4-methylumbelliferone enrichment material and application thereof

A methylumbelliferone and enrichment technology, applied in the field of microbial detection, can solve the problems of unfavorable fluorescence results and interpretation, achieve accurate detection results and save detection time

Active Publication Date: 2018-12-07
SOUTH CENTRAL UNIVERSITY FOR NATIONALITIES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

If two detection fluorescent / color substrates are added to one culture tube at the same time, the color product is usually a better fluorescence quencher, which will quench the possible fluorescence generation, which is not conducive to the interpretation of fluorescence results

Method used

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  • 4-methylumbelliferone enrichment material and application thereof
  • 4-methylumbelliferone enrichment material and application thereof
  • 4-methylumbelliferone enrichment material and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] Accurately weigh 5 g of agarose and place it in a 250 mL three-neck round bottom flask. Add 60mL NaOH solution (containing NaOH2.4g) and 30mL dimethyl sulfoxide, then place it in a constant temperature water bath at 38°C and stir slowly while slowly adding 5mL 1-chlorooctane, then increase the stirring speed to 300rpm, and react at constant temperature for 2h . After the reaction, the agarose was washed 5 times with acetone and deionized water respectively, centrifuged, dried, and set aside.

[0019] Take 1g of 4-methylumbelliferone-enriched material, 4g of agarose, add 200mL of water, melt the agarose in a microwave oven, stir evenly, add 500μL of the above-mentioned agarose mixture melted to each 24-well plate well, add 75% ethanol after solidification After sterilization, blow it on the ultra-clean bench until there is no obvious water trace. Use Colilert-18 reagent for sterile filtration, add 2 mL to each well, add 100 μL each of diluted Escherichia coli DH5α and ...

Embodiment 2

[0022] Accurately weigh 5 g of agarose and place it in a 250 mL three-neck round bottom flask. Add 60mL of NaOH solution (containing 2.4g of NaOH) and 30mL of dimethyl sulfoxide, then place it in a constant temperature water bath at 50°C and stir slowly while adding 5mL of 6-chlorohexanoic acid slowly, then increase the stirring speed to 300rpm, and react at constant temperature for 2h . After the reaction, the agarose was washed 5 times with acetone and deionized water respectively, centrifuged, dried, and set aside.

[0023] Take 2g of 4-methylumbelliferone enrichment material, 3g of agarose, add 100mL of water, melt the agarose in a microwave oven, stir evenly, add 500μL of the above-mentioned agarose mixture melted into each 24-well plate well, add 75% ethanol after solidification After sterilization, blow it on the ultra-clean bench until there is no obvious water trace. Add 2 mL to each well after aseptic filtration using the medium in the table below, insert 100 μL ea...

Embodiment 3

[0028] Accurately weigh 5 g of agarose and place it in a 250 mL three-neck round bottom flask. Add 60mL of NaOH solution (containing 4.8g of NaOH) and 30mL of dimethyl sulfoxide, then place it in a constant temperature water bath at 70°C and stir slowly while slowly adding 5mL of 1-chloroheptane, then increase the stirring speed to 300rpm, and react at constant temperature for 2h . After the reaction, the agarose was washed 5 times with acetone and deionized water respectively, centrifuged, dried, and set aside.

[0029]Take 3g of 4-methylumbelliferone enrichment material, 2g of agarose, add 200mL of water, melt the agarose in a microwave oven, stir evenly, add 500μL of the above-mentioned agarose mixture melted to each 24-well plate well, add 75% ethanol after solidification After sterilization, blow it on the ultra-clean bench until there is no obvious water trace. Use Colilert-18 reagent for sterile filtration, add 2 mL to each well, add 100 μL each of diluted Escherichia...

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Abstract

The invention provides a 4-methylumbelliferone enrichment material. The enrichment material is prepared by the following method: mixing agarose and a NaOH solution and dimethyl sulfoxide at the mass ratio of agarose to the NaOH solution to dimethyl sulfoxide beign 1:0.2-5:1-10, placing the mixture under the thermostatic waterbath condition, stirring while adding halogenated hydrocarbon or halogenated acid at the mass ratio of agarose to halogenated hydrocarbon or halogenated acid being 1: 0.2-5, continuously stirring after addition and carrying out a thermostatic reaction until the end of thereaction, cleaning with acetone and deionized water, centrifuging and drying to prepare the 4-methylumbelliferone enrichment material. By introducing the material to the bottom of a culture tube, simultaneous determination of Escherichia coli and Escherichia coli can be simultaneously determined in the same culture tube when respective chromogenic / fluorogenic substrate mediums of Escherichia coliand Escherichia coli are added into the culture tube for culture.

Description

technical field [0001] The invention provides a 4-methylumbelliferone enrichment material, which is used for simultaneous detection of Escherichia coli and Escherichia coli, and belongs to the technical field of microbial detection. Background technique [0002] Coliform bacteria is an important indicator to measure food hygiene status, especially the potential threat of enteropathogenic bacteria, so it is also an important microbial item that must be tested for almost any food. The traditional detection method is based on the fermentation of lactose, acid and gas production, and the incubation time is 24-96h. However, due to the discovery of anaerobic strains of Escherichia coli (Appl EnvironMicrobiol.1982 43(6):1320-1329.), it is meaningless to some extent to use gas production as the basis for identification. [0003] The test of coliform bacteria adopts the method of measuring β-galactosidase instead of the lactose fermentation test, and controls the culture temperature...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C08B37/12C12Q1/10C12Q1/06
CPCC08B37/0039C12Q1/06C12Q1/10C12Q2334/22G01N2333/245
Inventor 梁晓声
Owner SOUTH CENTRAL UNIVERSITY FOR NATIONALITIES
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