CN103185774B - A kind of based on Fe 3o 4the Methods for Fast Detection of Foodborne Pathogenic Bacteria of nano particle indirect enrichment immunity Magneto separate - Google Patents
A kind of based on Fe 3o 4the Methods for Fast Detection of Foodborne Pathogenic Bacteria of nano particle indirect enrichment immunity Magneto separate Download PDFInfo
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- CN103185774B CN103185774B CN201310083586.8A CN201310083586A CN103185774B CN 103185774 B CN103185774 B CN 103185774B CN 201310083586 A CN201310083586 A CN 201310083586A CN 103185774 B CN103185774 B CN 103185774B
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Abstract
A kind of based on Fe
3o
4the Methods for Fast Detection of Foodborne Pathogenic Bacteria of nano particle indirect enrichment immunity Magneto separate, belongs to technical field of quick detection of pathogenic bacteria for food safety.The present invention depends on the detection method that may be used for pathogenic bacteria in food liquid sample of foundation, utilizes 1 anti-specific binding object bacteria, utilizes Fe
3o
4nanometer particle material prepares the aimed strain of the anti-mark of 2 anti-immunomagnetic beads enrichment pointedly 1, the nanometer magnetic bead of object bacteria by separating trap, nitration reaction makes it to be converted into ferric ion again, and whether inspection ferric ion thus indirect detection go out in sample containing target pathogenic bacteria.The amount of ferric ion and object bacteria demonstrate linear relationship under certain condition, quantitatively can detect object bacteria in certain limit.The method may be used for the quick detection of harmful pathogenic bacteria in food samples, thus can as the rapid screening of large quantities of measuring samples.
Description
Technical field
The present invention relates to the quick detection side of a kind of pathogenic bacteria, particularly relate to a kind of based on Fe
3o
4the Methods for Fast Detection of Foodborne Pathogenic Bacteria of nano particle indirect enrichment immunity Magneto separate.
Background technology
Ultimate principle: monoclonal antibody or antigen molecule are combined by covalent bond with enzyme molecule, 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.Its main principle steps: 1. the 1st antibody adding object bacteria in sample to be checked, if there is object bacteria, then forms 1 anti-compound with 1 anti-binding; 2. utilize magnetic Fe
3o
4nano particle, realizes surface-functionalized by modified antibodies, that is 1 anti-antibody 2 is anti-ly coupled at magnetic bead surfaces, forms specific immunity magnetic bead, and closed by unnecessary avtive spot.2. adopt certain method to be fixed on surface of solid phase carriers the anti-monoclonal antibody of specificity 1 of another part object bacteria, and unnecessary avtive spot is closed.3. the 2 anti-specific immunity magnetic beads the 2nd step prepared join in the sample to be checked of the 1st step, for capturing enrich target bacterium, by externally-applied magnetic field by Beads enrichment out, now, the 1 anti-compound combining object bacteria and there is no combining target bacterium excessive 1 anti-all can with 2 anti-bindings of magnetic bead surfaces, still mix.4. by the above-mentioned magnetic bead mixed, be added to the surface of solid phase carriers of the 2nd step, the magnetic bead then having captured object bacteria will generation specific binding anti-with surface of solid phase carriers 1, forms double antibodies sandwich, and the magnetic bead wash-out combined can not occur by aseptic washed with de-ionized water.5. adopt eluant, eluent to be washed by the specific nano immunomagnetic beads of the combination on immobilization carrier again, Magneto separate washes ion, solvent.If this part magnetic bead exists, capture the magnetic bead of object bacteria exactly.6. add nitric acid and sulfuric acid (chloroazotic acid) carries out nitration reaction, if this part magnetic bead exists, then Fe
3o
4be converted into ferric ion and ferrous ion.In all food standards, pathogenic bacteria all must not detect.Whether there is ferric ion by detection and just can detect in sample whether there is object bacteria.By mark-on, quantitatively object bacteria can be detected within the specific limits.Fe in the method
3o
4magnetic bead is the means of separation and concentration, is also the carrier quantitatively detected simultaneously, plays the effect that a signal amplifies.The major advantage of the method be exactly fast, sensitivity is relatively high.2-3 days even time of several days is cultivated relative to the microorganism of pathogenic bacteria.The method depends primarily on the pretreatment time of sample.Therefore, can do the positive-selecting of extensive sample to be checked by the method, the positive sample detected also needs to cultivate with microorganism to confirm.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 do not adopt the method for inspection ferric ion to do further detection.
Summary of the invention
The object of this invention is to provide a kind of based on Fe
3o
4the Methods for Fast Detection of Foodborne Pathogenic Bacteria of nano particle indirect enrichment immunity Magneto separate, for evaluating various different food samples, the method is a kind of objective method effectively detecting harmful pathogenic bacteria in food, thus greatly reduces the screening time of food samples harmful pathogenic bacteria to a certain extent.
A kind of based on Fe
3o
4the immune Magneto separate Methods for Fast Detection of Foodborne Pathogenic Bacteria of nano particle indirect enrichment, by the immunomagnetic beads of indirect enrichment, separating trap, making it be converted into ferric ion, the correlation metric of mover iron ion and object bacteria, carrying out indirect quantification object bacteria by detecting ferric ion.Different pathogenic bacteria detect lower limit difference.
What the method depended on foundation can be used for the enrichment of harmful pathogenic bacteria characteristic immunomagnetic beads, separation in food samples.Adopt the coupling immunomagnetic beads of monoclonal antibody specific, the specific pathogenetic bacterium in sample can be carried out enrichment; By the magnetic bead of design double antibodies sandwich separating trap to object bacteria, then magnetic bead nitration reaction is converted into ferric ion and ferrous ion.Adopting Phen absorption photometry, potassium rhodanide colourimetry etc. can detect the amount of ferric ion, thus the amount of magnetic bead can be calculated, going out the amount of object bacteria by adding scalar quantity magnetic bead to a certain degree indirect quantification.By the magnetic bead content of quantitative test target acquisition bacterium, thus detrimental bacterial content in food samples can be gone out by indirect quantification.The pathogenic bacteria content detected and magnetic bead content linear correlation, degree of fitting is better.Final with the corresponding relation between immunomagnetic beads and pathogenic bacteria for tie, determine the pathogenic bacteria clump count in food samples.
The present invention is achieved in that step is as follows:
1) in sample to be checked, add the 1st antibody of object bacteria, if there is object bacteria, then form 1 anti-compound with 1 anti-binding;
2) preparation of the 2 anti-i.e. antibody immune magnetic beads of the 1st antibody;
3) anti-for object bacteria specificity 1 monoclonal antibody is fixed on surface of solid phase carriers;
4) immunomagnetic beads enrich target bacterial strain, and be separated: the 2 anti-immunomagnetic beadses the 2nd step obtained add the measuring samples of the 1st step, abundant mixing concussion, by applying externally-applied magnetic field after capturing object bacteria, then magnetic bead is just pooled to magnetic field on one side, if siphon away supernatant then can isolate in magnetic bead sample to be checked and have object bacteria, then by the enrichment of magnetic bead institute, add the magnetic bead suspension that deionized water aseptic on a small quantity then forms object bacteria; Now, the 1 anti-compound combining object bacteria and there is no combining target bacterium excessive 1 anti-all can with 2 anti-bindings of magnetic bead surfaces, still mix.
4) the magnetic bead suspension of enrichment is added on the solid phase carrier securing 1 anti-monoclonal antibody of the 3rd step making, if there is object bacteria, forms double antibodies sandwich; By aseptic washed with de-ionized water, then the magnetic bead not grabbing object bacteria is just washed off, if there is not object bacteria, then all magnetic beads are all washed off;
5) after, wash with the magnetic bead of eluant, eluent by the double antibodies sandwich on fixed head, the method being separated magnetic bead with externally-applied magnetic field drops off son and solvent by aseptic washed with de-ionized water, if also there is magnetic bead is exactly the magnetic bead catching object bacteria;
6) add nitric acid and sulfuric acid (chloroazotic acid) carries out nitration reaction, if this part magnetic bead exists, be then converted into ferric ion and ferrous ion by reaction.All food standards, pathogenic bacteria all must not detect, if now detected ferric ion just indirectly detected target pathogenic bacteria.After excessive acid is neutralized, adopting Phen absorption photometry, potassium rhodanide colourimetry etc. can detect the amount of ferric ion, thus the amount of magnetic bead can be calculated, going out the amount of object bacteria by adding scalar quantity magnetic bead to a certain degree indirect quantification.
Described nano immune magnetic bead core material Fe
3o
4material, nanometer particle size is less than 1000 nanometers.
The evaluation method that finally detects of described object bacteria is concentration based on whether detecting ferric ion and ferric ion.
Beneficial effect of the present invention: the invention provides a kind of method objectively detecting the harmful pathogenic bacteria in food fast, is characterized in that indirectly detecting target pathogenic bacteria by inspection ferric ion.The method can objectively detect harmful pathogenic bacteria in food effectively, confirms compared to the biological culture of pathogenic bacteria, and the method has the advantage detected fast, may be used for the rapid screening of extensive sample.Can quantitatively detect to a certain extent.
Embodiment
Example 1
Whether it is measured containing harmful pathogenic bacteria---Listeria in inspection food samples.
1.1.2 anti-immunomagnetic beads preparation: 1 anti-employing listerial rabbit anti-igg monoclonal antibody, 2 resist for listerial goat anti-rabbit igg, can be monoclonal antibody also can be resist more.2 anti-immunomagnetic beads preparations: the coupling of magnetic bead and monoclonal antibody.Adopt the carboxylated magnetic bead of commercial Ao Run company, PM3-020(180nm) and 2 anti-couplings.Coupling step: 1. clean: get the carboxylated magnetic bead of 5mg respectively in 5mL centrifuge tube, 2mLPB(0.02M, pH=6.0) wash 2 times, wash rear 2mLMES(0.05M, pH=6.0) resuspended; 2. activate: the ultrasonic magnetic bead that makes disperses, then adds 1mgEDC respectively, 1mgNHSS(0.05M, pH=6.0MES dissolve), 25 DEG C of activation 2h, gyroscope 15r/min keep suspended state; 3. coupling: Magneto separate, sucks supernatant, 2mLPB(0.02M, pH=7.4) wash once and be transferred to new centrifuge tube, 3mLPB(0.02M, pH=7.4) resuspended, the ultrasonic magnetic bead that makes disperses.Joined by 275 μ g the 2nd antibody goat anti-rabbit iggs respectively more in activated magnetic beads, 25 DEG C of coupling 3h, gyroscope 15r/min keep suspended state; 4. close: Magneto separate, take out supernatant (protein content in supernatant is to be checked), add 1mg/mL monoethanolamine and 1mg/mLBSA closed (PB of 0.02M, pH=7.4 dissolves, and regulates pH to neutral), 25 DEG C of closed 2h, gyroscope 15r/min keep suspended state; 5. preserve: 0.01MPBS washs magnetic bead 2 times, and Magneto separate, removes supernatant, with 3mLpH7.4PB(0.02M, 0.02%NaN3,0.5%BSA) resuspended, be stored in 4 DEG C.The immunomagnetic beads of preparation is for subsequent use.
2.2.1 anti-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 sprays one deck Cr (2 – 4nm) in order to help fixing gold.Be used in surface sputtering again and spray one deck nm of gold, then adopt 200 microlitre 2mmol disulfide group-succinimide-propionic esters (DSP) to modify (DMSO, dimethyl sulfoxide (DMSO) dilution DSP) nm of gold.Add listerial rabbit anti-igg monoclonal monoclonal antibody, fix on a glass also 37 C by 100 μ L100 μ g/mL monoclonal antibodies by e and hatch 45min.Add 1% bovine serum albumin (BSA), 22 C, 1 hour, avtive spot remaining on plate is carried out close and dry.
3.3. food samples is carried out pre-service, adopt FDA enrichment if desired, sample is filtered, increases the pre-service such as bacterium activation, obtain sample to be checked.The 1st antibody will be added, listerial rabbit anti-igg in sample to be checked.If there is Listeria in sample to be checked, will with the anti-compound of 1 anti-formation 1.Fully shake after the listerial goat anti-rabbit igg magnetic bead of the 2nd antibody that 1st step is obtained is added.Upper magnetic frame is separated magnetic bead, adds the suspension that deionized water aseptic on a small quantity obtains magnetic bead.Now, if having target Listeria in sample to be checked, then pass through the interaction of the 2nd antibody and the 1st antibody, thus catch this compound, reach the object of object bacteria enrichment.Now, the 1 anti-compound combining object bacteria and there is no combining target bacterium excessive 1 anti-all can with 2 anti-bindings of magnetic bead surfaces, still mix.The magnetic bead suspension of this enrichment is added on 1 anti-fixed head prepared by the 2nd step, then combine in magnetic bead emulsion listerial magnetic bead can further with the monoclonal antibody generation specific binding on fixed head, form double antibodies sandwich structure.Now by aseptic washed with de-ionized water, just can will not wash away in conjunction with listerial magnetic bead.What fixed head was left 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, is separated magnetic bead and cleans 1-2 time, ion, solvent being washed away.Add nitric acid and sulfuric acid carries out nitration reaction, if there is magnetic bead, then 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 target pathogenic bacteria.Neutralization reaction is neutral to pH value, adding reductive agent oxammonium hydrochloride makes ferric ion all be converted into ferrous ion, adopt Phen absorption photometry can detect the amount of ferric ion, thus the amount of magnetic bead can be calculated, by adding scalar quantity magnetic bead and going out the amount of object bacteria to a certain degree indirect quantification.
Embodiment
example 2
Whether measure food samples containing harmful pathogenic bacteria Escherichia coli O 157: H7.
1.2 anti-immunomagnetic beadses preparations: the rabbit anti-igg monoclonal antibody of 1 anti-employing O157:H7,2 resist the goat anti-rabbit igg for O157:H7, can be monoclonal antibody also can be resist more.2 anti-immunomagnetic beads preparations: the coupling of magnetic bead and monoclonal antibody.Adopt the carboxylated magnetic bead of commercial Ao Run company, PM3-020(180nm) and 2 anti-couplings.Coupling step: 1. clean: get the carboxylated magnetic bead of 5mg respectively in 5mL centrifuge tube, 2mLPB(0.02M, pH=6.0) wash 2 times, wash rear 2mLMES(0.05M, pH=6.0) resuspended; 2. activate: the ultrasonic magnetic bead that makes disperses, then adds 1mgEDC respectively, 1mgNHSS(0.05M, pH=6.0MES dissolve), 25 DEG C of activation 2h, gyroscope 15r/min keep suspended state; 3. coupling: Magneto separate, sucks supernatant, 2mLPB(0.02M, pH=7.4) wash once and be transferred to new centrifuge tube, 3mLPB(0.02M, pH=7.4) resuspended, the ultrasonic magnetic bead that makes disperses.Joined by 275 μ g the 2nd antibody respectively more in activated magnetic beads, 25 DEG C of coupling 3h, gyroscope 15r/min keep suspended state; 4. close: Magneto separate, take out supernatant (protein content in supernatant is to be checked), add 1mg/mL monoethanolamine and 1mg/mLBSA closed (PB of 0.02M, pH=7.4 dissolves, and regulates pH to neutral), 25 DEG C of closed 2h, gyroscope 15r/min keep suspended state; 5. preserve: 0.01MPBS washs magnetic bead 2 times, and Magneto separate, removes supernatant, with 3mLpH7.4PB(0.02M, 0.02%NaN3,0.5%BSA) resuspended, be stored in 4 DEG C.The immunomagnetic beads of preparation is for subsequent use.
2.1 anti-monoclonal antibodies are fixed: can adopt conventional ELISA Plate fixing means, also can adopt following methods.With clean cover glass 5 × 5mm
2square, coating machine first sprays one deck Cr (2 – 4nm) in order to help fixing gold.Be used in surface sputtering again and spray one deck nm of gold, then adopt 200 microlitre 2mmol disulfide group-succinimide-propionic esters (DSP) to modify (DMSO, dimethyl sulfoxide (DMSO) dilution DSP) nm of gold.Add O157:H7 rabbit anti-igg monoclonal antibody, fix on a glass also 37 C by 100 μ L100 μ g/mL monoclonal antibodies by e and hatch 45min.Add bovine serum albumin avtive spot remaining on plate is carried out close and dry.
3. food samples carried out pre-service, sample is filtered, increase the pre-service such as bacterium activation, obtain sample to be checked.The 1st antibody will be added, the rabbit anti-igg of O157:H7 in sample to be checked.If there is O157:H7 in sample to be checked, will with the anti-compound of 1 anti-formation 1.Fully shake after the goat anti-rabbit igg antibody magnetic bead of the 2nd antibody O157:H7 the 1st step obtained adds.Upper magnetic frame is separated magnetic bead, adds the emulsion that a small amount of water obtains magnetic bead.Now, if having target Escherichia coli O 157 in sample to be checked: H7, then pass through the interaction of the 2nd antibody and the 1st antibody, thus catch this compound, reach the object of object bacteria enrichment.Now, the 1 anti-compound combining object bacteria and there is no combining target bacterium excessive 1 anti-all can with 2 anti-bindings of magnetic bead surfaces, still mix.The magnetic bead suspension of this enrichment is added on 1 anti-fixed head prepared by the 2nd step, then combines Escherichia coli O 157 in magnetic bead suspension: the magnetic bead of H7 can further with the monoclonal antibody generation specific binding on fixed head, form double antibodies sandwich structure.Now by aseptic washed with de-ionized water, just can by conjunction with Escherichia coli O 157: the magnetic bead of H7 washes away.What fixed head was left just only combines Escherichia coli O 157: the magnetic bead of H7.
4. fixed head will combine Escherichia coli O 157 with eluent (methyl alcohol etc.): the magnetic bead of H7 elutes.Upper magnetic frame, is separated magnetic bead and cleans 1-2 time, ion, solvent being washed away.Add nitric acid and sulfuric acid (chloroazotic acid) carries out nitration reaction, if there is magnetic bead, then 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 target pathogenic bacteria.Neutralization reaction is neutral to pH value, adding reductive agent oxammonium hydrochloride makes ferric ion all be converted into ferrous ion, adopt Phen absorption photometry can detect the amount of ferric ion, thus the amount of magnetic bead can be calculated, by adding scalar quantity magnetic bead and going out the amount of object bacteria to a certain degree indirect quantification.
Claims (5)
1. one kind based on Fe
3o
4the Methods for Fast Detection of Foodborne Pathogenic Bacteria of nano particle indirect enrichment immunity Magneto separate, its characterization step is as follows:
1) 1 anti-binding detecting object bacteria and object bacteria is formed 1 anti-compound;
2) 1 antiantibody of object bacteria is detected, i.e. the preparation of 2 anti-immunomagnetic beadses;
3) 1 anti-monoclonal antibody fixing in ELISA Plate is separately got;
4) immunomagnetic beads enrich target bacterial strain, and be separated: by the 2nd) the obtained 2 anti-immunomagnetic beadses of step join the 1st) step combines 1 anti-measuring samples, abundant mixing concussion, by applying externally-applied magnetic field after capturing object bacteria, then magnetic bead is just pooled to magnetic field on one side, siphons away supernatant and then can isolate magnetic bead; If have object bacteria in sample to be checked, first object bacteria forms compound with 1 anti-binding, after adding 2 diamagnetic pearls, anti-and the 1 anti-interaction by 2, thus catch 1 anti-compound, by the enrichment of magnetic bead institute, add the magnetic bead suspension that deionized water aseptic on a small quantity then forms object bacteria;
5) the magnetic bead suspension of enrichment being added to the 3rd) step secures in the ELISA Plate of 1 anti-monoclonal antibody, if there is object bacteria, form double antibodies sandwich, by aseptic washed with de-ionized water, the magnetic bead then not grabbing object bacteria is just washed off, if there is not object bacteria, then all magnetic beads are all washed off;
6) after, wash with the magnetic bead of eluant, eluent by the double antibodies sandwich in ELISA Plate, the method being separated magnetic bead with externally-applied magnetic field drops off son and solvent by aseptic washed with de-ionized water, if also there is magnetic bead is exactly the magnetic bead catching object bacteria;
7) this part magnetic bead, adds analytically pure red fuming nitric acid (RFNA) and concentrated hydrochloric acid carries out nitration reaction, and the volume ratio 1:3 of red fuming nitric acid (RFNA) and concentrated hydrochloric acid makes magnetic bead Fe
3o
4change ferric ion and ferrous ion into; In and adopt Phen absorption photometry or potassium rhodanide colourimetry can detect the amount of ferric ion after excess acid, thus the amount of magnetic bead can being calculated, going out the amount of object bacteria by adding scalar quantity magnetic bead to a certain degree indirect quantification.
2. according to claim 1 based on Fe
3o
4the Methods for Fast Detection of Foodborne Pathogenic Bacteria of nano particle indirect enrichment immunity Magneto separate, is characterized in that described nano particle is Fe
3o
4nano particle, nanometer particle size is less than 1000 nanometers.
3. according to claim 1 based on Fe
3o
4the Methods for Fast Detection of Foodborne Pathogenic Bacteria of nano particle indirect enrichment immunity Magneto separate, is characterized in that by nitration reaction, the magnetic bead of target acquisition bacterium is changed into ferric ion, by detecting that the amount of ferric ion carrys out quantitative magnetic bead amount and indirect quantification object bacteria.
4. according to claim 1 based on Fe
3o
4the Methods for Fast Detection of Foodborne Pathogenic Bacteria of nano particle indirect enrichment immunity Magneto separate, it is characterized in that object bacteria first forms compound with 1 anti-binding, with 2 anti-immunomagnetic beads enrichments, separation, 1 has resisted signal amplification.
5. according to claim 1 based on Fe
3o
4the Methods for Fast Detection of Foodborne Pathogenic Bacteria of nano particle indirect enrichment immunity Magneto separate, 1 anti-finger object bacteria monoclonal antibody, the antibody that 2 anti-fingers 1 are anti-can be monoclonal also can be polyclonal antibody.
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| CN110596383A (en) * | 2019-09-24 | 2019-12-20 | 珠海市德灏生物科技有限公司 | Fluorescent dyeing-based rapid magnetic separation and identification method and kit for general pathogenic microorganisms |
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| CN101509919A (en) * | 2009-03-12 | 2009-08-19 | 湖南工业大学 | Method for producing water- soluble magnetic nanoparticle for detecting SQUID |
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| CN101509919A (en) * | 2009-03-12 | 2009-08-19 | 湖南工业大学 | Method for producing water- soluble magnetic nanoparticle for detecting SQUID |
Non-Patent Citations (2)
| Title |
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| Nanoparticle labelling-based magnetic immunoassay on chip combined with electrothermal vaporization - inductively coupled plasma mass spectrometry for the determination of carcinoembryonic antigen in human serum;Beibei Chen 等;《Analyst》;20110822;第136卷;摘要，3937页右栏最后一段至3938页左栏第一段，Scheme 1，Fig 7 * |
| 超小的羧甲基壳聚糖超顺磁氧化铁纳米粒制备及处方优化;范彩霞等;《中国现代应用药学》;20100928;第27卷(第9期);摘要、2.2.5节、3.2.5节 * |
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