DIGITAL BIOMOLECULES DETECTION AND/OR QUANTIFICATION USING ISOTHERMAL AMPLIFICATION

1 . A digital method for detecting and/or quantifying at least one target biomolecule in a sample comprising the following steps:

a) mixing said sample with a mixture including a buffer, enzymes, a first oligonucleotide which is an amplification oligonucleotide, a second oligonucleotide which is a leak absorption oligonucleotide, and a third oligonucleotide which is a target-specific conversion oligonucleotide;

b) partitioning the mixture obtained in step a) into several compartments so that a fraction of the compartments does not contain the target biomolecule;

c) converting the target biomolecule into a signal;

d) amplifying the signal, and

e) detecting and/or measuring said signal in each compartment.

2 . The digital method of claim 1 , wherein the target biomolecule is a nucleic acid or protein.

3 . The method according to claim 2 , wherein the target biomolecule is a nucleic acid selected from the group consisting of DNA, cDNA, RNA, mRNA, and micro RNA.

4 . The method according to claim 1 , wherein the enzymes used in step a) are selected from the group consisting of polymerase, nicking enzyme or restriction enzyme, and exonuclease.

5 . The method according to claim 1 , wherein the first oligonucleotide includes a partial repeat structure containing a nicking enzyme recognition site, and the second oligonucleotide is able to bind, extend, deactivate, and slowly release the products of polymerization along the first oligonucleotide, thereby inducing a threshold effect.

6 . The method of claim 1 , further comprising adding a fourth oligonucleotide which is a reporting probe.

7 . The method of claim 1 , further comprising adding a fifth oligonucleotide which is a cross inhibiting oligonucleotide for detecting and/or quantifying two or more biomolecules.

8 . The method according to claim 1 , wherein the mixture obtained in step a) is partitioned in step b) into droplets.

9 . The method according to claim 8 , wherein the size of droplet is between 0.001 and 100 pL.

10 . The method according of claim 1 , wherein said signal is labelled.

11 . The method according to claim 1 , wherein the step d) of detecting and/or measuring said signal comprises detecting and/or counting the compartments emitted a fluorescence.

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

13 . The method according to claim 1 , wherein the target biomolecule is used as a biomarker.

14 . An in vitro method for diagnosis of a disease selected from the group comprising cancer, neuronal diseases, cardiovascular diseases, inflammatory diseases, autoimmune diseases, diseases due to a viral or bacterial infection, skin diseases, skeletal muscle diseases, dental diseases, and prenatal diseases comprising the use of the method according to claim 1 .

15 . An in vitro method for agro diagnosis of a disease selected from the group comprising:

diseases caused by biotic stress, or

diseases caused by abiotic stress,

said method comprising the use of the digital method according to claim 1 .

16 . A kit for detecting and/or quantifying at least one target biomolecule comprising:

a) a mixture of enzymes, selected from the group consisting of polymerase, nicking enzyme or restriction enzymes and exonuclease;

b) a mixture of oligonucleotides comprising a first oligonucleotide which is an amplification oligonucleotide, a second oligonucleotide which is a leak absorption oligonucleotide, and a third oligonucleotide which is a target-specific conversion oligonucleotide and optionally a fourth oligonucleotide which is a reporting probe, and

c) a partitioning agent.

17 . The method according to claim 1 , wherein the converted signal in step c) is a DNA single strand.

18 . The method according to claim 6 , wherein the reporting probe is a fluorescent probe.

19 . The method according to claim 8 , wherein the droplets are water-in-oil emulsion droplets.

20 . The in vitro method according to claim 15 , wherein:

the diseases caused by biotic stress have an infectious and/or parasitic origin, or

the diseases caused by abiotic stress are caused by nutritional deficiencies and/or unfavorable environment.