Kanamycin rapid testing test paper for aptamer recognition and functionalized magnetic microsphere separating pretreatment and preparation and application thereof

A nucleic acid aptamer and magnetic microsphere technology, used in food and medical analysis and environmental fields, can solve problems such as being unsuitable for on-site detection and scale-up production, and achieve a convenient expansion and application, enhanced practical application effect, and improved detection efficiency. Effect

Active Publication Date: 2018-05-08
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There are some shortcomings in the application of these detection methods, and they are not suitable for on-site detection and scale-up production. Therefore, a convenient, fast, economical, accurate and high-sensitivity method that can be used to detect kanamycin residues in food should be established as soon as possible. Detection method that can effectively improve the safety of food of animal origin

Method used

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  • Kanamycin rapid testing test paper for aptamer recognition and functionalized magnetic microsphere separating pretreatment and preparation and application thereof
  • Kanamycin rapid testing test paper for aptamer recognition and functionalized magnetic microsphere separating pretreatment and preparation and application thereof
  • Kanamycin rapid testing test paper for aptamer recognition and functionalized magnetic microsphere separating pretreatment and preparation and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1 Kanamycin rapid detection test paper based on nucleic acid aptamer and magnetic microsphere separation pretreatment

[0041] Including pretreatment of magnetic microspheres, preparation and functionalization of gold nanoparticles, pretreatment of gold standard pads and sample pads, pretreatment of nitrocellulose membranes, assembly of test strips, and detection of color development of test strips; The steps are:

[0042] (1) Preparation of functionalized magnetic microspheres

[0043] Firstly, a cDNA complementary to the aptamer Apt was designed, and the cDNA and Apt were placed in a metal bath at 95°C for 3 min to open the complementary part of the cDNA to form a single strand, which facilitated the complementary hybridization of the cDNA and Apt in the next step. The cDNA and Apt were mixed thoroughly, and incubated in a constant temperature incubator at 37°C for 1 h to fully combine the cDNA and Apt. Since the surface of the magnetic microspheres is modif...

Embodiment 2

[0055] Embodiment 2 Determination of kanamycin standard solution with test strips

[0056] (1) Pretreatment of magnetic microspheres

[0057] First, design a section of cDNA that is complementary to the aptamer Apt, and place the cDNA and Apt in a metal bath at 95°C for 3 minutes to open the complementary part of the cDNA to form a single strand, which is convenient for the complementary hybridization of the cDNA and Apt in the next step. The cDNA and Apt were thoroughly mixed, and incubated in a constant temperature incubator at 37°C for 1 h to fully combine the cDNA and Apt. Since the surface of the magnetic microspheres is modified with streptavidin and the 5' end of Apt is modified with biotin, Apt can be coupled to the surface of the magnetic microspheres by streptavidin-biotin interaction. The magnetic microspheres were washed 5 times with 10 mmol / L, pH 7.4 PBS buffer, combined with the above-mentioned Apt-cDNA mixture, and shaken at 37°C for 1 h to avoid the magnetic m...

Embodiment 3

[0059] Example 3 Specificity Verification of Test Strip Detection Method

[0060] The test strips prepared under the same conditions were used to detect streptomycin (STR), ampicillin (AMP), neomycin (NEO), gentamicin (GEN), and kanamycin at a concentration of 5 mmol / L. Mycin (KAN) and water, after detection, compare the color development and peak area of ​​the detection line to verify the specificity of the test strip detection. Different types of antibiotics are used for the detection line peak area diagram after detection, such as Figure 4 shown. exist Figure 4It can be seen that the peak area of ​​the detection line is significantly higher than that of several other antibiotics when detecting kanamycin, thus proving that the detection method has high specificity for the detection of kanamycin.

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Abstract

The invention discloses Kanamycin rapid testing test paper for aptamer recognition and functionalized magnetic microsphere separating pretreatment and preparation and application thereof, and belongsto the field of environment, food and medicine analysis. The good separating effect of magnetic microspheres, high affinity between an aptamer (Apt) and drones and indication mark features of Au nanoparticles (AuNPs) are utilized, quantitative analysis of Kanamycin can be achieved by observing the developing shallowness of a detecting line, and meanwhile, a colloidal gold quantometer is utilized for quantitative analysis. cDNA can be mediated to form a sandwich structure to capture AuNPs at the detecting line, the number of captured AuNPs and cDNA are then in linear correlation with the concentration of Kanamycin, and then the content of Kanamycin can be judged according to the developing shallowness of the detecting line. A test paper method for detecting Kanamycin is simple and convenient, the detection is not limited by environment conditions, the result is visualized, it takes only 20 min for detection, and application and popularization are facilitated.

Description

technical field [0001] The invention relates to a rapid detection test paper for kanamycin based on nucleic acid aptamer recognition and functionalized magnetic microsphere separation pretreatment and its preparation and application, belonging to the fields of environment, food and medicine analysis. Background technique [0002] Antibiotics are secondary metabolites metabolized by bacteria, fungi, animals and plants, and have anti-pathogen properties. Aminoglycoside antibiotics are widely used due to their relatively low cost compared with other types of antibiotics, better water-soluble effect, broad antibacterial spectrum, and good therapeutic effect on common bacterial diseases. Kanamycin (kanamycin) is one of the most frequently used aminoglycoside antibiotics, and kanamycin is passed through Streptomyces ( Streptomyces kanamyceticus ) produced by fermentation and widely used in the treatment of infections caused by bacteria. The mechanism of action of kanamycin is to...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/558G01N33/531G01N33/532
CPCG01N33/531G01N33/532G01N33/558
Inventor 周楠迪刘晶曾静怡田亚平
Owner JIANGNAN UNIV
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