MULTIANALYTE ASSAY FOR THE SIMULTANEOUS DETECTION OF NUCLEIC ACID AND ANALYTES

Abstract

The present invention relates to a method and a kit to detect the presence or absence of at least two different target molecules form one sample, wherein at least one target molecule is a target analyte of interest and at least one other target molecule is a target nucleic acid of interest, wherein the method combines performing an isothermal amplification reaction, wherein the target nucleic acid or its amplicon is labeled with at least two affinity labels and a ligand binding assay, wherein affinity molecules are used, which can capture and detect the presence of target analytes and/or labeled target nucleic acid via signal generation. The invention also relates to the use of this method or kit in various fields.

Claims

1. A method to detect the presence or absence of at least two different target molecules from one sample, wherein at least one target molecule is a target analyte of interest and at least one other target molecule is a target nucleic acid of interest, the method comprising performing an isothermal amplification reaction, comprising contacting a sample to be analyzed for the presence or absence of at least one target nucleic acid and/or at least one target analyte to at least one set of amplification primers, wherein the two amplification primers can hybridize with the target nucleic acid, wherein at least one of the amplification primers comprises a first affinity label, wherein a second affinity label is provided in a way that in can be incorporated into an amplicon of the target nucleic acid, simultaneously performing a ligand binding assay, wherein affinity molecules are used, which can capture and detect the presence of target analytes and/or labeled target nucleic acid via signal generation, detecting the presence or absence of said target analytes and/or target nucleic acids, wherein the target analyte is a protein, peptide, antibody, hormone, enzyme, small molecule, carbohydrate or any other substance, but not a nucleic acid, wherein the sample is not split and no separate assay procedures and/or protocols are required.

2. The method according to claim 1, wherein the target nucleic acid is amplified and simultaneously at least the first and the second affinity labels are introduced into the amplified target nucleic acid.

3. The method according to claim 1, wherein a specific probe is provided which hybridizes with a sequence localized between the at least two amplification primer hybridization sites, and wherein the specific probe comprises the second affinity label.

4. The method according to claim 1, wherein the isothermal amplification reaction leads to a primary single labeled product, which is recognized by the specific probe, which hybridizes to a sequence localized between the at least two amplification primers, leading to second double labeled product.

5. The method according to claim 1, wherein the second label differs from the first label.

6. The method according to claim 1, wherein the signal for the target nucleic acid is generated via the binding of affinity labels by affinity molecules.

7. The method according to claim 1, wherein the target nucleic acid is a DNA molecule, or a RNA molecule.

8. The method according to claim 1, wherein the isothermal amplification reaction occurs in conditions, which are compatible with analytes.

9. The method according to claim 1, wherein the first label is an affinity tag, preferred a biotin, FAM, digoxigenin or dinitrophenyl (DNP), tetramethylrhodamine (TAMRA), texas red, cascade blue, streptavidin and derivatives, Cy5, dansyl, fluorescein, azide, alkyne, or other bio-orthogonal functional groups and/or tags.

10. The method according to claim 3, wherein the specific probe further comprises at least one functional site, preferred an abasic residue or a polymerase extension blocking group.

11. The method according to claim 1, wherein the affinity molecules are antibodies, aptamers, functional groups, proteins, ligand binding polymer structures, (macro-) molecules which can contain a functional group and/or molecularly imprinted polymers (MIPs) and/or wherein signalling tools, preferred marker are attached to the affinity molecules.

12. A Kit for performing a method according to claim 1, the kit comprising at least one set of amplification primers, which hybridize with a target nucleic acid, wherein at least one of the amplification primers comprises a first affinity label, optionally a specific probe, which can hybridize with the target nucleic acid, a second affinity label which is either associated with the second primer or with the specific probe, reagents for performing an isothermal amplification reaction and affinity molecules.

13. (canceled)

14. The method of claim 7, wherein the DNA molecule is selected from the group consisting of ssDNA, dsDNA, cDNA, rDNA, mtDNA, cpDNA and plasmid DNA.

15. The method of claim 7, wherein the RNA molecule is selected from the group consisting of mRNA, circulating RNA, miRNA, snRNA, snoRNA, rRNA, tRNA, asRNA, circRNA, hnRNA, siRNA, shRNA, snoRNA, snRNA, lncRNA, piRNA, and tracrRNA.

16. An in vitro diagnostic, drug development, food safety or environmental safety method that detects the presence or absence of at least two different target molecules from one sample, wherein at least one target molecule is a target analyte of interest and at least one other target molecule is a target nucleic acid of interest, the method comprising performing an isothermal amplification reaction, comprising contacting a sample to be analyzed for the presence or absence of at least one target nucleic acid and/or at least one target analyte to at least one set of amplification primers, wherein the two amplification primers can hybridize with the target nucleic acid, wherein at least one of the amplification primers comprises a first affinity label, wherein a second affinity label is provided in a way that in can be incorporated into an amplicon of the target nucleic acid, simultaneously performing a ligand binding assay, wherein affinity molecules are used, which can capture and detect the presence of target analytes and/or labeled target nucleic acid via signal generation, detecting the presence or absence of said target analytes and/or target nucleic acids wherein the target analyte is a protein, peptide, antibody, hormone, enzyme, small molecule, carbohydrate or any other substance, but not a nucleic acid, wherein the sample is not split and no separate assay procedures and/or protocols are required.

Description

[0117] FIG. 1 shows a conventional detection of multiple target molecules versus simultaneous detection of multiple target molecules in one go from one sample. (Top) Conventional detection of multiple target molecules. (Bottom) Simultaneous detection of the target molecules via Multianalyte Assay according to the invention.

[0118] FIG. 2 shows the simultaneous detection of P. aeruginosa gDNA and IL-6 within within a microtiter plate.

[0119] FIG. 3 shows the simultaneous detection of P. aeruginosa gDNA and IL-6 via lateral flow assay.

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