathogens such as parasites in environmental samples.

[0018] This invention enables the detection of a target nucleic acid and its extraction from the original culture medium to be performed in a limited number of steps. The proposed test is highly sensitive, with a detection limit of approximately 1 fM. This invention does not require amplification by PCR or the use of enzymes. This invention makes it possible to detect nucleic acids, from short microRNAs to long nucleic acids, using direct detection of luminescence signals.

[0019] Finally, the fluorescence signal generated in the test according to the present invention can be read using a plate reader, thereby accelerating the detection of nucleic acids in large samples.

[0020] In certain embodiments, the method according to the present invention utilizes the synergistic effect of a combination of capture using bead-based, particularly magnetic bead-based, solid surface hybridization and ultra-high-brightness fluorescent particles.

[0021] In another embodiment, the kits and methods according to the invention utilize the synergistic effect of a combination of capture using solid surface-based hybridization and detection using ultra-high brightness fluorescent DNA nanoparticles (DNA NPs) as disclosed in WO2017 / 220453.

[0022] In a first aspect, the invention relates to a kit for the detection of a target nucleic acid in a sample by a sandwich test. In a second aspect, the invention relates to a method of using the kit of the invention for the detection of a target nucleic acid in a sample by a sandwich test.

Mode for Carrying Out the Invention

[0023] In a first aspect, the invention relates to a kit for detecting a target nucleic acid in a sample, the kit comprising at least: i) A probe P1 comprising or consisting of a nucleic acid fragment NA1 bound to a functional unit F1, wherein NA1 comprises or consists of a nucleotide sequence complementary to the nucleotide sequence of a region T1 of the target nucleic acid; ii) A functional unit F2 bound to a solid surface, wherein F2 exhibits a high affinity for F1 or is covalently bound to F1; iii) Nucleic acid fragments NA2 bound to ultra-high brightness luminescent particles exhibiting a brightness of at least 10 7 M -1 cm -1 , preferably at least 2x10 7 M -1 cm -1 , at least 5x10 7 M -1 cm -1 , at least 10x10 7 M -1 cm -1 , or at least 40x10 7 M -1 cm -1 and comprising or consisting of a probe P2.

[0024] Figure 1A shows the elements of the kit according to the present invention and an unrestricted diagram of their respective interactions.

[0025] A “kit” refers to a set containing two or more distinct components housed together in conventional materials such as packaging. These distinct components interact for a specific purpose to achieve a particular result. It should be understood that the term “comprising” is not limiting. The term “consisting of” is considered a preferred embodiment of the term “comprising.”

[0026] The term "sample" refers to any small amount of a medium that may contain the target nucleic acid. In particular, biological samples containing cells or tissues of any origin are included. Environmental samples, such as those obtained from extraction or removal from an environmental matrix like water, are also included.

[0027] In certain embodiments, the present invention relates to a kit for in vitro detection of a target nucleic acid in a sample.

[0028] The term "in vitro" retains its original meaning, referring to experiments performed on biological molecules or living cells provided as samples extracted from a normal environment for analysis.

[0029] The term "nucleic acid" means deoxyribonucleotide or ribonucleotide polymer and includes known analogues of natural nucleotides that can function similarly to natural nucleotides and naturally occurring nucleotides. Non-limiting examples of such analogues of natural nucleotides are locked nucleic acids (LNAs) and 2'-O-methylated nucleotides. Target nucleic acid molecules detected by the kit or method according to the present invention are any nucleic acid molecules, including single-stranded and double-stranded DNA molecules, and single-stranded and double-stranded RNA molecules, including microRNA molecules.

[0030] The nucleic acid fragment of probe P1 or probe P2 is preferably an oligonucleotide, where “oligonucleotide” means a nucleic acid containing or consisting of a sequence of at least 5 nucleotides, preferably containing or consisting of a sequence of at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or at least 20 to at least 150 nucleotides.

[0031] "Complementary" means that the nucleic acid sequences described above can form a stable hybridization complex under appropriate stringency conditions. Sequence-specific hybridization uses an oligonucleotide probe complementary to the sequence of interest and captures it through hybridization. "Hybridization" means that a stable complex is formed between complementary nucleic acid molecules. Hybridization may occur between partially complementary molecules or between fully complementary molecules. The stability of the formed hybridization complex depends on the reaction conditions and the proportion of complementary nucleic acid sequences within the nucleic acid molecule. Oligonucleotide sequences are designed to ensure the formation of a stable hybridization complex.

[0032] The length of the nucleotide sequence of probe P1, P2, or P3, which is complementary to the nucleic acid, is preferably at least 8 nucleotides, more preferably at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or at least 20 to at least 150 nucleotides.

[0033] Within probe P1, the nucleotide acid fragment NA1 is bound to the functional unit F1 via covalent or non-covalent bonds, preferably via covalent bonds. Within probe P2, the nucleotide acid fragment NA2 is bound to the ultra-high brightness luminescent particle (ULP) via covalent or non-covalent bonds, preferably via covalent bonds.

[0034] In a particular embodiment of the kit according to the present invention, probe P1 comprises or consists of nucleic acid fragment NA1 and functional unit F1, wherein NA1 and F1 are linked via a linker. In another particular embodiment of the kit according to the present invention, probe P2 comprises or consists of nucleic acid fragment NA2 and ULP, wherein NA2 and ULP are linked via a linker.

[0035] "Linker" refers to any suitable compound selected by those skilled in the art, including: - For example, a non-coding nucleotide sequence selected from a DNA linker such as the "A10" or "A20" linker (if present, the DNA linker contains nucleotides that are not involved in the complementary hybridization sequence), and / or - For example, non-nuclear elements such as chemical elements like tetraethylene glycol (TEG).

[0036] A "functional unit" refers to any molecule, biomolecule, or chemical group that can selectively form non-covalent or covalent bonds with another functional unit.

[0037] Functional units commonly used by those skilled in the art include biotin (BIO), avidin (A), neutraavidin (N), and streptavidin (SV). Biotin is known for its very high affinity for avidin, neutraavidin, and streptavidin. Functional units can also be complementary nucleic acid sequences of at least 12 bases.

[0038] In certain embodiments, the first functional unit is biotin (BIO), and the second functional unit is selected from the group consisting of streptavidin (SV) and streptavidin analogs, particularly from the group consisting of avidin (A) and neutraavidin (N).

[0039] In a first embodiment of the present invention, F2 is bound to F1 via a non-covalent bond and exhibits high affinity for F1. "Non-covalent bond" means a bond that does not involve electron sharing, but rather involves other electromagnetic interactions. Examples of non-covalent bonds include hydrophobic effects, van der Waals forces, electrostatics, and the pi effect. Among non-covalent bonds, for example, a non-covalent bond π between complementary nucleic acid molecules can be cited. "High affinity" means a binding complex affinity characterized by a kD of at least 1 nM (upper limit), preferably at least 1 pM. In a specific example, the first functional unit is biotin (BIO), and the second functional unit is streptavidin, neutraavidin, or avidin. In this embodiment, F1 and F2 can form a high-affinity binding complex together through non-covalent (electron-non-sharing) interactions. For example, the very high affinity between biotin and streptavidin allows for the formation of a stable non-covalent complex, and this stability is observed even when an external physical force is applied to the complex. In further specific embodiments, functional unit F1 is biotin, and functional unit F2 is avidin or streptavidin.

[0040] In a second embodiment of the present invention, F2 is bonded to F1 via a covalent bond. A “covalent bond” refers to a chemical bond in which electrons are shared, forming at least one pair of electrons between two atoms. In the case of a covalent bond, the functional unit is a chemically reactive group. In certain embodiments, the functional group is an azide, which can react with acetylene derivatives by a “click” cycloaddition reaction. In certain embodiments, the functional group may be a carboxylate, which can react with an amine to form a covalent amide bond.

[0041] In the kit or method according to the present invention, F1 can covalently or noncovalently bind to nucleic acid fragment NA1 and to F2, and F2.

[0042] Functional unit F2 is bound to a solid surface via either covalent or non-covalent bonds by any technique known to those skilled in the art. For example, established methods for functionalizing a glass surface with streptavidin are based on the adsorption of BSA-biotin, followed by the binding of streptavidin, and finally, the addition of a corresponding DNA capture having a biotin unit (Figure 2A).

[0043] In a first specific embodiment of the kit according to the present invention, the functional unit F2 is bonded to a solid immobilized surface, such as a glass surface, a reaction support surface, a multiwell plate surface, a microfluidic device surface, or any surface suitable for covalent or non-covalent functionalization.

[0044] In a second specific embodiment of the kit according to the present invention, the functional unit F2 is bonded to a surface which is the surface of a solid particle. This solid particle may be, for example, a magnetic bead or a glass bead. Those skilled in the art can select any suitable bead commonly used in this type of kit. The solid particle exists as a suspension and is sensitive to the application of external physical force.

[0045] The solid particles of the kit according to the present invention react to external physical forces. Among these physical forces, for example, the following can be cited: - Applicable via centrifugal separation process, gravity - Electromagnetic force, or applicable in the presence of a magnet. - Shear force applied by fluidization or filtration.

[0046] In certain embodiments, the kit according to the present invention includes magnetic beads covalently bonded to a functional unit F2. The magnetic beads bonded to the functional unit F2 are sensitive to magnetic force.

[0047] Ultra-high brightness emitting particles exhibit very high levels of brightness. Brightness is defined as the product of the absorption coefficient and the fluorescence quantum yield. In the kit and method according to the present invention, ultra-high brightness emitting particles (ULPs) are at least 10 7M -1 cm -1 Preferably at least 2x10 7 M -1 cm -1 , at least 5x10 7 M -1 cm -1 , at least 10x10 7 M -1 cm -1 , at least 40x10 7 M -1 cm -1 It is any particle characterized by its brightness.

[0048] The brightness of the particles can also be defined by the ratio of the dye weight to the total particle weight. In the case of ultra-bright particles in the kit or method according to the present invention, the above ratio is preferably at least 1% by weight of dye retention, preferably at least 5% by weight of dye retention, at least 10% by weight of dye retention, at least 20% by weight of dye retention, at least 30% by weight of dye retention, at least 40% by weight of dye retention, or at least 50% by weight of dye retention.

[0049] In the kit according to the present invention, the ultraluminescent particles (ULPs) may be selected from any luminescent particles known to those skilled in the art. The particles are preferably selected from the group consisting of fluorescent particles, phosphorescent particles, chemiluminescent particles, and bioluminescent particles. In a particular embodiment, the ultraluminescent particles are ultraluminescent fluorescent particles.

[0050] In a particular embodiment of the kit according to the present invention, the probe P2 is at least 10 7 M -1 cm -1 Preferably at least 2x10 7 M -1 cm -1 , at least 5x10 7 M -1 cm -1 , at least 10x10 7 M -1 cm -1 or at least 40x10 7 M -1 cm-1 The kit comprises, or consists of, nucleic acid fragments NA2 bound to ultra-high brightness fluorescent particles exhibiting a brightness of . More specifically, the kit according to the present invention comprises at least 10 7 M -1 cm -1 Preferably at least 2x10 7 M -1 cm -1 , at least 5x10 7 M -1 cm -1 , at least 10x10 7 M -1 cm -1 or at least 40x10 7 M -1 cm -1 The present invention includes nucleic acid fragments NA2 bound to ultra-high brightness fluorescent polymer nanoparticles exhibiting a high brightness, or a probe P2 consisting of the same.

[0051] In another specific embodiment, the ultra-high-brightness light-emitting particles are ultra-high-brightness light-emitting nanoparticles. In yet another specific embodiment, the ultra-high-brightness light-emitting particles are ultra-high-brightness light-emitting polymer nanoparticles.

[0052] Dye-retaining polymer nanoparticles (NPs) are a promising, highly sensitive alternative for detecting nucleic acids in solution due to their high brightness and modularity. Previously, to ensure high brightness while addressing the quenching problem caused by the aggregation of dyes encapsulated within these NPs, the concept of insulating ionic dyes with bulky hydrophobic counterions was proposed, resulting in NPs approximately 100 times brighter than semiconductor quantum dots (QDs) of comparable size (Melnychuk & Klymchenko, 2018), (Reisch et al., 2014), (Reisch et al., 2017). Furthermore, charge-controlled nanoprecipitation of hydrophobic polymers with a small number of charged groups was introduced to achieve size control of polymer NPs (Reisch et al., 2015). Strategies based on charged amino acids to expose azide groups on the NP surface have enabled further modification of nucleic acids by click chemistry. The resulting DNA-NP complexes yielded ultra-bright nanoprobes for smartphone-based sensing-compatible DNA / RNA detection amplification in picomolar detection-limited solutions and on surfaces with single-molecule sensitivity. These DNA-functionalized NPs have already been proven effective for the detection of microRNAs in cell extracts and for the direct detection of intracellular RNA using RNA-fluorescence in situ hybridization protocols. Their application to enzyme-free amplification of RNA in solution using simple one-step protocols with total RNA cell extracts or serum media is being investigated.

[0053] Ultra-high brightness dye-retaining polymer nanoparticles refer to particles containing a fluorescent dye, and containing at least 200, 500, 1000, 2000, or 10000 fluorescent dye molecules, wherein each fluorescent molecule in the particle contains at least 4 x 10⁶ molecules. 4 M -1 cm -1The particles exhibit high brightness. The ultra-high brightness particles may consist of polymers in particular. Non-limiting examples of polymers include polymethacrylates, aliphatic polyesters, polystyrenes, and polyurethanes. Examples of polymethacrylates include, but are not limited to, poly(methyl methacrylate), poly(ethyl methacrylate), and poly(butyl methacrylate). Examples of derivatives of polymethacrylates include poly(methyl methacrylate-co-methacrylic acid) (PMMA-MA) and poly(methyl methacrylate-co-2-methacrylamide ethanesulfonic acid) (PMMA-SO3). Examples of aliphatic polyesters include, but are not limited to, polycaprolactone (PCL), polylactic acid (PLA), polyglycolic acid (PGA), and poly(lactide-co-glycolide) (PLGA).

[0054] Furthermore, in certain embodiments, the kit according to the present invention includes ultra-high brightness dye-retaining fluorescent nanoparticles, particularly as described in EP3536806.

[0055] In a particular embodiment of this first aspect, the present invention relates to a kit for detecting a target nucleic acid in a sample, the kit comprising at least: i) A probe P1 comprising or consisting of a nucleic acid fragment NA1 bound to a functional unit F1, wherein the nucleic acid fragment NA1 comprises a nucleotide sequence complementary to the nucleotide sequence of region T1 of the target nucleic acid molecule, ii) Functional unit F2 bonded to a solid surface, iii) A probe P2 comprising or consisting of a nucleic acid fragment NA2 bound to an ultra-high brightness dye-retaining light-emitting particle, wherein the nucleic acid fragment NA2 comprises a nucleotide sequence complementary to the nucleotide sequence of region T2 of the target nucleic acid, or the probe P2 consisting of such a fragment.

[0056] In another specific embodiment of the present invention, the kit of the present invention includes at least the following: i) A probe P1 comprising or consisting of a nucleic acid fragment NA1 covalently bonded to a functional unit F1, wherein NA1 comprises a nucleotide sequence complementary to the nucleotide sequence of region T1 of the target nucleic acid, ii) Functional unit F2 bonded to a solid surface, iii) At least one probe P2 comprising or consisting of a nucleic acid fragment NA2 bound to an ultra-high brightness light-emitting particle, and iv) A probe P3 comprising, or consisting of, a first portion comprising a nucleic acid fragment NA4 having a nucleotide sequence complementary to the nucleotide sequence of NA2, and a second portion comprising, or consisting of, a nucleic acid fragment NA3 having a nucleotide sequence complementary to the nucleotide sequence of region T3 of the target nucleic acid, wherein the first portion and the second portion are linked via a nucleotide linker.

[0057] Figure 1B shows the elements of the kit according to the present invention and a non-limiting diagram of their respective interactions.

[0058] In this embodiment of the kit according to the present invention, probe P3, also named "post-it capture sequence" or "post-it probe," comprises at least the following two parts: - A first portion comprising or consisting of a nucleic acid fragment NA4 having a nucleotide sequence complementary to the nucleotide sequence of NA2 of probe P2, and - A nucleic acid fragment NA3 having a nucleotide sequence complementary to the nucleotide sequence of region T3 of the target nucleic acid, or a second portion consisting thereof, wherein T3 differs from T1, The first and second portions are linked via a nucleotide linker.

[0059] The first and second portions of P3 are preferably oligonucleotides, and probe P3 is preferably a nucleic acid comprising or consisting of a sequence of at least 10 nucleotides, and preferably a nucleic acid comprising or consisting of a sequence of at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 50, 80, 100, 120, or 150 nucleotides.

[0060] In certain embodiments, the first and second portions of P3 are linked via a linker preferably having a length of at least 5 nucleotides, more preferably at least 10, 15, 20, or 40 nucleotides.

[0061] In this particular embodiment, the nucleotide sequence of the nucleic acid fragment NA2 of probe P2 is independent of the nucleotide sequence of the target nucleic acid. This embodiment presents the advantage of including probe P2, which contains a nucleic acid fragment NA2 whose nucleotide sequence is independent of the target to be detected.

[0062] In another specific embodiment, the kit of the present invention may include at least two probes P1, for example, 2, 3, 4, 5, or n probes P1, where n is the total number of P1 probes in the kit. Each of the above P1 probes is named P1-1, P1-2, or P1-n and contains a nucleic acid fragment NA1 named NA1-1, NA1-2, or NA1-n, respectively, whose nucleotide sequence is complementary to the nucleotide sequence T1-1, T1-2, or T1-n of the target nucleic acid, respectively. The kit according to the present invention may include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more probes.

[0063] In this embodiment, the kit of the present invention includes at least the following: i) A probe P1 comprising or consisting of a nucleic acid fragment NA1 covalently bonded to a functional unit F1, wherein NA1 contains a nucleotide sequence complementary to the nucleotide sequence of region T1 of the target nucleic acid, ii) Functional unit F2 bonded to a solid surface, iii) At least one probe P2 comprising or consisting of a nucleic acid fragment NA2 bound to an ultra-high brightness light-emitting particle, and iv) A probe P3 comprising, or consisting of, a first portion comprising a nucleic acid fragment NA4 having a nucleotide sequence complementary to the nucleotide sequence of NA2, and a second portion comprising, or consisting of, a nucleic acid fragment NA3 having a nucleotide sequence complementary to the nucleotide sequence of region T3 of the target nucleic acid, wherein the first portion and the second portion are linked via a nucleotide linker.

[0064] In another specific embodiment, the kit of the present invention may include at least two, three or more probe P3s. Each of the above P3 probes differs from the other P3 probes in the nucleotide sequence of its first portion and / or the nucleotide sequence of its second portion.

[0065] For example, probe P3-NA4-1 contains a nucleotide sequence complementary to the nucleotide sequence NA2-1 on probe P2, or contains a nucleic acid fragment NA4-1 consisting of such a sequence.

[0066] For example, probe P3-NA3-1 contains a nucleotide sequence complementary to the nucleotide sequence T3-1 on the target nucleic acid, or contains a nucleic acid fragment NA3-1 consisting of such a nucleotide sequence.

[0067] In this embodiment, the kit of the present invention may include 1, 2, 3, or n different P3 probes, where n is the number of P3 probes. The kit of the present invention may include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more P3 probes.

[0068] In this embodiment, the kit of the present invention includes at least the following: i) A probe P1 comprising or consisting of a nucleic acid fragment NA1 covalently bonded to a functional unit F1, wherein NA1 comprises or consists of a nucleotide sequence complementary to the nucleotide sequence of region T1 of the target nucleic acid. ii) Functional unit F2 bonded to a solid surface, iii) A probe P2 comprising or consisting of nucleic acid fragment NA2 bound to ultra-high brightness light-emitting particles, and iv) At least two probes P3 comprising, or consisting of, a first part containing or comprising a nucleic acid fragment NA4 having a nucleotide sequence complementary to the nucleotide sequence of NA2, and a second part containing or consisting of a nucleic acid fragment NA3 having a nucleotide sequence complementary to the nucleotide sequence of region T3 of the target nucleic acid, wherein the first part and the second part are linked via a nucleotide linker.

[0069] The nucleotide sequences of multiple P1 probes and multiple P3 probes are each capable of binding to different regions of the target nucleic acid, which is a long-sized nucleic acid molecule, where "long-sized" refers to target nucleic acids of at least 35 nucleotides, at least 50 nucleotides, at least 100 nucleotides, at least 1000 nucleotides, and more. The presence of one, two, three, or n probes allows for the detection of this long-sized nucleic acid molecule present in the sample in an untreated or fragmented form.

[0070] In another specific embodiment, the kit of the present invention may include at least two probes P2, for example, 2, 3, 4, 5, or n probes P2, where n is the total number of P2 probes in the kit.

[0071] Each of the above P2 probes is named P2-1, P2-2, or P2-n and contains a nucleic acid fragment NA2 named NA2-1, NA2-2, or NA2-n, respectively, the nucleotide sequence of which is complementary to the sequence of the nucleotide fragment NA4-1, NA4-2, or NA4-n of probe P3, respectively. The kit according to the present invention may contain probes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more probes P2.

[0072] In this embodiment, each P2 probe may differ from other P2 probes in terms of the properties of the superluminescent particle. In fact, the use of P2 probes containing different ULPs can enable detection multiplexing by using the kit according to the present invention.

[0073] In certain embodiments, in the presence of two or more different P3 probes, the kit according to the present invention may include one or more different P2 probes. Conversely, in certain embodiments, in the presence of two or more different P2 probes, each of the P3 probes must be adapted to hybridize with each of the P2 probes, so the kit according to the present invention must include two or more different P2 probes.

[0074] In this embodiment, the kit of the present invention includes at least the following: i) A probe P1 comprising or consisting of a nucleic acid fragment NA1 covalently bonded to a functional unit F1, wherein NA1 comprises a nucleotide sequence complementary to the nucleotide sequence of region T1 of the target nucleic acid, ii) Functional unit F2 bonded to a solid surface, iii) At least one probe P2, preferably at least two probe P2s, comprising or consisting of nucleic acid fragment NA2 bound to ultra-high brightness light-emitting particles, and iv) A probe P3 comprising or consisting of a nucleic acid fragment NA3 having a nucleotide sequence complementary to the nucleotide sequence of NA2 and a nucleotide sequence complementary to the nucleotide sequence of region T3 of the target nucleic acid.

[0075] This is important for the simultaneous multiplexing of different regions of nucleic acids. In this case, ULPs preferably have different spectral characteristics.

[0076] The kit according to the present invention may also include any suitable elements for carrying out a detection reaction, such as a buffer.

[0077] A second aspect of the present invention relates to a method for detecting a target nucleic acid molecule in a sample prepared from a biological sample by a sandwich test, the method comprising at least the following steps: a) Under conditions suitable for hybridization of complementary nucleic acid sequences and formation of non-covalent complexes, at least, - A probe P1 comprising or consisting of a first nucleic acid fragment NA1 bound to functional unit F1, wherein NA1 comprises a nucleotide sequence complementary to the nucleotide sequence of region T1 of the target nucleic acid, - Functional unit F2 bonded to a solid surface, which exhibits high affinity for F1 or is covalently bonded to F1, and - At least 10 7 M -1 cm -1 Preferably at least 2x10 7 M -1 cm -1 , at least 5x10 7 M -1 cm -1 , at least 10x10 7 M -1 cm -1 or at least 40x10 7 M -1 cm -1 A step of contacting a probe P2 containing or consisting of nucleic acid fragment NA2 bound to ultra-high brightness light-emitting particles exhibiting a certain brightness, b) A step of applying an external physical force to the mixture from step a) in order to isolate the formed composite, c) A step of measuring the luminescence intensity of the above non-covalent complex of nucleic acids.

[0078] Thus, the present invention relates to a method belonging to the group of sandwich-type tests for detecting a target nucleic acid in a sample. In the method according to the present invention, the physical force applied is adapted to the nature of the solid surface of the kit and is easily selected by a person skilled in the art.

[0079] In a particular embodiment, the present invention relates to a method for detecting a target nucleic acid in a sample in vitro.

[0080] Among physical forces, in particular, for example, the following can be mentioned: - Gravity, applicable via a centrifugation step, - Electromagnetic force, applicable in the presence of a magnet, or - Shearing force, exerted by flow or filtration.

[0081] In a particular embodiment, the method according to the present invention comprises applying a centrifugation step, applying an electromagnetic force, or applying at least a filtration step. In the method according to the present invention, "applying a physical force" includes applying one, two, three or more of the above physical force steps.

[0082] In a particular embodiment, the present invention relates to a method for detecting a target nucleic acid molecule in a sample, wherein the above functional unit F2 is bound to a solid surface, and the solid surface is the surface of solid particles. In a further particular embodiment, the solid particles are magnetic beads or glass beads.

[0083] In the method according to the present invention, the ultra-high brightness luminescent particles (ULP) are at least 10 7 M -1 cm -1 , preferably at least 2x10 7 M -1 cm -1 , at least 5x10 7 M -1 cm -1 , at least 10x10 7 M -1 cm -1 , at least 40x10 7 M-1 cm -1 Any luminescent particles characterized by a luminance of -1 , or a ratio of dye weight to total particle weight of at least 1% dye retention, preferably at least 5% dye retention, at least 10% dye retention, at least 20% dye retention, at least 30% dye retention, at least 40% dye retention or at least 50% dye retention.

[0084] In the method according to the invention, the ultra-high luminance luminescent particles (ULP) may be selected from any luminescent particles known to those skilled in the art. The particles are preferably selected from the group consisting of fluorescent particles, phosphorescent particles, chemiluminescent particles and bioluminescent particles. In certain embodiments, the ultra-high luminance luminescent particles are ultra-high luminance fluorescent particles.

[0085] In a particular embodiment of the method according to the invention, the probe P2 is at least 10 7 M -1 cm -1 comprises or consists of a nucleic acid fragment NA2 bound to ultra-high luminance fluorescent particles exhibiting a luminance of -1 . More specifically, in the method according to the invention, the probe P2 is at least 10 7 M -1 cm -1 comprises or consists of a nucleic acid fragment NA2 bound to ultra-high luminance fluorescent polymer nanoparticles exhibiting a luminance of -1 .

[0086] In another particular embodiment, the ultra-high luminance luminescent particles are ultra-high luminance luminescent nanoparticles. In a more particular embodiment, the ultra-high luminance luminescent particles are ultra-high luminance luminescent polymer nanoparticles.

[0087] In a more particular embodiment, the method according to the invention comprises ultra-high luminance dye-retaining fluorescent nanoparticles, as described in particular in EP3536806.

[0088] In a particular embodiment of the method according to the invention, the probe P2 is at least 10 7 M -1 cm -1The device contains, or comprises, a nucleic acid fragment NA2 bound to ultra-high-brightness fluorescent particles exhibiting a certain brightness, wherein the nucleic acid fragment NA2 contains, or comprises, a nucleotide sequence complementary to the nucleotide sequence of region T2 of the target nucleic acid.

[0089] In another embodiment, the present invention relates to a method for detecting a target nucleic acid molecule in a sample, wherein step a) is at least, - A probe P1 comprising or consisting of a nucleic acid fragment NA1 bound to a functional unit F1, wherein NA1 comprises or consists of a nucleotide sequence complementary to the nucleotide sequence of region T1 of the target nucleic acid. - Functional unit F2 bonded to a solid surface, wherein F2 exhibits high affinity for F1 or is covalently bonded to F1, and - A probe P2 containing or consisting of nucleic acid fragment NA2 bound to ultra-high brightness light-emitting particles, - At least one probe P3 comprising or consisting of a nucleotide sequence complementary to the nucleotide sequence of region T3 of the target nucleic acid, wherein T3 is different from T1 and is a nucleotide sequence complementary to the nucleotide sequence of NA2, Make contact, - Including forming a mixture.

[0090] In another specific embodiment, the present invention relates to a method for detecting a target nucleic acid molecule in a sample, wherein step a) comprises at least: - At least two probes P1, each of which contains or consists of a nucleic acid fragment NA1, each of which is named NA1-1 or NA1-2, and is complementary to the nucleotide sequence T1-1 or T1-2 of the target nucleic acid, - A probe P2 containing or consisting of nucleic acid fragment NA2 bound to ultra-high brightness light-emitting particles, - At least one probe P3, *A nucleotide sequence complementary to the nucleotide sequence of region T3 of the target nucleic acid (T3 is different from T1), and *A nucleotide sequence complementary to the nucleotide sequence of NA2, By bringing into contact at least one probe P3, which includes or consists of, - Including forming a mixture.

[0091] In another specific embodiment, the present invention relates to a method for detecting a target nucleic acid molecule in a sample, wherein step a) comprises at least: - At least two probes P1, each of which contains or consists of a nucleic acid fragment NA1, each of which is named NA1-1 or NA1-2, and is complementary to the nucleotide sequence T1-1 or T1-2 of the target nucleic acid, - A probe P2 containing or consisting of nucleic acid fragment NA2 bound to ultra-high brightness light-emitting particles, - At least two probes P3, each of which probes P3 is *A nucleic acid fragment NA3 containing or consisting of nucleotide sequences NA3-1 or NA3-2 that are complementary to the nucleotide sequences T3-1 and T3-2 of the target nucleic acid, respectively, wherein T3-1 or T3-2 is different from T1, and *A nucleotide sequence complementary to the nucleotide sequence of NA2, By bringing at least two probes P3, which include or consist of, - Including forming a mixture.

[0092] In a third aspect, the present invention relates to the use of a kit according to the present invention for the detection of a target nucleic acid.

[0093] In the third aspect described above, the present invention also relates to the use of the method according to the present invention for the detection of target nucleic acids.

[0094] The kits and methods according to the present invention are suitable for detecting target nucleic acids having variable size. In fact, the kits and methods according to the present invention are suitable for detecting target nucleic acids such as microRNA or long-sized RNA, and single-stranded or double-stranded DNA of different sizes. Therefore, the kits and methods according to the present invention can be used to detect the size of target nucleic acids, preferably consisting of at least 15 nucleotides.

[0095] In a fourth aspect, the present invention relates to an in vitro diagnostic method for a pathology or disease condition, the method comprising the use of a kit or method according to the present invention for the detection and / or extraction of a target nucleic acid specific to the pathology or disease condition.

[0096] In particular, the present invention relates to an in vitro diagnostic method for pathologies resulting from the presence of the SARS-Cov-2 virus, the method comprising the use of a kit or method according to the present invention for the detection and / or extraction of a target nucleic acid from the virus.

[0097] The present invention will be described in more detail by the following figures and embodiments. [Brief explanation of the drawing]

[0098] [Figure 1A-B] Figure 1A illustrates a specific embodiment of the nucleic acid detection method according to the present invention, showing ultraluminescent particles (ULPs) functionalized with nucleic acid fragment NA2 that can directly hybridize with region T2 of the target nucleic acid. Figure 1B illustrates a specific embodiment of the nucleic acid detection method according to the present invention, showing ultraluminescent particles (ULPs) functionalized with nucleic acid fragment NA2 that can bind to region T2 of the target nucleic acid via an intermediate, i.e., a "Post-it" probe P3. [Figure 2A] This represents a specific embodiment of the present invention in the presence of a target nucleic acid, where the P1 probe is immobilized on a solid surface and comprises a functional unit F1 and nucleic acid fragment NA1, and the P2 probe comprises nucleic acid fragment NA2 and ultraluminescent particles (NPs). [Figure 2B]This figure shows the quantification of particle numbers at increasing concentrations of the DNA target (SEQ ID NO: 6) (0, 0.001, 0.01, 0.1, 1, and 10 pM). PBS buffer containing 0.01 g / L Tween 80 was systematically used for incubation and imaging. [Figure 3A-B] Figure 3A shows a specific embodiment of the present invention in the presence of a target nucleic acid, where the P1 probe is immobilized on solid particles in a suspension and contains functional unit F1 and nucleic acid fragment NA1, and the P2 probe contains nucleic acid fragment NA2 and ultraluminescent particles (NPs). Figure 3B shows the quantification of fluorescence intensity as the concentration of the DNA target (SEQ ID NO: 6) increases (0, 0.001, 0.01, 0.1, 1, and 10 pM). PBS buffer containing 0.01 g / L Tween 80 was systematically used for incubation and detection. [Figure 4A-D] Figure 4A illustrates the detection of target nucleic acids of different lengths, showing the quantification of particle counts in detection by immobilized glass surface for increasing concentrations (0, 1, 10, 100, and 1000 pM) of 100-nucleotide RNA targets (left bars for each concentration) or 1000-nucleotide RNA targets (right bars for each concentration). Figure 4B illustrates the detection of target nucleic acids of different lengths, showing the quantification of fluorescence intensity by magnetic bead sandwich detection of 100-nucleotide RNA targets (left bars for each concentration) or 1000-nucleotide RNA targets (right bars for each concentration) spiked at increasing concentrations (0, 1, 10, 100, and 1000 pM) in 2.5 μg of total RNA extract. RNA Secure 1x buffer containing 0.01 g / L Tween 80 was systematically used for incubation and detection. Figure 4C shows 100 and 1000-nucleotide RNA targets with different target sequence positions complementary to DNA-NPs. Figure 4D illustrates the potential difficulty of direct hybridization between DNA-NPs and target sequences not located at the target end. [Figure 5A]Representing a specific embodiment of the present invention in the presence of a target nucleic acid, the P1 probe (biotin capture) is immobilized on solid particles in a suspension and contains functional unit F1 and nucleic acid fragment NA1 which hybridizes with the target. The P2 probe contains nucleic acid fragment NA2 and ultraluminescent particles (NPs). The P3 probe (DNA Post-it) partially hybridizes with the target nucleic acid and the other half hybridizes with nucleic acid fragment NA2 and ultraluminescent particles (NPs). The inclined arrow on the right indicates that there is no steric hindrance at the target edges after DNA-NP hybridization. [Figure 5B] This figure shows the quantification of fluorescence intensity in detecting spiked increases in the concentration (0, 0.01, 0.1, 1, and 10 pM) of a 48-nucleotide DNA target (SEQ ID NO: 6) using either direct hybridization of DNA-NPs (white bars) or indirect hybridization via a Post-it probe (P3) (dark bars) by magnetic bead sandwich detection in 2.5 μg of total RNA extract. PBS buffer containing 0.01 g / L Tween 80 was systematically used for incubation and detection. [Figure 6A] This study demonstrates the effect of NP characteristics (NP size) on sandwich hybridization sensitivity: In indirect hybridization via a P3 probe, DNA-NPs with core sizes of 20 nm or 50 nm were used, and the fluorescence intensity in detecting spiked increases in concentration (0, 0.1, 1, and 10 pM) of a 48-nucleotide DNA target (SEQ ID NO: 6) was quantified by magnetic bead sandwich detection in 2.5 μg of total RNA extract. [Figure 6B]This study demonstrates the effect of NP characteristics (dye retention) on sandwich hybridization sensitivity: DNA-NPs retaining 0.1, 1, 10, and 50 wt% dye were used, and fluorescence intensity was quantified in the detection of spiked increases in concentration (0, 0.1, 1, and 10 pM) of a 48-nucleotide DNA target (SEQ ID NO: 6) by sandwich detection using magnetic beads in 2.5 μg of total RNA extract via indirect hybridization using a Post-it probe (P3 probe). RNA Secure 1x buffer containing 0.01 g / L Tween 80 was systematically used for incubation and detection. [Figure 7A] This diagram illustrates a method for detecting long RNA targets, showing a workflow in which long RNAs are sandwich-detected by indirectly hybridizing DNA-NPs in suspended magnetic beads via Post-it probe capture (P3). [Figure 7B] This figure illustrates the detection method for long RNA targets, showing the quantification of fluorescence intensity detected by magnetic bead sandwiching using 50 nm core-sized DNA-NPs with spiked, increasing concentrations of 100 nucleotide RNA targets (SEQ ID NO: 23) or 1000 nucleotide RNA targets (SEQ ID NO: 24) (0, 0.01, 0.1, 1, and 10 pM) in 2.5 μg of total RNA extract, and biotin capture with or without a TEG motif in a 20 adenine linker. RNA Secure 1x buffer containing 0.01 g / L Tween 80 was systematically used for incubation and detection. [Figure 8] This report describes the detection of SARS-CoV-2 RNA in RNA extracts derived from clinical samples. Infected sample CS7 (CT=18.75) was diluted several-fold with negative sample CS1 (CT=37), and the fluorescence intensity was quantified using a 50 nm core-size DNA-NP, three biotin-captures (P1), and three Post-it probe (P3) captures, while maintaining a constant total RNA amount (0.25 μg). RNA Secure 1x buffer containing 0.01 g / L Tween 80 was systematically used for incubation and detection. [Figure 9A] This figure shows the results of directly detecting SARS-CoV-2 RNA from clinical samples without RNA extraction, and quantifies the fluorescence intensity in detecting SARS-CoV-2 RNA in clinical samples collected from a supply source (CS series) using a 50nm core-sized DNA-NP with three biotin-captures (P1) and three DNA Post-it probes (P3). [Figure 9B] This figure shows the results of directly detecting SARS-CoV-2 RNA from clinical samples without RNA extraction, and quantifies the fluorescence intensity in detecting SARS-CoV-2 RNA in clinical samples collected from a source (CH series) using a 50nm core-sized DNA-NP with three biotin-captures (P1) and three DNA Post-it probes (P3). [Figure 9C] The results show the direct detection of SARS-CoV-2 RNA from clinical samples without RNA extraction, with negative CS2 and positive CS6 samples diluted several times with buffer. RNA Secure 1x buffer containing 0.01 g / L Tween 80 was systematically used for incubation and detection. [Figure 10] This graph shows the concentration-dependent response of a glass surface test method for detecting microRNA targets: miR200a (left bar) and miR21 (right bar). Target concentrations range from 0 to 100 pM, and NP quantification ranges from 0 to 25,000. PBS buffer containing 0.01 g / L Tween 80 was systematically used for incubation and detection.

[0099] Examples: Example 1: Detection of short nucleic acids 1.1. Materials and Methods Chemicals were purchased from either Sigma Aldrich, Alfa Aesar, or Thermofisher Scientific. The polymer PMMA-AspN3-5% was synthesized as previously described (Melnychuk et al, 2020) (Melnychuk and Klymchenko, 2018). Rhodamine B octadecyl ester trakis(pentafluorophenyl) borate (R18 / F5) was synthesized by ion exchange as previously described and purified by column chromatography (Reish et al, 2014).

[0100] Preparation of NPs NP-PEMA-MA-5% A polymer solution in 50 μL of acetonitrile (containing 2 mg / mL of R18 / F5, or 50 wt% of the polymer) was rapidly added to 450 μL of 20 mM phosphate buffer and pH 7.4 50 mM NaCl at 21°C under shaking (Thermomixer comfort, Eppendorf, 1100 rpm) using a micropipette. While continuing to stir, 500 μL of 20 mM phosphate buffer (pH 7.4 50 mM NaCl) was added. The residual acetonitrile was then evaporated.

[0101] General protocols for functionalizing nanoparticles with DNA Lyophilized single-stranded DNA sequences were purchased from Biomers or Eurogentec, dissolved in Milli-Q water, aliquoted, and stored at -20°C for further experiments. A fixed amount of dibenzocyclooctyne (DNA-DBCO)-bound corresponding nucleic acid fragment (SEQ ID NO: 1, SEQ ID NO: 8, SEQ ID NO: 14, SEQ ID NO: 19, SEQ ID NO: 20, or SEQ ID NO: 21) was added to 200 μl of the corresponding nanoparticles at a concentration of 20 μM in the reaction mixture. The reaction mixture was mixed, protected from light, and allowed to stand overnight at 40°C without shaking. The reaction mixture was then cooled to room temperature. The mixture was diluted to 4 mL in 20 mM phosphate buffer, and the NPs were purified by centrifugation at 1000 g, 20°C, for 5 minutes using a centrifugal filter (Amicon, 0.5 mL, 100 kD, Sigma-Aldrich). To remove unreacted oligonucleotides, centrifugation was repeated six times in the corresponding buffer. After each centrifugation, the overflow was discarded, and the filtrate was resuspended in 4 mL of 20 mM phosphate buffer. This high level of purification is crucial for achieving the high sensitivity of this method. The resulting functionalized DNA-NPs were stored in the dark at 4°C.

[0102] Characterization of nanoparticles Dynamic light scattering (DLS) measurements were performed using a Zetasizer Nano ZSP (Malvern Instruments SA). Zetasizer software, which provides particle size distributions by standard cumulative and volumetric analysis, was used for DLS characterization of nanoparticles. The following parameters were used for data analysis: solvent (water), temperature 25°C, refractive index RI 1.33, viscosity 0.8872 cP. All nanoparticles were assumed to be homogeneous and spherical. Absorption spectra were recorded using a Cary 5000 scan UV-Vis spectrophotometer (Varian). Excitation, emission spectra, and anisotropy were recorded using an FS5 Spectrofluoremeter (Edinburg Instruments). For standard fluorescence spectroscopy recording, the excitation wavelength was set to 530 nm. Fluorescence spectra were corrected for detector response and lamp fluctuations.

[0103] Transmission electron microscope (TEM) A 300-mesh carbon-coated copper-rhodium electron microscope grid (Euromedex, France) was surface-treated with glow discharge (0.45 mbar, 5-5.3 mA, 25 seconds) in an amylamine atmosphere using an Elmo glow discharge system (Cordouan Technologies, France). Then, 5 μL of NP solution was deposited onto the grid and left for 2 minutes. The grid was then stained with a 2% uranyl acetate solution for 1 minute. These grids were observed using a Tecnai F20 electron microscope equipped with a 200 keV FEG. Areas covered with the target nanoparticles were recorded at 29,000x magnification using a GATAN CCD 2K*2K "US10001" camera. Image analysis was performed using Fiji software.

[0104] Nucleic acid detection on glass surfaces A LabTek chamber (borosilicate coverslip, 8 wells, Thermofisher Scientific) was pre-treated with 1M KOH for 30 minutes, washed three times with PBS, and then 100 μL of 0.5 mg / mL BSA-biotin (Sigma-Aldrich) was added to PBS. -1 The mixture was incubated with the solution for 20 minutes. Then, the BSA-biotin solution was removed, and the chamber was washed three times with 500 μL of PBS. Next, the chamber was filled with 100 μL of nutraavidin (ThermoFisher Scientific) solution (0.5 mg / mL in PBS). -1 The chamber was incubated with the solution for 15 minutes and washed three times with 500 μL of PBS. Next, the chamber was incubated with 100 μL of a 1 μM solution of the P1 biotin-capture sequence (SEQ ID NO: 2) in PBS for 20 minutes and washed three times with PBS. Finally, 100 μL of detection solution containing 200 pM probe P2 and synthetic targets of different concentrations in PBS containing 0.01 g / L Tween 80 was added dropwise and incubated at 40°C in the dark for 1 hour. The chamber was washed three times before measurement and covered with 200 μL of the same buffer.

[0105] Single-particle measurements were performed using a Nikon Ti-E inverted microscope (Apo TIRF, air, NA 1.4, Nikon) equipped with a 20x objective lens, in epifluorescence mode. Excitation was performed at 550 nm using a light-emitting diode (SpectraX, Lumencor). The exposure time was set to 500 ms per image frame. The fluorescence signal was recorded with a Hamamatsu Orca Flash 4 camera.

[0106] Single-particle analysis was performed using Fiji software. Particle positions were detected by applying a Fiji routine to the projection (maximum intensity) of all frames obtained for each experiment. After automatic background removal, the number of target particles in a 10-pixel diameter area was measured. At least 10 image sequences (1024 pixels x 1024 pixels) were analyzed for each condition.

[0107] Magnetic bead nucleic acid detection Synthetic target solutions of different concentrations (SEQ ID NO: 6, Figures 3, 5, and 6) were incubated in PBS containing 0.01 g / L Tween 80 with 50 nM P1 biotin-capture sequences and, where indicated, 10 nM Post-it probe (P3) capture sequences. Cell lysates and RNA extracts were diluted with RNA Secure (Sigma) containing 0.01 g / L Tween 80 and incubated with 20 nM of each biotin-capture sequence P1 and 10 nM of each Post-it (P3) capture sequence P3, as shown. The solutions were heated at 95°C for 30 seconds, cooled on ice for 5 minutes, and then incubated at 40°C for 20 minutes without stirring. Next, 0.1 mg of streptavidin magnetic beads, pre-washed twice with PBS, were added. This suspension was incubated at 40°C for 1 hour with gentle stirring. The complex consisting of magnetic beads, target sequences, and capture sequences was separated from non-reactive nucleic acids by a magnetic field. The beads were washed three times with the same incubation buffer. A 200 pM nanoprobe (containing SEQ ID NO: 19, SEQ ID NO: 20, or SEQ ID NO: 21) was added, and the mixture was incubated at 40°C for 1 hour with gentle agitation. The complex, consisting of magnetic beads, target, capture sequence, and nanoprobe, was separated from unreactive nanoparticles by a magnetic field. The beads were washed three times with the same incubation buffer, and the complex was resuspended in 50 μL of the same buffer before being transferred to a 96-well black plate. Nucleic acids on the bead surface were directly detected by measuring fluorescence intensity using a microplate (SPARK, TECAN).

[0108] The oligonucleotides and their respective nucleic acid sequences used in the examples are listed in Table 1 and in the sequence list attached to this patent application. N1, SPIKE, and ORF1 represent regions of the SARS-CoV-2 virus used as targets, respectively, from which the nucleotide sequences of probes P1, P2, and P3 are specified. B, C, and NS represent the respective non-coding sequences of probe P2 that bind to NP. NP stands for ultrabright nanoparticle. When using P3, NP is the same to detect all target sequences, but P2 is not complementary to the target, so the P3 probe differs depending on the target to be detected.

[0109] [Table 1]

[0110] 1.2.Results This embodiment discloses an embodiment of the present invention that combines a single recognition event with the binding of ultra-high brightness NPs to a solid support. Due to the high brightness from numerous encapsulated dye R18 having bulky counterions F5-TPB that minimize self-quenching, a dye-retaining polymer NP was selected. To form the NPs, the polymer PEMA-AspN3 having azide and carboxylate functional groups was used (Melnychuk et al., 2020). The hydrophobic PEMA block of this polymer provides good optical properties to the encapsulated dye, and the carboxylate ensures the formation of small NPs and the exposure of azide-reactive groups on the NP surface. NPs were formed by nanoprecipitation of an acetonitrile solution of PEMA-AspN3 in phosphate buffer (pH 7.4). The R18 / F5-TPB dye was retained inside the NPs at 50% by weight relative to the polymer (i.e., 33% by weight relative to the total particle mass) by premixing with the polymer in acetonitrile before nanoprecipitation, according to a previously developed method (Melnychuk et al., 2020). The obtained nanoparticles were 24.5 nm in size and showed good polydispersity. Next, DNA (SEQ ID NO: 1) was grafted onto the NP surface using a click reaction between the DBCO group of the DNA oligonucleotide and the azide group of the polymer NP. After purification by ultrafiltration, the obtained NP was 31.5 nm in size according to DLS. An increase of approximately 7 nm corresponds to twice the thickness of the DNA shell on the NP surface. The absorption and fluorescence spectra of the obtained DNA-NP corresponded well to the encapsulated R18 / F5-TPB dye. The fluorescence quantum yield was 28%, which was consistent with previous studies on similar NPs. Given that the dye retention was 50 wt% relative to the polymer and the NP size was 24.5 nm, the number of dye molecules per NP is estimated to be approximately 1100. Therefore, the brightness of the NP corresponds to approximately 1000 dye molecules with high quantum yield.

[0111] To detect the target, two methods were proposed that were based on similar hybridization concepts in which the target DNA binds NPs to a solid support.

[0112] In the first method, the glass surface and the NP surface were functionalized with two different capture DNA sequences (SEQ ID NO: 2 and SEQ ID NO: 1, respectively) (Figure 2A). To functionalize the glass surface, an established method was used, based on BSA-biotin adsorption, further streptavidin binding, and finally the addition of the corresponding DNA capture containing the biotin unit (Figure 2A). In the presence of the target DNA / RNA, the capture sequence hybridizes with the target, immobilizing the NP on the glass surface. After a washing step, the NPs were visualized using a fluorescence microscope. Given the high brightness of these NPs, they could be directly visualized using a simple epifluorescence microscope with LED excitation. The N1 region of SARS-CoV-2 RNA was selected as the target. Without the addition of a short target, virtually no NPs were detected on the NP surface (data not shown). Next, upon addition of a short target (SEQ ID NO: 6), fluorescent dots appeared on the surface, and the signal clearly increased with the amount of target (Figure 2B). Thus, while DNA-NPs did not bind nonspecifically to the DNA-functionalized glass surface, the target oligonucleotide immobilized the NPs on the glass surface. Importantly, the number of immobilized NPs correlated well with the concentration of the target oligonucleotide (Figure 2B). The detection limit in these measurements was 1–10 fM, which is remarkably low. Low signals in experiments targeting non-coding DNA indicated that detection was sequence-specific (data not shown). These experiments provided proof of concept for nucleic acid detection using a target-driven DNA-NP immobilization strategy.

[0113] Inspired by the results obtained with glass surfaces, we replaced the glass surface with streptavidin-functionalized magnetic beads to simplify the detection scheme, eliminating the need for a fluorescence microscope for DNA / RNA detection. Using magnetic beads offers two key advantages. First, magnetic beads are well-established in RNA extraction. Second, their ease of handling via magnetic processes allows for efficient washing and further use in simpler detection schemes using plate readers, etc. Here, the target oligonucleotide (SEQ ID NO: 6) hybridizes with captured DNA-biotin (as in the glass-based method) and is then immobilized on the magnetic bead surface (Figure 3A). After washing the beads with a magnetic stand, the resuspended beads were mixed with DNA-NPs (as in the glass-based method). In this case, the presence of the target is expected to immobilize the DNA-NPs on the bead surface, while excess unreacted DNA-NPs can be washed away using magnetic separation. The result is magnetic beads hybridized with fluorescent NPs via the target. Resuspending this complex and detecting it with a fluorescence plate reader avoids the need for expensive microscopes. The results showed that increasing the target concentration increased the fluorescence signal recorded by the plate reader (Figure 3B). In the absence of the target, the signal was very low, close to the signal obtained with magnetic beads alone. Therefore, without the target, DNA-NPs do not interact with the beads, but the presence of the target induces hybridization of DNA-NPs on the bead surface. The signal was already clearly observed at 1 fM, and the detection limit was estimated to be 0.3 fM. The results obtained are consistent with those obtained with glass immobilization, suggesting that target-induced immobilization of DNA-NPs is a robust method for nucleic acid detection. However, with magnetic beads, equivalent or better sensitivity could be obtained using a simpler and less expensive fluorescence apparatus. To verify the sequence specificity of oligonucleotide detection, the same bead-based experiments were performed in the presence of total RNA lysates from cells containing a wide variety of RNA sequences.Importantly, a similar dose-dependent response was observed even in the presence of total RNA extracted from the cell lysate, indicating that the numerous RNA sequences present in the lysate did not interfere with the hybridization method. These results confirmed that the detection of the target nucleic acid was sequence-specific. The obtained detection limit was 1 fM, which was close to the value obtained without using a lysate.

[0114] Example 2: Detection of target nucleic acids of different lengths (in the presence of a third probe if necessary) Viral RNA is a long, folded molecule and has inaccessible regions, even if it is commonly present in biological or clinical samples in the form of fragments resulting from the degradation of relatively unstable RNA molecules. Therefore, longer RNA sequences were investigated. The first target (SEQ ID NO: 23) was 100 mer (100 nt), with the sequence complementary to the biotin-capture DNA of SEQ ID NO: 2 located inside the sequence, and the sequence complementary to the DNA-NP of SEQ ID NO: 1 located at its end. The second target (SEQ ID NO: 24) was 1000 nt, with both target sequences located inside the entire sequence. These were tested using both glass surface and magnetic bead-based methods. The results showed that the 100 nt RNA target was readily detectable by both methods (Figures 4A and 4B) and exhibited clear dose-dependence in particle count (glass surface method) or fluorescence intensity (magnetic bead method). However, both particle count and fluorescence intensity were decreased compared to 48 nt in both cases. In fact, the values ​​obtained when detecting a 48-nucleotide target were 10 to 100 times higher than when detecting a 100-nucleotide nucleic acid at the same concentration. Given the relatively small difference in target length, this significant decrease in the performance of these two methods may be related to the fact that the biotin-capture complementary sequence is located inside the entire 100-nt sequence, causing steric problems in its interaction with streptavidin on the magnetic beads. On the other hand, with a 1000-nt RNA target, while an increase in signal was observed in the targeted sample compared to the control, no clear reaction was seen (Figures 4A and 4B). Therefore, the combination of long targets and the internal sequence complementary to the DNA-NP degrades the performance of our hybridization method, regardless of the properties of the immobilization surface (glass or beads). It can be inferred that hybridizing DNA-NPs with this type of target is sterically difficult because it requires unfavorable steric-hindrance turns in the RNA strand. Therefore, in the next step, we performed a series of optimizations on both the hybridization mechanism and the particles themselves, focusing on a simpler method based on magnetic beads.

[0115] To improve the hybridization of the target sequence and NP located inside the entire sequence, we designed a third probe, named "Post-it" sequence, named P3, which is linked with an A20 linker, one end complementary to the target and the other to the DNA-NP (Figure 5A). In this case, the steric problem of the DNA-NP is completely resolved. To validate this method, we tested it using a 48-nucleotide sequence from the N1 region of the SARS-CoV-2 virus (SEQ ID NO: 6) as the target, and the signal showed a clear dose-dependent relationship, as shown in Figure 5B. The application of this method to longer RNA sequences is shown below.

[0116] Example 3: Effect of NP properties on sandwich hybridization sensitivity To increase the signal obtained with this test method, the number of encapsulated dyes per particle was varied. First, the size of the NPs was increased. This was achieved by nanoprecipitation of the same polymer with a high concentration of salt. In the presence of 50 mM NaCl in phosphate buffer, NPs with a polymer core of 52.8 nm were obtained by DLS measurement. According to TEM images, the particle size was 35 ± 8 nm, which corresponds to approximately 3200 dyes per particle. The absorption and fluorescence spectra of these NPs were similar to those of smaller parent analogs. Considering that these NPs showed a good fluorescence quantum yield (41%), it is expected that they will be about three times brighter than the original method. Its fluorescence intensity is 3200 x 0.41 x 125000 = 1.6 x 10⁻¹⁰ 8 M -1 cm -1This corresponds to [the above]. When these bright NPs were tested using the Post-it (Probe P3) DNA hybridization method, a significant improvement in signal intensity was observed at all tested 48nt target concentrations (Figure 6A). Thus, larger particles actually produce a stronger signal. Next, the importance of dye retention was investigated using larger NPs (Figure 6B). Using the same DNA Post-it method, NPs with low dye retention of 0.1 wt% and 1 wt% were observed to produce insufficient signals at target concentrations below 10 pM. In contrast, with retention of 10 wt%, and especially 50 wt%, signals were clearly observed at all tested 48nt target concentrations, including 0.1 pM. In the magnetic bead method, it can be concluded that high brightness of DNA-NPs is most important: approximately 64 dyes per NP (1 wt% dye retention) was insufficient for good performance of the test method, while approximately 640 dyes per NP (10 wt% retention) yielded good results. This is likely because, at low dye retention levels, the scattering signal from the magnetic beads is comparable to the signal from the bound NP. However, in the case of ultra-high brightness NPs, a very low concentration of target is required, as the signal can be detected with just a few hybridized DNA-NPs. Therefore, in the following experiment, we focused on large NPs with a dye retention level of 50 wt%.

[0117] Example 4: Optimization Conditions The importance of linkers between biotin and capture sequences was determined by testing A10 and A20 linkers (Haider et al., 2016). Longer linkers demonstrated advantages in that they reduced background noise for controls without the target, improving the sensitivity of the method.

[0118] Finally, the optimized conditions included the following improvements: (1) increasing the particle size to 50 nm while maintaining a 50 wt% dye retention; (2) using DNA Post-its (probe P3); and (3) using an A20 linker for biotin-captured DNA. Using this optimized method (Figure 7A), 100 nt and 1000 nt RNA targets were tested and added (spiked) to RNA lysates.

[0119] Furthermore, the importance of a tetraethylene glycol (TEG) linker between oligonucleotides and biotin was validated, as it was reported to improve biotin capture by beads. For a 100nt target, a superior response was observed with the optimized test method, regardless of the presence of the TEG linker (Figure 7B). The response was better than the original method: a detectable signal was clearly observed for a 0.01 pM target, compared to 1 pM with the original method. Most importantly, the new method clearly detected a 1000nt target at 0.01 pM, while biotin capture with TEG showed a clearer dose response. These results provide a robust proof of concept for viral RNA detection using a novel method combining ultra-high-brightness DNA-NPs and magnetic bead capture.

[0120] Example 5: Testing of clinical samples Next, clinical samples were tested using an optimized RNA detection assay. SARS-CoV-2 RNA is long and contains many potential target sequences that could be detected. Three pairs of target sequences were identified in different parts of the viral genome. These SARS-CoV-2 sequences are as follows: N1 (SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 23, SEQ ID NO: 24), SPIKE (SEQ ID NO: 13), and ORF1a (SEQ ID NO: 22). Three biotin capture probes (SEQ ID NO: 2, SEQ ID NO: 9, SEQ ID NO: 15) and three DNA Post-it probes (SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18) were designed (including those mentioned above). It was expected that using three pairs of primers would significantly increase the probability of capturing viral RNA as well as the hybridization of DNA-NPs.

[0121] The clinical samples used were collected from the Department of Microbiology of University Hospital Center of Strasbourg (CHU) or purchased from CliniSciences. A total of 10 nasopharyngeal swab samples were from qRT-PCR-positive SARS-CoV-2 patients, and 10 nasopharyngeal swab samples were from qRT-PCR-negative patients. All samples arrived in inactivated transport medium. RNA extracts were prepared from all 20 samples using a minispin column kit.

[0122] First, the optimized test method was tested for the detection of SARS-CoV-2 RNA in patient-derived RNA extracts. The target concentration was varied by diluting the RNA extract from SARS-CoV-2-positive samples with the RNA extract from SARS-CoV-2-negative samples, while simultaneously maintaining a constant total RNA concentration. It was clearly observed that the SARS-CoV-2-positive RNA extract showed a much stronger fluorescence signal than the negative RNA extract (Figure 8). As the positive sample was diluted, the signal gradually decreased, indicating a dose-dependent reaction. Notably, a significant reaction was observed even after diluting the positive sample 10,000-fold (CT=18.75), theoretically demonstrating that this method can detect SARS-CoV-2 RNA in RNA extracts with a sensitivity equivalent to approximately 30 PCR cycles.

[0123] Ultimately, an attempt was made to detect SARS-CoV-2 RNA directly from clinical samples without RNA extraction. The hypothesis was that magnetic beads, commonly used for RNA extraction, could effectively capture viral RNA with the help of biotin-capture DNA. Furthermore, the protocol included magnetic separation with washing before applying DNA-NPs to ensure that the buffer used in the original clinical samples would not harm the DNA-NPs. Two series of clinical samples were used from two different sources: commercially available samples (CliniSciences, CS series) and university hospital CHU samples (CH series). When these samples were detected directly, all three SARS-CoV-2 positive samples tested in the CS series showed a higher signal compared to the three negative samples (Figure 9A). In the CH series, the response of the five positive samples tested showed a systematically higher signal compared to four of the five controls (Figure 9B), with only one of the ten CH samples tested showing a false positive result. Next, during the preparation of sample dilutions in buffer, it was found that the signal for SARS-CoV-2 decreased with dilution, whereas this was not the case for the control (Figure 9C). These results confirm the ability of the DNA-NP-based assay to directly detect SARS-CoV-2 in clinical samples without a dedicated RNA extraction step.

[0124] In conclusion, the kit and method according to the present invention demonstrate a highly sensitive nucleic acid detection method. Target-driven immobilization of DNA-NPs onto a solid surface enables the detection of RNA / DNA targets. The kit and method of the present invention enable the detection of nucleic acids, particularly RNA, in biological samples in a simple, automated, and high-throughput manner. The brightness of the NPs plays an essential role in obtaining a good fluorescence signal even at low concentrations of RNA, enabling LOD in the lower fM (sub-fM) range.

[0125] The detection of long nucleic acids is challenging, likely due to their slow diffusion rate and the fact that RNA folding can prevent access to the target sequence from within the entire base sequence. Therefore, the presence of a third probe, designated as an intermediate between the long target nucleic acid and a hyperluminescent particle, and a linker of sufficient length for the biotin-capture DNA are crucial. The optimized test method enabled the detection of 1000nt RNA fragments of SAR-CoV-2 with fM sensitivity. Furthermore, it demonstrated the ability to detect SARS-CoV-2 in clinical samples with sensitivity comparable to 19–30 cycles of RT-PCR.

[0126] Ultimately, the combination of DNA-NPs and magnetic beads enabled the direct detection of SARS-CoV-2 in clinical samples without a dedicated RNA extraction step. This study presents a novel method for simple and rapid RNA detection, where ultra-high-brightness DNA-NPs provide ultimate sensitivity, while the use of magnetic beads avoids RNA extraction and allows for a simple detection mode using a plate reader.

[0127] In conclusion, the inventors designed a highly sensitive RNA detection method based on ultra-high brightness DNA functionalization dye-retaining polymers (NPs), magnetic beads, and corresponding primers. Immobilizing DNA-NPs on a solid support in a target-driven manner enables detection of RNA / DNA targets on glass surfaces using fluorescence microscopy and direct detection of RNA / DNA targets on magnetic beads using a fluorescence plate reader. The latter method is particularly interesting because it allows for simple, automated, and high-throughput RNA detection in biological samples. The brightness of the NPs plays a crucial role in obtaining good fluorescence signals even at low RNA concentrations, enabling LODs in the fM range and even lower fM ranges. Furthermore, detection of long RNAs proved difficult. This is likely due to their slow diffusion rate and the possibility that RNA folding may prevent access to the target sequence from within the entire sequence. Therefore, a DNA Post-it (Probe P3) capture sequence was introduced as an intermediate between long RNAs and our DNA-NPs to ensure sufficient linker length for the biotin-captured DNA. The optimized test method enabled the detection of 1000nt RNA fragments of SARS-CoV-2 with fM sensitivity. Furthermore, it demonstrated the ability to detect SARS-CoV-2 in clinical samples with sensitivity equivalent to 19-30 cycles of RT-PCR. Ultimately, the combination of DNA-NPs and magnetic beads allowed the method to directly detect SARS-CoV-2 in clinical samples without a dedicated RNA extraction step. This study presents a novel method for simple and rapid RNA detection, where ultra-high-brightness DNA-NPs provide ultimate sensitivity, while the use of magnetic beads avoids RNA extraction and enables a simple detection mode using a plate reader. This method is easily extendable to other types of RNA, enabling rapid molecular diagnosis of various diseases, particularly viral infections and cancer.

[0128] Example 6: Detection of microRNA targets To detect microRNA (miRNA) targets with a length of 20-22 nucleotides, the inventors designed a simple system based on hybridization between complementary sequences. This system consists of a glass surface capture sequence (10-12 nucleotides) complementary to one side of the miRNA target and a non-transparent polyploid (ULP) coated with a nucleic acid (10-12 nucleotides) complementary to the other side of the miRNA target, which functions as a detection probe. To enhance the stability of the short double helix, locked nucleic acid (LNA) was incorporated into the glass surface capture sequence. The formed double helix was sufficiently stable, likely due to the cooperative phenomenon of adjacent DNA grafted onto the NP, so the LNA was not incorporated into the complementary oligonucleotide on the NP surface.

[0129] The oligonucleotides used in the examples and their respective nucleic acid sequences (RNA or DNA, the DNA target is shown in Figure 10), biotin capture, and recognition sequences (DBCO miR) are referenced in Table 2 and the sequence list attached to this patent application.

[0130] In this patent application, the symbol "T" represents thymine in DNA, and the symbol "U" represents uracil in RNA. In the accompanying sequence list, as required by the ST26 standard, the symbol "T" is interpreted as thymine in DNA and uracil in RNA.

[0131] In this patent application, locked nucleotides in Sequence ID No. 27 and Sequence ID No. 31 in Table 2 are represented in bold and underlined text. As required by the ST26 standard, modified nucleotides such as locked nucleotides are represented in the sequence as the corresponding unmodified nucleotides in the attached sequence list.

[0132] [Table 2]

[0133] The target oligonucleotide hybridizes with capture DNA-biotin and is then immobilized on the glass surface. After washing the glass surface, the particles were observed under a microscope.

[0134] If a target is present, it hybridizes with the surface capture sequence on one side and with the complementary DNA of the NP on the other side, thus immobilizing the NP and enabling microscopic detection. If no target is present, the NP remains in the solution and is consequently washed away.

[0135] For the targets shown in Figure 10, the LOD (Limit of Detection) was calculated to be 4.5 fM for miR200a and 2 fM for miR21 (DNA version). The results for the RNA version of microRNA were very similar.

[0136] These results suggest that relatively short oligonucleotides (10–12 nucleotides) bound to NPs may be sufficient to detect microRNAs using the sandwich test method.

[0137] References: Cai H. et al. Optofluidic analysis system for amplification-free, direct detection of Ebola infection. Sci Rep 5, 14494 (2015). Egloff S. et al. Enzyme-free amplified detection of cellular microRNA by light-harvesting fluorescent nanoparticle probes. Biosens Bioelectron 179, 113084 (2021) Haider M. et al. A Double-Hybridization Approach for the Transcription- and Amplification-Free Detection of Specific mRNA on a Microarray, Microarrays, Mar. 5(1):5, 2016. Lim S. H. et al Quantitative Analysis of Nucleic Acid Hybridization on Magnetic Particles and Quantum Dot-Based Probes. Sensors 9, 5590-5599 (2009). Melnychuk & Klymchenko, DNA-Functionalized Dye-Loaded Polymeric Nanoparticles: Ultrabright FRET Platform for Amplified Detection of Nucleic Acids. J. Am. Chem. Soc. 140, 10856-10865 (2018). Melnychuk et al. Light-Harvesting Nanoparticle Probes for FRET-Based Detection of Oligonucleotides with Single-Molecule Sensitivity. Angewandte Chemie International Edition 59, 6811-6818 (2020). Ngo H. T. et al. Direct Detection of Unamplified Pathogen RNA in Blood Lysate using an Integrated Lab-in-a-Stick Device and Ultrabright SERS Nanorattles. Sci Rep 8, 4075 (2018). Reisch A. et al. Collective fluorescence switching of counterion-assembled dyes in polymer nanoparticles. Nat Commun 5, 4089 (2014). Reisch A. et al. Tailoring Fluorescence Brightness and Switching of Nanoparticles through Dye Organization in the Polymer Matrix. ACS Appl. Mater. Interfaces 9, 43030-43042 (2017). Reisch A. et al. Charge-Controlled Nanoprecipitation as a Modular Approach to Ultrasmall Polymer Nanocarriers: Making Bright and Stable Nanoparticles. ACS Nano 9, 5104-5116 (2015). Severi C. et al. Smartphone-assisted detection of nucleic acids by light-harvesting FRET-based nanoprobe. Biosensors and Bioelectronics 168, 112515 (2020) Zheng Z. et al. Sensitive and quantitative measurement of gene expression directly from a small amount of whole blood. Clin Chem 52, 1294-1302 (2006).

Claims

1. A kit for detecting target nucleic acids in a sample, wherein at least, i) A probe P1 comprising or consisting of a nucleic acid fragment NA1 bound to a functional unit F1, wherein NA1 comprises a nucleotide sequence complementary to the nucleotide sequence of region T1 of the target nucleic acid, ii) Functional units F2 bonded to a solid surface, which exhibit high affinity for F1, or are covalently bonded to F1. iii) at least 10 －１ M －１ cm －１ , preferably at least 2×10 ７ M －１ cm －１ , more preferably at least 5x10 ７ M －１ cm －１ , 10x10 ７ M －１ cm －１ , or at least 40x10 ７ M －１ cm －１ A kit comprising, or consisting of, a probe P2 comprising a nucleic acid fragment NA2 bound to ultra-high brightness luminescent particles exhibiting a brightness of

2. The kit according to claim 1, wherein the solid surface is selected from among an immobilized surface and the surface of solid particles, preferably magnetic beads or glass beads.

3. The kit according to claim 1 or 2, wherein the ultra-high brightness light-emitting particles are ultra-high brightness dye-retaining fluorescent polymer nanoparticles.

4. The kit according to any one of claims 1 to 3, wherein the nucleic acid fragment NA2 contains or consists of a nucleotide sequence complementary to the nucleotide sequence of region T2 of the target nucleic acid.

5. A kit according to any one of claims 1 to 3, comprising or consisting of a nucleic acid fragment NA3 having a nucleotide sequence complementary to the nucleotide sequence of region T3 of a target nucleic acid, wherein region T3 comprises a first portion different from region T1 and a second portion comprising or consisting of a nucleic acid fragment NA4 having a nucleotide sequence complementary to the nucleotide sequence of NA2, and the first portion and the second portion are linked via a nucleotide linker.

6. It includes at least two probes P1, each of which probes P1 is - The kit according to claim 5, comprising, or consisting of, at least one nucleic acid fragment NA1, named as NA1-1 or NA1-2, and complementary to the nucleotide sequence T1-1 or T1-2 of the target nucleic acid.

7. i) A first part comprising or consisting thereof a nucleic acid fragment having a nucleotide sequence complementary to the nucleotide sequence of NA2, and a second part comprising or consisting thereof a nucleic acid fragment NA3 having a nucleotide sequence complementary to the nucleotide sequence of region T3 of the target nucleic acid, wherein the first part and the second part are linked via a nucleotide linker, and ii) The kit according to claim 5 or 6, comprising a nucleic acid fragment NA2 bound to an ultra-high brightness light-emitting particle, or at least one probe P2 consisting of the same.

8. A method for detecting a target nucleic acid molecule in a sample, the method comprising at least the following steps: a) Under conditions suitable for hybridization of complementary nucleic acid sequences and formation of non-covalent complexes of nucleic acids, at least, - A probe P1 comprising or consisting of a nucleic acid fragment NA1 bound to a functional unit F1, wherein NA1 comprises a nucleotide sequence complementary to the nucleotide sequence of region T1 of the target nucleic acid, - Functional unit F2 bonded to a solid surface, wherein F2 exhibits high affinity for F1 or is covalently bonded to F1, and - Containing or comprising nucleic acid fragments NA2 bound to ultra-high brightness light-emitting particles, the particles are at least 10 ７ M －１ cm －１ Probe P2, which shows the brightness. A step of bringing the two into contact to form a mixture, b) A step of applying an external physical force to the mixture in step a) in order to isolate the non-covalent complex of nucleic acids, and c) A method comprising the step of measuring the luminescence intensity of the non-covalent complex of nucleic acids.

9. The method according to claim 8, wherein the solid surface is the surface of solid particles, preferably magnetic beads or glass beads.

10. The method according to claim 8 or 9, wherein the ultra-high brightness light-emitting particles are ultra-high brightness dye-retaining fluorescent polymer nanoparticles.

11. The method according to any one of claims 8 to 10, wherein the nucleic acid fragment NA2 includes or consists of a nucleotide sequence complementary to the nucleotide sequence of region T2 of the target nucleic acid.

12. Step a) is at least, - A probe P1 comprising or consisting of a nucleic acid fragment NA1 bound to functional unit F1, wherein NA1 contains or consists of a nucleotide sequence complementary to the nucleotide sequence of region T1 of the target nucleic acid. - Functional units F2 bonded to a solid surface, wherein F2 exhibits high affinity for F1, or is covalently bonded to F1, and - A probe P2 comprising or consisting of a nucleic acid fragment NA2 covalently bonded to an ultra-high-brightness fluorescent particle, wherein the nucleic acid fragment NA2 comprises or consists of a nucleotide sequence complementary to probe P3, and - A first part comprising or consisting of a nucleic acid fragment NA4 having a nucleotide sequence complementary to the nucleotide sequence of NA2, and a second part comprising or consisting of a nucleic acid fragment NA3 having a nucleotide sequence complementary to the nucleotide sequence of region T3 of the target nucleic acid, wherein the first part and the second part are linked via a nucleotide linker, and at least one probe P3 The method according to any one of claims 8 to 11, comprising bringing into contact with

13. Step a) is at least, The method according to claim 12, comprising contacting two probes P1, each of which probes P1 comprises or consists of a nucleic acid fragment NA1, each of which NA1 is named NA1-1 or NA1-2 and is complementary to the nucleotide sequence T1-1 or T1-2 of the target nucleic acid, respectively.

14. Step a) is, i) A first part comprising or consisting of a nucleic acid fragment NA4 having a nucleotide sequence complementary to the nucleotide sequence of NA2, and a second part comprising or consisting of a nucleic acid fragment NA3 having a nucleotide sequence complementary to the nucleotide sequence of region T3 of the target nucleic acid, wherein the first part and the second part are linked via a nucleotide linker, and ii) The method according to claim 12 or 13, comprising contacting at least one probe P2 comprising or consisting of a nucleic acid fragment NA2 bound to an ultra-high brightness light-emitting particle.

15. Use of the kit according to any one of claims 1 to 7, or the method according to any one of claims 8 to 14, for the detection of a target nucleic acid.

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