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 the problems of increasing the detection cost, low sensitivity, and failing to meet the requirements of nucleic acid detection, and achieve the effect of high specificity and low cost.

Inactive Publication Date: 2012-12-19
HUADONG RES INST FOR MEDICINE & BIOTECHNICS
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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 la

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 10 mmol / L MOPS, pH 7.5, 0.05% Tween-20, 0.05% Nonidet P40, 0.1 μmol / L upstream probe (sequence: 5'-TGGTTCCTCCTGAGGCCTCC-3', namely SEQ ID NO.1 ), 0.2 μmol / L downstream probe (seque...

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 having one base difference with the target nucleic acid at different positions (sequences are: a: 5'-TCTACAACCCAAGGAGTGAGGCCTCAGGAGGAACCA-3', 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;) There is only one base difference between the sequence of each mutation template and the target nucleic acid sequence in Example 1 at different pos...

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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 amplification product is identical to the target nucleic acid sequence, such as polymerase chain reaction technology (Polymerase Chain Reaction, PCR), loop-mediated nucleic acid isothermal amplification technology (Loop- Mediated Isothermal Amplification (LAMP), Rolling Cycle Amplification (RCA), Nucleic Acid S...

Claims

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Application Information

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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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