Method for detecting a target substance by using a nucleic acid probe

• US 20060234253A1
• Filed: 10/06/2005
• Published: 10/19/2006
• Est. Priority Date: 10/08/2004
• Status: Abandoned Application

• Current Assignee: Sysmex Corporation
• Original Assignee: Sysmex Corporation
• Inventors: Yamagata, Koichi, Abe, Shigeki, Daito, Motonari, Hasui, Yasushi
• Application Number:
  US11/244,045
• Publication Number:
  US 20060234253A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6834 Enzymatic or biochemical coupling of nucleic acids to a solid phase
• CPC Class Codes: C12Q 2537/125 Sandwich assay format
• CPC Class Codes: C12Q 2537/1373 Displacement by a nucleic acid
• CPC Class Codes: C12Q 2537/1376 Displacement by an enzyme
• CPC Class Codes: C12Q 2565/519 characterised by the capture moiety being a single stranded oligonucleotide

Abstract

A method for detecting a target substance is described, which comprises the steps of: forming a first complex comprising (a) a first nucleic acid probe immobilized on a first solid phase and having a single-strand portion, (b) a nucleic acid ligand which has a binding section as a base sequence hybridizable with the single-strand portion of the first nucleic acid probe and which has a target-binding substance capable of binding to the target substance, (c) the target substance, and (d) a labeling substance capable of binding to the target substance; separating, from the first complex, a second complex comprising the nucleic acid ligand, the target substance and the labeling substance; and detecting the target substance on the basis of the labeling substance in the second complex.

Claims

1. A method for detecting a target substance, comprising the steps of:

forming a first complex comprising

(a) a first nucleic acid probe immobilized on a first solid phase and having a single-strand portion,

(b) a nucleic acid ligand which has a binding section as a base sequence hybridizable with the single-strand portion of the first nucleic acid probe and which has a target-binding substance capable of binding to the target substance,

(c) the target substance, and

(d) a labeling substance capable of binding to the target substance;

separating, from the first complex, a second complex comprising the nucleic acid ligand, the target substance and the labeling substance; and

detecting the target substance on the basis of the labeling substance in the second complex.

2. The method according to claim 1, comprising the step of forming a third complex comprising the separated second complex and a second nucleic acid probe which is immobilized on a second solid phase and has a single-strand portion hybridizable with the binding section of the nucleic acid ligand,

and wherein the detecting step is carried out by detecting the target substance on the basis of the labeling substance in the second complex included in the third complex.

3. The method according to claim 1, wherein said target substance is selected from the group consisting of protein, nucleic acid, sugar, lipid, hormone, heptene, toxin, viruse and a microorganism.

4. The method according to claim 1, wherein said first nucleic acid probe, said binding section and said second nucleic acid probe are DNAs.

5. The method according to claim 1, wherein the binding section is DNA or RNA, and the target-binding substance is bound to the 3′

end of the binding section.

6. The method according to claim 1, wherein said target-binding substance is selected form the group consisting of a base sequence, an antibody, a receptor, and an aptamer.

7. The method according to claim 1, comprising a step of washing the first solid phase after formation of the first complex.

8. The method according to claim 1, wherein the formation of the first complex is carried out by binding the target substance and the labeling substance to a complex comprising the first nucleic acid probe and the nucleic acid ligand.

9. The method according to claim 1, wherein the formation of the first complex is carried out by binding the first nucleic acid probe to a complex comprising the target substance, the target substance bound and the nucleic acid ligand.

10. The method according to claim 1, wherein said labeling substance is selected from the group consisting of a radioisotope, a fluorescent substance, an enzyme, a coloring substance and a luminescent substance.

11. The method according to claim 10, wherein said labeling substance has a base sequence, an antibody, a receptor, and an aptamer.

12. The method according to claim 1, wherein the labeling substance comprises a first substance containing a second target-binding substance capable of binding to the target substance, and a second substance capable of binding to the first substance and having a label selected from the group consisting of an enzyme, a fluorescent substance, a luminescent substance, a coloring substance and a radioisotope.

13. The method according to claim 1, wherein the separating step is carried out by heating the first complex.

14. The method according to claim 1, wherein the separating step is carried out by heating the first complex until reaching a temperature in the presence of a single-strand competitive nucleic acid hybridizable with the first nucleic acid probe and longer than the binding section, the temperature being higher than a melting temperature (Tm) value of a hybrid of the first nucleic acid probe and the binding section, and lower than a melting temperature (Tm) value of a hybrid of the first nucleic acid probe and the competitive nucleic acid.

15. The method according to claim 1, wherein the separating step is carried out by strand displacement reaction as a template of the single-strand portion of the first nucleic acid probe with a DNA polymerase with strand displacement activity and nucleotide(s) selected from the group consisting of DATP, dGTP, dTTP and dCTP.

16. A method for collecting a target substance, comprising the steps of:

forming a first complex comprising

(a) a first nucleic acid probe immobilized on a first solid phase and having a single-strand portion,

(b) a nucleic acid ligand which has a binding section hybridizable with the single-strand portion of the first nucleic acid probe and which has a target-binding substance capable of binding to the target substance,

(c) the target substance, and

(d) a labeling substance capable of binding to the target substance;

separating, from the first complex, a second complex comprising the nucleic acid ligand, the target substance and the labeling substance; and

collecting the separated second complex.

17. A reagent kit for detection of a target substance, comprising:

a first solid phase on which a first nucleic acid probe having a single-strand portion is immobilized;

a nucleic acid ligand having both a binding section hybridizable with the single-strand portion of the first nucleic acid probe and a target-binding substance capable of binding to the target substance;

a second solid phase on which a second nucleic acid probe having a single-strand portion hybridizable with the binding section of the nucleic acid ligand is immobilized; and

a labeling substance capable of binding to the target substance.

18. The reagent kit according to claim 17, further comprising a washing solution for washing the first solid phase.

19. The reagent kit according to claim 17, further comprising DNA polymerase with strand displacement activity and nucleotide(s) selected from the group consisting of DATP, dGTP, dTTP and dCTP.

20. The reagent kit according to claim 17, wherein said first nucleic acid probe, said binding section and said second nucleic acid probe are DNAs.