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DNA sensor for detecting staphylococcus aureus as well as preparation method and application of DNA sensor

A sensor and biosensor technology, applied in instruments, measuring devices, scientific instruments, etc., can solve the problems of false negatives in experiments, difficult to popularize and apply, expensive advanced detection equipment and operating costs, to improve dispersion and promote the transfer of electrons , the effect of reducing the detection limit

Active Publication Date: 2015-05-20
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] At present, the detection methods of Staphylococcus aureus generally include traditional bacterial culture methods and biochemical identification, immunological methods, fluorescent quantitative PCR method, fluorescence in situ hybridization (FISH, Fluorescent in Situ hybridization) and gene chip method, etc. Each method has its own advantages but at the same time there are deficiencies at different levels. For example, the FISH method can be directly applied to the detection of complex samples without extracting the DNA of the sample, directly retaining the integrity of the sample DNA; but some of the samples Factors such as the autofluorescence of the bacteria itself, the attenuation of the fluorescent probe signal, and too few fixed microbial cells may lead to false negative results in the experiment; the gene chip method is a nucleic acid detection technology developed rapidly in recent years, which is fast, convenient and simple. and other advantages have become the bright spot of this technology, but because this technology requires expensive advanced detection equipment and operating costs, it is difficult to popularize and apply

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  • DNA sensor for detecting staphylococcus aureus as well as preparation method and application of DNA sensor
  • DNA sensor for detecting staphylococcus aureus as well as preparation method and application of DNA sensor
  • DNA sensor for detecting staphylococcus aureus as well as preparation method and application of DNA sensor

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Embodiment 1

[0036] The preparation method of embodiment 1 DNA sensor

[0037] Dissolve 5 mg of carbon nanotubes in 5 mL of 1% chitosan, disperse ultrasonically for 2 h to obtain a carbon nanotube solution with a concentration of 0.001 (m / v), drop 5 μL onto the surface of a clean gold electrode, and incubate at 37° C. for 30 min. 1% (v / v) chloroauric acid was dissolved in 0.1M potassium nitrate to prepare a 3.3mM nano-gold solution, and electroplating was carried out by controlled potential electrolytic coulomb method to prepare a nano-material modified electrode. figure 1 It is a transmission electron microscope image of gold nanoparticles on the surface of the electrode, from which it can be seen that the gold nanoparticles are evenly dispersed on the surface of the electrode.

[0038] On the surface of the modified gold electrode, drop 5 μL of 1.0x10 -6 One end of M contains thiol-containing probe ssDNA (probe ssDNA sequence is shown in SEQ ID NO.1) 5′-HS-C6-GCGAGG GCGATT GAT GGT GATAC...

Embodiment 2

[0040] The electrochemical characterization of embodiment 2DNA sensor

[0041] Take out the prepared DNA sensor electrode, add 5 μL of different concentrations of complementary ssDNA standard substances successively for hybridization, and incubate at 37°C for 30 minutes, use cyclic voltammetry and AC impedance method to investigate the changes of the working electrode interface, and the electrode surface characterization. The result is as figure 2 shown.

[0042] CV (cyclic voltammetry) test conditions: voltage 0.2-0.6V, scan rate 0.1V / s; EIS (electrochemical impedance spectroscopy) test conditions: initial amplitude 0.05V, frequency 1-100kHz, rest time 2s. The reaction medium liquid is 2.5mmol Fe(CN) 6 3- / 4- solution (all tests were performed at room temperature).

[0043] from figure 2 In (A), it can be seen that the current values ​​of the three lines a, b, and c gradually increase, indicating that the dropped carbon nanotubes and the gold nanometers on the electrop...

Embodiment 3

[0044] Application of embodiment 3 DNA sensor

[0045] The application method of the sensor prepared by the present invention is as follows:

[0046] Specifically: (1) Drop different concentrations of ssDNA complementary to the probe ssDNA onto the DNA sensor, wash with PBS after incubation, and then wash at 5.0×10 -5 Soak in the methylene blue solution of M concentration for 12min, wash and dry with nitrogen, and measure the reduction current change by differential pulse voltammetry (DPV) in PBS to obtain the standard curve of complementary ssDNA and current value change; (2) the sample to be tested Drop onto the DNA sensor, wash with PBS after incubation, and then in 5.0×10 -5 Soak in methylene blue solution of M concentration for 12min, wash with nitrogen and blow dry, measure the change of current value in PBS, calculate the concentration of target ssDNA in the sample according to the standard curve obtained in the previous step, so as to determine the presence and qualit...

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Abstract

The invention discloses a DNA sensor for detecting staphylococcus aureus as well as preparation method and application thereof, belonging to the technical field of rapid pathogenic bacteria detection. A electrochemical carbon nano tube / gold nano particle composite membrane DNA sensor is prepared by utilizing the biological fixation effect and electrochemical electron transfer rate enhancing function of the gold nano particle, and is capable of effectively improving the detection sensitivity. According to the invention, a detection method, having high universality, high sensitivity and high accuracy, and detection conditions for staphylococcus aureus are preliminarily established through a detection technology in which specific staphylococcus aureus gene sequence is synthesized to be used as probe ssDNA so as to form a hybridization system with a complementation target ssNDA segment and methylene blue is used as a hybridization indicator.

Description

technical field [0001] The invention relates to a DNA sensor for detecting Staphylococcus aureus and its preparation and application, belonging to the technical field of rapid detection of pathogenic bacteria. Background technique [0002] Staphylococcus aureus exists widely in nature, and can be found in air, water, dust, and human and animal excrement. Staphylococcus aureus is an important pathogenic microorganism. It can produce more virulence factors such as toxins and enzymes, and is highly virulent. Infection by Staphylococcus aureus can cause various diseases, including food poisoning, pseudomembranous Acute enteritis, scalded skin syndrome, toxic shock syndrome, suppurative inflammation and sepsis, etc. have caused great threats and losses to human beings, and have attracted widespread attention. In recent years, the U.S. Centers for Disease Control reported that infections caused by Staphylococcus aureus accounted for the second place, second only to Escherichia co...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C25D15/00G01N27/48G01N27/327
Inventor 孙秀兰孙艳格张银志
Owner JIANGNAN UNIV
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