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Detection method for kanamycin A enzyme-linked aptamer and application of kanamycin A enzyme-linked aptamer

An enzyme-linked aptamer and kanamycin technology, which is applied in the direction of biochemical equipment and methods, microbial measurement/inspection, etc., can solve the detection method and application of kanamycin A enzyme-linked aptamer Convenience and lack of sensitivity of the method, etc., to achieve good accuracy, easy operation, and strong specificity

Active Publication Date: 2013-09-11
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, research on the surveillance screening method for kanamycin A is mainly focused on immunoassays, such as "Immunocolloidal gold test strips for detecting kanamycin residues and their preparation methods" (Application No. : 200810116854.0) discloses a colloidal gold immunochromatographic test paper method; "Kanamycin Residual ELISA Kit and Its Application" (application number: 200710064345.3) discloses an enzyme immunoassay method, which is easy to use and The sensitivity of the method is limited
There is no research report on the detection method of kanamycin A enzyme-linked aptamer

Method used

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  • Detection method for kanamycin A enzyme-linked aptamer and application of kanamycin A enzyme-linked aptamer
  • Detection method for kanamycin A enzyme-linked aptamer and application of kanamycin A enzyme-linked aptamer
  • Detection method for kanamycin A enzyme-linked aptamer and application of kanamycin A enzyme-linked aptamer

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] S1. Enzyme-linked aptamer coating.

[0034] Dilute streptavidin with carbonate buffer (pH 9.6, 0.01 mol / L) to obtain 8 μg / mL coating buffer, add the coating buffer to the microplate, 100 μL / well, coat overnight , shake off the liquid in the well, pat dry, add 0.01 mol / L phosphate buffer saline (PBST) containing 0.5% Tween-20 to wash twice, and pat dry.

[0035] The formula of the phosphate buffered saline (PBST) is: Tween-20 500 μL, NaCl 8.5 g, NaCl 2 HPO 4 12H 2 O 2.9g, KCl 0.2g, KH 2 PO 4 0.2g, dilute to 1L with deionized water;

[0036] Add 150 μL / well of blocking buffer (0.1% casein), block in a 37°C water bath for 3 h, shake off the liquid in the wells, pat dry, and dry in a 37°C drying oven for later use.

[0037] S2. The 5' end of the ssDNA aptamer of kanamycin A was modified with biotin (synthesized by Shanghai Sangon Biotechnology Co., Ltd.). The sequence of the ssDNA aptamer of kanamycin A is 5'-TGGGGGTTGAGGCTAAGCCGA-3' (as shown in SEQ ID NO: 1). ...

Embodiment 2

[0045] S1. Enzyme-linked aptamer coating.

[0046] Dilute streptavidin with carbonate buffer (pH 9.6, 0.01 mol / L) to obtain 1 μg / mL coating buffer, add the coating buffer to the microplate, 200 μL / well, and coat overnight , shake off the liquid in the well, pat dry, add 0.01 mol / L phosphate buffer saline (PBST) containing 0.5% Tween-20 to wash twice, and pat dry.

[0047] The formula of the phosphate buffered saline (PBST): same as Example 1.

[0048] Add 150 μL / well of blocking buffer (0.1% casein), block in a 37°C water bath for 3 h, shake off the liquid in the wells, pat dry, and dry in a 37°C drying oven for later use.

[0049] S2. The modified aptamer (the aptamer is the same as in Example 1) was used to contain 5 mM MgCl 2 0.01 mol / L pH 7.4 PBS buffer solution to obtain the aptamer with a concentration of 0.5 nmol / L, add 100 μL to each well, incubate in a water bath at 37 °C for 1 h, wash the plate 5 times, and pat dry to make the aptamer and The streptavidin coat...

Embodiment 3

[0055] S1. Enzyme-linked aptamer coating.

[0056] Dilute streptavidin with carbonate buffer (pH 9.6, 0.01 mol / L) to obtain 32 μg / mL coating buffer, add the coating buffer to the microplate, 100 μL / well, and coat overnight , shake off the liquid in the well, pat dry, add 0.01 mol / L phosphate buffer saline (PBST) containing 0.5% Tween-20 to wash twice, and pat dry.

[0057] The formula of the phosphate buffered saline (PBST): same as Example 1.

[0058] Add 150 μL / well of blocking buffer (0.1% casein), block in a 37°C water bath for 3 h, shake off the liquid in the wells, pat dry, and dry in a 37°C drying oven for later use.

[0059] S2. The aptamer modified with biotin at the 5' end (the aptamer is the same as in Example 1) was treated with 5 mM MgCl 2 0.01 mol / L pH 7.4 PBS buffer to obtain an aptamer solution with a concentration of 4 nmol / L, add 100 μL to each well, incubate in a water bath at 37 °C for 1 h, wash the plate 5 times, and pat dry to make the aptamer Con...

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Abstract

The invention relates to the field of food safety detection, and in particular discloses a detection method for a kanamycin A enzyme-linked aptamer and an application of the kanamycin A enzyme-linked aptamer. By a streptavidin-biotin-aptamer mode, kanamycin A can be detected; furthermore, a detection condition is optimized; and finally a condition with the lowest detection limit is obtained and the condition is high in sensitivity and specificity. According to the method, the kanamycin A in milk, pork, chicken, pork liver and honey can be detected according to the specificity; and the method has a wide application prospect.

Description

technical field [0001] The invention relates to the field of food safety detection, in particular to a kanamycin A enzyme-linked aptamer detection method and application thereof. Background technique [0002] Kanamycin A (Kanamycin A, KaA) is an aminoglycoside antibiotic drug with broad-spectrum antibacterial properties, effective against both Gram-negative bacteria and Gram-positive bacteria, and can promote the growth of livestock, so it is widely used in agriculture , animal husbandry, aquaculture. However, kanamycin A has severe nephrotoxicity, ototoxicity, blockage of neuromuscular junctions and allergic reactions, and the residual hazards in livestock, poultry and aquatic products have increasingly attracted people's attention. Therefore, many countries and regions have stipulated their maximum residue limits. The European Union clearly prohibits the use of aminoglycoside antibiotics as growth promoters for livestock. The detection limit of kanamycin is 10 μg / kg as s...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/28
Inventor 雷红涛孙远明刘信嘉王弘沈玉栋杨金易徐振林
Owner SOUTH CHINA AGRI UNIV
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