A method of detection campylobacter jejuni
阅读说明：本技术 一种检测空肠弯曲杆菌的方法 (A method of detection campylobacter jejuni ) 是由 吴正宗 崔波 袁超 刘鹏飞 于滨 郭丽 赵海波 陶海腾 于 2019-09-06 设计创作，主要内容包括：本发明涉及一种检测空肠弯曲杆菌的方法,包括以下步骤：(1)制备检测线包被空肠弯曲杆菌抗体、质控线包被羊抗鼠IgG抗体的试纸条；(2)在试纸条检测区域下方放置磁铁；(3)检测空肠弯曲杆菌含量。本发明的试纸条基于在检测区域聚集的Fe<Sub>3</Sub>O<Sub>4</Sub>@Au所产生的磁信号,可定量测定样品中的空肠弯曲杆菌含量。本发明的试纸条检测限低(1cfu/mL、10 cfu/mL)、特异性好、成本低廉、检测时间短。此外,该类型试纸条不需要任何额外设备即可实现对目标物的快速灵敏检测,能够满足现场快速检测的需求,适用于液体样品中致病菌及各种有毒有害物质的检测,极具实用价值。(The present invention relates to a kind of methods for detecting campylobacter jejuni, comprising the following steps: (1) prepares the test strips of detection line coating campylobacter jejuni antibody, nature controlling line coating sheep anti-mouse igg antibody；(2) magnet is placed below test strips detection zone；(3) campylobacter jejuni content is detected.Test strips of the invention are based on the Fe assembled in detection zone 3 O 4 Magnetic signal caused by@Au can quantitative determine the campylobacter jejuni content in sample.Test strips detection of the invention limits that low (1cfu/mL, 10 cfu/mL), specificity is good, low in cost, detection time is short.It can be realized in addition, the type test strips do not need any extras to the detection of the rapid sensitive of object, can satisfy the demand of field quick detection, the detection of pathogenic bacteria and various poisonous and harmful substances suitable for fluid sample, great practical value.)
1. a kind of method for detecting campylobacter jejuni, which comprises the following steps:
(1) test strips of preparation detection line coating campylobacter jejuni antibody, nature controlling line coating sheep anti-mouse igg antibody；
(2) magnet is placed below test strips detection zone；
(3) preparation and modification of golden magnetic nano particle: preparation Fe3O4@Au nano particle and in its surface modification campylobacter jejuni Antibody detects campylobacter jejuni content using test strips；Or by sample solution and Fe3O4@AuNPs solution, avidin solution It is sufficiently mixed with liposome, reacts at room temperature, after being then added drop-wise to test strips chromatography, Triton X-100 solution is added, finally Gold nano grain solution is added in test strips detection zone, detects campylobacter jejuni content.
2. a kind of method for detecting campylobacter jejuni according to claim 1, which is characterized in that the magnetic of the magnet Field intensity is in 20mT-500mT.
3. a kind of method for detecting campylobacter jejuni according to claim 1, which is characterized in that the test strips packet Include liner plate, sample pad, nanoparticle marker bonding pad, nitrocellulose filter and blotting paper, the sample pad, magnetic Nano Grain marker bonding pad, nitrocellulose filter and blotting paper are successively pasted from back to front to be adsorbed on the liner plate, the nitric acid Drawing on cellulose membrane has a detection line and a nature controlling line；The detection line is coated with campylobacter jejuni antibody；The Quality Control Line is coated with sheep anti-mouse igg antibody.
4. a kind of method for detecting campylobacter jejuni according to claim 3, which is characterized in that the preparation side of test strips Method the following steps are included:
(1) preparation of golden magnetic nano particle: synthesizing magnetic nano particle Fe3O4, in one layer of gold of its pan coating, obtain Fe3O4@Au Nano particle；Fe3O4@Au surface modification campylobacter jejuni antibody；
(2) example enrichment pre-processes: will modify the Fe of campylobacter jejuni antibody3O4It is molten [email protected] nanoparticles solution is added to sample It sufficiently reacts in liquid, campylobacter jejuni present in enriched sample, then outside plus under magnetic field condition, isolates in conjunction with jejunum The immunomagnetic nanoparticles of campylobacter, are re-dissolved in PBS solution；
(3) processing of nitrocellulose film: campylobacter jejuni antibody-solutions being sprayed on nitrocellulose film and form detection line, will Sheep anti-mouse igg antibody solution spraying forms detection nature controlling line on nitrocellulose film, and drying is to get to treated nitrocellulose Film；
(4) assembling of test strips: successively overlapping on liner plate and sample pad, bonding pad and nitrocellulose after gluing treatment Film, then overlap blotting paper in the side of acid cellulose film, is cut to 60mm long after assembling, the test strips of 3.8mm wide are to get arriving institute Test strips obtained, are packed into that be protected from light bag close by the immuno-chromatographic test paper strip for stating Quantitative Detection of Campylobacter jejuni together with desiccant Envelope saves stand-by.
5. a kind of method for detecting campylobacter jejuni according to claim 3, which is characterized in that the preparation side of test strips Method the following steps are included:
(1) preparation of golden magnetic nano particle: synthesizing magnetic nano particle Fe3O4, in its pan coating golden shell, obtain Fe3O4@ AuNPs nano particle, in Fe3O4@AuNPs surface modification campylobacter jejuni antibody；
(2) liposome of load horseradish peroxidase HRP the preparation of liposome: is prepared using reverse phase evaporation；Liposome is straight Diameter is 150-500nm；
(3) processing of nitrocellulose film: campylobacter jejuni antibody-solutions being sprayed on nitrocellulose film and form detection line, will Sheep anti-mouse igg antibody solution spraying forms detection nature controlling line on nitrocellulose film, and drying is to get to treated nitrocellulose Film；
(4) assembling of test strips: successively overlapping on liner plate and sample pad, bonding pad and nitrocellulose after gluing treatment Film, then overlap blotting paper in the side of acid cellulose film, is cut to 60mm long after assembling, the test strips of 3.8mm wide are to get arriving institute Test strips obtained, are packed into that be protected from light bag close by the immuno-chromatographic test paper strip for stating Quantitative Detection of Campylobacter jejuni together with desiccant Envelope saves stand-by.
6. a kind of method for detecting campylobacter jejuni according to claim 1, which is characterized in that test strips further include mould The golden magnetic nano particle Fe of campylobacter jejuni antibody is modified on nanoparticle marker bonding pad3O4@Au。
7. a kind of method for detecting campylobacter jejuni according to claim 1, which is characterized in that golden magnetic nano particle Fe3O4@Au diameter is in 20-500nm.
8. a kind of method for detecting campylobacter jejuni according to claim 7, which is characterized in that the test strips packet Include liner plate, sample pad, nanoparticle marker bonding pad, nitrocellulose filter and blotting paper, the sample pad, magnetic Nano Grain marker bonding pad, nitrocellulose filter and blotting paper are successively pasted from back to front is adsorbed on the nitric acid fibre on the liner plate It ties up to draw on plain film and has a detection line and a nature controlling line；The detection line is coated with campylobacter jejuni antibody；The nature controlling line It is coated with sheep anti-mouse igg antibody；The magnetic nanoparticle marker combines the Jin Cina for being lined with modification campylobacter jejuni antibody Rice grain Fe3O4@Au。
9. a kind of method for detecting campylobacter jejuni according to claim 7 or 8, which is characterized in that the system of test strips Preparation Method the following steps are included:
(1) preparation of golden magnetic nano particle: synthesizing magnetic nano particle Fe3O4, in one layer of gold of its pan coating, obtain Fe3O4@Au Nano particle, in Fe3O4@Au surface modification campylobacter jejuni antibody；
(2) Fe of campylobacter jejuni antibody will the processing of bonding pad: be modified3O4@Au nanoparticles solution is sprayed on bonding pad, The marker bonding pad that obtains that treated；
(3) processing of nitrocellulose film: campylobacter jejuni antibody-solutions being sprayed on nitrocellulose film and form detection line, will Sheep anti-mouse igg antibody solution spraying forms detection nature controlling line on nitrocellulose film, and drying is to get to treated nitrocellulose Film；
(4) assembling of test strips: successively overlapping on liner plate and sample pad, bonding pad and nitrocellulose after gluing treatment Film, then overlap blotting paper in the side of acid cellulose film, is cut to 60mm long after assembling, the test strips of 3.8mm wide are to get arriving institute Test strips obtained, are packed into that be protected from light bag close by the immuno-chromatographic test paper strip for stating Quantitative Detection of Campylobacter jejuni together with desiccant Envelope saves stand-by.
10. a kind of method of detection campylobacter jejuni described in one of -8 according to claim 1, which is characterized in that jejunum is curved Curved bar bacterium is monoclonal antibody or polyclonal antibody.
Technical field
The invention belongs to analysis detection fields, and in particular to a method of detection campylobacter jejuni.
Background technique
Campylobacter jejuni, a kind of zoonosis pathogen are one of the main pathogenic fungis for threatening human health.Due to It is widely present in the animal bodies such as bird, fowl, dog, cat, can cause to feel by pollution meat, milk and the water of non-sterile processing Dye, often crowd's quantity is more for the disease caused by it, and distribution is wide, brings huge threat to food safety and health of people.Cause This, the detection of campylobacter jejuni has very important significance in food.
It is currently used detection food-borne pathogens method mainly have colony counting method, recombinase polymeric enzymatic amplification method, Instant Polymerase Chain Reaction method, enzyme-linked immunosorbent assay, Surface Plasmon Resonance, quartz crystal microbalance sensing Device, impedance type immunosensor, color sensor, fluoroimmunoassay etc..These existing detection methods usually require pair Sample is separated, purifies culture, enrichment, and step is various, experimental period is long, causes entire detection process time-consuming and laborious, cannot Meet the needs of on-site rapid inspection.
Immunochromatography technique in field of food safety has huge answer because of the features such as its is at low cost, quick, simple, portable Use prospect.However, traditional immunochromatography technique based on colloidal gold haves the defects that sensitivity is low, its application is strongly limited Range and commercialized possibility.Therefore, the bottleneck problem for overcoming immunochromatography technique sensitivity low using new strategy, for Its application range is expanded, improving the practicality just becomes particularly necessary.
Summary of the invention
In order to solve this obstruction immunochromatography technique application bottleneck, the present invention in, we by by classics use Colloidal gold is replaced into magnetic gold nano grain, reduces it by the way of the rate travel on nitrocellulose film gram using externally-applied magnetic field The low defect of immunochromatography technique sensitivity is taken.The strategy magnetic nanoparticle it is highly selective, can enriching and flow velocity Controllability is combined with high-throughput, quick, the simple and direct advantage of immunochromatography technique, and detection sensitivity greatly improved.
The basic principle of method of the present invention is as follows: being different from classical colloidal gold immunity chromatography, the present invention proposes The immunochromatographic method based on " magnetic focusing " strategy use Fe3O4@Au nano particle is being tried instead of Au colloidal nanoparticles One piece of small magnet is placed below paper slip detection zone.
In method 1: the Fe of campylobacter jejuni antibody will be modified3O4@Au nano particle is sprayed on bonding pad.Jejunum is curved The specific region that curved bar bacteria antibody and sheep anti-mouse igg are fixed on nitrocellulose film is respectively formed detection line and nature controlling line.When by sample When product solution is added drop-wise in sample pad, sample flows in test strips under capillary action, when being moved to bonding pad, based on anti- The Fe of antibody is modified in immune response between original antibody3O4@Au is formed in conjunction with the campylobacter jejuni contained in sample Fe3O4@Au- antibody-target bacterium compound.Sample continuation continues to flow on nitrocellulose film, when compound flows to detection line When region, due to the effect in magnetic field, magnetic Fe3O4@Au nano particle can make flow velocity obviously slow down, and be fixed on nitre in this way Antibody on base tunica fibrosa can have longer time and Fe3O4@Au- antibody-target bacterium compound, which combines, forms " sandwich " folder Core structure.Without combine campylobacter jejuni compound then continue to move along, at nature controlling line with two anti-bindings.
Method 2: in the present invention, we can also be rich using immunomagnetic nanoparticles first before immunochromatography progress Collect the object bacteria in sample solution, to achieve the purpose that the final detection sensitivity of enhancing.It is first before the progress of immunochromatography process First using the Fe of modification campylobacter jejuni antibody3O4@Au nano particle is mixed with sample solution, and the jejunum in enriched sample is curved Curved bar bacterium.Then outside plus under magnetic field condition, the Fe of campylobacter jejuni will be combined3O4@Au nano particle and supernatant separation, And it is re-dissolved in PBS solution and solution is used for immunochromatography detection.Campylobacter jejuni antibody and sheep anti-mouse igg are consolidated The specific region for being scheduled on nitrocellulose film is respectively formed detection line and nature controlling line.Fe will be dissolved3O4@Au- antibody-jejunum campylobacter bar The PBS solution of bacterium compound is added drop-wise in sample pad, and sample flows in test strips under capillary action, when compound flows to When detection line region, due to the effect in magnetic field, magnetic Fe3O4@Au nano particle can make flow velocity obviously slow down, solid in this way The antibody being scheduled on nitrocellulose film can have longer time and Fe3O4@Au- antibody-target bacterium compound combines and forms " three Mingzhi " interlayer structure.Without combine campylobacter jejuni compound then continue to move along, at nature controlling line with two resistive connections It closes.
Due to the extension in reaction time, correspondingly, compared with the immune chromatography method for not applying magnetic field, just have more The antibody that object is fixed on detection zone is captured.Therefore, final testing result is obviously more preferable, i.e., detection limit it is lower, Sensitivity is higher.It is sandwich in " sandwich " that detection line goes out aggregation if the campylobacter jejuni concentration in sample is higher Structural composites are more, can show Fe3O4The color [email protected] nano particle.On the contrary, if campylobacter jejuni in sample Concentration is lower, then it is fewer in " sandwich " interlayer structure compound that detection line goes out aggregation, then it will not show Fe3O4@Au The color of nano particle.After chromatography process, test strips are put into the dedicated card slot of magnetic signal detector, test strip detection The magnetism intensity in region arrange to data and draws standard curve, and campylobacter jejuni content in sample is determined in realization It is fixed to measure.
The present invention greatly improves the sensitivity of immune chromatography method, the detection of method 1 by introducing " magnetic focusing " strategy For limit down to 10cfu/mL, sensitivity can reach 10000 times of traditional colloidal gold method.The detection of method 2 is limited down to 1cfu/mL, Sensitivity can reach 100000 times of traditional colloidal gold method.And because this method is the magnetism based on golden magnetic nano particle, inspection Surveying result is hardly influenced by color sample.Because magnetic nanoparticle magnetism is difficult to change, testing result can also It saves for a long time.The present invention is that the rapid field detection of harmful constituent in food system specifies new direction.
Preferably, the magnetic field strength of the magnet is 20mT-200mT in the method.Golden magnetic nano particle (Fe3O4@ Au) diameter is 100-500nm.
Method 3: we load auxiliary letter from the angle of " signal of detection zone is made to be easier to read ", by introducing The liposome of number amplification factor (i.e. L-cysteine) by object in Traditional immunochromatographic method and detects the ratio of signal 1:1 Example relationship changes into 1:n, further improves the sensitivity of immunochromatography technique.
The basic principle of method of the present invention is as follows: being different from classical colloidal gold immunity chromatography, the present invention proposes The double mode immunochromatographic method based on " magnetic focusing " strategy use Fe3O4@AuNPs nano particle and load auxiliary signal amplify Compound (liposome-the Fe of the liposome of the factor (L-cysteine)3O4@AuNPs) instead of Au colloidal nanoparticles, and One piece of small magnet is placed below test strips detection zone.Campylobacter jejuni antibody and sheep anti-mouse igg are fixed on nitrocellulose The specific region of film is respectively formed detection line and nature controlling line.By the Fe of sample solution and modification campylobacter jejuni antibody3O4@ AuNPs and liposome mixing, when being added drop-wise in sample pad, sample flows in test strips under capillary action, ties when being moved to When closing pad, based on the immune response between antigen-antibody, Antibody Liposomes-Fe is modified3O4@AuNPs by with the jejunum that contains in sample Campylobacter combines, and forms liposome-Fe3O4@AuNPs- antibody-target bacterium compound.Sample continue on nitrocellulose film after Afterflow is dynamic, when compound flows to detection line region, due to the effect in magnetic field, and magnetic liposome-Fe3O4@AuNPs meeting So that flow velocity obviously slows down, the antibody being fixed on nitrocellulose film in this way can have longer time and liposome-Fe3O4@ AuNPs- antibody-target bacterium compound combines and forms " sandwich " interlayer structure.Without combining the compound of campylobacter jejuni Object then continues to move along, at nature controlling line with two anti-bindings.Due to the extension in reaction time, correspondingly, and do not apply magnetic field Immune chromatography method compare, just have more objects and be fixed on the antibody of detection zone and captured.Therefore, finally Testing result is obviously more preferable, i.e., detection limit is lower, sensitivity is higher.If the campylobacter jejuni concentration in sample is higher, that It is more in " sandwich " interlayer structure compound that detection line goes out aggregation, it can show liposome-Fe3O4@AuNPs is compound The color of object.On the contrary, going out " sandwich " folder assembled in detection line if the campylobacter jejuni concentration in sample is lower Core structure compound is fewer, then will not show liposome-Fe3O4The color [email protected] compound.After chromatography process, It is firstly added Triton X-100 and destroys liposome structure, release the L-cysteine loaded in its cavity.Then plus Enter gold nano grain, under the conditions of existing for the L-cysteine, gold nano grain is combined together, and is generated aggregation, is led to test paper Detection line goes out the red color signal enhancing generated.Finally, test strips are able to detect the jejunum campylobacter of low concentration in sample Bacillus.
The present invention is added significantly to the sensitivity of immune chromatography method, detection by introducing " magnetic focusing " strategy and liposome For limit down to 10cfu/mL, sensitivity can reach 10000 times of traditional colloidal gold method.The present invention be food system in pathogenic bacteria and The rapid field detection of poisonous and harmful chemical constituent specifies new direction.
Preferably, the magnetic field strength of the magnet is 100mT-500mT in the method.Liposomal diameter is 150- 500nm。
Beneficial effect
Based on example enrichment pretreatment and " magnetic focusing " strategy, the sensitivity of immuno-chromatographic test paper strip is greatly improved, is solved The low bottleneck problem of its sensitivity.Golden magnetic nano particle (Fe used in the invention3O4@Au) both in sample pretreatment It is enriched with campylobacter jejuni in step, and reduces operation step for constructing signal probe in subsequent immunochromatography detection Suddenly, it reduces costs.
Test strips of the invention are based on the Fe assembled in detection zone3O4Magnetic signal caused [email protected] can quantitative determine sample Campylobacter jejuni content in product.Also by the liposome of introducing " magnetic focusing " strategy and embedding L-cysteine, substantially mention The sensitivity for having risen immuno-chromatographic test paper strip solves the low bottleneck problem of its sensitivity.Test strips detection limit of the invention is low (1cfu/mL, 10 cfu/mL, 10 cfu/mL), specificity is good, low in cost, detection time is short.In addition, the type test strips Not needing any extras can be realized to the detection of the rapid sensitive of object, can satisfy the demand of field quick detection, The detection of pathogenic bacteria and various poisonous and harmful substances suitable for fluid sample, great practical value.
Detailed description of the invention
Mold of the Fig. 1 for novel immune chromatography detection；
Fig. 2 campylobacter jejuni concentration range is 20-105The standard curve that magnetic data when cfu/mL based on acquisition is established；
Fig. 3 campylobacter jejuni concentration range is 10-105The standard curve that magnetic data when cfu/mL based on acquisition is established；
Fig. 4 campylobacter jejuni concentration range is 25-105The mark that color data when cfu/mL based on acquisition is established is bent.
Specific embodiment
It elaborates below to the embodiment of the present invention, the present embodiment carries out under the premise of the technical scheme of the present invention Implement, the detailed implementation method and specific operation process are given, but protection scope of the present invention is not limited to following implementation Example.