Methods for detecting and / or quantifying target nucleotide sequences

Abstract

The present invention relates to a method for detecting and / or quantifying a target nucleotide sequence in a sample, the method comprising the following steps: providing a target nucleotide sequence in a single-stranded form; hybridizing the target nucleotide sequence with a first probe of a single-stranded nucleic acid and a second probe of a single-stranded nucleic acid, wherein one end of the first probe is fixed on a solid support, and the other end contains a sequence complementary to a first part of the target nucleotide sequence, one end of the second probe is labeled, and the other end contains a sequence complementary to a second part of the target nucleotide sequence, wherein the first part and the second part of the target nucleotide sequence have 2 to 10 nucleotides in total, thereby forming a first probe-target nucleotide sequence-second probe complex; and detecting the complex.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This patent application claims the priority benefit of Italian Patent Application No. 102018000008017 filed on August 9, 2018, the disclosure of which is incorporated herein by reference in its entirety. Technical Field

[0003] The present invention relates to a method for detecting and / or quantifying a target nucleotide sequence in a sample based on a system having two nucleic acid probes. Background Art

[0004] The analysis of nucleic acids in different biological samples (biospies, serum, plasma, cell cultures) is fundamental for the study and early diagnosis of many diseases (e.g. Alzheimer's disease, cancer, osteoarthritis, infectious diseases) and has increasing applications in veterinary medicine, agriculture and food.

[0005] The target nucleic acids analyzed are genomic DNA, messenger RNA (mRNA), ribosomal RNA (rRNA), transfer RNA (tRNA), heterogeneous nuclear RNA (hnRNA, pre-mRNA) and small sequence RNA (small RNA). Small RNA is split into: microRNA (miRNA), small nuclear RNA (snRNA), short interfering RNA (siRNA), piwi interacting RNA (piRNA).

[0006] For the research and early diagnosis of many diseases, microRNA is the most concerned nucleic acid to be analyzed.MicroRNA is a short sequence RNA (ribonucleic acid) with 18-24 nucleotides, which regulates gene expression at the transcription and post-transcription levels, and is combined with target mRNA by suppressing it. Very many miRNAs in different species (particularly mammals) have been recorded, and the difference of the usual sequence (therefore the difference of the effect) is due to the change of a single nucleotide. Their numerousness and similarity need to develop a highly specific method that can identify differences at the single nucleotide level.MicroRNA has the potential to become a new generation of biomarkers for early diagnosis of many diseases. In fact, many new biomarkers found today are microRNAs, which participate in regulating many biological systems and diseases, including various types of cancer, neurodegenerative diseases and musculoskeletal diseases (such as osteoarthritis, Alzheimer's disease).

[0007] The main techniques used to analyze nucleic acids so far are PCR (polymerase chain reaction) and direct sequencing. These techniques take a long time to get results, require highly qualified personnel to carry out, and are very expensive. Another limitation of these techniques is that they are easily changed by the components present in the biological sample, such as Ca+, proteases, nucleases, and / or the quality and type (such as the concentration of EDTA, SDS) of the buffer that is easily used to collect and extract samples. Therefore, another limitation of these technologies is to prepare, extract and purify samples before actual analysis, which is easy to extend time and increase costs. Specifically, for microRNA, the technologies (PCR and sequencing) used today and the products being sold are not satisfactory because these technologies are complex, difficult to repeat, not specific enough (not optimized for short sequences), expensive, require a long time and highly qualified personnel, and are incompatible with instruments and diagnostic-clinical workflows.

[0008] A method that avoids the use of PCR has been described in EP 2 639 312. This method involves the use of oligonucleotides with DNA and antibodies capable of recognizing DNA / RNA hybrids, but it still does not allow results to be obtained in a sufficiently simple, reproducible and sensitive manner.

[0009] Hideyuki A.et al., (2012), The Analyst, Vol.137, No.14, p.3234 discloses a method for detecting miRNA, which includes sandwich hybridization with a capture probe and a labeled fluorescent probe, wherein the two probes are completely adjacent to provide a coaxial stacking effect. This method shows limited sensitivity.

[0010] Therefore, the object of the present invention is to provide a method for detecting and / or quantifying a target nucleotide sequence in a sample, which method solves the above-mentioned problems in a simple and effective manner. Specifically, the method must show high sensitivity, specificity, affinity, stability, ease of standardization, low cost and rapid execution.

[0011] This object is achieved by the invention in that it is associated with the method defined in claim 1 .

[0012] definition

[0013] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those of ordinary skill in the art involved in the present invention. Although in practice or in the assessment of the present invention, many materials and methods similar or equivalent to those described herein can be used, preferred materials and methods are as follows. Unless otherwise indicated, the technology described herein for the present invention is standard method known to those of ordinary skill in the art. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 A schematic diagram showing elements used in the method according to the invention is shown. DETAILED DESCRIPTION

[0015] refer to Figure 1 , the method for detecting and / or quantifying target nucleotide sequence 3 in a sample comprises the following steps.

[0016] In the first step, a single-stranded target nucleotide sequence 3 is provided.

[0017] Pre-amplification of the target nucleotide sequence 3 is not essential, but it must be provided in a single-stranded form.

[0018] The target nucleotide sequence can be genomic DNA, messenger RNA (mRNA), ribosomal RNA (rRNA), transfer RNA (tRNA), heterogeneous nuclear RNA (hnRNA, pre-mRNA) and small sequence RNA (small RNA): microRNA (miRNA), small nuclear RNA (snRNA), short interfering RNA (siRNA), piwi interacting RNA (piRNA).

[0019] Preferably, the target nucleotide sequence 3 is microRNA (miRNA).

[0020] Typically, the length of the target nucleotide sequence 3 is 10 to 50 nucleotides, preferably 18 to 30 nucleotides.

[0021] The sample containing the target nucleotide sequence 3 can be composed of biological fluids (including blood, serum, plasma, synovial fluid, saliva, or the above fluids diluted in buffer) as well as cell cultures, biopsies, tissues, which require a single homogenization and lysis stage without extraction / purification and amplification stages. Preferably, the sample is a living cell.

[0022] In the second step, the single-stranded target nucleotide sequence 3 is hybridized with the first probe 1 of the single-stranded nucleic acid and the second probe 2 of the single-stranded nucleic acid.

[0023] One end of the first probe 1 is fixed to a solid support 4 , and the other end comprises a sequence complementary to a first part 5 of the target nucleotide sequence 3 .

[0024] Solid carrier 4 is selected from cellulose, nitrocellulose, cellulose acetate, cross-linked dextran, agarose, polystyrene, polypropylene, nylon, ceramic, glass, metal (such as gold and silver, magnetite) and their combination. Solid carrier 4 can also be composed of cells, liposomes and vesicles of phospholipids. Preferably, solid carrier 4 is composed of microplates with 48, 96, 384 holes, magnetic beads, gold beads or silver beads, microparticles, strips, chromatographic sheets, tubes, rods, and filter materials.

[0025] Probe 1 can be conjugated with molecules adapted for solid phase conjugation, such as lysine, arginine, thiol, cysteine, and glutathione.

[0026] One end of the second probe 2 is labeled, and the other end contains a sequence complementary to the second part 6 of the target nucleotide sequence 3 .

[0027] The second probe 2 can be conjugated with one or more molecules, for example: glycine, lysine, arginine.

[0028] Preferably, the second probe 2 is labeled with biotin, a fluorescent molecule (eg, cyanine, alexafluor), or fluorescein.

[0029] Advantageously, the first part 5 and the second part 6 of the target nucleotide sequence have 2 to 10 nucleotides in total.

[0030] The result of the second step is the formation of a first probe-target nucleotide sequence-second probe complex.

[0031] Preferably, the first probe 1 and the second probe 2 are composed of a nucleic acid selected from the following: PNA (peptide nucleic acid), LNA (locked nucleic acid), morpholino, orn-PNA (PNA with ornithine backbone), aep-PNA (aminoethylprolyl-PNA), aepone-PNA (aminoethylpyrrolidone-PNA), GNA (ethylene glycol nucleic acid), HNA (hexitol nucleic acid), TNA (threose nucleic acid), ENA (2'-O,4'-C-ethylene-bridged nucleic acid), ANA (arabinoic acid), F-ANA (2'-F-arabinoic acid). Even more preferably, the first probe 1 and the second probe 2 are composed of PNA (peptide nucleic acid), even more preferably of PNA with a modified backbone structure (wherein the α carbon has a side chain of arginine or lysine as a substituent).

[0032] The use of probes composed of PNAs 1, 2 allows to improve the stability, specificity and sensitivity of the method. Similar properties can also be obtained using other artificial oligonucleotides (such as LNA and morpholinos).

[0033] The uncharged nature of the PNA backbone is an important property, due to the absence of electrostatic repulsion, which means that the affinity bond between PNA and the two chains of DNA or RNA is much stronger than that between the two chains of DNA or RNA. The higher stability of the double strand containing PNA translates into a higher melting temperature. The complex of PNA with DNA or RNA is more sensitive to the presence of a single mismatch than the complex formed by the two chains of DNA or RNA. PNA has high biological stability; in fact, they are resistant to attack by nucleases and proteases in human serum and cells or cell extracts, thereby extending their lifespan in vitro and in vivo. Resistance to enzymatic degradation is accompanied by high chemical stability. Due to these characteristics of PNA and the possibility of operating at physiological pH (pH 7.2) as shown in Example 3, the method can be used for living cells.

[0034] The method can also be used at saline concentrations of 0 mM to 1000 mM.

[0035] Preferably, the first probe 1 and / or the second probe 2 consists of 10 to 18 nucleotides.

[0036] Preferably, when the method is used to detect and / or quantify miRNA, probe 2 comprises a region of 2-6 nucleotides in addition to the N-terminal region to increase the specificity only for mature miRNA excluding pre-miRNA, such as in SEQ ID NO: 13 (PNA 2d), wherein 4Timine (T) is added to the N-terminal region (see Examples).

[0037] In the third step, the first probe-target nucleotide sequence-second probe complex is detected.

[0038] Preferably, the complex is detected with a system selected from streptavidin / antibody conjugated to: peroxidase (single HRP, HRP20, HRP40, HRP80), PE (phycoerythrin), fluorescent molecules (eg cyanine, alexafluor, fluorescein).

[0039] The kit for use in the method according to the invention comprises:

[0040] - a first probe 1 as described above;

[0041] - a second probe 2 as described above;

[0042] -Optionally at least one buffer solution selected from the group consisting of: SSC5x, SSC05x, SSC5x Tween 0.05 / 0.01%, SSC 0.5x Tween 0.05 / 0.01%, SSC2.5x SDS 1 / 0.5%, SSC2.5x SDS 0.1 / 0.05% BSA 5 / 10%, SSC5x SDS 0.1 / 0.05% BSA 5 / 10%, PBS, PBS Tween 0.05 / 0.1%, phosphate buffer at pH 5.8 to pH 7.2, phosphate buffer SDS 0.1 / 0.05% at pH 5.8 to pH 7.2.

[0043] Example

[0044] The target nucleotide sequence 3 and probes 1 and 2 used in the examples are as follows.

[0045] Target nucleotide sequence. 22 single-stranded bases of DNA and RNA, purchased from Sigma-Aldrich. (Mismatches are underlined)

[0046]

[0047] PNA-containing probes 1 and 2 were produced by Bioridis using automated peptide synthesis.

[0048]

[0049] Keywords: K: lysine; G: glycine; AEEA: aminoethylethanolamine; ac: acetylated; K Biot: lysine containing biotin conjugated to the side chain.

[0050] Preparation of solid phase. Two solid phases were tested with different conjugation systems to evaluate the ability of the method on different solid supports: a) Nunc Immobilizer Amino (Thermo-Fischer), a 96-well microtiter plate with stable electrophilic groups on the surface to bind positively charged groups; b) Nunc Maxisorp (Thermo-Fischer), a 96-well microtiter plate with a hydrophobic / hydrophilic surface that has been modified to be suitable for binding many types of different molecules (charged or neutral). Probe 1 was conjugated to the solid phase according to the supplier's protocol.

[0051] Buffer. SSC5x (750mM NaCl, 75mM sc pH 7.0); SSC5x T (750mM NaCl, 75mM sc pH7.0, Tween 0.05); SSC5x S (750mM NaCl, 75mM sc EDTA 0.5mM; SDS 0.05%, pH 7.0); SSC2.5x S (375mM NaCl, 37.5mM sc EDTA 0.5mM; SDS 0.05% pH 7.0;); SSC0.5x (75mM NaCl, 7.5mM sc pH 7.0); SSC0.5x T (75mM NaCl, 7.5mM sc pH 7.0; Tween 0.05); PBS (8.1mM Na2HPO4;KH2PO4 1.7mM; KCl 0.27mM; NaCl 137mM; pH 7.2); PBST (8.1mM Na2HPO4; KH2PO4 1.7mM; KCl 0.27mM; NaCl 137mM; pH 7.2; Tween 0.05 / 0.1); Phosphate buffer, BF5.8 (100mM NaP; 100mM NaCl EDTA 0.5mM pH 5.8) BF5.8 S (100mM NaP; 100mM NaCl EDTA0.5mM SDS 0.05% pH 5.8); Phosphate buffer, BF7.2 (100mM NaP; 100mM NaCl EDTA 0.5mM pH7.2); Phosphate buffer, BF7.2 S (100mM NaP; 100mM NaCl EDTA 0.5mM SDS 0.05% pH 7.2)

[0052] Detection system.

[0053] The detection systems tested in the examples and used in the methods are: chemiluminescence, colorimetry and fluorescence, used as directed by the supplier's protocol. Data were acquired using the BIOTEK Synergy HT following the instructions for use and setup. For chemiluminescence and colorimetric systems, measurements were expressed in RLU (Relative Luminescence Units) (maximum for chemiluminescence systems 4.5*10 6 RLU; maximum value of the colorimetric system 4.500 RLU); for fluorescence, measurements were expressed in RFU (Relative Fluorescence Units, maximum value 99998 RFU) (the latter experimental results are not shown).

[0054] Example 1

[0055] Method. Dilute RNA NA1 to a concentration of 150fmol / ml in SSC 0.5T0.1%. Place 100μl of the previously prepared solution containing the probe in the wells and leave for hybridization for 3 hours. Then remove the RNA and wash the wells. Add 10nM oligonucleotides to BF5.8 containing SDS 0.1% and hybridize overnight (on). Then remove the oligonucleotides and wash the wells. Add 100ul of SA-HRP40 at a concentration of 0.1μg / ml to PBST for 60 minutes. Then remove the SA-HRP40 and wash the wells. Add 100μl of chemiluminescent substrate (Clarity, Biorad). Collect after 5 minutes.

[0056] result

[0057]

[0058] This example shows the dominance of hybridization between probes 1 and 2 and target probe 3 depending on the nucleotides involved in the overlapping region.

[0059] Example 2 (Effect of Salt Concentration)

[0060] Method. Dilute RNA NA1 in two parts to a concentration of 150fmol / ml in SSC 0.5 (NaCl 75mM) T0.1% and SSC 5x (NaCl 750nM) T0.1%. 100μl of the previously prepared solution containing probe 1b is spread in each well and left for hybridization for 3 hours. The RNA is then removed and the wells are washed. 10nM of probe 2c is added to BF5.8 containing SDS 0.1% and hybridized overnight (on). The oligonucleotides are then removed and the wells are washed. 100μl of SA-HRP40 at a concentration of 0.1μg / ml is added to PBST for 60 minutes. SA-HRP40 is then removed and the wells are washed. 100μl of chemiluminescent substrate (Clarity, Biorad) is added. Collection is performed after 5 minutes.

[0061] Buffer δ (signal minus noise) SSC0.5 777.338 SSC 5 1.509.413

[0062] This example shows the ability of the method to work over a wide range of ionic strengths (750 mM to 75 mM NaCl concentration).

[0063] Example 3 (Stability as pH changes)

[0064] Method. Dilute RNA NA1 in two parts to a concentration of 150fmol / ml in SSC 5x. Place 100μl of the previously prepared solution containing probe 1b in each well and leave for hybridization for 3 hours. Then remove the RNA and wash the wells. Add 10nM of probe 2c to BF5.8 or BF7.2 containing SDS 0.05% and hybridize overnight (on). Then remove the oligonucleotides and wash the wells. Add 100μl of SA-HRP40 at a concentration of 0.1μg / ml to PBST for 60 minutes. Then remove SA-HRP40 and wash the wells. Add 100μl of chemiluminescent substrate (Clarity, Biorad). Collect after 5 minutes.

[0065] Buffer δ (signal minus noise) BF5.8 802.804 BF7.2 933.631

[0066] This example shows the ability of the method to work over a wide pH range, in particular at physiological and cellular pH (pH 7.2).

[0067] Example 4 (reduced hybridization time, no need for overnight)

[0068] Method. Dilute RNA NA1 in two parts in SSC 5x to a concentration of 150fmol / ml, 15fmol / ml, and 1.5fmol / ml. Spread 100μl of the previously prepared solution containing probe 1b in each well and leave for hybridization for 90 minutes. Then remove the RNA and wash the wells. Add 10nM of probe 2c to BF7.2 containing SDS 0.05% and hybridize for 2 hours. Then remove the oligonucleotides and wash the wells. Add 100μl of SA-HRP40 at a concentration of 0.1μg / ml to PBST for 60 minutes. Then remove SA-HRP40 and wash the wells. Add 100μl of chemiluminescent substrate (Clarity, Biorad). Collect after 5 minutes.

[0069] RNA concentration δ (signal minus noise) 150fmol / ml Off scale 15fmol / ml 1.082.246 1.5fmol / ml 66.998

[0070] This example shows the ability of the method to work quickly (5 hours) without the need for an overnight phase.

[0071] Example 5 (Effect of Positive Charge)

[0072] Method. Dilute RNA NA1 in two parts in SSC 5x to a concentration of 15fmol / ml. Spread 100μl of the previously prepared solution containing probe 1b in each well and leave for hybridization for 90 minutes. Then remove the RNA and wash the wells. Add 10nM of probe 2c or 2d or 2e to BF7.2 containing SDS 0.05% and hybridize for 2 hours. Then remove the oligonucleotides and wash the wells. Add 100μl of SA-HRP40 at a concentration of 0.1μg / ml in PBST for 60 minutes. Then remove SA-HRP40 and wash the wells. Add 100μl of chemiluminescent substrate (Clarity, Biorad). Collect after 5 minutes.

[0073] Oligonucleotide probe 1 Oligonucleotide probe 2 Charge δ (signal minus noise) 1c 2d 0 753855 1c 2c 1 725035 1c 2e 3 841788

[0074] This example shows the effect of positive charges added to the sequence (intercalated with lysine) on hybridization ability.

[0075] Example 6 (Use in serum and living cells without extraction / purification)

[0076] Methods. Serum and extracts were treated with ribonuclease inhibitor (Thermo) and diluted 1:1 with SSC5x SDS 0.1% EDTA 1mM. RNA NA1 was diluted in portions to a concentration of 15fmol / ml. 100μl of the previously prepared solution containing probe 1b was spread in each well and left for hybridization for 90 minutes. The RNA was then removed and the wells were washed. 10nM of probe 2c was added to BF7.2 containing SDS 0.05% and hybridized for 2 hours. The oligonucleotides were then removed and the wells were washed. 100μl of SA-HRP40 at a concentration of 0.1μg / ml was added to PBST for 60 minutes. SA-HRP40 was then removed and the wells were washed. 100μl of chemiluminescent substrate (Clarity, Biorad) was added. Collection was performed after 5 minutes.

[0077] Matrix δ (signal minus noise) Ssc5x 2038134 serum 2085434 cell 1214368

[0078] This example shows the ability of the method to act directly on biological samples: biological fluids (eg serum) or living cells, without the need for extraction and / or purification stages.

[0079] Example 7 (single step)

[0080] Method. RNA NA1 was diluted in two parts to a concentration of 15fmol / ml in SSC 5x. For one series, 100μl of the previously prepared solution containing probe 1b was spread in each well and left for hybridization for 90 minutes. The RNA was then removed and the wells were washed. In one series, 10nM of probe 2c was added to BF7.2 containing SDS 0.05% for hybridization, and in another series, a solution containing 15fmol / ml RNA NA1 and 10nM 2c was added to BF7.2 containing SDS0.05% and left for hybridization for 2 hours. The oligonucleotides were then removed and the wells were washed. 100μl of SA-HRP40 at a concentration of 0.1μg / ml was added to PBST for 60 minutes. SA-HRP40 was then removed and the wells were washed. 100μl of chemiluminescent substrate (Clarity, Biorad) was added. Collection was performed after 5 minutes.

[0081] Number of steps δ (signal minus noise) 2 steps (5 hours) 1090550 1 step (3 hours) 1227210

[0082] This example demonstrates the ability of the method to work rapidly (3 hours) and use a single hybridization step.

[0083] Example 8 (Specificity of the method)

[0084] Method. Dilute RNA NA1, NA2, NA3, and NA4 in portions to a concentration of 150 fmol / ml in SSC2.5x S, and add 20 nM probe 2d. Spread 100 μl of the previously prepared solution containing probe 1d in each well and leave for hybridization for 2 hours. Then remove the oligonucleotides and wash the wells. Add 100 μl of SA-HRP40 at a concentration of 0.1 μg / ml in PBST for 60 minutes. Then remove SA-HRP40 and wash the wells. Add 100 μl of chemiluminescent substrate (Clarity, Biorad). Collect after 5 minutes.

[0085] RNA δ (signal minus noise) NA1 2070630 NA2 0 NA3 0 NA4 0

[0086] This example shows how the method is highly specific also at the single mismatch level.

[0087] Example 9 (Comparison of RNA and DNA)

[0088] Method. Dilute RNA NA1 and DNA NA5 in two parts to a concentration of 150fmol / ml in SSC2.5x S, and add 20nM probe 2d. Spread 100μl of the previously prepared solution containing probe 1b in each well and leave for hybridization for 4 hours. Then remove the oligonucleotides and wash the wells. Add 100μl of SA-HRP40 at a concentration of 0.1μg / ml in PBST for 60 minutes. Then remove SA-HRP40 and wash the wells. Add 100μl of chemiluminescent substrate (Clarity, Biorad). Collect after 5 minutes.

[0089] Nucleic Acids δ (signal minus noise) NA1(RNA) 2300299 NA5(DNA) 904829

[0090] The method can also be used to detect DNA. It can be advantageously used for RNA, as the lower affinity for DNA ensures a lower risk of non-specificity from any DNA fragments.

[0091] Example 10 (colorimetric detection)

[0092] Method. Dilute RNA NA1 and NA2 in two parts to a concentration of 150fmol / ml and 15fmol / ml (NA1 only) in SSC2.5x S, and add 20nM probe 2d. 100μl of the previously prepared solution containing probe 1b is spread in each well and left for hybridization for 4 hours. The oligonucleotides are then removed and the wells are washed. 100μl of SA-HRP40 at a concentration of 0.1μg / ml is added to PBST for 60 minutes. SA-HRP40 is then removed and the wells are washed. 100μl of colorimetric substrate (TMB high sensitivity substrate, Biolegend) is added. After 10 minutes, 100μl of stop solution (Biolegend) is added to block the reaction. Read the plate at 450nm according to the protocol.

[0093] δ (signal minus noise) NA1 150fmol / ml 3.846 NA1 15fmol / ml 0.600 NA2 150fmol / ml 0.000

[0094] The method can also be used for colorimetric detection and on solid supports different from those used in previous experiments.

[0095] Example 11 (Sensitivity)

[0096] Method. Dilute RNA NA1 in two parts in SSC2.5x S to a concentration of 150fmol / ml, 15fmol / ml, 1.5fmol / ml, 150amol / ml, 15amol / ml, 1.5amol / ml, and add 10nM probe 2d. For each concentration, 100μl of the previously prepared solution containing probe 1b was spread in each well and left for hybridization for 2 hours. The oligonucleotides were then removed and the wells were washed. 100μl of SA-HRP40 at a concentration of 0.1μg / ml was added to PBST for 60 minutes. SA-HRP40 was then removed and the wells were washed. 100μl of chemiluminescent substrate (Clarity, Biorad) was added. Collection was performed after 5 minutes.

[0097] The limit of detection (LOD), calculated as noise + (3*standard deviation), was below a concentration of 1.5 amol / ml (last point of the example).

[0098] It is obvious from the results shown in the examples that the method according to the present invention has high sensitivity, specificity, affinity, and is easy and fast to perform.

[0099] In particular, the use of partial superposition of three oligonucleotides (two probes and the target nucleic acid) offers advantages that have never been achieved before. In fact, triplex complexes have greater stability than duplex complexes.

[0100] Finally, it is obvious that modifications and variations may be made to the method described and illustrated without departing from the scope of protection of the appended claims. Sequence Listing <110> Bioridis Srl <120> Methods and kits for detecting and / or quantifying target nucleotide sequences <130> 428-18 <160> 14 <170> BiSSAP 1.3.6 <210> 1 <211> twenty two <212> RNA <213> Artificial sequence <220> <223> RNA mir 146a full match <400> 1 ugagaacuga auuccauggg uu 22 <210> 2 <211> twenty two <212> RNA <213> Artificial sequence <220> <223> mir 146b mismatch <400> 2 ugagaacuga auuccauagg cu 22 <210> 3 <211> twenty two <212> RNA <213> Artificial sequence <220> <223> 1 mismatch <400> 3 ugacaacuga auuccauggg uu 22 <210> 4 <211> twenty two <212> RNA <213> Artificial sequence <220> <223> 4 mismatches <400> 4 ugagaacuga auuucacuug uu 22 <210> 5 <211> twenty two <212> DNA <213> Artificial sequence <220> <223> DNA Full Match <400> 5 tgagaactga attccatggg tt 22 <210> 6 <211> 10 <212> DNA <213> Artificial sequence <220> <223> PNA 1A <220> <221> misc_feature <222> 1 <223> / Standard Name="N-terminal acetilato" <220> <221> misc_structure <222> 1..10 <223> / Standard Name="PNA" <220> <221> misc_feature <222> 10 <223> / Standard Name="AEEA-K-CONH2" <400> 6 tcagttctca 10 <210> 7 <211> 12 <212> DNA <213> Artificial sequence <220> <223> PNA 1B <220> <221> misc_feature <222> 1 <223> / Standard Name="N-terminal acetilato" <220> <221> misc_feature <222> 1..12 <223> / Standard Name="PNA" <220> <221> misc_feature <222> 12 <223> / Standard Name="AEEA-K-CONH2" <400> 7 attcagttct ca 12 <210> 8 <211> 16 <212> DNA <213> Artificial sequence <220> <223> PNA 1C <220> <221> misc_feature <222> 1 <223> / Standard Name="N-terminal acetilato" <220> <221> misc_feature <222> 1..16 <223> / Standard Name="PNA" <220> <221> misc_feature <222> 16 <223> / Standard Name="AEEA-AEEA-K-CONH2" <400> 8 tggaattcag ttctca 16 <210> 9 <211> 12 <212> DNA <213> Artificial sequence <220> <223> PNA 1D <220> <221> misc_feature <222> 1 <223> / Standard Name="NH2-AEEA-AEEA-AEEA" <220> <221> misc_feature <222> 1..12 <223> / Standard Name="PNA" <220> <221> misc_feature <222> 12 <223> / Standard Name="G-CONH2" <400> 9 actcttgact ta 12 <210> 10 <211> 16 <212> DNA <213> Artificial sequence <220> <223> PNA 2A <220> <221> misc_feature <222> 1 <223> / Standard Name="N-terminal acetilato-5(K(Biot)-AEEA)" <220> <221> misc_feature <222> 1..16 <223> / Standard Name="PNA" <220> <221> misc_feature <222> 16 <223> / Standard Name="K-CONH2" <400> 10 aacccatgga attcag 16 <210> 11 <211> 16 <212> DNA <213> Artificial sequence <220> <223> PNA 2B <220> <221> misc_feature <222> 1 <223> / Standard Name="N-terminal acetilato-5(K(Biot)-AEEA)" <220> <221> misc_feature <222> 1..16 <223> / Standard Name="PNA" <220> <221> misc_feature <222> 16 <223> / Standard Name="K-CONH2" <400> 11 catggaattc agttct 16 <210> 12 <211> 12 <212> DNA <213> Artificial sequence <220> <223> PNA 2C <220> <221> misc_feature <222> 1 <223> / Standard Name="N-terminal acetilato-5(K(Biot)-AEEA)" <220> <221> misc_feature <222> 1..12 <223> / Standard Name="PNA" <220> <221> misc_feature <222> 12 <223> / Standard Name="K-CONH2" <400> 12 aacccatgga at 12 <210> 13 <211> 16 <212> DNA <213> Artificial sequence <220> <223> PNA 2D <220> <221> misc_feature <222> 1 <223> / Standard Name="N-terminal acetilato-5(K(Biot)-AEEA" <220> <221> misc_feature <222> 1..16 <223> / Standard Name="PNA" <220> <221> misc_feature <222> 16 <223> / Standard Name="G-CONH2" <400> 13 ttttaaccca tggaat 16 <210> 14 <211> 12 <212> DNA <213> Artificial sequence <220> <223> PNA 2E <220> <221> misc_feature <222> 1 <223> / Standard Name="N-terminal acetilato-5(K(Biot)-AEEA)" <220> <221> misc_feature <222> 1..12 <223> / Standard Name="DNA" <220> <221> misc_feature <222> 12 <223> / Standard Name="KKK-CONH2" <400> 14 aacccatgga at 12

Claims

1. A method for detecting and / or quantifying a target nucleotide sequence (3) in a sample, the method comprising the following steps: - providing a target nucleotide sequence in single-stranded form (3); - hybridizing the single-stranded target nucleotide sequence (3) with a first probe (1) in the form of a single-stranded nucleic acid and with a second probe (2) in the form of a single-stranded nucleic acid, One end of the first probe (1) is fixed on a solid support (4), and the other end contains a sequence complementary to the first part (5) of the target nucleotide sequence (3); one end of the second probe (2) is labeled, and the other end contains a sequence complementary to the second part (6) of the target nucleotide sequence (3), wherein the overlapping region of the first part (5) and the second part (6) of the target nucleotide sequence has 2 to 10 nucleotides in total, thereby forming a first probe-target nucleotide sequence-second probe complex; - Detecting the first probe-target nucleotide sequence-second probe complex.

2. The method according to claim 1, wherein the first probe (1) and the second probe (2) are formed by nucleic acids selected from the group consisting of: PNA (peptide nucleic acid), LNA (locked nucleic acid), morpholino, orn-PNA (PNA with an ornithine backbone), aep-PNA (aminoethylprolyl-PNA), aepone-PNA (aminoethylpyrrolidone-PNA), GNA (ethylene glycol nucleic acid), HNA (hexitol nucleic acid), TNA (threose nucleic acid), ENA (2'-O,4'-C-ethylene-bridged nucleic acid), ANA (arabinoic acid), and F-ANA (2'-F-arabinoic acid).

3. The method according to claim 2, wherein the first probe (1) and the second probe (2) are formed of PNA (peptide nucleic acid).

4. The method according to claim 1, wherein the target nucleotide sequence (3) is a microRNA (miRNA) nucleotide sequence.

5. The method according to claim 1, wherein the first probe (1) and / or the second probe (2) is formed of 10 to 18 nucleotides. The method according to claim 1 , wherein the sample is living cells.

7. The method according to claim 1, wherein the second probe (2) is labeled with biotin or a fluorescent molecule.

8. The method according to claim 1, wherein the second probe (2) is labeled with fluorescein.

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