Method for simultaneously detecting two types of HIV DNA through DNA-directed color-variable silver nano-clusters

A silver nano-cluster and detection method technology, applied in the fields of molecular biology and nucleic acid chemistry, can solve the problems of complicated operation, high modification cost, increased difficulty, etc., and achieve the effects of simple preparation, large emission range, and economic simplicity.

Active Publication Date: 2018-12-25
XIANGTAN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the high cost of modification, the time-consuming separation and purificati

Method used

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  • Method for simultaneously detecting two types of HIV DNA through DNA-directed color-variable silver nano-clusters
  • Method for simultaneously detecting two types of HIV DNA through DNA-directed color-variable silver nano-clusters
  • Method for simultaneously detecting two types of HIV DNA through DNA-directed color-variable silver nano-clusters

Examples

Experimental program
Comparison scheme
Effect test

Example Embodiment

[0045] Example 1

[0046] Design of fluorescent probe based on color-changing silver nanoclusters

[0047] (1) Design of DNA probe in this embodiment:

[0048] 1) The DNA probe is composed of two partially complementary ssDNA, namely cDNA and P strand;

[0049] 2) The cDNA chain consists of four parts: G-rich sequence, C-rich sequence of synthesized AgNCs, sequence complementary to HIV-2 sequence and base sequence paired with HIV-1 complementary chain part;

[0050] 3) The P chain is composed of four parts: the sequence of AgNCs that can synthesize yellow-green fluorescence, the sequence of AgNCs that can synthesize orange-red light, the sequence complementary to the HIV-1 sequence and the base sequence paired with the complementary chain of HIV-2. .

[0051] The DNA sequence involved in this example is as figure 2 Shown.

[0052] (2) Synthesis of DNA templated AgNCs:

[0053] 10 μL of sequence P (500 nM) was dissolved in 170 μL of PBS buffer (20 mM, pH 7.0). Then, the P solution was he...

Example Embodiment

[0054] Example 2

[0055] Verification of the feasibility of detecting two kinds of HIV DNA simultaneously with fluorescent probe based on silver nanocluster

[0056] The present invention simultaneously uses luminescent AgNCs with 565nm and 630nm emission peaks as templates to detect HIV-1 and HIV-2. The working principle is as follows figure 1 .

[0057] In this example, we use fluorescence spectroscopy to verify the feasibility of the principle.

[0058] The conditions of fluorescence spectroscopy detection are:

[0059] The concentration of the test substance: HIV-1 and HIV-2 are 500nM respectively

[0060] Instrument: RF-5301PC Fluorescence Spectrophotometer

[0061] Instrument parameters: excitation wavelength: 500nm and 580nm, emission wavelength: 565nm and 630nm, excitation slit: 5nm, emission slit: 5nm

[0062] The specific experimental operations are:

[0063] (1) First, in the absence of target DNA (HIV-1 and HIV-2), hybridize 10 μL of P (500 nM) and 10 μL of cDNA solution for mi...

Example Embodiment

[0067] Example 3

[0068] Optimization of experimental conditions

[0069] (1) Optimization of PH

[0070] Since the pH of the buffer has a certain influence on the synthesis of AgNCs, the pH environment of the experiment must be optimized. We chose five pH values ​​of 6.4, 6.8, 7.0, 7.2, and 7.6 to make a detailed optimization. The difference in fluorescence difference under different pH values ​​is from Figure 4 It can be clearly seen that when the pH is 7.0, the obtained fluorescence difference is the highest, so in the next experiment, choose 7.0 as the optimal pH of the system. (I 0 Is the fluorescence intensity before adding the target DNA, I is the fluorescence intensity after adding the target).

[0071] (2) Optimization of the dosage ratio of reagents and DNA templates

[0072] After the pH is determined, a series of optimization work is done on the dosage ratio of reagents and DNA templates, so that the synthesized AgNCs have the best fluorescence effect. In the experiment...

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Abstract

The invention discloses a method for simultaneously detecting two types of HIV DNA by using silver nano-clusters (AgNCs) as a label-free fluorescence biosensing platform. The fluorescence biosensing platform is based on two features of the silver nano-clusters: (1) fluorescence of the silver clusters can be enhanced in multiples by a rich guanine (G) sequence; and (2) the fluorescence can be remarkable enhanced by mutual extrusion of two neighboring AgNCs. Different templates of the synthetic AgNCs are designed at two ends of a single chain, and two different signals with different luminous peaks are obtained so as to avoid signal interference. A DNA chain mispaired with the single chain is designed as a template of which one end is a rich-G sequence, and the other end is a synthetic silver cluster extrusion pair. A target object is completely paired with complementary DNA, high fluorescence of the AgNCs is damaged, and a fluorescence signal is weakened. Only one type of the color-variable silver nano-clusters needs to be used as a fluorescence probe so that two wider detection ranges (0.2-700 nm) can be obtained. A limit of detection is as low as 0.2 nm, and high sensitivity detection of multiple types of DNA or MiRNA can be further realized, so accuracy and practicability of bioanalysis are improved.

Description

technical field [0001] The invention belongs to the fields of molecular biology and nucleic acid chemistry, and relates to a method for simultaneously detecting two kinds of HIV DNA by DNA-guided color-changing silver nanoclusters. Background technique [0002] When human immunodeficiency virus (HIV) infects and destroys the immune system of the host, the function of the immune system will gradually lose and be accompanied by the occurrence of many diseases, and even cause death in severe cases. Therefore, accurate detection of HIV genes is of great significance for early detection and timely treatment of infected persons. [0003] For decades, many methods for single-target HIV gene detection have been developed, including surface-enhanced Raman scattering (SERS), fluorescence, colorimetry, and electrochemical techniques. Among them, the fluorescence method is widely used because of its simple operation and intuitive observation. Das et al. designed a new type of fluoresc...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/6813
CPCC12Q1/6813C12Q1/701C12Q2563/107C12Q2563/137
Inventor 蔡昌群邹容龚行韩云鹏
Owner XIANGTAN UNIV
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