Method for detecting target numerator based on nucleic acid aptamer and PCR amplification

A nucleic acid aptamer and target detection technology, which is applied in the direction of biochemical equipment and methods, microbial measurement/inspection, etc., can solve the problems of limited application, achieve the effect of few influencing factors, simple operation and steps, and reliable results

Inactive Publication Date: 2009-02-18
QINGDAO UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are many factors affecting immuno-PCR, such as linker molecules, selection of display system, concentration of DNA reporter molecules, number of PCR cycles, etc., which all affect its sensitivity and specificity. At present, the technology is not yet fully mature, which limits its further development. application

Method used

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  • Method for detecting target numerator based on nucleic acid aptamer and PCR amplification
  • Method for detecting target numerator based on nucleic acid aptamer and PCR amplification
  • Method for detecting target numerator based on nucleic acid aptamer and PCR amplification

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1: Use Aptamer based PCR to detect trace amounts of cocaine in serum or plasma (small molecule detection) (without magnetic bead enrichment and separation)

[0033] DNA sequences used in this experiment:

[0034] A chain sequence: (39 bases)

[0035] 3-HS-(CH2) 6 -CTG GGT GAA GTA ACT TCC TAA AAG GAA CAG AGG GGGAGT-5

[0036] B chain sequence: (58bases)

[0037] 5-GTGTGAAGCGGCATCTAAAGCGTCTCCCCCCTCAAGACTAGGTCCAGCCCAGGGTGTC-biotin-3

[0038] The part in italics is the hybridization site of the template

[0039] B chain primer:

[0040] S: 5-GTG TGA AGC GGC ATC TAA AGC-3 (21bases)

[0041] A: 5-GAC ACC TCT GGG CTG GAC C-3 (19bases)

[0042] Experimental steps:

[0043] 1. Treatment of gold electrodes

[0044] 1. First drop a drop of separation reagent on the gold electrode, after 15 minutes, wash it off with double distilled water.

[0045] Separation reagent: H 2 o 2 : Concentrated H 2 SO 4 =7:3 (volume ratio)

[0046] 2. Use Al 2 o 3The slurry pol...

example 2

[0089] Example 2: Using Aptamer based PCR to detect thrombin in serum (protein detection)

[0090] DNA sequences used in this experiment:

[0091] A chain sequence:

[0092] 5-HS-(CH2) 6 -ATATAGGTTGGTGTGGTTGG-3

[0093] B chain sequence:

[0094] 5-CAG TGA AGC GGC ATC TAA AGC ACG AGT CTG AGT CCA ACC ACA-3

[0095] Primer for B chain: A: 5-TGT GGT TGG ACT CAG AC TCG-3

[0096] S: 5-GTG TGA AGC GGC ATC TAA AGC-3

[0097] Experimental steps:

[0098] A gold electrode treatment:

[0099] 1 First drop a drop of separation reagent on the gold electrode, after 15 minutes, wash it off with double distilled water.

[0100] Separation reagent: H 2 o 2 : Concentrated H 2 SO 4 =7:3 (volume ratio)

[0101] 2 with Al 2 o 3 The slurry polishes the gold electrodes. When grinding the gold electrode, the force should be uniform, and the gold electrode must be vertical.

[0102] 3 Rinse the gold electrode with double distilled water, and dry the electrode with filter paper and e...

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Abstract

The invention discloses a PCP (aptamer based polymerase chain reaction, A-PCR) based on an aptamer, which relates to a high-sensitivity technology for detecting a micro-target matter. In the technology, the high specificity shown in the integration of a nucleic acid aptamer and a ligand and the high sensibility of the aptamer reacted with a polymerase chain are organically combined; the character of the technology is a method which augments a section of DNA reporter molecule through PCP so as to amplify the combination rate of the aptamer and the ligand, thereby detecting a target ligand. The ligand PCR is one of the most sensitive detecting modes at the moment and can detect a single target molecule in theory. Compared with immuno PCR, the technology has the characteristics of simple operation and process, less affection factor, more reliable result, and the like; the sensibility is improved by 10<2> to 10<8> times than the existing ELISA method; the technology can be broadly applied to the micro detection of protein molecules and small molecules.

Description

technical field [0001] The invention relates to a method for identifying and combining target molecules in a sample with nucleic acid molecules, and using PCR method to amplify to quantify and detect target molecules. Background technique [0002] Nucleic acid aptamer (aptamer) refers to the oligonucleotide fragments that can specifically bind proteins or other small molecular substances screened by in vitro screening technology (Systematic evolution of ligand by exponential enrichment, SELEX), and have a certain effect on the binding ligands. Strict recognition ability and high affinity. Since 1990, Tuerk et al. successfully screened phage T for the first time using SELEX technology. 4 Since the RNA-type aptamer of DNA polymerase, as of June 8, 2004, according to the report of the Aptamer Database website, various laboratories have successfully screened out 2,882 nucleic acid aptamers through this technology, and many aptamer sequences for patent protection Not counted ye...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
Inventor 石超马翠萍张书圣孔建美
Owner QINGDAO UNIV OF SCI & TECH
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