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Cascade intrusion signal amplification reaction combined nanogold-oligonucleotide probe visual nucleic acid detection method

An oligonucleotide probe and signal amplification technology, applied in the field of molecular biology, can solve problems such as increased detection cost, low sensitivity, and failure to meet the requirements of nucleic acid detection, achieving high specificity and low cost

Inactive Publication Date: 2014-05-28
HUADONG RES INST FOR MEDICINE & BIOTECHNICS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Among them, the B-DNA method and the nucleic acid invasion signal amplification experiment have high sensitivity, but they all need to synthesize probes with special structures or special fluorescent labels, which increases the detection cost; while the signal amplification method based on electrochemical detection Often the sensitivity is low and cannot meet the requirements of nucleic acid detection

Method used

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  • Cascade intrusion signal amplification reaction combined nanogold-oligonucleotide probe visual nucleic acid detection method
  • Cascade intrusion signal amplification reaction combined nanogold-oligonucleotide probe visual nucleic acid detection method
  • Cascade intrusion signal amplification reaction combined nanogold-oligonucleotide probe visual nucleic acid detection method

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Embodiment 1

[0027] Example 1 Detection of Artificially Synthesized Oligonucleotide Fragments Using Cascade Invasion Signal Amplification Reaction Combined with Nanogold-Oligonucleotide Probe Visual Nucleic Acid Detection Method

[0028] This embodiment detects different concentrations of artificially synthesized single-stranded DNA templates, which are used to verify the feasibility of the nucleic acid detection method stated in the present invention and examine the sensitivity of the method. Six oligonucleotides with one nucleotide difference are used to verify the sequence specificity of the method stated in the present invention.

[0029] The reaction conditions and reaction process are:

[0030] 1) Cascade invasion signal amplification reaction

[0031]The nucleic acid invasion reaction system consists of 10mmol / L MOPS, pH7.5, 0.05%Tween-20, 0.05%Nonidet P40, 0.1μmol / L upstream probe (sequence: 5'-TGGTTCCTCCTGAGGCCTCC-3', which is SEQ ID NO. 1), 0.2 μmol / L downstream probe (sequence...

Embodiment 2

[0035] Example 2 Using Cascade Invasion Signal Amplification Reaction Combined with Gold Nano-Oligonucleotide Probe Visual Nucleic Acid Detection Method to Realize Differential Detection of Single Base Difference Template

[0036] In this example, six artificially synthesized mutation templates (sequences: a:5'-TCTACAACCCAAGGAGTGAGGCCTCAGGAGGAACCA-3', which have one base difference with the target nucleic acid at different positions, are detected, namely SEQ ID NO.7;

[0037] b: 5'-TCTACAACCCAAGGACAGAGGCCTCAGGAGGAACCA-3', namely SEQ ID NO.8;

[0038] c: 5'-TCTACAAGCCAAGGAGAGAGGCCTCAGGAGGAACCA-3', namely SEQ ID NO.9;

[0039] d: 5'-TCTACAACCCAAGGAGACAGGCCTCAGGAGGAACCA-3', namely SEQ ID NO.10;

[0040] e: 5'-TCTACAACCCAAGGAGAGAGCCCTCAGGAGGAACCA-3', namely SEQ ID NO.11;

[0041] f: 5'-TCTACAACCCAAGGAGAGAGGCCTCAGGACGAACCA-3', namely SEQ ID NO.12;)

[0042] The sequence of each mutation template and the target nucleic acid sequence in Example 1 have only one base difference at d...

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Abstract

The invention belongs to the field of molecular biology and relates to cascade intrusion signal amplification reaction combined nanogold-oligonucleotide probe visual detection. The cascade intrusion signal amplification reaction combined nanogold-oligonucleotide probe visual nucleic acid detection method provided by the invention comprises the following steps of: 1) first-step intrusion signal amplification reaction, during which accumulation of flap fragments is formed; 2) flap fragment circulation phase, during which complementary hybridization of the flap fragments and hairpin probes is performed to form a flap fragment-hairpin probe special structure, and the hairpin probes are cut after the structure is recognized by enzymes; and 3) nanogold-oligonucleotide probe detection phase, during which results of the cascade intrusion signal amplification reaction are visually detected by the utilization of two different oligonucleotide modified nanogold probes. By the utilization of the detection method provided by the invention, visual detection of a nucleic acid target sequence can be carried out. In addition, as the intrusion signal amplification reaction has high specificity, differentiated detection of the nucleic acid sequences with single-base differences near intrusion sites can be realized.

Description

technical field [0001] The invention belongs to the field of molecular biology, and relates to a nucleic acid invasion signal amplification method and nano gold-oligonucleotide probes. Specifically relates to a cascade invasion signal amplification reaction combined with nano gold-oligonucleotide probe visualization nucleic acid detection method. Background technique [0002] Nucleic acid detection has been widely used in clinical diagnosis, environmental monitoring, prevention and control of infectious diseases and many other aspects. At present, most commonly used nucleic acid detection technologies are based on the principle of template amplification, that is, the amplified product is identical to the target nucleic acid sequence, such as polymerase chain reaction (Polymerase Chain Reaction, PCR), loop-mediated nucleic acid isothermal amplification technology (Loop- Mediated Isothermal Amplification (LAMP), Rolling Cycle Amplification (RCA), Nucleic Acid Sequence Based A...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68
Inventor 周国华马寅姣邹秉杰
Owner HUADONG RES INST FOR MEDICINE & BIOTECHNICS
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