METHOD AND KIT FOR DETECTING TARGET NUCLEIC ACID

• US 20180148779A1
• Filed: 05/17/2016
• Published: 05/31/2018
• Est. Priority Date: 05/22/2015
• Status: Active Grant

• Current Assignee: PaxGenBio Co., Ltd., Young Suk Park
• Original Assignee: PaxGenBio Co., Ltd.
• Inventors: Park, Young Suk
• Granted Patent: US 10,640,821 B2
• Time in Patent Office: Days
• CPC Class Codes: C12Q 1/68 involving nucleic acids
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6811 Selection methods for production or design of target specific oligonucleotides or binding molecules
• CPC Class Codes: C12Q 1/682 Signal amplification
• CPC Class Codes: C12Q 1/6825 Nucleic acid detection involving sensors
• CPC Class Codes: C12Q 1/6832 Enhancement of hybridisation reaction
• CPC Class Codes: C12Q 1/6834 Enzymatic or biochemical coupling of nucleic acids to a solid phase
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 1/6846 Common amplification features
• CPC Class Codes: C12Q 1/6853 using modified primers or templates
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 1/6883 for diseases caused by alterations of genetic material
• CPC Class Codes: C12Q 2537/143 Multiplexing, i.e. use of multiple primers or probes in a single reaction, usually for simultaneously analyse of multiple analysis
• CPC Class Codes: C12Q 2565/514 characterised by the use of the arrayed oligonucleotides as identifier tags, e.g. universal addressable array, anti-tag or tag complement array
• CPC Class Codes: G01N 33/5308 for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites
• CPC Class Codes: G01N 33/538 by sorbent column, particles or resin strip , i.e. sorbent materials

Abstract

The present disclosure relates to a method and a kit for analyzing a nucleic acid amplification product, and more particularly to a method and kit of analyzing a nucleic acid amplification product using a labeled primer comprising (i) a binding region complementary to the target nucleic acid, (ii) a nucleic acid oligomer containing nucleotides non-complementary to the target nucleic acid, and (iii) a label, and using a membrane having immobilized thereon a probe that binds complementarily to the nucleic acid oligomer.

Claims

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

(a) amplifying the target nucleic acid by reacting a sample comprising the target nucleic acid with a primer comprising (i) a binding region complementary to the target nucleic acid and (ii) a nucleic acid oligomer comprising nucleotides non-complementary to the target nucleic acid, and (iii) a primer with a label;

(b) reacting a product amplified in step (a) with a membrane having immobilized thereon a probe that binds complementarily to the nucleic acid oligomer; and

(c) confirming whether or not a reaction is shown from the label to detect whether or not the target nucleic acid would be amplified.

2. The method of claim 1, wherein the nucleic acid oligomer of step (a) is 10-40 bp in length.

3. The method of claim 1, wherein the nucleic acid oligomer of step (a) comprises one or more sequences selected from the group consisting of SEQ ID NOs:

1 to 3.

4. The method of claim 1, wherein the step (b) comprises injecting the amplification product of step (a) into a lateral side of the membrane so that the nucleic acid oligomer comprised in the amplification product can complementarily hybridize to the probe immobilized on the membrane, while the amplification product moves.

5. The method of claim 1, wherein the probe that binds complementarily to the nucleic acid oligomer of step (b) comprises one or more sequences selected from the group consisting of SEQ ID NOs:

10 to 12.

6. The method of claim 1, wherein the probe of step (b) additionally comprises a nucleic acid oligomer for immobilization on the membrane.

7. The method of claim 6, wherein the additional nucleic acid oligomer comprises a sequence of SEQ ID NO:

13.

8. The method of claim 1, wherein the label is biotin, Cy5, Cy3, FITC, EDANS (5-(2′

-aminoethyl)amino-1-naphthalenesulfonic acid), tetramethylrhodamine (TMR), tetramethylrhodamine isocyanate (TMRITC), x-rhodamine, DIG, or an antibody.

9. The method of claim 1, wherein step (c) further comprises a reaction with a binding agent that elicits a chromogenic signal of the label.

10. The method of claim 9, wherein the binding agent is streptavidin.

11. A kit for detecting a target nucleic acid, comprising:

(a) a primer comprising (i) a binding region complementary to the target nucleic acid and (ii) a nucleic acid oligomer comprising nucleotides non-complementary to the target nucleic acid, and (iii) a primer with a label; and

(b) a membrane having immobilized thereon a probe that binds complementarily to the nucleic acid oligomer.

12. The kit of claim 11, wherein the nucleic acid oligomer is 10-40 bp in length.

13. The kit of claim 11, wherein the nucleic acid oligomer comprises one or more sequences selected from the group consisting of SEQ ID NOs:

1 to 3.

14. The kit of claim 11, wherein the probe that binds complementarily to the nucleic acid oligomer comprises one or more sequences selected from the group consisting of SEQ ID NOs:

10 to 12.

15. The kit of claim 11, wherein the probe further comprises a nucleic acid oligomer for immobilization on the membrane.

16. The kit of claim 15, wherein the additional nucleic acid oligomer comprises a sequence of SEQ ID NO:

13.