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Urine microalbumin detection method based on DNA aptamer and kit thereof

A technology for urinary microalbumin and nucleic acid aptamers, which is applied in the biological field and achieves the effects of wide application, high affinity and specificity, and simple and fast operation.

Active Publication Date: 2019-12-17
CAPITAL NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

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Method used

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  • Urine microalbumin detection method based on DNA aptamer and kit thereof
  • Urine microalbumin detection method based on DNA aptamer and kit thereof
  • Urine microalbumin detection method based on DNA aptamer and kit thereof

Examples

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Embodiment 1

[0032] Embodiment 1. according to the method of the present invention, adopt longer HSA nucleic acid aptamer (table 1, Cy3-HSA-apt) to the determination of the working curve that different concentrations of HAS are detected

[0033] Specific methods include such as figure 1 The steps shown are as follows:

[0034] (1) Hybridization of nucleic acid aptamers and complementary probes: Cy3-HSA-apt (final concentration is 0.1 micromoles per liter (μM) and biotin-EG18-c-pool (Table 1, final concentration is 0.15 μM) Place in 1-fold concentrated binding and washing buffer solution (1×B&W) (5 millimolar per liter (mM) trishydroxymethylaminomethane (Tris), 0.5 mM ethylenediaminetetraacetic acid (EDTA), 1 mole per liter liter (M) sodium chloride (NaCl)), hybridization at a material ratio of 1:1.5: 95°C for 10 minutes, slowly cooled to room temperature.

[0035] (2) Immobilization of the nucleic acid aptamer on the magnetic beads: the glass bottle containing the streptavidin-coated mag...

Embodiment 2

[0040] Example 2. According to the method of the present invention, using a longer HSA nucleic acid aptamer (Table 1, Cy3-HSA-apt) to perform fluorescence detection on normal human urine with standard additions of 30nM and 300nM HSA respectively

[0041] (1) hybridization of nucleic acid aptamer and complementary probe: same as step (1) of embodiment 1;

[0042] (2) Immobilization of the nucleic acid aptamer on the magnetic beads: same as step (2) of Example 1;

[0043] (3) Divide the magnetic beads obtained in (2) into two groups: 1×Abb group: add HSA to the urine diluted 10 times (the final concentration of HSA is 0nM, 30nM, 300nM) to make the solution in each tube The final volume was 110 μL, mixed slowly, and incubated at room temperature for 1 hour.

[0044] (4) Fluorescence measurement: same as step (4) of Example 1.

[0045] The normal reference value range of HSA takes 24h urine albumin as the measurement standard, and its normal reference range is 30-300mg / 24h. The...

Embodiment 3

[0046] Embodiment 3. According to the method of the present invention, the determination of the working curve of detecting different concentrations of HSA using shorter HSA nucleic acid aptamers (Table 1, Cy3-HSA-apt-sh)

[0047] The experimental operation is the same as in Example 1, except that the HSA aptamer (Cy3-HSA-apt) used in Example 1 is replaced by a truncated HSA aptamer (Cy3-HSA-apt-sh), and The final concentration of HSA in the system is 0nM, 30nM, 80nM, 100nM, 300nM, 600nM, 800nM.

[0048] Such as Figures 4A-4B As shown, the fluorescence signal increases with the increase of HSA concentration, close to a linear relationship within 800nM HSA, and the detection limit is 80nM. The sensitivity of the detection was slightly lower than that of using a longer HSA aptamer (Cy3-HSA-apt).

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Abstract

The invention relates to a urine microalbumin detection method based on a DNA aptamer and a kit thereof. The method comprises the following steps: (1), carrying out hybridization of HSA aptamer with acomplementary chain; (2), immobilizing the hybridization chain on a solid phase; (3), incubating a magnetic bead with the immobilized hybrid chain with a to-be-detected sample; (4), carrying out fluorescence testing. According to the method disclosed by the invention, on the basis of the characteristic of large conformational structure change under the condition of combination of the structural switch type HSA aptamer with the HAS as well as relatively high affinity and specificity, when the urine contains high-concentration HAS, the aptamer of the HSA bound to the solid phase makes to conformation change due to combination with the HAS and thus is separated from the solid phase to enter a solution; the HSA in the urine can be quantitatively detected according to the intensity of a fluorescence signal of the solution and the detection sensitivity is 40nM. The detection method is simple to operate and does not require expensive instrument, so that the cost is low; the detection sensitivity meet the requirement of clinical detection; and the urine microalbumin detection method has the great practical application value.

Description

technical field [0001] The invention relates to a method for detecting urine microalbumin based on a DNA nucleic acid aptamer and a kit thereof, belonging to the field of biotechnology. Background technique [0002] Albumin is one of the important plasma proteins with a molecular weight of 69 kilodaltons (kD). Under normal circumstances, albumin cannot cross the glomerular basement membrane, and its concentration in the urine of healthy people is very low. However, when the glomerular capillary endothelium is damaged, albumin can leak out of the blood and into the urine. There are three common ways to express the normal reference range of human serum albumin (HSA): (1) urine albumin / creatinine at one point, reference range: 30-300 mg / g (mg / g); (2) 24-hour ( h) Reference range of urine albumin: 30-300 mg / 24h; (3) Reference range of 4-hour urine albumin: 20-200 micrograms / minute (μg / min). Microalbuminuria (MAU) refers to the presence of microalbumin (HSA) in the urine in exc...

Claims

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Application Information

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IPC IPC(8): G01N33/50G01N33/68
CPCG01N33/50G01N33/68G01N2800/347
Inventor 娄新徽乔娜
Owner CAPITAL NORMAL UNIVERSITY
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