Magnetic fluorescent kit for rapidly detecting microbes as well as preparation method and use method thereof

A technology of fluorescence kits and microorganisms, used in biological testing, fluorescence/phosphorescence, measurement devices, etc.

Inactive Publication Date: 2011-11-23
EMERGING THERAPEUTICS SHANGHAI CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0008] The technical problem to be solved by the present invention is to provide a magnetic fluorescent kit fo

Method used

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  • Magnetic fluorescent kit for rapidly detecting microbes as well as preparation method and use method thereof
  • Magnetic fluorescent kit for rapidly detecting microbes as well as preparation method and use method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Example 1 Preparation of Nanomagnetic Microspheres Containing Amino Groups

[0052] The immunomagnetic microsphere is a magnetic polymer composite microsphere with a core-shell structure, and the polymer material includes polystyrene and silicon dioxide, preferably a magnetic silicon dioxide composite microsphere with a surface functionalized core-shell structure.

[0053] In a 500ml three-necked bottle, add 5.0g of ferric oxide powder washed with deionized water, add 200ml of deionized water, stir and disperse at 600rpm, add 100ml of nitric acid solution (3.0M), and stir at room temperature 10 minutes. Then separate with a magnet and wash 3-5 times with deionized water. Stir and disperse the washed iron ferric oxide slurry in 400ml of sodium citrate solution (0.2M), then separate it with a magnet and wash it 3-5 times with deionized water, and finally the obtained iron ferric oxide is dispersed in 200ml of deionized water. In ionized water, a magnetic fluid with a so...

Embodiment 2

[0055] Example 2 Preparation of Nano Fluorescent Microspheres Containing Amino Groups

[0056] Immunofluorescent microspheres use fluorescent materials to covalently bind nanospheres. Usually, immunofluorescence microspheres are embedded in nano-microspheres with fluorescent materials. The immunofluorescent microspheres are polymer composite microspheres containing fluorescent materials, and the polymer materials include polystyrene and silicon dioxide, and are preferably surface functionalized silicon dioxide composite microspheres.

[0057] In a 25ml single-neck round bottom flask, dissolve 0.0356g of fluorescein FITC with 10ml of absolute ethanol, then add 0.0183g of triethoxyaminopropylsilane (APS), and stir for 48 hours in the dark to obtain A silane coupling agent that binds the fluorescent molecule FITC.

[0058] In a 500ml three-necked bottle, add 5ml deionized water and 250ml absolute ethanol, then add 8.5ml of concentrated ammonia water under high-speed stirring, a...

Embodiment 3

[0059] Example 3 Preparation of immunomagnetic microspheres against Salmonella surface lipopolysaccharide

[0060] 1. Take about 25 mg of the above-prepared amino magnetic microspheres in 5 ml of phosphate buffer (pH 7.4) containing 5% glutaraldehyde, mix and shake, and react at room temperature for 3 hours.

[0061] 2. Magnetically separate the microspheres and wash them thoroughly with phosphate buffer solution. After magnetic separation, discard the supernatant to remove unreacted glutaraldehyde.

[0062] 3. Place the activated amino magnetic microspheres in 4ml of phosphate buffer, add 2ml of phosphate buffer solution containing anti-Salmonella lipopolysaccharide antibody (10mg / ml), shake, and react at 37°C for 6h.

[0063] 4. Magnetically separate the microspheres, fully wash with phosphate buffer solution, discard the supernatant after magnetic separation, and remove unreacted coupled antibodies to obtain antibody-coupled anti-Salmonella-immunomagnetic microspheres.

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Abstract

The invention discloses a magnetic fluorescent kit for rapidly detecting microbes as well as a preparation method and a use method thereof. The kit comprises two components: 1) immunomagnetic microspheres specifically bound with the microbes to be detected; and 2) immunofluorescent microspheres specifically bound with the microbes to be detected. The preparation method comprises the following steps: (1) preparation of the immunomagnetic microspheres; and (2) preparation of the immunofluorescent microspheres. The use method comprises the following steps: (1) adding the sample to be detected, lyophilized powder of the immunomagnetic microspheres and the immunofluorescent microspheres to a buffer solution; (2) ensuring the surfaces of the identified microbes to have antigenic determinants simultaneously bound with the immunomagnetic microspheres and the immunofluorescent microspheres; (3) enriching the microbes bound with the immunomagnetic microspheres through magnetic separation of the immunomagnetic microspheres; and (4) qualitatively and quantitatively judging the microbes by measuring the fluorescence intensity of the immunofluorescent microspheres bound with the separated and enriched microbes. The kit, the preparation method and the use method have the advantages of rapidness, quantitative property and wide scope of application.

Description

technical field [0001] The present invention relates to a magnetic fluorescent reagent kit for rapid detection of microorganisms and its preparation method and use method, in particular to the composition of a magnetic fluorescent reagent kit capable of specific and rapid quantitative detection of microorganisms that cause infectious diseases, Preparation and method of use. Background technique [0002] The rapid detection method of microorganisms is a rapidly developing field in applied microbiology. Whether it is the control and treatment of infectious diseases, or the monitoring and management of food safety, there is an urgent need for rapid detection methods of pathogenic bacteria. [0003] At present, the methods of microbiology, chemistry, biochemistry, molecular biology and immunology are mainly used for the rapid detection of pathogenic bacteria to isolate, detect, identify and count pathogenic bacteria. At present, these pathogenic bacteria detection methods still...

Claims

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Application Information

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IPC IPC(8): G01N33/53G01N33/533G01N33/531G01N21/64
CPCG01N33/569G01N33/54326G01N33/582
Inventor 李莉杨武利府寿宽府宇雷
Owner EMERGING THERAPEUTICS SHANGHAI CO LTD
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