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Method for high specificity detection of target DNA in sample, and application thereof

A technology for detecting samples and high specificity, applied in the field of nucleic acid detection, can solve the problems of false positive detection results, high hybridization background, restricting the wide application of non-isotope labeled probe methods, etc., to improve specificity, improve detection efficiency, avoid The effect of false positives

Inactive Publication Date: 2016-03-02
BEIJING BIOLKEY BIOLOGICAL TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although biomarker probes are stable and inactive, during the hybridization reaction, avidin and labeled probes are easy to be non-specifically adsorbed directly to the surface of the solid phase support, and in severe cases, a high hybridization background will be generated , leading to false-positive detection results, which restricts the widespread application of non-isotope-labeled probe methods in the detection of target nucleic acids
[0005] For this consideration, the inventors of the present invention have carried out research with the aim of solving the problems exposed by the prior art in the related field, expecting to provide a method for detecting target DNA in a sample with high specificity, and improving target DNA detection. Efficiency can effectively avoid false positive detection

Method used

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  • Method for high specificity detection of target DNA in sample, and application thereof
  • Method for high specificity detection of target DNA in sample, and application thereof
  • Method for high specificity detection of target DNA in sample, and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0086] (1) Immobilization of target DNA on the surface of solid support

[0087] 1.1 Experimental materials:

[0088] Solid support: nitrocellulose membrane, produced by Millipore, 0.45μm pore size, cut into 4 membranes with a size of 2cm×1cm for use, and 4 nitrocellulose membranes are labeled A1, B1, and C1 And D1.

[0089] Test sample containing target DNA: human papillomavirus (HPV) type 16 (1pg / μl and 0.1pg / μl), type 18 (1pg / μl and 0.1pg / μl), type 6 (1pg / μl and 0.1pg / μl) and type 11 (1pg / μl and 0.1pg / μl) genome-wide plasmid standards, provided by Shanghai Jereh Company.

[0090] Denaturing liquid: 0.4mol / L NaOH solution.

[0091] 20×SSC buffer solution: 3.0mol / L NaCl, 0.3mol / L sodium citrate, pH 7.0.

[0092] In addition, the 20×SSC buffer solution was diluted to 15×SSC buffer solution and 10×SSC buffer solution for use.

[0093] 1.2 Experimental steps

[0094] 1.2.1 Pretreatment of nitrocellulose membrane: Use tweezers to take the nitrocellulose membrane and soak it in 15×SSC buffe...

Embodiment 2

[0153] Same as Example 1, except that the composition of the pretreatment solution in Example 1 is adjusted to: PH7.0, 0.1mol / LTris-HCl, 1mol / L NaCl, 3% BSA, 0.05% TritonX-100, 0.1% SLS and 0.1% APAM, the balance is water. Other conditions remain unchanged.

[0154] In this example, the 4 nitrocellulose membranes are marked as A2, B2, C2, and D2, and the color results are as follows figure 2 Shown.

Embodiment 3

[0156] Same as Example 1, except that the composition of the pretreatment solution in Example 1 is adjusted to: PH8.0, 0.1mol / LTris-HCl, 1mol / L NaCl, 4% BSA, 0.2% Tween-20, 0.2% SLS, 0.3% APAM, the balance is water. Other conditions remain unchanged.

[0157] In this example, the 4 nitrocellulose membranes are marked as A3, B3, C3 and D3, and the color rendering results are as follows image 3 Shown.

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Abstract

The invention discloses a method for high specificity detection of target DNA in a sample. The method comprises the steps of pretreatment, preliminary treatment, a one-step reaction, post-treatment, and a color development reaction. Traditional molecular hybridization methods are substantially improved, nucleic acid hybridization and enzyme linking reactions are carried out in a combined manner, and synergistic cooperation treatment of solid sustenance surfaces before and after the hybridization reaction are carried out through pretreatment, preliminary treatment and post-treatment, so the detection efficiency of the target DNA in the sample and the solid sustenance surface DNA molecule hybridization specificity are improved, and the false positivity of a detection result is effectively avoided.

Description

Technical field [0001] The invention belongs to the field of nucleic acid detection, and specifically relates to a method for detecting target DNA in a sample with high specificity. Background technique [0002] Nucleic acid molecular hybridization technology is currently one of the most widely used technologies in biochemistry and molecular biology research. It is complementary nucleotide sequences that form stable hybrid double-stranded molecular DNA through Walson-Crick base pairing. Because molecular hybridization technology has high sensitivity and high specificity, it has been widely used in the field of molecular biology for the screening of cloned genes, the production of restriction maps, and the qualitative and quantitative determination of specific gene sequences in the genome. Detection and diagnosis of diseases. It is not only widely used in the field of molecular biology, but also increasingly used in clinical diagnosis. [0003] The two sides of molecular hybridiza...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/70C12R1/94
Inventor 杨华卫曾冀
Owner BEIJING BIOLKEY BIOLOGICAL TECH
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