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Kit for detecting methylation degree of DNA (Deoxyribonucleic Acid) based on gold nanoparticle probe as well as detection method and application of kit

A gold nanoparticle and probe detection technology, applied in the fields of biological detection and medicine, can solve the problems of complex precision instruments and difficult detection results

Inactive Publication Date: 2017-02-01
周宏灏
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, the methods used for DNA methylation detection mainly include pyrosequencing, BSP, and methylation-specific PCR. However, the existing DNA methylation detection methods require the help of complex and sophisticated instruments, which are difficult to detect in real time and quickly. result

Method used

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  • Kit for detecting methylation degree of DNA (Deoxyribonucleic Acid) based on gold nanoparticle probe as well as detection method and application of kit
  • Kit for detecting methylation degree of DNA (Deoxyribonucleic Acid) based on gold nanoparticle probe as well as detection method and application of kit
  • Kit for detecting methylation degree of DNA (Deoxyribonucleic Acid) based on gold nanoparticle probe as well as detection method and application of kit

Examples

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Effect test

Embodiment 1

[0047] The kit for detecting the degree of DNA methylation provided in this example is aimed at the detection of the degree of DNA methylation of the PAX1 gene. PAX1 is a tumor suppressor gene, and hypermethylation of its promoter region is often used in the diagnosis and treatment of some cancers. For screening, especially in cervical cancer, studies have shown that the combination of PAX1 gene methylation detection and HPV detection can reduce the misdiagnosis rate of cervical cancer and greatly improve the diagnostic level of cervical cancer, so the PAX1 gene is selected. The kit includes gold nanoparticle probe, gold nanoparticle sol and downstream primers.

[0048] The preparation method of gold nanoparticle sol is: used glassware is soaked overnight through aqua regia; During preparation, add 100mL deionized water and 4.8mL mass fraction in the 250mL three-neck flask and be 1.0% chloroauric acid aqueous solution, the flask is placed On a magnetic stirrer, add a condensin...

Embodiment 2

[0068] The kit and detection method for detecting the degree of DNA methylation provided in this example are the same as those in Example 1, except that the DNA to be tested used in this example is obtained from cervical exfoliated cells of patients with cervical cancer. Through colorimetric detection, the color change of the obtained system after adding NaCl solution is not obvious, and it is still wine red after 6 minutes, which is very close to the color of 100% methylation percentage in the color comparison card, judging the DNA to be tested. The degree of methylation is close to 100%. The methylation percentage of the DNA to be tested was detected by ultraviolet-visible light spectrophotometry, and the calculated methylation percentage was 85.23%.

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Abstract

The invention relates to a kit for detecting the methylation degree of DNA (Deoxyribonucleic Acid) based on a gold nanoparticle probe as well as a detection method and application of the kit. The kit comprises the gold nanoparticle probe, wherein the gold nanoparticle probe comprises gold nanoparticles and an upstream primer for performing methylation specific polymerease chain reaction; sulfydryl is modified at the 5'-terminal of the upstream primer; the upstream primer is coupled with the gold nanoparticles by the sulfydryl; the particle sizes of the gold nanoparticles are 10 to 15nm. According to the kit as well as the detection method and application thereof disclosed by the invention, by designing a standard color chart, the methylation degree of to-be-detected DNA can be detected quickly, simply, conveniently and economically in real time according to the color of the system; in addition, a standard curve of ultraviolet absorbancy degree and DNA methylation percentage is designed; related data are substituted into the standard curve by an ultraviolet visible light absorbance spectrum of a test system, so that an accurate DNA methylation percentage can be obtained; no valuable instrument is needed, and a detection method is simpler, more convenient and more economical; higher practical value is obtained.

Description

technical field [0001] The invention relates to the fields of biological detection and medicine, in particular to a kit for detecting the degree of DNA methylation based on a gold nanoparticle probe, a detection method and an application thereof. Background technique [0002] As one of the main forms of epigenetics, DNA methylation is closely related to the occurrence and development of cancer. DNA methylation is the selective addition of a methyl group to the fifth C atom of cytosine in the CpG dinucleotide in the genome under the catalysis of methyltransferase to form a 5-methylcytosine structure, which is common in genes The 5'-CG-3' sequence. DNA methylation does not change the base sequence of the gene, but it can affect the expression of the gene. Studies have shown that the occurrence and development of cancer is closely related to abnormal DNA methylation of tumor suppressor genes. In normal cells, tumor suppressor genes regulate cell growth, repair DNA, and contr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/6858C12Q1/6886C12Q2600/154C12Q2563/137C12Q2563/155
Inventor 黄金谭志荣俞竟周宏灏
Owner 周宏灏
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