Nucleic acid cross flow test strip-based method for detecting single nucleotide polymorphism

A single nucleotide polymorphism, horizontal technology, applied in the direction of biochemical equipment and methods, microbial determination/inspection, etc., can solve the problems of difficult storage, increased cost and steps, etc.

Active Publication Date: 2011-07-27
SHANGHAI INST OF MICROSYSTEM & INFORMATION TECH CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The test strip has certain advantages, but because of the use of protein antigen-antibody reactions, more labels are required, and the number of high-affinity antibody / hapten pairs limits the detection of gene multi-targets, increasing costs and steps , and less easily stored

Method used

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  • Nucleic acid cross flow test strip-based method for detecting single nucleotide polymorphism
  • Nucleic acid cross flow test strip-based method for detecting single nucleotide polymorphism
  • Nucleic acid cross flow test strip-based method for detecting single nucleotide polymorphism

Examples

Experimental program
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Effect test

Embodiment 1

[0048] Establishment of a method for detecting KIF single nucleotide polymorphisms based on nucleic acid lateral flow test strips

[0049] (1) Preparation of sulfhydryl-labeled oligonucleotide gold nanoprobes

[0050] ① Preparation of nano-gold colloid

[0051] Prepare a 1mM / L HAuCl4 solution and a 38.8mM / L trisodium citrate solution. Heat the HAuCl4 solution to 130°C and stir it fully during the heating process. After about 20 minutes, quickly add 25mL of the newly prepared trisodium citrate solution (pay attention to heat preservation during this process, do not let the HAuCl4 solution cool down), and continue stirring until the solution After cooling to room temperature, a wine-red solution was formed. Filter the solution with a 0.22 μm nitrocellulose nylon membrane to obtain a nano-gold solution with uniform particles.

[0052] ② Preparation of DNA-labeled gold nanoprobes

[0053] Take 1.0mL of 13nm nano-gold solution with a concentration of 12nM / L, centrifuge at 9500r...

Embodiment 2

[0074] Detection and judgment of single nucleotide polymorphism in clinical samples of KIF6DNA

[0075] (1) Preparation of sulfhydryl-labeled oligonucleotide gold nanoprobes

[0076] Method is as described in embodiment 1 / (1);

[0077] (2) Design and synthesis of KIF6 DNA detection probe

[0078] The invention adopts a double-probe detection method to design a detection probe and a connection probe according to the DNA sequence of the 719Arg allele (Arg / Trp or Arg / Arg) of the KIF6 gene. Firstly, a sequence (30 bp) containing the single nucleotide polymorphism site of the 719Arg allele of the KIF6 gene was selected as Target DNA, including alleles KIF6 (719Arg) and KIF6 (719Trp). Using the upstream sequence of the single nucleotide polymorphism site as a template, its complementary nano-gold detection probe (KIF-P-PS) was designed, and its 5' end was modified with thiol functionalization to bind to gold nanoparticles. Design two connection probes (27bp), the first base at th...

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Abstract

The invention relates to a nucleic acid cross flow test strip-based method for detecting single nucleotide polymorphism, comprising the following steps: firstly, preparing the nucleic acid cross flow test strip; secondly, obtaining a sample to be tested, denaturing and annealing; obtaining water, a nano-gold probe solutions, a connecting probe, Taq DNA ligase buffer solutions and Taq DNA ligase, and blending uniformly to obtain a mixed solution; adding KIF-1 and KIF-2 or the mixed solution of the two to the mixed solution, and blending uniformly; adding the sample to be tested, carrying out hybrid connection, denaturing, and annealing; and finally dropping obtained solutions on the binding area of the nucleic acid cross flow test strip, immersing the immersion area of the test strip into the running buffer solutions, and observing. The method achieves easy operation and low cost, is characterized by specificity, fastness as well as high resolution and sensitivity, and can be applied to the detection on the single nucleotide polymorphism and gene type as well as the identification on different pathogenic microorganisms of genes in hereditary diseases, communicable diseases, tumour and angiocardiopathy in clinical medicines.

Description

technical field [0001] The invention belongs to the detection field of single nucleotide polymorphisms, in particular to a method for detecting single nucleotide polymorphisms based on nucleic acid lateral flow test strips. Background technique [0002] Cardiovascular disease is the number one killer of modern people. Now nearly a quarter of the world's population is threatened by cardiovascular and related diseases, and throughout their lives, about one-third of the population is shrouded in the shadow of cardiovascular diseases, and finally one-fifth of the population dies from Cardiovascular related diseases. KIF6 (kinesin-like protein 6) gene plays an important role in the occurrence, diagnosis and treatment of cardiovascular diseases (such as coronary heart disease). KIF6 (kinesin-like protein 6) protein is a protein related to intracellular transport. KIF6 genotype provides important information beyond traditional risk factors to help identify high-risk patients wit...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
Inventor 毛红菊邹能利金庆辉赵建龙
Owner SHANGHAI INST OF MICROSYSTEM & INFORMATION TECH CHINESE ACAD OF SCI
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