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Netilmicin nucleic acid aptamer and its screening and application in the detection of netilmicin

A technology of mixing nucleic acid and netilmicin, which is applied in the field of chemical substance detection, can solve the problem of rapid, real-time, effective, and simple qualitative and quantitative analysis of netilmicin, which requires high detection time and is not suitable for fast Detection and other problems, to achieve the effect of simple sample pretreatment, good selectivity and low cost

Active Publication Date: 2022-07-26
浙江首信检测有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, it has high requirements for equipment, professional knowledge, and detection time, and is not suitable for on-site rapid detection and other shortcomings.
Most of these methods have complex sample processing, expensive analytical instruments, and some methods have low detection sensitivity
Therefore, there are limitations in the current traditional analytical methods for detecting netilmicin, and it is difficult to achieve fast, real-time, effective, and simple qualitative and quantitative analysis of netilmicin. Therefore, a cheap, real-time Fast, simple, efficient, accurate, and stable netilmicin detection technology is an urgent problem for today's analysts

Method used

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  • Netilmicin nucleic acid aptamer and its screening and application in the detection of netilmicin
  • Netilmicin nucleic acid aptamer and its screening and application in the detection of netilmicin
  • Netilmicin nucleic acid aptamer and its screening and application in the detection of netilmicin

Examples

Experimental program
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Effect test

Embodiment 1

[0119] The present embodiment provides a method for screening and obtaining a netilmicin nucleic acid aptamer, comprising the following steps:

[0120] A. Pipette 100 μL of carboxyl magnetic beads into a 1.5 mL centrifuge tube, place it on a magnetic separation rack, and let it stand for 2 min. Carefully aspirate the supernatant and discard it. Then take out the centrifuge tube, add 100 μL of ultrapure water, mix by suction, place it on a magnetic separation rack, let it stand for 2 min, carefully aspirate the supernatant and discard. Add 100 μL of 2-(N-morpholinyl)ethanesulfonic acid solution to the centrifuge tube, mix by pipetting, and repeat this step. Use 2-(N-morpholinyl)ethanesulfonic acid to wash the carboxyl magnetic beads. After 5 washes , carefully aspirate the supernatant and discard.

[0121]B. Weigh 10 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride into 1 ml of 2-(N-morpholinyl)ethanesulfonic acid (25 mM). Meanwhile, 50 mg of N,N-hydroxysucci...

Embodiment 2

[0149] The present embodiment provides a netilmicin detection method, comprising the following steps:

[0150] A. The netilmicin nucleic acid aptamer obtained in Example 1 was used to prepare a 100 nM stock solution.

[0151] B. Add SYBR Green I solution to a certain amount of the netilmicin nucleic acid aptamer solution, mix and incubate, so that the SYBR Green I fluorescent probe is embedded in the double strand formed by the netilmicin nucleic acid aptamer Among them, add the aqueous solution of netilmicin to be tested and 3-propanesulfonic acid buffer, mix and incubate, and measure the fluorescence intensity F at the excitation wavelength of 485 nm and the emission wavelength of 535 nm; The aqueous solution of tilmicin was used as the control group, and the fluorescence intensity F was measured. 0 ; Calculate △F=F according to the fluorescence intensity 0 -F size to judge the content of netilmicin in the solution to be tested.

[0152] In the present embodiment, the SYB...

Embodiment 3

[0160] Based on the method of Example 2, this example optimizes the type of system buffer

[0161] Set the test group (F) with 1μM netilmicin and the blank group (F0) without netilmicin, add 20nM ofloxacin aptamer solution to a 1.5mL centrifuge tube, and choose 10mM pH respectively 7.0 MOPS buffer, 10mM pH 7.4 PBS buffer, 50mM pH 7.4 Tris-HCl buffer and ultrapure water ddH2O were used as buffer systems. The systems were all routinely prepared, supplemented the system volume and incubated the reaction at constant temperature for 30 minutes, and then added the concentration SYBR GreenI fluorescent dye with grade 1x, continue to incubate for 10 minutes, sample in a 96-well black microplate plate, measure the fluorescence intensity with an Infinite M200 Pro microplate reader, obtain F and F0 and calculate ΔF, which is calculated after repeating the test three times. The average value of the difference ΔF of the fluorescence intensity is the ordinate, and the name of the buffer sys...

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Abstract

The invention belongs to the technical field of chemical substance detection, and relates to a netilmicin nucleic acid aptamer and its screening and application in netilmicin detection; specifically discloses a netilmicin nucleic acid aptamer, a netilmicin nucleic acid A screening method for aptamers, a biosensor for netilmicin detection, and a netilmicin detection method. Compared with the prior art, the detection method of the present invention has the advantages of low detection limit, high sensitivity, strong specificity, visual and rapid qualitative analysis, simple sample pretreatment, low instrument requirements and low cost. The detection method provided by the invention can be used to detect the content of netilmicin in the water sample, and the detection concentration range is 1.95-200 nmol / L.

Description

technical field [0001] The invention belongs to the technical field of chemical substance detection, and in particular relates to a netilmicin nucleic acid aptamer and its screening and application in netilmicin detection. Background technique [0002] Netilmicin is a semi-synthetic aminoglycoside antibiotic with a chemical composition of 3-N-ethylsisomicin, which is white or off-white powder or loose lumps. The antibacterial effect is basically similar to that of gentamicin, and its characteristic is that it is stable to aminoglycoside acetyltransferase AAC (3), and has good antibacterial effect on gram-negative bacteria and some gram-positive bacteria. Because of its smaller adverse reactions than other similar products, it has been used in clinical practice abroad and successfully trial-produced in China. It is used to prevent, treat and diagnose diseases, and at the same time purposefully regulate animal physiological functions and promote growth and development. It is ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/115C12N15/10G01N33/58G01N33/53
CPCC12N15/115G01N33/9446G01N33/582G01N33/5308C12N2310/16C12N2330/31
Inventor 王鲁梅潘超强沈国清耿雪青
Owner 浙江首信检测有限公司
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