CN102253193A - Magnetic fluorescent kit for rapidly detecting microbes as well as preparation method and use method thereof - Google Patents
Magnetic fluorescent kit for rapidly detecting microbes as well as preparation method and use method thereof Download PDFInfo
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- CN102253193A CN102253193A CN2010101786579A CN201010178657A CN102253193A CN 102253193 A CN102253193 A CN 102253193A CN 2010101786579 A CN2010101786579 A CN 2010101786579A CN 201010178657 A CN201010178657 A CN 201010178657A CN 102253193 A CN102253193 A CN 102253193A
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- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
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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
The present invention relates to magnetic fluorescence kit and preparation method thereof and the using method of a kind of fast detecting microorganism, but be specifically related to a kind of magnetic fluorescence kit composition, preparation and using method that inrichment specificity, fast quantification detect the microorganism that causes infectious diseases that have.
Background technology
The method for quick of microorganism is field that develops rapidly in the applied microbiology, no matter is control and treatment to infectious diseases, or the monitoring of food security and management, all presses for pathogenic bacteria detection method fast.
At present pathogenic bacteria being determined, mainly employing microbiology, chemistry, biological chemistry, molecular biology, immunologic method are separated, detect, are identified and count pathogenic bacteria in the speed detection.Still there is limitation in these pathogenic bacteria detection methods at present, up to the present, also do not have a kind of method separation and concentration pathogenic bacteria from multiple biological specimen easily; And then in good time answer goes out following two problems: 1, having does not have specific pathogenic bacteria, as the salmonella that threatens food security, staphylococcus aureus etc., and the Much's bacillus of serious threat human life quality, streptococcus pneumonia (checking qualitatively); If 2 have, what are there? (quantitative detection) this for definite pathogenic bacteria and adopt the corresponding treatment method most important.
The immunomagnetic isolation method, be that specific antibody is coupled at the magnetic-particle surface, combine with tested pathogenic microorganisms generation specificity in the sample, the magnetic-particle that is loaded with pathogenic microorganisms is under the effect of externally-applied magnetic field, assemble to pole orientation, and accumulate in the inwall of container, after a while sample liquid is shifted out, spread demagnetizing field, collect magnetic microsphere, microorganism can be disintegrated down from magnetic microsphere, this method can make pathogenic microorganisms not only be able to from several samples, separate as obtaining in pedotheque, foodstuff samples, the clinical sample, and obtain enrichment method.
The immunomagnetic isolation technology is isolated salmonella from milk and milk products, meat and vegetables, its detection is limited to every gram 1 * 10
2Individual bacterium.Seo etc. combine fluoroimmunoassay (FIA) with immunomagnetic isolation, measure the low concentration Escherichia coli that are inoculated in beef, cider and the raw milk, only have 4 Escherichia coli to be detected in every gram beef.As seen with immunomagnetic isolation technology and other method of inspection, as Enzyme Linked Immunoadsorbent Assay (ELISA), polymerase chain reaction (PCR), fluoroimmunoassay (FIA), electron chemistry luminous (ECL) combines, and can improve separation efficiency and detection limit in several times ground.And at present in microorganism detection, evaluation that all combine with immunomagnetic isolation and detection method include follow-up biological chemistry, molecular biology, immunologic inspection method, complex operation, and appointed condition requires high, and application prospect allows of no optimist.
Fluorescent microsphere is a kind of functional microsphere that is loaded with fluorescence molecule and since its in single microballoon enrichment molecule that can emitting fluorescence, be with a wide range of applications in a lot of fields.The fluorescent microsphere of binding specificity antibody has been used for mark and the various antigens of identification at present, comprises polysaccharide, protein, cell etc.The fluorescence intensity that is detected then with the testing concentration linear dependence, thereby realize the qualitative and detection by quantitative of determined antigen.The advantage that this method is the most outstanding is to carry out the analysis of many targets simultaneously in same system, and high specificity can be realized quick diagnosis.
Therefore utilize the inrichment of magnetic Nano microsphere and the modern nanometer technologies such as qualitative, quantitative of fluorescent microsphere, seek the various microorganisms of a kind of simple effective method fast enriching in conjunction with modern biological detecting method, and specificity, fast qualitative, detection by quantitative microorganism are the main contents that the present invention need solve.
Summary of the invention
Technical matters to be solved by this invention is to provide the magnetic fluorescence kit of a kind of fast detecting microorganism, to solve the existing existing many weak points of microorganism detection reagent box.This invention is applicable to hospital diagnosis, the check of food hygiene department, and perhaps scientific research institution's research is used.
One of technical issues that need to address of the present invention are the magnetic fluorescence kits that discloses a kind of fast detecting microorganism.
Two of the technical issues that need to address of the present invention provide the preparation method of this magnetic fluorescence kit.
Three of the technical issues that need to address of the present invention provide the using method of this magnetic fluorescence kit.
Principle of the present invention, referring to Fig. 1:
Fluorescent microsphere is because the surface of microballoon indicates the microballoon that fluorescent material or microsphere inner structure contain fluorescent material, and being subjected to outside energy stimulates and can inspire fluorescence.In recent years, people can prepare the fluorescent microsphere of various particle diameters from the nanoscale to the submicron order.The immunofluorescence microballoon has more stable morphosis and luminous behavior, is subjected to the influence of external conditions such as solvent, heat, electricity, magnetic little more a lot of than pure fluorescent chemicals.Fluorescence molecule or luminescent material are wrapped up or connection by macromolecule layer or silicon dioxide shell material, and the surface is coupling target biology molecule again, then can form the fluorescent nano particles with target; The Nano microsphere that has prepared fluorescent dyeing, by specific chemical modification, form immune magnetic microsphere, thousands of fluorescent nano particles are combined with the surface antigen of a microorganism, the detection of this enlarge-effect can realize the ultra-high sensitive of microorganism is detected.
Immune magnetic microsphere has the characteristic of paramagnetism and macromolecule particle in magnetic field.The paramagnetism of immune magnetic microsphere makes Separation of Solid and Liquid easier, can save numerous and diverse traditional operations such as filtration; And the immune magnetic microsphere particle is little, and specific surface area is big, and big with other material coupling capacity, suspension stability is good, helps the antigen-antibody coupling reaction and successfully carries out.During with testing sample and immune magnetic microsphere and the mixing of immunofluorescence microballoon, microorganism can combine with immune magnetic microsphere that is coated with the microorganism specific antibody and immunofluorescence microballoon simultaneously and form new compound in the testing sample.During by magnetic field, carry out magnetic sorting, this compound can be detained, and is separated with other component, to carrying out microbial morphology observation or fluorescent strength determining by fluoroscopic examination through compound after the enrichment, its phosphor dot quantity and fluorescence intensity are relevant with microbial numbers in the sample.Immunomagnetic isolation is simple and easy to do, and the immunomagnetic beads isolation technics is used in the microorganism detection aspect can accurately isolate out microorganism in the sample fast.
The present invention is by the inrichment of immune magnetic microsphere to sample, the quantitative detection effect of binding immunoassay fluorescent microsphere sensitivity, the immunofluorescence microballoon that can isolate the microorganism in the sample simultaneously efficiently and combine with the microorganism specificity, by fluorescence intensity, fast microorganism is carried out qualitative, quantitative test.This has great importance for food hygiene, prevention from suffering from the diseases and treatment.
The technical matters that will solve required for the present invention can be achieved through the following technical solutions:
As a first aspect of the present invention, the magnetic fluorescence kit of a kind of fast detecting microorganism comprises following constituent:
1) immune magnetic microsphere that combines with microorganism specificity to be measured;
2) the immunofluorescence microballoon that combines with microorganism specificity to be measured.
Further, described immune magnetic microsphere is the Nano microsphere with magnetic responsiveness, and its particle diameter is 20-150nm, and the magnetic response time is less than 30min.
The preferred magnetic responsiveness time of described immune magnetic microsphere is less than 10min.
The preferred magnetic responsiveness time of described immune magnetic microsphere is less than 3min.
Further, described immune magnetic microsphere is the super-paramagnetism nano microballoon, and magnetic material is iron, cobalt, nickel and alloy nano particle thereof, comprises Fe
3O
4, Fe
2O
4, Fe
xPt
y, Co
xPt
y, MnFe
xO
y, CoFe
xO
y, NiFe
xO
y, CuFe
xO
y, ZnFe
xO
y, and CdFe
xO
y, wherein x and y are 1-6.
Described immune magnetic microsphere has the magnetic polymer composite microspheres of nucleocapsid structure, and polymeric material comprises polystyrene, silicon dioxide etc.
Described immune magnetic microsphere is preferably the magnetic silicon dioxide composite microsphere with surface-functionalized nucleocapsid structure.
Described immune magnetic microsphere is preferably functionalization hydrophilic magnetic microballoon, monoclonal antibody or polyclonal antibody that surface combination combines with the microorganism specificity.
Further, described immunofluorescence microballoon is the Nano microsphere of energy emitting fluorescence, and its particle diameter is 10-150nm.
Further, the preferred microspherulite diameter of described immunofluorescence microballoon is 5-50nm.
Described immunofluorescence microballoon comprises inorganic fluorescent material, organic fluorescence materials and quantum dot light emitting material.
Described fluorescent material comprises compounds such as fluoresceins, rhodamine class, O-phthalic aldehydes, as fluorescein isothiocynate (F1TC), rhodamine etc.
Described immunofluorescence microballoon adopts fluorescent material and Nano microsphere covalent bond.
Described immunofluorescence microballoon adopts fluorescent material to be embedded in Nano microsphere inside.
Described immunofluorescence microballoon is the polymer composite microsphere that contains fluorescent material, and polymeric material comprises polystyrene, silicon dioxide.
Described immunofluorescence microballoon is preferably has surface-functionalized SiO 2 composite microsphere.
As a second aspect of the present invention, the preparation method of the magnetic fluorescence kit of a kind of fast detecting microorganism is characterized in that, comprises the steps:
(1) preparation of immune magnetic microsphere:
With magnetic Nano microsphere and the coupling of microbial antigen specific antibody, magnetic separates by difunctional coupling reagent, fully after the washing, adds the buffer solution that contains protective agent and spreading agent, makes the magnetic Nano microsphere of coupling antibody after the freeze drying;
(2) preparation of immunofluorescence microballoon:
With fluorescent nanometer microsphere and the coupling of microbial antigen specific antibody, centrifugal or ultrafiltration dialysis separates unconjugated antibody by difunctional coupling reagent, adds the buffer solution that contains protective agent and spreading agent, makes the fluorescent nanometer microsphere of coupling antibody after the freeze drying.
As a third aspect of the present invention, the using method of the magnetic fluorescence kit of a kind of fast detecting microorganism, this kit will be converted into the detection to fluorescence intensity to the detection of microorganism signal, and its operation steps is:
(1) testing sample, immune magnetic microsphere freeze-dried powder and immunofluorescence microballoon are added in the buffer solution;
(2) institute Identifying micro-organisms surface has the surface antigen determinant that combines with immune magnetic microsphere and immunofluorescence microballoon simultaneously; So the microorganism surface is distribution immune magnetic microsphere and immunofluorescence microballoon evenly;
(3) pass through the magnetic resolution immune magnetic microsphere enrichment microorganism of combination with it;
(4) by measure with separate, the fluorescence intensity of immunofluorescence microballoon that the microorganism of enrichment combines makes qualitative and quantitative judgement to microorganism.
Further, immune magnetic microsphere and immunofluorescence microballoon ratio are 1～2: 1～2.
Beneficial effect of the present invention:
Compare with the pathogenic bacteria detection method of present routine, as microbe growth, immune ELISA method and pcr amplification method, compare, this method has fast enriching microorganism, easy to use, characteristics such as accuracy is high, susceptibility is high, false positive is low, equipment requirements is low, can be widely used in fields such as infectious disease detection, prevention from suffering from the diseases and treatment, food security.
Description of drawings
Further specify the present invention below in conjunction with the drawings and specific embodiments.
Fig. 1 is a principle schematic of the present invention.
Embodiment
In order to make technological means of the present invention, creation characteristic, to reach purpose and effect is easy to understand,, further set forth the present invention below in conjunction with concrete diagram.
The preparation of the nano-magnetic microsphere of embodiment 1 amido-containing group
Immune magnetic microsphere has the magnetic polymer composite microspheres of nucleocapsid structure, and polymeric material comprises polystyrene, silicon dioxide, is preferably the magnetic silicon dioxide composite microsphere with surface-functionalized nucleocapsid structure.
In the 500ml three-necked bottle, add the ferroferric oxide powder that 5.0g crosses through deionized water wash, add the deionized water of 200ml, after dispersed with stirring under the 600rpm rotating speed, add 100ml salpeter solution (3.0M), stirred 10 minutes under the room temperature.Separate with magnet then and with deionized water wash 3-5 time.With the tri-iron tetroxide mud dispersed with stirring (0.2M) in the sodium citrate solution of 400ml after the washing, then separate and with deionized water wash 3-5 time with magnet, last resulting tri-iron tetroxide is dispersed in the 200ml deionized water, and the preparation solid content is about the magnetic fluid of 2.0wt%.
In the three-necked bottle of a 500ml, add the good magnetic fluid of 5.0g prepared beforehand, and with the dilution of 40mL deionized water and 200ml absolute ethyl alcohol, the strong aqua that under high-speed stirred, adds 5ml then, the ethyl orthosilicate that adds 4ml, after keeping stirring 6h, in this system, add the triethoxy aminopropyl silane (APS) of 0.5ml again, continue reaction 12h.Reaction finishes the back centrifuge washing, and the particle diameter of prepared immune magnetic microsphere is 80-90nm, and magnetic saturation intensity is 8.0emu/g, and the magnetic responsiveness time of immune magnetic microsphere is less than 3min.
The preparation of the nanometer fluorescent microspheres of embodiment 2 amido-containing groups
The immunofluorescence microballoon adopts fluorescent material and Nano microsphere covalent bond.Usually the immunofluorescence microballoon adopts fluorescent material to be embedded in Nano microsphere inside.The immunofluorescence microballoon is the polymer composite microsphere that contains fluorescent material, and polymeric material comprises polystyrene, silicon dioxide, is preferably to have surface-functionalized SiO 2 composite microsphere.
In single neck round-bottomed flask of a 25ml, with the fluorescein FITC of the anhydrous alcohol solution 0.0356g of 10ml, add the triethoxy aminopropyl silane (APS) of 0.0183g then, lucifuge stirring reaction 48h makes the silane coupling agent of bonding fluorescence molecule FITC.
In the three-necked bottle of a 500ml, add 5ml deionized water and 250ml absolute ethyl alcohol, the strong aqua that under high-speed stirred, adds 8.5ml then, the ethyl orthosilicate that adds 7.5ml, after keeping stirring 4h, add the silane coupling agent of the bonding fluorescence molecule fluorescein isothiocynate (F1TC) of the above-mentioned preparation of 2ml, in this system, add the ethyl orthosilicate of 5ml simultaneously again, in this system, add the triethoxy aminopropyl silane (APS) of 0.5ml behind the reaction 12h again, continue stirring reaction 12h.Reaction finishes the back centrifuge washing, and the particle diameter of prepared fluorescent microsphere is 60-70nm.
The preparation of the immune magnetic microsphere of embodiment 3 anti-salmonellas surface lipopolysaccharides
1, the amino-magnetic microballoon of getting the above-mentioned preparation of about 25mg contains in the phosphate buffer (pH7.4) of 5% glutaraldehyde in 5ml, mixes vibration, room temperature reaction 3h.
2, magnetic separates microballoon, fully washs with the phosphate-buffered liquor, and magnetic is abandoned supernatant after separating, and removes unreacted glutaraldehyde.
3, will activate back amino-magnetic microballoon and place the 4ml phosphate buffer, add the phosphate-buffered liquor that 2ml contains anti-salmonella surface lipopolysaccharides antibody (10mg/ml), vibration, 37 ℃ of reaction 6h.
4, magnetic separates microballoon, fully washs with the phosphate-buffered liquor, and magnetic is abandoned supernatant after separating, and removes the antibody of unreacted coupling, makes the anti-salmonella-immune magnetic microsphere of antibody coupling.
The preparation of embodiment 4 anti-salmonellas surface lipopolysaccharides immunofluorescence microballoon
1, the amino fluorescent microsphere of getting the above-mentioned preparation of about 25mg contains in the phosphate buffer (pH7.4) of 5% glutaraldehyde in 5ml, mixes vibration, room temperature reaction 3h.
2, centrifuging microballoon fully washs with the phosphate-buffered liquor, centrifugal after, abandon supernatant.
3, will activate the amino fluorescent microsphere in back and place the 4ml phosphate buffer, add the phosphate-buffered liquor that 2ml contains anti-salmonella surface lipopolysaccharides antibody (10mg/ml), vibration, 37 ℃ of reaction 6h.
4, centrifuging immunofluorescence microballoon fully washs with the phosphate-buffered liquor, and is centrifugal, abandons supernatant,
Make the anti-salmonella-immunofluorescence microballoon of antibody coupling.
Embodiment 5 fluorescent microscopes detect salmonella
1, salmonella is inoculated in the nutrient culture media 37 ℃ of shaken overnight, count of bacteria.
2, get 10
3The microballoon lyophilized powder of individual salmonella, 10mg immune magnetic microsphere freeze-dried powder and 10mg immunofluorescence adds 10ml incubation buffer (phosphate buffer of 10mM, pH value 7.4,0.05% polysorbas20s), and the room temperature gentleness is swayed 5min.
3, the magnetic resolution magnetic microsphere is abandoned supernatant.
4, with 5ml incubation buffer washing three times, magnetic separates, and removes all unconjugated anti-salmonella-immunofluorescence microballoons.
5, remove magnetic field, the gained sample is suspended from a small amount of incubation buffer.
6, pass through the formed phosphor dot of fluorescent microsphere of fluorescence microscope salmonella surface adsorption, and carry out count of bacteria.
Embodiment 6 fluorescence intensity detection by quantitative salmonellas
1, salmonella is inoculated in the nutrient culture media 37 ℃ of shaken overnight, count of bacteria.
2, get the different bacterium number salmonella, add 10ml and contain the immune magnetic microsphere of anti-salmonella surface lipopolysaccharides and the incubation buffer (phosphate buffer of 10mM, pH value 7.4,0.05% polysorbas20s) of immunofluorescence microballoon, the room temperature gentleness is swayed 5min.
3, the magnetic resolution magnetic microsphere is abandoned supernatant.
4, with 5ml incubation buffer washing three times, magnetic separates, and removes all unconjugated anti-salmonella-immunofluorescence microballoons.
5, remove magnetic field, add 1ml 8M urea solution metaprotein, the immunofluorescence microballoon is separated with immune magnetic microsphere.
6, magnetic resolution immune magnetic microsphere is collected supernatant.
7, measure the supernatant fluorescence intensity by the fluorescence calculating instrument, and the drawing standard curve.
8, fluorescence intensity and the typical curve that testing sample produced compared, promptly get testing sample salmonella content.
More than show and described ultimate principle of the present invention, principal character and advantage of the present invention.The technician of the industry should understand; the present invention is not restricted to the described embodiments; that describes in the foregoing description and the instructions just illustrates principle of the present invention; the present invention also has various changes and modifications without departing from the spirit and scope of the present invention, and these changes and improvements all fall in the claimed scope of the invention.The claimed scope of the present invention is defined by appending claims and equivalent thereof.
Claims (19)
1. the magnetic fluorescence kit of a fast detecting microorganism comprises following constituent:
1) immune magnetic microsphere that combines with microorganism specificity to be measured;
2) the immunofluorescence microballoon that combines with microorganism specificity to be measured.
2. kit according to claim 1 is characterized in that, described immune magnetic microsphere is the Nano microsphere with magnetic responsiveness, and its particle diameter is 20-150nm, and the magnetic response time is less than 30min.
3. kit according to claim 2 is characterized in that, the magnetic responsiveness time of described immune magnetic microsphere is less than 10min.
4. kit according to claim 3 is characterized in that, the magnetic responsiveness time of described immune magnetic microsphere is less than 3min.
5. kit according to claim 1 is characterized in that, described immune magnetic microsphere is the super-paramagnetism nano microballoon, and magnetic material is iron, cobalt, nickel and alloy nano particle thereof, comprises Fe
3O
4, Fe
2O
4, Fe
xPt
y, Co
xPt
y, MnFe
xO
y, CoFe
xO
y, NiFe
xO
y, CuFe
xO
y, ZnFe
xO
y, and CdFe
xO
y, wherein x and y are 1-6.
6. kit according to claim 1 is characterized in that described immune magnetic microsphere has the magnetic polymer composite microspheres of nucleocapsid structure, and polymeric material comprises polystyrene, silicon dioxide.
7. kit according to claim 1 is characterized in that, described immune magnetic microsphere is the magnetic silicon dioxide composite microsphere with surface-functionalized nucleocapsid structure.
8. kit according to claim 1 is characterized in that, described immune magnetic microsphere is a functionalization hydrophilic magnetic microballoon, monoclonal antibody or polyclonal antibody that surface combination combines with the microorganism specificity.
9. kit according to claim 1 is characterized in that, described immunofluorescence microballoon is the Nano microsphere of energy emitting fluorescence, and its particle diameter is 5-150nm.
10. kit according to claim 9 is characterized in that, described immunofluorescence microspherulite diameter is 5-50nm.
11. kit according to claim 1 is characterized in that, described immunofluorescence microballoon comprises inorganic fluorescent material, organic fluorescence materials and quantum dot light emitting material.
12. kit according to claim 11 is characterized in that, described fluorescent material comprises fluoresceins, rhodamine class, O-phthalic aldehyde compound.
13. kit according to claim 1 is characterized in that, described immunofluorescence microballoon adopts fluorescent material and Nano microsphere covalent bond.
14. kit according to claim 1 is characterized in that, described immunofluorescence microballoon adopts fluorescent material to be embedded in Nano microsphere inside.
15. kit according to claim 1 is characterized in that, described immunofluorescence microballoon contains the polymer composite microsphere of fluorescent material, and polymeric material comprises polystyrene, silicon dioxide.
16., it is characterized in that described immunofluorescence microballoon is for having surface-functionalized SiO 2 composite microsphere according to the described kit of claim 15.
17. the preparation method of the magnetic fluorescence kit of an a kind of fast detecting microorganism as claimed in claim 1 is characterized in that, comprises the steps:
(1) preparation of immune magnetic microsphere:
With magnetic Nano microsphere and the coupling of microbial antigen specific antibody, magnetic separates by difunctional coupling reagent, fully after the washing, makes the magnetic Nano microsphere of coupling antibody after the freeze drying;
(2) preparation of immunofluorescence microballoon:
With fluorescent nanometer microsphere and the coupling of microbial antigen specific antibody, centrifugal or ultrafiltration dialysis separates unconjugated antibody, makes the fluorescent nanometer microsphere of coupling antibody after the freeze drying by difunctional coupling reagent.
18. the using method of the magnetic fluorescence kit of an a kind of fast detecting microorganism as claimed in claim 1, this kit will be converted into the detection to fluorescence intensity to the detection of microorganism signal, and its operation steps is:
(1) testing sample, immune magnetic microsphere and immunofluorescence microballoon are added in the buffer solution;
(2) institute Identifying micro-organisms surface has the surface antigen determinant that combines with immune magnetic microsphere and immunofluorescence microballoon simultaneously;
(3) by the enrichment of magnetic resolution immune magnetic microsphere with it combination microorganism and be incorporated into immunofluorescence microballoon on the microorganism;
(4) by measure with separate, the fluorescence intensity of immunofluorescence microballoon that the microorganism of enrichment combines makes qualitative and quantitative judgement to microorganism.
19. using method according to claim 17 is characterized in that, the ratio of immune magnetic microsphere and immunofluorescence microballoon is 1～2: 1～2.
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| CN2010101786579A CN102253193A (en) | 2010-05-20 | 2010-05-20 | Magnetic fluorescent kit for rapidly detecting microbes as well as preparation method and use method thereof |
| PCT/CN2011/074138 WO2011144012A1 (en) | 2010-05-20 | 2011-05-17 | Magnetic kit for rapid detection of microorganisms and its preparation and usage |
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| CN2010101786579A CN102253193A (en) | 2010-05-20 | 2010-05-20 | Magnetic fluorescent kit for rapidly detecting microbes as well as preparation method and use method thereof |
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