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Preparation and detection method for electrochemical quantitative polymerase chain reaction detecting chip

A chain reaction and detection chip technology, which is applied in the field of electrochemical detection, can solve the problems of high cost, affecting the reliability of detection, and complicated operation of the detection process, and achieve the effects of low cost, reduced detection cost and high detection sensitivity

Inactive Publication Date: 2005-04-13
SOUTHEAST UNIV
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  • Abstract
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  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The detection process is complicated to operate, which affects the reliability of detection
In addition, fluorescence detection requires better optical detection system support, and the cost is relatively high in clinical applications.

Method used

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  • Preparation and detection method for electrochemical quantitative polymerase chain reaction detecting chip
  • Preparation and detection method for electrochemical quantitative polymerase chain reaction detecting chip
  • Preparation and detection method for electrochemical quantitative polymerase chain reaction detecting chip

Examples

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Effect test

example 1

[0037] Example 1: Combining electrochemical and PCR to detect the methylation status of p16 gene

[0038] 1. Preparation of electrodes and their probes: immobilize specific probes that can detect methylation status on gold electrodes. Firstly, the electrode was pretreated. The gold electrode was soaked in Piranha solution, acetone, and absolute ethanol for 0.5 min, then polished with a 0.05 μm Al2O3 abrasive pad, ultrasonically cleaned for about 1 min, and dried with nitrogen. Dip the gold electrode into the solution containing 100 μg / ml HS 2 ssDNA in TE buffer, placed at 4°C for 12 hours to obtain a single-stranded DNA modified electrode (HS 2 ssDNA / Au electrode).

[0039] 2. PCR amplification: After the DNA of the sample to be tested is extracted, an electrochemical indicator is added to it for ordinary PCR amplification.

[0040] 3. Detection of the amplified product: After the amplified product is combined with the probe on the electrode, under the action of the electro...

Embodiment 2

[0041] Implementation 2: Electrochemical PCR chip detection of fetal DNA in maternal plasma

[0042]1. Design several groups of gene probes and primer sequences to amplify and detect fetal specificity (specific segment on Y chromosome), and use common electrophoresis detection to screen out the best combination of primers and probes for electrochemical real-time detection.

[0043] 2. The method of making and fixing electrodes and fetal DNA-specific probes is the same as above

[0044] 3. Sample processing and PCR amplification: first extract and purify the DNA from the plasma sample, and then amplify it under a suitable temperature cycle (94°C, 30s-50°C, 30s-72°C, 30s). After the cycle, the relevant signals were collected and processed with an electrochemical instrument, and the results showed that the fetal DNA gradually increased with the increase of the number of PCR cycles. (Such as Figure 6 , a is negative control b, c, d, e are different samples)

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Abstract

The present invention is the electrochemical preparation process of quantitative PCR detection chip, and relates to the electrochemical detection technology for quantitative nucleic acid PCR chip. The preparation process includes preparing a micro electrode array on the surface of solid carrier; fixing nucleic acid capturing molecular probes on the electrodes of the micro electrode array; preparing closed micro cavity for carrying liquid in the electrode area of molecular probes; and connecting wires to the electrodes on the solid carrier. The detection process includes adding nucleic acid, enzyme, deoxyribonucleotide, electrochemically active matter and other reaction components into the micro cavity; setting the chip in large cavity for controlling temperature; detecting the current and voltage variance caused by reaction of the captured DNA probe molecule with constant potential instrument; and detecting the variance rule of PGR circulating process in different electrodes for PCR detection of genes.

Description

technical field [0001] The invention relates to an electrochemical detection technology of a nucleic acid quantitative polymerase chain reaction (PCR) chip, and the novel electrochemical PCR detection chip and detection method can be used for parallel quantitative amplification detection of multiple nucleic acids in the fields of biological science and medicine . Background technique [0002] With the development of life sciences, genome research is gradually deepening, and it will become possible to understand the differences in life, the occurrence and development of diseases, and the interaction between drugs and living organisms at the genetic level. The high-throughput detection and analysis technology of nucleic acid sequence information will become one of the core technologies in the field of life sciences such as medicine. People need to develop high-throughput, accurate, and low-cost genetic information detection methods. Recently, gene c...

Claims

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Application Information

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IPC IPC(8): G01N27/26G01N27/417
Inventor 陆祖宏葛芹玉刘全俊白云飞
Owner SOUTHEAST UNIV
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