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A homogeneous biosensing method for detecting kanamycin and its application

A kanamycin and biosensing technology, applied in the field of biological analysis, can solve the problems of poor repeatability, cumbersome operation and high cost, and achieve the effects of high analytical sensitivity, overcoming cumbersome operation and short detection time.

Active Publication Date: 2021-03-16
HUBEI NORMAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] The purpose of the present invention is to provide a homogeneous biological method for simple and rapid detection of kalamycin content in samples, aiming at the problems of cumbersome operation, high cost, long time and poor repeatability of the current detection method for kalamycin. Sensitive method, the method is easy to operate, low in cost, high in sensitivity, good in stability, short in analysis time, and low in requirements for operators. Value

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  • A homogeneous biosensing method for detecting kanamycin and its application

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Embodiment 1

[0018] A homogeneous biosensing method for detecting kanamycin of the present embodiment comprises the following steps:

[0019] (1) G-quadruplex characteristic sequence and kanamycin (Kana) nucleic acid aptamer hybridization double-strand preparation

[0020] Add 990 µL of 10 mM pH=7.4 tris(hydroxymethyl)aminomethane buffer solution to the PV tube, which contains 200 mM NaCl, 10 mM KCl, 1% DMSO and 0.05% Triton X- 100, then add 5 µL 10 µM G-quadruplex DNA strand (S1) and 5 µL 10 µM Kanamycin (Kana) nucleic acid aptamer (S2) solution, the G-quadruplex DNA strand (S1) The base sequence is 5'-GGG TAG GGC GGG TTG GGA ACC TCAAGA CCA CTT GGA CAT TTT-3', and the base sequence in the kanamycin nucleic acid aptamer (S2) is 5'-TGT CCAAGT GGT CTT GAG GTT TTTT-3', shake at room temperature for 45 minutes to obtain aptamer hybrid double-stranded complex solution for use;

[0021] (2) Homogeneous reaction to determine the content of kanamycin in the standard solution

[0022] Take 100 µ...

Embodiment 2

[0025] The detection of kanamycin content in the milk powder sample of embodiment 2

[0026] According to the working curve obtained in Example 1, the content of kanamycin in the milk powder sample was detected. Weigh 1 g of commercially available milk powder and dissolve it in 5 mL of 20 mM pH 7.4 Tris-HCl buffer solution, which contains 100 mM NaCl, 2 mM MgCl 2 and 5mM KCl, take 100µL of the dissolved milk powder solution, add 20% acetic acid to it to adjust the pH to 4.6, centrifuge after 20min to remove the coagulated protein and fat in the sample, and filter the sample with a 0.22μm filter membrane solution, and re-adjust the sample solution to pH = 7.4; take 50 µL of the treated sample solution, add 100 µL aptamer hybridization double-stranded complex solution, 5 µL 2 µM kanamycin nucleic acid aptamer partial complementary sequence ( S3), 5 µL 5U / µL exonuclease III (Exo III) and 5 µL 1 µM hemin (hemin), the base sequence in the partially complementary sequence (S3) of t...

Embodiment 3

[0031] Example 3 Detection of Kanamycin Content in Honey Samples

[0032] According to the working curve obtained in Example 1, the content of kanamycin in the honey sample was detected. Weigh 2 g of commercially available honey and dissolve it in 4 mL of 20 mM pH=7.4 Tris-HCl buffer solution, which contains 100 mM NaCl, 2 mM MgCl 2 and 5mM KCl; then filter the sample solution with a 0.22μm filter membrane; take 50μL of the filtered sample solution, add 100μL aptamer hybridization double-strand complex solution, 5μL 2μM kanamycin nucleic acid aptamer partially complementary sequence (S3), 5 µL 5U / µL exonuclease III (Exo III), and 5 µL 1 µM hemin (hemin), the bases in the partial complementary sequence (S3) of the kanamycin nucleic acid aptamer The sequence is 5'-AAA AAA CCT GAC ACT AC-3'; after vortex mixing at 37°C for 70 minutes, increase the temperature to 65°C, keep it for 5 minutes and then lower it to 25°C; then add 70µL containing 0.4mM TMB, 0.4mM H 2 o 2 The pH=5.0...

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Abstract

The invention discloses a homogeneous phase biosensing method for detecting kanamycin and application thereof. The activity of a DNA enzyme is suppressed by a double-strand hybridization reaction between G-quadruplex DNA (S1) and kanamycin nucleic acid aptamer, the corresponding quantity of S1 is released via specific binding of the kanamycin and the nucleic acid aptamer thereof, and is combined with chlorhematin to form the DNA enzyme and perform catalytic chromogenic signal transduction; in addition, after a complementary chain S3 is introduced for hybridization, a hairpin structure formed by the kanamycin and the nucleic acid aptamer thereof can catalyze target analyte circulation via exonuclease III, and thus the sensitivity of the method is improved; a quantitative relation between the absorbancy of a chromogenic product and the concentration of kanamycin analyte is built by using a catalytic chromogenic reaction of the DNA enzyme released by specific recognition of the nucleic acid aptamer. The method provided by the invention is convenient to operate, low in cost, high in sensitivity and good in stability, and has good application value for quick detection of kanamycin residual in complex mediums such as foods and water.

Description

technical field [0001] The invention relates to the technical field of bioanalysis, in particular to a homogeneous biosensing method for detecting kanamycin and its application. Background technique [0002] Kanamycin is an aminoglycoside high-efficiency broad-spectrum antibiotic against most Enterobacteriaceae bacteria such as Escherichia coli, Klebsiella, Enterobacter, Proteus, Shigella, Salmonella , Citrobacter, Providencia, Yersinia, Haemophilus influenzae, Brucella, Neisseria meningitidis, Neisseria gonorrhoeae, etc. all have good antibacterial effects, so they are widely used It is used for the treatment of various Gram-positive and Gram-negative bacterial infectious diseases in humans and animals. However, excessive use of kanamycin can not only cause hearing loss, tinnitus, and even serious side effects such as nephrotoxicity and drug allergy, but also cause serious drug resistance. In recent years, excessive antibiotic residues in food, water, and soil caused by t...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N21/31
CPCG01N21/31
Inventor 赖国松陈志超
Owner HUBEI NORMAL UNIV
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