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Ultra-sensitive circulating nucleic acid detection system, kit and method for constructing multi-dimensional DNA enzyme matrix based on DNA three-chain mediation

A DNA enzyme and system technology, applied in the field of ultra-sensitive circulating nucleic acid detection system, can solve problems such as numerous influencing factors and complex blood environment, and achieve the effects of strong anti-interference, good detection performance and simple operation

Pending Publication Date: 2022-03-01
HUNAN INSTITUTE OF ENGINEERING
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Problems solved by technology

However, because the concentration of ctDNA in the blood is too small, and the blood environment is complex, and there are many influencing factors, it is still necessary to find a new method with better specificity, high sensitivity, easy operation, and economy. Of course, further clinical research and practice are needed.

Method used

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  • Ultra-sensitive circulating nucleic acid detection system, kit and method for constructing multi-dimensional DNA enzyme matrix based on DNA three-chain mediation
  • Ultra-sensitive circulating nucleic acid detection system, kit and method for constructing multi-dimensional DNA enzyme matrix based on DNA three-chain mediation
  • Ultra-sensitive circulating nucleic acid detection system, kit and method for constructing multi-dimensional DNA enzyme matrix based on DNA three-chain mediation

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Embodiment 1

[0064] Source of probes: Shanghai Sangon Bioengineering Co., Ltd. was commissioned to synthesize the probes and ctDNA shown in Table 1.

[0065] 1. Formation of multi-dimensional network DNA nanowire structure

[0066] The capture probe capture and the hairpin probes H1, H2, and L3 were heated to 90°C and incubated for 10 minutes, and slowly gradient annealed to room temperature to ensure the formation of the hairpin structure, and stored at 4°C before use. Take a clean and sterilized 0.2mL EP tube, add ctDNA 0.4μL (10μM), 1.2μL capture probe Capture (10μM), 1.2μL H1 (10μM), 1.2μL H2 (10μM), 0.8μL L3 (10μM), 15.2 μL of Tris-Ac buffer (20 mM), mixed and shaken, incubated at 37°C for 3 hours, and then incubated at 30°C for 4 hours, to form a multidimensional three-dimensional network DNA nanowire structure.

[0067] 2. Construction of intermolecular split G-quadruplex-heme DNase matrix

[0068] Add 6 μL L1 (10 μM), 6 μL L2 (10 μM), and 16 μL Tris-Ac buffer (20 mM) to the above...

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Abstract

The invention provides an ultra-sensitive circulating nucleic acid detection system, kit and method for constructing a multi-dimensional DNA enzyme matrix based on DNA three-chain mediation, and relates to the technical field of gene detection. According to the invention, three-chain DNA is taken as the core of a signal amplification technology, and hybridization chain reaction (HCR) amplification and G-quadruplex-heme DNA enzyme enzymatic signal amplification are integrated. In the presence of ctDNA, a multi-dimensional mesh hybridization chain reaction is initiated through three-chain DNA, a three-dimensional mesh DNA nanowire structure is formed, the structure has obvious refined layering and spatial distribution rules, low-level branches extend from branched chains, and each branch can further extend to form lower-level sub-branches. On the basis, an intermolecular split G-quadruplex-heme DNA enzyme matrix is constructed through three-chain DNA mediation, so that ultra-sensitive detection of circulating nucleic acid is realized, and the kit has the characteristics of non-enzymatic signal amplification and visual detection and has great development potential.

Description

technical field [0001] The invention belongs to the technical field of gene detection, and in particular relates to an ultrasensitive circulating nucleic acid detection system, kit and method based on the construction of a multi-dimensional DNA enzyme matrix mediated by DNA triple strands. Background technique [0002] In 2020, the most new cancer in the world is breast cancer. At the same time, about 10 million people will die from cancer in 2020, of which 685,000 patients will die from breast cancer. Most breast cancer patients are already in the middle and late stages when they are discovered, and their mortality rate is as high as 50%, while the long-term cure rate of early breast cancer can reach more than 90%. Therefore, the early detection, diagnosis and treatment of breast cancer are the key to improving the cure rate and reducing the mortality rate. [0003] At present, breast cancer diagnosis methods mainly include: mammography, ultrasound imaging, biopsy, infrare...

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Application Information

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IPC IPC(8): C12Q1/6886C12Q1/6825C12Q1/682C12N15/11
CPCC12Q1/6886C12Q1/6825C12Q1/682C12Q2525/301C12Q2545/114C12Q2563/125Y02A50/30
Inventor 张何张培柔马思元傅昕
Owner HUNAN INSTITUTE OF ENGINEERING
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