CN103116026A - Quick detection method for food-borne pathogens based on immunomagnetic separation of Fe3O4 nano materials - Google Patents
Quick detection method for food-borne pathogens based on immunomagnetic separation of Fe3O4 nano materials Download PDFInfo
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- CN103116026A CN103116026A CN2013100321099A CN201310032109A CN103116026A CN 103116026 A CN103116026 A CN 103116026A CN 2013100321099 A CN2013100321099 A CN 2013100321099A CN 201310032109 A CN201310032109 A CN 201310032109A CN 103116026 A CN103116026 A CN 103116026A
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Abstract
The invention relates to a quick detection method for food-borne pathogens based on immunomagnetic separation of Fe3O4 nano materials, belonging to the technical field of quick detection of food safety pathogens. Depending on an established quick screening method which can be used for pathogens in a food liquid sample, the invention prepares immunomagnetic beads from Fe3O4 nano materials, enriches target strains in a targeted manner, and indirectly detects whether the target pathogens are contained in the sample by separating the nano magnetic beads capturing the target bacteria, converting into iron ions through nitration reaction and inspecting the iron ions. The amount of the iron ions and the target bacteria show a linear relationship under certain conditions, and the target bacteria can be quantitatively detected in a certain range. The method can be used for the quick detection of harmful pathogens in a food sample, and thus can be used for quickly screening a large number of samples to be detected.
Description
Technical field
The present invention relates to the fast detecting side of a kind of pathogenic bacteria, relate in particular to a kind of based on Fe
3O
4The Methods for Fast Detection of Foodborne Pathogenic Bacteria that nano material immunity magnetic separates.
Background technology
Ultimate principle: monoclonal antibody or antigen molecule and enzyme molecule are by covalent bonds, and this combination can not change immunological characteristic and the biologically active of monoclonal antibody, antigen and enzyme, and specific monoclonal antibody only can be combined with specific antigen.The principle steps that it is main: 1. utilize Fe
3O
4Magnetic bead, coated one deck macromolecular compound adopts suitable coupling agent that the monoclonal antibody of object bacteria to be checked is coupled at magnetic bead surfaces after after modifying, and forms the specific immunity magnetic bead, and seals unnecessary avtive spot.2. the monoclonal antibody specific with another part object bacteria adopts certain method to be fixed on surface of solid phase carriers, and with unnecessary avtive spot sealing.3. the specific immunity magnetic bead with preparation is used for crawl enrichment object bacteria, by externally-applied magnetic field, magnetic bead is separated, and at this moment, combines the magnetic bead of object bacteria and does not have the magnetic bead of combining target bacterium still to mix.4. with the above-mentioned magnetic bead that mixes, be added to the surface of solid phase carriers in the 2nd step, grasped object bacteria magnetic bead will with surface of solid phase carriers monoclonal antibody generation specific binding, form double antibodies sandwich, with aseptic washed with de-ionized water can will the magnetic bead wash-out of combination not occur, as there not being object bacteria, all magnetic beads are all washed off.5. adopt again eluant, eluent that the specific nano immunomagnetic beads of combination on solid phase carrier is washed, with externally-applied magnetic field, this part magnetic bead is separated, wash ion, solvent.If this part magnetic bead exists, grasped exactly the magnetic bead of object bacteria.6. add nitric acid and sulfuric acid to carry out nitration reaction, if this part magnetic bead existence, Fe
3O
4Be converted into ferric ion and ferrous ion.In all food standards, pathogenic bacteria all must not detect.Whether exist ferric ion just can detect by detection and whether have object bacteria in sample.By mark-on, can quantitatively detect object bacteria within the specific limits.Fe in the method
3O
4Magnetic bead is the means of separation and concentration, is also simultaneously the carrier that quantitatively detects, and has played a signal and has amplified.The major advantage of the method be exactly fast, sensitivity is relatively high.Cultivate 2-3 days time of several days even with respect to the microorganisms of pathogenic bacteria.The method depends primarily on the pretreatment time of sample.Therefore, can make the positive-selecting of extensive sample to be checked of the method, the positive sample that detects also needs to cultivate with microorganism and proves conclusively.At present, also there is no this method of bibliographical information both at home and abroad, but adopt immunomagnetic beads to carry out the report of the enrichment of pathogenic bacteria, the enrichment of object etc. or a lot, but adopt the method for check ferric ion to do further detection.
Summary of the invention
The purpose of this invention is to provide a kind of based on Fe
3O
4The immune magnetic of nano material separates Methods for Fast Detection of Foodborne Pathogenic Bacteria, be used for various food samples is estimated, the method is a kind of objective method that effectively detects harmful pathogenic bacteria in food, thereby has greatly reduced to a certain extent the screening time of the harmful pathogenic bacteria of food samples.
A kind of based on Fe
3O
4Nano immune magnetic separate fast detecting and go out the method for the harmful pathogenic bacteria in food, by the immunomagnetic beads of separating trap, make it be converted into ferric ion, the correlativity index of mover iron ion and object bacteria is by detecting the indirect quantitative objective bacterium of ferric ion.Different pathogenic bacteria detect the lower limit difference.
The method depends on harmful pathogenic bacteria characteristic immunomagnetic beads enrichment, the separation in food samples of can be used for of foundation.The immunomagnetic beads of monoclonal antibody specific that adopted coupling can carry out enrichment with the specificity pathogenic bacteria in sample; By the magnetic bead of design double antibodies sandwich separating trap to object bacteria, then the magnetic bead nitration reaction is converted into ferric ion and ferrous ion.Adopt Phen absorption photometry, potassium rhodanide colourimetry etc. can detect the amount of ferric ion, thereby can calculate the amount of magnetic bead, to a certain degree indirectly quantifying the amount of object bacteria by adding the scalar quantity magnetic bead.By the magnetic bead content of quantitative test target acquisition bacterium, thereby can indirectly quantify harmful pathogenic bacteria content in food samples.Detected pathogenic bacteria content and magnetic bead content linear dependence, degree of fitting is better.Final take the corresponding relation between immunomagnetic beads and pathogenic bacteria as tie, determine the pathogenic bacteria clump count in food samples.
The present invention is achieved in that step is as follows:
1) detect the preparation of the specific immunity magnetic bead of object bacteria;
2) the object bacteria monoclonal antibody specific is fixed on surface of solid phase carriers;
3) immunomagnetic beads enrichment aimed strain, and separate: the immunomagnetic beads that the 1st step was made adds sample to be checked, fully mix concussion, the crawl object bacteria after by applying externally-applied magnetic field, magnetic bead just is pooled to magnetic field on one side, siphons away supernatant and can isolate magnetic bead, if in sample to be checked, object bacteria is arranged, by the enrichment of magnetic bead institute, add the magnetic bead suspension that a small amount of aseptic deionized water forms object bacteria;
4) the magnetic bead suspension with enrichment is added on the solid phase carrier of having fixed monoclonal antibody of the 2nd step making, if exist object bacteria to form double antibodies sandwich; With aseptic washed with de-ionized water, the magnetic bead that does not grab object bacteria is just washed off, if there is not object bacteria, all magnetic beads are all washed off;
5) afterwards, wash with the magnetic bead of eluant, eluent with the double antibodies sandwich on fixed head, separate the method for magnetic bead with externally-applied magnetic field and separate magnetic bead, drop off son and solvent with aseptic washed with de-ionized water, if also there is magnetic bead, this part magnetic bead is exactly the magnetic bead of catching object bacteria;
6) add nitric acid and sulfuric acid to carry out nitration reaction, if this part magnetic bead exists, be converted into ferric ion and ferrous ion by reaction.All food standards, pathogenic bacteria all must not detect, and have just indirectly detected the target pathogenic bacteria if detected ferric ion this moment.Adopt Phen absorption photometry, potassium rhodanide colourimetry etc. can detect the amount of ferric ion, thereby can calculate the amount of magnetic bead, to a certain degree indirectly quantifying the amount of object bacteria by adding the scalar quantity magnetic bead.
Described NMR nano immune magnetic bead core material Fe
3O
4Material, nanometer particle size are less than 1000 nanometers, and capsulating material is that glucosan, shitosan etc. can be by nitrated organic polymers.
The evaluation method that finally detects of described object bacteria is based on the concentration that whether detects ferric ion and ferric ion.
Beneficial effect of the present invention: the invention provides a kind of objective fast detecting and go out the method for the harmful pathogenic bacteria in food, it is characterized in that indirectly detecting the target pathogenic bacteria by the check ferric ion.The method can objectively detect harmful pathogenic bacteria in food effectively, confirms than the biological culture of pathogenic bacteria, and the method has the advantage of fast detecting, can be used for the rapid screening of extensive sample.Can quantitatively detect to a certain extent.
Embodiment
Example 1
Measure it in the detection food samples and whether contain harmful pathogenic bacteria---Listeria.
1. immunomagnetic beads preparation: the coupling of magnetic bead and monoclonal antibody.Adopt the profit commercial carboxylated magnetic bead PM3-020(180nm of company difficult to understand), or adopt microemulsion method to prepare voluntarily Fe
3O
4Nano microsphere is by coated carboxylated magnetic bead or the amination magnetic bead of being modified into.Monoclonal antibody adopts that biological reagent company buys commodity Listeria monoclonal antibody, or prepare voluntarily monoclonal antibody and separation and purification.Take carboxylated magnetic bead as example, coupling step: 1. clean: get respectively the carboxylated magnetic bead of 5mg in the 5mL centrifuge tube, 2mL phosphate buffer (PB) 0.02M, pH=6.0 washes 2 times, and after washing, with 2mL water morpholino b acid (MES) 0.05M, pH=6.0 is resuspended; 2. activation: the ultrasonic magnetic bead that makes disperses, then adds respectively 1mg carbodiimide (EDC), 1mg N-hydroxy-succinamide (NHSS) (using 0.05M, pH=6.0 MES dissolving), and 25 ℃ of activation 2h, gyroscope 15r/min keeps suspended state; 3. coupling: magnetic separates, and sucks supernatant, 2mL PB(0.02M, pH=7.4) wash once and be transferred to new centrifuge tube, 3mL phosphate buffer (PBS) 0.02M, pH=7.4 is resuspended, and the ultrasonic magnetic bead that makes disperses.Respectively 275 μ g antibody are joined again and activate in magnetic bead, 25 ℃ of coupling 3h, gyroscope 15r/min keeps suspended state; 4. sealing: magnetic separates, (protein content in supernatant detects with BCA determination of protein concentration kit to take out supernatant, measuring and calculating coupling amount), add 1mg/mL monoethanolamine and 1mg/mL bovine serum albumin(BSA) (BSA) sealing (0.02M, the PB dissolving of pH=7.4, and regulate pH to neutral), 25 ℃ of sealing 2h, gyroscope 15r/min keeps suspended state; 5. preserve: 0.01M PBS washing magnetic bead 2 times, magnetic separates, and removes supernatant, with 3mL pH7.4 PB(0.02M, 0.02% NaN3,0.5%BSA) resuspended, be stored in 4 ℃.The immunomagnetic beads of preparation is standby.
2. monoclonal antibody is fixed: can adopt conventional ELISA Plate fixing means, also can adopt following methods.With clean cover glass 5 * 5mm
2Square, coating machine first spray one deck Cr (2 – 4 nm) in order to help fixing gold.Be used in again surface sputtering and spray one deck nm of gold, then adopt 200 microlitre 2 mmol disulfide group-succinimide-propionic esters (DSP) to modify (DMSO, dimethyl sulfoxide (DMSO) dilution DSP) to nm of gold.Add Listeria monoclonal monoclonal antibody, be about to
Monoclonal antibody be fixed on glass plate by e and 37 ℃ hatch 45 min.Add 1% bovine serum albumin (BSA), 22 ℃, 1 hour, remaining avtive spot on plate is sealed and drying.
3. food samples is carried out pre-service, obtain liquid sample to be checked.After adding, the immune monoclonal antibody magnetic bead that the first step is made carries out fully concussion several minutes.Upper magnetic frame separates magnetic bead, adds the suspension that a small amount of aseptic deionized water obtains magnetic bead.At this moment, if the target Listeria is arranged in sample to be checked, just reached the purpose of object bacteria enrichment by the specific reaction of immunomagnetic beads.The magnetic bead suspension of this enrichment is added on prepared monoclonal antibody fixed head of the 2nd step, reaction a period of time combine in the magnetic bead emulsion listerial magnetic bead can be further with fixed head on monoclonal antibody generation specific binding, form the double antibodies sandwich structure.Just can will in conjunction with listerial magnetic bead do not wash away with aseptic washed with de-ionized water this moment.That is left on fixed head just only combines listerial magnetic bead.
4. with eluent (methyl alcohol etc.), the listerial magnetic bead that combines on fixed head is eluted.Upper magnetic frame separates magnetic bead and cleans 1-2 time, and ion, solvent are washed away.Add nitric acid and sulfuric acid to carry out nitration reaction, if there is magnetic bead, reaction makes it to be converted into ferric ion and ferrous ion.All food standards, pathogenic bacteria all must not detect, and have detected ferric ion and have just indirectly detected the target pathogenic bacteria.Add the reductive agent oxammonium hydrochloride to make ferric ion all be converted into ferrous ion, adopt the Phen absorption photometry can detect the amount of ferric ion, thereby can calculate the amount of magnetic bead, to a certain degree indirectly quantify the amount of object bacteria by adding the scalar quantity magnetic bead.
Embodiment
Example 2
Measure food samples and whether contain harmful pathogenic bacteria Escherichia coli O 157: H7.
1. immunomagnetic beads preparation: the coupling of magnetic bead and monoclonal antibody.Adopt the profit commercial carboxylated magnetic bead PM3-020(180nm of company difficult to understand), or adopt microemulsion method to prepare voluntarily Fe
3O
4Nano microsphere is by coated carboxylated magnetic bead or the amination magnetic bead of being modified into.Monoclonal antibody adopts the Escherichia coli O 157 of commodity: the H7 monoclonal antibody, or prepare voluntarily monoclonal antibody and separation and purification.Take carboxylated magnetic bead as example, coupling step: 1. clean: get respectively the carboxylated magnetic bead of 5mg in the 5mL centrifuge tube, 2mL PB(0.02M, pH=6.0) wash 2 times, use 2mL MES(0.05M, pH=6.0 after washing) resuspended; 2. activation: the ultrasonic magnetic bead that makes disperses, then adds respectively 1mg EDC, 1mg NHSS(0.05M, pH=6.0 MES dissolving), 25 ℃ of activation 2h, gyroscope 15r/min keeps suspended state; 3. coupling: magnetic separates, and sucks supernatant, 2mL PB(0.02M, pH=7.4) washing once and be transferred to new centrifuge tube, 3mL PB(0.02M, pH=7.4) resuspended, the ultrasonic magnetic bead that makes disperses.Respectively 275 μ g monoclonal antibodies are joined again and activate in magnetic bead, 25 ℃ of coupling 3h, gyroscope 15r/min keeps suspended state; 4. sealing: magnetic separates, and takes out supernatant (protein content in supernatant is to be checked), adds 1mg/mL monoethanolamine and 1mg/mL BSA sealing (0.02M, the PB of pH=7.4 dissolves, and regulates pH to neutral), 25 ℃ of sealing 2h, and gyroscope 15r/min keeps suspended state; 5. preserve: 0.01M PBS washing magnetic bead 2 times, magnetic separates, and removes supernatant, with 3mL pH7.4 PB(0.02M, 0.02% NaN3,0.5%BSA) resuspended, be stored in 4 ℃.The immunomagnetic beads of preparation is standby.
2. monoclonal antibody is fixed: can adopt conventional ELISA Plate fixing means, also can adopt following methods.With clean cover glass 5 * 5mm
2Square, coating machine first spray one deck Cr (2 – 4 nm) in order to help fixing gold.Be used in again surface sputtering and spray one deck nm of gold, then adopt 200 microlitre 2 mmol disulfide group-succinimide-propionic esters (DSP) to modify (DMSO, dimethyl sulfoxide (DMSO) dilution DSP) to nm of gold.Add the O157:H7 monoclonal antibody, be about to
Monoclonal antibody be fixed on glass plate by e and 37 ℃ hatch 45 min, form following structure.Add bovine serum albumin remaining avtive spot on plate to be sealed and drying.
3. food samples is carried out pre-service, sample is filtered, increases the pre-service such as bacterium activation, obtain sample to be checked.After adding, the immune monoclonal antibody magnetic bead that the first step is made carries out fully concussion several minutes.Upper magnetic frame separates magnetic bead, adds the emulsion that a small amount of water obtains magnetic bead.At this moment, if target Escherichia coli O 157: H7 is arranged in sample to be checked, just reached the purpose of object bacteria enrichment by the specific reaction of immunomagnetic beads.The magnetic bead suspension of this enrichment is added on prepared monoclonal antibody fixed head of the 2nd step, combine in the magnetic bead suspension magnetic bead of Escherichia coli O 157: H7 can be further with fixed head on monoclonal antibody generation specific binding, form the double antibodies sandwich structure.This moment is with aseptic washed with de-ionized water, and just can be with not in conjunction with Escherichia coli O 157: the magnetic bead of H7 washes away.That is left on fixed head just only combines the magnetic bead of Escherichia coli O 157: H7.
4. with eluent (methyl alcohol etc.), the magnetic bead that combines O157:H7 on fixed head is eluted.Upper magnetic frame separates magnetic bead and cleans 1-2 time, and ion, solvent are washed away.Add nitric acid and sulfuric acid to carry out nitration reaction, if there is magnetic bead, reaction makes it to be converted into ferric ion and ferrous ion.All food standards, pathogenic bacteria all must not detect, and have detected ferric ion and have just indirectly detected the target pathogenic bacteria.Add the reductive agent oxammonium hydrochloride to make ferric ion all be converted into ferrous ion, adopt the Phen absorption photometry can detect the amount of ferric ion, thereby can calculate the amount of magnetic bead, to a certain degree indirectly quantify the amount of object bacteria by adding the scalar quantity magnetic bead.
Claims (4)
1. one kind based on Fe
3O
4The Methods for Fast Detection of Foodborne Pathogenic Bacteria that nano material immunity magnetic separates, its characterization step is as follows:
1) detect the preparation of the specific immunity magnetic bead of object bacteria;
2) the object bacteria monoclonal antibody specific is fixed on solid phase carrier;
3) immunomagnetic beads enrichment aimed strain and separation: the immunomagnetic beads that the 1st step was made adds sample to be checked, fully mix concussion, the crawl object bacteria after by applying externally-applied magnetic field, magnetic bead just is pooled to magnetic field on one side, siphon away supernatant and can isolate magnetic bead, if in sample to be checked, object bacteria is arranged, by the enrichment of magnetic bead institute, add the magnetic bead suspension that a small amount of aseptic deionized water forms object bacteria;
4) the magnetic bead suspension with enrichment is added on the solid phase carrier of having fixed monoclonal antibody, if exist object bacteria to form double antibodies sandwich, with aseptic washed with de-ionized water, the magnetic bead that does not grab object bacteria is just washed off, if there is not object bacteria, all magnetic beads are all washed off;
5) afterwards, wash with the magnetic bead of eluant, eluent with the double antibodies sandwich on fixed head, separate the method for magnetic bead with externally-applied magnetic field and separate magnetic bead, drop off son and solvent with aseptic washed with de-ionized water, if also there is magnetic bead, this part is exactly the magnetic bead of catching object bacteria;
6) this part magnetic bead adds analytically pure nitric acid and sulfuric acid to carry out nitration reaction, makes magnetic bead Fe
3O
4Change ferric ion and ferrous ion into, adopt Phen absorption photometry, potassium rhodanide colourimetry etc. can detect the amount of ferric ion, thereby can calculate the amount of magnetic bead, to a certain degree indirectly quantify the amount of object bacteria by adding the scalar quantity magnetic bead.
2. according to claim 1 based on Fe
3O
4The Methods for Fast Detection of Foodborne Pathogenic Bacteria that nano material immunity magnetic separates is characterized in that described immunomagnetic beads is Fe
3O
4Material, finishing are that glucosan, shitosan etc. can be by the organic polymers of nitration reaction, and the nanometer magnetic bead particle diameter is less than 1000 nanometers.
3. according to claim 1 based on Fe
3O
4The Methods for Fast Detection of Foodborne Pathogenic Bacteria that nano material immunity magnetic separates is characterized in that it is by analyzing the magnetic bead Fe of target acquisition bacterium that described object bacteria finally detects evaluation method
3O
4Amount, the amount of indirect calculation object bacteria.
4. according to claim 1 based on Fe
3O
4The Methods for Fast Detection of Foodborne Pathogenic Bacteria that nano material immunity magnetic separates is characterized in that by nitration reaction, and the magnetic bead of target acquisition bacterium is changed into ferric ion, comes quantitative magnetic bead amount and quantitative objective bacterium indirectly by the amount that detects ferric ion.
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Cited By (6)
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| CN105784998A (en) * | 2016-03-09 | 2016-07-20 | 南昌大学 | Rapid virus detecting method based on Fe2O3 nanoparticles |
| CN106526197A (en) * | 2016-10-10 | 2017-03-22 | 中南大学 | Method for detecting human IgG concentration |
| CN110873791A (en) * | 2018-08-29 | 2020-03-10 | 中国农业大学 | Indirect background fluorescent colloidal gold immunochromatographic test strip based on double-labeled signal amplification and application thereof |
| CN110988349A (en) * | 2019-11-12 | 2020-04-10 | 西北农林科技大学 | Two-channel detection method of capture probe and Escherichia coli O157: H7 and application thereof |
| CN112964702A (en) * | 2021-02-08 | 2021-06-15 | 南京工业大学 | Method for rapidly detecting salmonella based on visual multicolor electrochemiluminescence technology |
| CN112964701A (en) * | 2021-02-08 | 2021-06-15 | 南京工业大学 | Method for detecting escherichia coli based on visual BPE-ECL technology |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105652007A (en) * | 2016-03-09 | 2016-06-08 | 南昌大学 | Fe3O4 nano particle based rapid virus detection method |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105784998A (en) * | 2016-03-09 | 2016-07-20 | 南昌大学 | Rapid virus detecting method based on Fe2O3 nanoparticles |
| CN106526197A (en) * | 2016-10-10 | 2017-03-22 | 中南大学 | Method for detecting human IgG concentration |
| CN110873791A (en) * | 2018-08-29 | 2020-03-10 | 中国农业大学 | Indirect background fluorescent colloidal gold immunochromatographic test strip based on double-labeled signal amplification and application thereof |
| CN110988349A (en) * | 2019-11-12 | 2020-04-10 | 西北农林科技大学 | Two-channel detection method of capture probe and Escherichia coli O157: H7 and application thereof |
| CN110988349B (en) * | 2019-11-12 | 2023-02-28 | 西北农林科技大学 | Capture probe, two-channel detection method of Escherichia coli O157: H7 and application thereof |
| CN112964702A (en) * | 2021-02-08 | 2021-06-15 | 南京工业大学 | Method for rapidly detecting salmonella based on visual multicolor electrochemiluminescence technology |
| CN112964701A (en) * | 2021-02-08 | 2021-06-15 | 南京工业大学 | Method for detecting escherichia coli based on visual BPE-ECL technology |
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| CN103116026B (en) | 2015-04-15 |
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