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DNA or RNA detection system, detection method and application thereof

A detection method and system technology, applied in the field of chemical and biological sensing, can solve the problems of material toxicity, limited wide application, poor stability, etc., and achieve the effect of low detection limit

Inactive Publication Date: 2020-01-10
CIXI INST OF BIOMEDICAL ENG NINGBO INST OF MATERIALS TECH & ENG CHINESE ACAD OF SCI +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Although these methods have certain advantages in some aspects, they also have certain limitations, such as fluorescent or electrochemical group labeling, the need for expensive equipment, high toxicity of materials used, and poor stability, etc., which greatly limit the their wide application in clinical

Method used

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  • DNA or RNA detection system, detection method and application thereof
  • DNA or RNA detection system, detection method and application thereof
  • DNA or RNA detection system, detection method and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0051] Embodiment 1 Synthesis of carbon quantum dots and gold nanoparticles

[0052] (1) Carbon quantum dots (CQDs) synthesis: the final concentration of 10mM malic acid (1390.9mg) and 10mM urea (600.6mg) were evenly mixed in 10mL of N,N-dimethylformamide (DMF), and then The mixed solution was poured into a 100 mL autoclave and placed in an oven at 200° C. for 15 hours. The dark brown solution obtained by the reaction was centrifuged at 10,000 rpm for 10 minutes, and the supernatant was treated with ddH in a dialysis bag (molecular weight cut-off=1000Da). 2 O dialyzed for one week, followed by freeze-drying to obtain solid powder of CQDs.

[0053] (2) Synthesis of gold nanoparticles (AuNPs): AuNPs were prepared by the method reported in the literature. The specific preparation method is as follows:

[0054] 1. According to HCl:HNO 3 = 3:1 Prepare aqua regia in a beaker.

[0055] 2. Take a 100mL three-neck round bottom flask, put a magnetic stirring bar into the flask, fill...

Embodiment 2

[0063] Example 2 Design and Synthesis of Nucleic Acids for Detection

[0064] In this example, the wild-type target DNA is the human tumor suppressor gene TP53. The DNA sequence of TP53: 5'-AGGTGCGTGTTT-3', the target DNA sequence containing the SNP site is: 5'-AGG A GCGTGTTT-3' (mutation 4), 5'-AGGTGCG A GTTT-3' (mutation 8), 5'-AGGTGCGTGTT A -3' (mutation 12), the above sequences can be used without further purification; manually synthesize a complementary PNA chain with a length of 12 bases by solid-phase synthesis: TCC ACG CAC AAA, after cleavage in a peptide synthesis tube to obtain crude PNA Wash three times with anhydrous ether, centrifuge and discard the supernatant, dry the sample with nitrogen gas and dissolve it with ultrapure water. The sample was purified on HPLC, and the target peak was taken for mass spectrometry identification. The product of the target peak was collected after multiple purifications, and the product was lyophilized and dissolved in ultrapur...

Embodiment 3

[0069] (1) Detection of wild-type target DNA:

[0070] Mix specific PNA probes (0.8 μM) with different concentrations (0, 5, 10, 20, 30, 40, 60, 80, 100, 150, 600, 800 nM) of target DNA in Tris-HCl buffer for 10 min, then add 50 μL of AuNPs (5 μM ) and 10 μL CQDs solution. The solution to be tested was allowed to stand for 1 h to allow it to fully react. Samples were vortexed one by one before testing to ensure good dispersion. Record and analyze the change of fluorescence intensity with a fluorescence spectrophotometer, the excitation wavelength is 345nm. AuNPs as a fluorescence quencher can effectively quench the fluorescence of CQDs through FRET. The absorption band of AuNPs overlaps with the fluorescence spectrum of CQDs, and fluorescence energy transfer can occur. The fluorescence of quantum dots is quenched by AuNPs. When PNA was added, PNA induced the aggregation of AuNPs, the fluorescence of CQDs was restored, and a new UV absorption peak appeared at 640 nm. The ag...

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Abstract

This application discloses a DNA or RNA detection system. The DNA or RNA detection system is a saline solution system, and comprises peptide nucleic acid, gold nanoparticles and carbon quantum dots. The peptide nucleic acid can be hybridized with target DNA or RNA to form double strands. This application further discloses a DNA or RNA detection method, a DNA or RNA detection system, and application of the detection method. The method has characteristics of being fast, economic, simple, and convenient. Neither large-scale instrument and equipment is required, nor precise temperature control anda strict washing step are required. According to the method, a detection limit on wild-type target DNA is relatively low, the method can be applied to detection of DNA in a cell lysis solution, and single-base mismatch can be distinguished.

Description

technical field [0001] The application belongs to the technical field of chemical and biological sensing, and specifically relates to a DNA or RNA detection system, a detection method and their application, more specifically, the DNA or RNA detection system and detection method are based on peptide nucleic acid regulation of gold nanoparticles / Carbon quantum dot fluorescence energy transfer to detect DNA or RNA. Background technique [0002] As people's understanding of cancer has gradually deepened to the gene level, more and more tumor-related genes (proto-oncogenes and tumor suppressor genes) have been discovered. A large number of clinical studies have shown that the amplification, mutation and expression status of specific genes are closely related to the occurrence, development and metastasis of tumors and the effectiveness of tumor treatment drugs. Simple, rapid, economical, and sensitive genetic detection methods can monitor the amplification, mutation, and express...

Claims

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Application Information

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IPC IPC(8): G01N21/64C12Q1/6818
CPCC12Q1/6818G01N21/6428G01N21/6486
Inventor 高婷婷赵超邢淑张晓康
Owner CIXI INST OF BIOMEDICAL ENG NINGBO INST OF MATERIALS TECH & ENG CHINESE ACAD OF SCI
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