Amplified nucleic acid detection method and detection device

Patent metadata

Field	Value
Publication number	US-9920356-B2
Application number	US-201113989207-A
Country	US
Kind code	B2
Filing date	Nov 24, 2011
Priority date	Nov 24, 2010
Publication date	Mar 20, 2018
Grant date	Mar 20, 2018

Abstract

An object of the invention is to provide a nucleic acid detection method which takes advantage of the high specificity of hybridization techniques, reduces the time length and the number of steps required for detection of PCR products, and allows for easy and highly accurate detection by visual observation without the need of special equipment; and a nucleic acid detection device or kit. The invention provides a method for detecting a target nucleic acid in a sample, which includes performing amplification of the target nucleic acid sequence to synthesize an amplification product having a partially double-stranded structure where a single-stranded region is added to each end of the target sequence, and hybridizing a nucleic acid sequence bound to a development medium and a nucleic acid sequence labeled with a labeling compound with the single-stranded regions of the amplification product to form a sandwich hybridization complex; and a detection device thereof.

First claim

The invention claimed is:

1. A nucleic acid detection method, comprising
performing a nucleic acid amplification reaction using two primers in the presence of a nucleic acid template and a DNA polymerase, thereby producing an amplified double-stranded DNA fragment consisting of two 5′ single-stranded regions respectively located at its opposite ends, wherein each of the primers contains a space structure comprising a polymerase reaction inhibitory region located between 5′ end and 3′ end of each of the primers and the polymerase reaction inhibitory region inhibits a nucleic acid extension reaction catalyzed by a polymerase,
detecting the amplified double-stranded DNA fragment by following steps:
hybridizing a first oligonucleotide probe immobilized on a solid phase with one of the two 5′ single-stranded regions of the amplified double-stranded DNA fragment,
hybridizing a second oligonucleotide probe labeled with a labeling substance with the other region of the two 5′ single-stranded regions of the amplified double stranded DNA fragment, and
detecting the presence of the amplified double-stranded DNA fragment.

2. The nucleic acid detection method according to claim 1 , wherein the labeling substance comprises a colored carrier that allows the amplified double-stranded DNA fragment to be visually detected.

3. The nucleic acid detection method according to claim 2 , wherein said detecting the amplified double-stranded DNA fragment comprises applying the amplified double-stranded DNA fragment on a nucleic acid detection device and said detecting the presence of the amplified DNA fragment is performed on the nucleic acid detection device and the solid phase is on the nucleic acid detection device.

4. The nucleic acid detection method according to claim 2 , wherein said detecting the amplified double-stranded DNA fragment further comprises diffusing the amplified double-stranded DNA fragment by chromatography such that the presence of the amplified double-stranded DNA fragment is detected by the chromatography.

5. The nucleic acid detection method according to claim 1 , wherein said detecting the amplified double-stranded DNA fragment comprises applying the amplified double-stranded DNA fragment on a nucleic acid detection device, said detecting the presence of the amplified double-stranded DNA fragment is performed on the nucleic acid detection device and the solid phase is on the nucleic acid detection device.

6. The nucleic acid detection method according to claim 5 , wherein said detecting the amplified double-stranded DNA fragment further comprises following steps (a) to (c):
(a) performing said applying the amplified double-stranded DNA fragment on a nucleic acid detection device by placing the amplified double-stranded DNA fragment in a zone on the solid phase of the nucleic acid detection device which is different from a zone where the first oligonucleotide probe is immobilized on the solid phase;
(b) diffusing the amplified double-stranded DNA fragment with a solvent on the device toward the zone where the first oligonucleotide probe is immobilized; and
(c) performing said hybridizing a first oligonucleotide probe immobilized on a solid phase with one of the two 5′ single-stranded regions of the amplified double-stranded DNA fragment by hybridizing the first oligonucleotide probe with the amplified double-stranded DNA fragment in the zone where the first oligonucleotide probe is immobilized.

7. The nucleic acid detection method according to claim 6 , wherein said hybridizing the second oligonucleotide probe labeled with a labeling substance with the amplified double-stranded DNA fragment is performed before the step (c).

8. The nucleic acid detection method according to claim 5 , wherein said detecting the amplified double-stranded DNA fragment further comprises following steps (a) to (e):
(a) performing said applying the amplified double-stranded DNA fragment on a nucleic acid detection device by placing the amplified double-stranded DNA fragment and the second oligonucleotide probe respectively in discrete zones on the solid phase of the nucleic acid detection device which are different from a zone where the first oligonucleotide probe is immobilized on the solid phase; then
(b) diffusing the amplified double-stranded DNA fragment with a solvent toward the zone of said discrete zones where the second oligonucleotide probe is placed; then
(c) performing said hybridizing a second oligonucleotide probe labeled with a labeling substance with the other region of the two 5′ single-stranded regions of the amplified double stranded DNA fragment by hybridizing the second oligonucleotide probe with the amplified double-stranded DNA fragment in the zone of said discrete zones where the second oligonucleotide probe is placed, thereby forming a hybridization complex;
(d) diffusing the hybridization complex obtained in the step (c) on the solid phase toward the zone where the first oligonucleotide probe is immobilized; and
(e) performing said hybridizing a first oligonucleotide probe immobilized on a solid phase with one of the two 5′ single-stranded regions of the amplified double-stranded DNA fragment by hybridizing the first oligonucleotide probe with the amplified double-stranded DNA fragment in the hybridization complex in the zone where the first oligonucleotide probe is immobilized.

9. The nucleic acid detection method according to claim 5 , wherein the nucleic acid detection device is a device comprising a chromatography and said detecting the amplified double-stranded DNA fragment further comprises diffusing the amplified double-stranded DNA fragment by the chromatography such that the presence of the amplified double-stranded DNA fragment is detected by the chromatography.

10. The nucleic acid detection method according to claim 1 , wherein said detecting the amplified double-stranded DNA fragment further comprises diffusing the amplified double-stranded DNA fragment by chromatography such that the presence of the amplified double-stranded DNA fragment is detected by the chromatography.

11. The nucleic acid detection method according to claim 1 , wherein each of the two 5′ single-stranded regions of the amplified double-stranded DNA fragment has a 5′ tag sequence from a 5′ tag sequence of each of the primers.

12. The nucleic acid detection method according to claim 1 , wherein the first primer of the two primers comprises a sequence capable of hybridizing to one strand of the nucleic acid template and a common sequence incapable of hybridizing to the template, and the second primer of the two primers comprises a sequence capable of hybridizing to other strand of the nucleic acid template and a tag region incapable of hybridizing to the template.

13. The nucleic acid detection method according to claim 12 , wherein the tag region comprises natural nucleotides.

Assignees

• Takahashi Koji
• Miyamoto Shigehiko
• Jikihara Takaaki
• Tomono Jun
• Kaneka Corp

Inventors

• Takahashi Koji
• Miyamoto Shigehiko
• Jikihara Takaaki
• Tomono Jun

Classifications

• G01N33/54313

  the carrier being characterised by its particulate form · CPC title

• C12Q1/686

  Polymerase chain reaction [PCR] · CPC title

• C12Q1/6832

  Enhancement of hybridisation reaction · CPC title

• C12Q1/6834 Primary

  Enzymatic or biochemical coupling of nucleic acids to a solid phase · CPC title

• C12N15/09

  Recombinant DNA-technology · CPC title