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Electrochemical method of detecting single-chain target DNA concentration based on G-quadruplex-heme compound and polymeric chain type amplification reaction

A polymer chain, heme technology, applied in the field of analytical chemistry, can solve the problems of radioactive contamination, low sensitivity, cumbersome operation, etc., and achieve the effect of high sensitivity detection, high sensitivity and strong specificity

Active Publication Date: 2017-03-22
ANHUI HUATENG AGRI TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Traditional DNA detection methods have certain disadvantages, such as cumbersome operation, possible radioactive contamination, need for expensive detection instruments, low sensitivity, etc.

Method used

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  • Electrochemical method of detecting single-chain target DNA concentration based on G-quadruplex-heme compound and polymeric chain type amplification reaction
  • Electrochemical method of detecting single-chain target DNA concentration based on G-quadruplex-heme compound and polymeric chain type amplification reaction
  • Electrochemical method of detecting single-chain target DNA concentration based on G-quadruplex-heme compound and polymeric chain type amplification reaction

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1 Detection of HIV DNA concentration based on G-quadruplex-heme complex and polymerization chain amplification reaction.

[0035] Not all HIV gene carriers are suffering from AIDS. Mutations in this gene will lead to the destruction of the human immune system, thereby causing epidemics. It is of great significance for the early screening of AIDS to develop a sensitive detection method using the gene fragment where the mutation site is located as the detection target sequence.

[0036] The HIV gene fragment is used as the target DNA, and the detection steps are the same as those described above.

[0037] The target DNA sequence is: 5'- GGCAGCAATT TCACCAGTAC TA -3'.

[0038] The designed capture probe sequence is: 5'-HS-(CH 2 ) 6 - TTTAGTACTG GTG -3', the 5'-end of the probe is modified with a thiol group for self-assembly onto the gold electrode surface.

[0039] The designed auxiliary probe sequence is: 5'- AAATTGCTGC CTTT GGGTAG GGCGGGTTGG G CTTAGTACTGGTG...

Embodiment 2

[0047] Example 2 Specific Analysis of HIV DNA Detection

[0048] Taking the above HIV gene fragment as an example, the original target DNA was replaced with single-stranded DNA with single-base mismatch and triple-base mismatch to participate in the hybridization reaction, and the specific steps were the same as in Example 1.

[0049] The target DNA sequence is: 5'- GGCAGCAATT TCACCAGTAC TA -3'

[0050] The single base mismatch sequence is: 5'- GGCAGCAATT T G ACCAGTAC TA-3’

[0051] The three-base mismatch sequence is: 5'- GGCAGCAATT AGT CCAGTAC TA -3' (Mismatched bases are in italics)

[0052] Compare the signal response in the presence of target DNA, single-base mismatch DNA, and triple-base mismatch DNA in the presence of three different DNAs. Such as Figure 4 As shown, the single-base (B) and triple-base mismatches (C) produced much lower signal intensities compared to the signal increase produced by unmismatched target DNA (A), thus validating the constructed elect...

Embodiment 3

[0053] Example 3 Electrochemical DNA biosensor detects target HIV DNA in actual samples

[0054] Still targeting the above HIV gene fragments, a series of different concentrations of target HIV DNA were detected in the actual sample human serum, and the specific steps were the same as in Example 1.

[0055] Add a certain concentration of HIV DNA to human serum samples to obtain a series of serum samples with different concentrations of target DNA. After performing the reaction with the same operations and reagents as in steps (1)-(4), determine the conditions of target DNA at different concentrations. under the DPV graph (as Figure 5 shown). Analyze the relationship between the peak current value and the target DNA concentration in the DPV curve, and draw a linear fitting curve (such as Figure 6 shown). With the increase of the target DNA concentration, the oxidation peak current signal also increases. When the target DNA concentration is in the range of 10 fM to 10 pM, t...

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Abstract

The invention relates to an electrochemical method of detecting single-chain target DNA concentration based on G-quadruplex-heme compound and polymeric chain type amplification reaction, and belongs to the technical field of analytical chemistry. A capture probe and an auxiliary probe are designed, the two ends of the auxiliary probe each contain a nucleotide sequence complemented with the target DNA, and the middle of the auxiliary probe contains a base sequence capable of forming G-quadruplex. The capture probe and the target DNA recognize each other and are subjected to continuous polymeric chain type reaction to form chain-shaped polymer, the chain-shaped polymer is fixed to an electrode through the capture probe on the surface of the gold electrode, and a great number of G-quadruplex structures are introduced onto the surface of the electrode. Then, G-quadruplex and heme are combined to form the compound with powerful electrochemical signals, and the target DNA is detected through the corresponding relation among the electrochemical signals obtained through differential pulse voltammetry (DPV) scanning, the G-quadruplex-heme compound on the surface of the electrode and the concentration of the target DNA added into the system. HIV DNA in the sample is detected through the method, and an ideal effect is obtained. The electrochemical method has the advantages of being high in sensitivity and specificity.

Description

technical field [0001] The invention relates to an electrochemical method for detecting single-stranded target DNA concentration based on G-quadruplex-heme complex and polymerization chain amplification reaction, belonging to the technical field of analytical chemistry. Background technique [0002] The detection of specific gene sequences is of great significance in clinical diagnosis, disease prevention and treatment, environmental detection, food safety detection and so on. Traditional DNA detection methods have certain disadvantages, such as cumbersome operations, possible radioactive contamination, expensive detection instruments, and low sensitivity. Compared with traditional gene detection technology, electrochemical DNA biosensing technology has the advantages of simple operation, fast response speed, high sensitivity, environmental friendliness, good portability, and no contamination and damage to detection samples. These above-mentioned advantages make electrochem...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N27/327
CPCG01N27/3277
Inventor 周楠迪孙笑凡王淑玲田亚平
Owner ANHUI HUATENG AGRI TECH CO LTD
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