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Dual-enzyme-mediated cascade-signal-amplified ampicillin detecting aptasensor

An aptamer sensor, ampicillin technology, applied in the direction of determination/inspection of microorganisms, instruments, biological tests, etc., can solve the problems of expensive reagents, precision instruments and technicians, low sensitivity and specificity, and cumbersome analysis process. Strong scalability, high sensitivity and specificity

Active Publication Date: 2020-12-15
HUNAN AGRICULTURAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Currently, conventional detection strategies for AMP, such as high-performance liquid chromatography (HPLC), gas chromatography (GC), liquid chromatography-tandem mass spectrometry (HPLC-MS), enzyme-linked immunosorbent assay (ELISA), colorimetry, etc., exist. The disadvantages are low sensitivity and specificity, cumbersome analysis process, expensive reagents, and dependence on precision instruments and technicians.

Method used

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  • Dual-enzyme-mediated cascade-signal-amplified ampicillin detecting aptasensor
  • Dual-enzyme-mediated cascade-signal-amplified ampicillin detecting aptasensor
  • Dual-enzyme-mediated cascade-signal-amplified ampicillin detecting aptasensor

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] The preparation of embodiment 1 probe

[0047] Take 50 μL of 10 μM DNA1, bio-Apt, and H-DNA priming strands respectively, place them in a metal constant temperature oscillator, and activate at 70 °C for 10 min. After mixing evenly, incubate at 4°C for 2 h to complete the preparation of the probe, and store at 4°C for use. Then 10 μL of SYBR Green I with a final concentration of 5× was added to it, incubated at 4 °C for 30 min, and the fluorescence intensity at 520 nm of the sample was detected under the irradiation of 490 nm excitation light. In order to verify the hybridization between DNA1, bio-Apt, and H-DNA. Select the following samples: a) DNA1 and bio-Apt of equal volume and concentration (10 μL, 10 μM), and 10 μL of water; b) DNA1 and H-DNA of equal volume and concentration (10 μL, 10 μM), and 10 μL of water. The mixture was incubated at 4°C for 2 h. Then, 10 μL of SYBR Green I with a final concentration of 5× was added to the two systems, incubated at 4 °C f...

Embodiment 2T4

[0049] Activity verification of embodiment 2T4 ligase and Exo III

[0050] Test procedure: a) Mix 10 μL of 100 U / mL T4 ligase with 100 μL of detection probe, add 90 μL of PBS buffer solution, incubate at 37 °C for 30 min, and then inactivate at 70 °C for 20 min. After cooling, 10 μL of SYBR Green I with a final concentration of 5× was added, and incubated at 4 °C for 30 min. b) Mix 10 μL of 100 U / mL Exo III with 100 μL of detection probe, add 90 μL of PBS buffer solution, incubate at 37 °C for 30 min, and then inactivate at 70 °C for 20 min. After cooling, 10 μL of SYBR Green I with a final concentration of 5× was added, and incubated at 4 °C for 30 min. c) Take 10 μL of 100 U / mL T4 ligase and 100 μL of detection probe, and incubate at 16 °C for 30 min. Add 10 μL of 100 U / mL Exo III and 80 μL of PBS buffer solution, incubate at 37 °C for 30 min, and then inactivate at 70 °C for 20 min. After cooling, 10 μL of SYBR Green I with a final concentration of 5× was added, and incu...

Embodiment 3

[0052] The optimization of the DNA degradation reaction experimental condition of embodiment 3 double enzyme mediation

[0053] Take 5, 10, 20, 30, and 40 μL of 100 U / mL T4 ligase, 60 μL of detection probe, and incubate at 16 °C for 30 min. Add 10 μL of 100 U / mL Exo III and mix with 80 μL of PBS buffer, incubate at 37 °C for 30 min, and then inactivate at 70 °C for 20 min. After cooling, 10 μL of SYBR Green I with a final concentration of 5× was added, and incubated at 4 °C for 30 min. Detect the fluorescence intensity value of the solution at 520 nm.

[0054] see image 3 A, When the volume of T4 ligase is 10 μL, the fluorescence intensity of the solution reaches a plateau, which is the optimal reaction volume.

[0055] Take 10 μL of 100 U / mL T4 ligase and 60 μL of detection probe, and incubate at 16 °C for 10, 30, 50, 70 and 90 min, respectively. Add 10 μL of 100 U / mL Exo III and mix with 80 μL of PBS buffer, incubate at 37 °C for 30 min, and then inactivate at 70 °C for...

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Abstract

The invention relates to an ampicillin aptasensor based on dual-enzyme mediated cascade signal amplification. The ampicillin aptasensor is prepared by the following steps: taking a double-stranded DNAformed through mutually hybridizing bio-AP, DNA1 and H-DNA as a detection probe, and acquiring a complete APH chain under the assistance of a T4 DNA ligase and an exonuclease III. Through specific binding of biotin-avidin, the APH chain is captured in a microporous plate as an HCR initiating chain, then, hairpin DNA H1 and H2 can open in a cross manner to produce a double-stranded DNA polymer, afluorescent dye SYBR Green I indicator is added, and obvious fluorescence is produced when the fluorescent dye SYBR Green I indicator is embedded into the double-stranded DNA. Based on specific binding between a nucleic acid aptamer and a target object, detection on ampicillin is achieved through changing of fluorescence intensity. The method is high in sensitivity, good in specificity and high inaccuracy and can be applied to the detection on the ampicillin in contaminated foods such as milk.

Description

technical field [0001] The invention belongs to the technical field of biosensing and nucleic acid detection, and relates to the detection of ampicillin (AMP) in food, in particular to an aptamer sensor for ampicillin detection with dual enzyme-mediated cascade signal amplification. Background technique [0002] As ampicillin, one of the broad-spectrum β-lactam antibiotics, AMP has been widely used in the fields of medicine and bacterial infection treatment, and has the advantages of low toxicity, low cost and good clinical effect. In recent years, although AMPs have promoted the rapid development of animal husbandry and feed processing industries as growth regulators or anti-infectives, the illegal and excessive use of AMPs in animals and livestock has directly led to the use of antifungal drugs in the production of human and animal-sourced food. Contamination and residues have caused increasingly prominent health and safety issues, such as the emergence of hypersensitivity...

Claims

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Application Information

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IPC IPC(8): C12Q1/682G01N33/53G01N21/64
CPCC12Q1/682G01N33/53G01N21/6428G01N2021/6439
Inventor 石星波赵倩张光胤
Owner HUNAN AGRICULTURAL UNIV
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