CN105974113A - Sandwiched immunochromatographic test paper used for detecting Escherichia coli O157:H7 - Google Patents
Sandwiched immunochromatographic test paper used for detecting Escherichia coli O157:H7 Download PDFInfo
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- CN105974113A CN105974113A CN201610288500.9A CN201610288500A CN105974113A CN 105974113 A CN105974113 A CN 105974113A CN 201610288500 A CN201610288500 A CN 201610288500A CN 105974113 A CN105974113 A CN 105974113A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- 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
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56911—Bacteria
- G01N33/56916—Enterobacteria, e.g. shigella, salmonella, klebsiella, serratia
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
- G01N2333/24—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
- G01N2333/265—Enterobacter (G)
Abstract
The invention relates to a sandwiched immunochromatographic test paper used for detecting Escherichia coli O157:H7. The sandwiched immunochromatographic test paper comprises a supporting layer, the upper side surface of the supporting layer is provided with adsorption layers, the adsorption layers sequentially comprise an adsorption fiber layer, a fluorescence antibody fiber layer, a cellulose film and a water absorbing material layer from a test end, the water absorbing material layer is arranged at a handle end, the cellulose film is provided with a detection line and a quality control line, the detection line is formed by an anti-Escherichia coli O157:H7 monoclonal antibody or polyclonal antibody, the quality control line is formed by a goat anti-mouse or rabbit anti-mouse IgG antibody, or a goat anti-rabbit IgG antibody, the fluorescence antibody fiber layer is adsorbed with a fluorescence antibody, and the fluorescence antibody is formed by an FITC labeled anti-Escherichia coli O157:H7 monoclonal antibody or polyclonal antibody. The invention also discloses a production method of the sandwiched immunochromatographic test paper. The test paper has the advantages of simple detection method, strong specificity, high sensitivity, visual property, high accuracy, and realization of lowest detection of 1CFU/mL of trace quantity pollution.
Description
Technical field
The invention belongs to bioengineering field, relate to a kind of immune chromatography test paper, particularly one and be used for detecting the sandwich type immune chromatography test paper of Escherichia coli O 157: H7.
Background technology
Escherichia coli O 157: H7 type (Escherichia coli O157:H7) is a kind of intestinal bleeding escherichia coli, is mainly to eat one of source pathogenic bacterium.By Escherichia coli O 157: the intestinal infection disease that H7 causes has become as the public health problem of global concern.Escherichia coli O 157: H7 infects can causing bleeding property colitis (HC), the serious gastrointestinal complication such as appendicitis and colonic perforation, the systemic complications such as serious caused thrombotic thrombocytic purpura (TTP) and hemolytic urinary tract syndrome (HUS), wherein 3-5% patient death, there are about 12% patient has serious sequela.And the infective dose of people is extremely low, takes in 10-100 viable bacteria and just can cause morbidity.In recent years, Escherichia coli O 157: H7 recall rate in China's food samples, especially animal meat product is higher.This bacterium is all detected in the numerous food of the provinces and cities such as Fujian, Beijing, Guangdong.Escherichia coli O 157: H7 environment to external world has relatively strong adaptability, to temperature, pH be dried and have certain resistance, can in food, sandy soil, water, fertilizer and artificial microenvironment survival up to the several months.
Escherichia coli O 157 at present: H7 method for quick mainly includes biochemical method, molecular biology method, biosensor, metabolism method and immunological method etc..Biochemical method takes time and effort, and easily missing inspection natural mutant.Molecular biology method is complicated high to equipment dependency degree due to its operation, and the accuracy in addition detected and repeatability such as have much room for improvement at the reason, therefore is not yet used widely at detection, the diagnostic field of pathogenic bacterium.Biosensor detection technique has the advantages such as selectivity is good, highly sensitive, it is fast to analyze speed, low cost, energy on-line checking compared with traditional detection method, but it generally requires optics or chemistry original paper, and detection needs instrument to assist, and testing cost is higher.Metabolism group method is convenient, sensitive, has great application potential, but is only used for total plate count and identifies, it is impossible to for strain identification.Immunological method is the serial detection method on the protein level of specific binding reaction development based on Ag-Ab, mainly includes enzyme linked immunosorbent assay (ELISA), latex agglutination test, immunochromatography technique and protein chip technology.Immune chromatography test paper technology is the most superior immunological detection method in detection speed and convenience.
The current main development direction of immune chromatography test paper is to improve sensitivity, detection by quantitative and multivariate detection.It is classified as two classes: use purity is high, affinity is strong monoclonal antibody and employing novel markings thing for the currently used method of sensitivity problem.The Novel emission spectral type label that upper conversion fluorescent nano particle, quantum dot and fluorescent microsphere etc. are representative, excellent optical characteristics, nano material luminous intensity is high, by amino coupled with pathogenic bacterium antibody after they functionalization, form stable illuminating complex, by detection light signal strength, sensitivity just can be able to a certain degree of raising by specific light source.But the preparation technology of these new materials is more complicated, relatively costly, raw material is difficult to obtain, from actual applications distances very away from.
Fluorescent marker FITC is the fluorescein being most widely used at present, its a length of 490-495nm of absorption maximum light wave, the a length of 525-530nm of emission maximum light wave, present bright yellow-green fluorescence, reaction can be carried out with amino under conditions of pH is 9.0-9.5 and form thiourea, FITC fluorescent labelling techniques is the most ripe, and labeling method is simple, but it is applied to immune chromatography test paper as label and does not study.
Summary of the invention
For above-mentioned technical problem of the prior art, the invention provides a kind of for detecting Escherichia coli O 157: the sandwich type immune chromatography test paper of H7, described is this for detecting Escherichia coli O 157: in the sandwich type immune chromatography test paper prior art to be solved of H7, in detection food, the method sensitivity of Escherichia coli O 157: H7 is the highest, detect time length, detect the technical problem that process is complicated.
nullThe invention provides a kind of for detecting Escherichia coli O 157: the sandwich type immune chromatography test paper of H7，Including a supporting layer，It is provided with adsorption layer on the upper side of described supporting layer，Described adsorption layer is followed successively by adsorbing fiber layer from test lead、Fluorescent antibody fibrous layer、Cellulose membrane layer and the absorbent material layer of handle end，Described adsorbing fiber layer and should antibody fibrous layer compact siro spinning technology，Described fluorescent antibody fibrous layer and described cellulose membrane layer compact siro spinning technology，Described cellulose membrane layer and described absorbent material layer compact siro spinning technology，Cellulose membrane layer is provided with detection line and nature controlling line，Described detection line is made up of monoclonal antibody or the polyclonal antibody of anti-Escherichia coli O 157: H7，Described nature controlling line is by goat-anti or rabbit anti-mouse IgG antibody、Or goat anti-rabbit igg antibody is constituted，It is adsorbed with fluorescent antibody on described fluorescent antibody fibrous layer，Described fluorescent antibody is made up of monoclonal antibody or the polyclonal antibody of the anti-Escherichia coli O 157 of FITC labelling: H7.
Further, the upper end of described adsorption layer is provided with protective layer,
Further, the anti-Escherichia coli O 157 of described FITC labelling: the monoclonal antibody of H7 or the preparation method of polyclonal antibody comprise the steps:
The step of (1) configuration cross-linking reaction liquid, adds 7.56g NaHCO in every liter of reactant liquor3, 1.06g Na2CO3With 7.36g NaCl, surplus is ultra-pure water, the anti-Escherichia coli O 157 by after purification: the monoclonal antibody of H7 in 4 DEG C to cross-linking reaction liquid in dialyse 2～4 times, to pH=9.0；
(2) reactant liquor after dialysis is transferred in reaction vessel, it is put on magnetic stirring apparatus, according to the DMSO solution of the ratio dropping FITC that the amount of the monoclonal antibody of anti-Escherichia coli O 157: H7: FITC is 1mg:150 μ g, lucifuge, 4 DEG C of reactions overnight, add the NH of 5mol/L4Cl 4 DEG C to final concentration of 5mmol/L, terminates reaction 2h；
(3) being dialysed in PBS more than 8 times by cross-linking agent, limpid to dialysis solution, 4 DEG C of dark place preservations are standby.
Further, the material of described adsorbing fiber layer is any one in glass fibre cotton, nylon membrane, PVDF membrane or polyester film；The fibrolaminar material of described fluorescent antibody is any one in glass fibre cotton, nylon membrane, PVDF membrane or polyester film；The material of described absorbent material layer is absorbent filter, and the material of described supporting layer is the toughness material not absorbed water；The material of described cellulose membrane layer is any one in nitrocellulose filter, pure cellulose film or carboxylated cellulose film.
Further, stealthy detection trace in described detection line and the stealthy comparison trace in nature controlling line are " " orthoscopic trace arranged in parallel, or be that " 10 " font arranges trace, or be that " ┬ ┬ " font arranges trace, or be that " ┴ ┴ " font arranges trace, or be that " ├ ├ " font arranges trace, or be " ┤ ┤ " font arrangement trace, or be " ● ● " point-like arrangement trace.
Further; described adsorbing fiber layer, fluorescent antibody fibrous layer and absorbent material layer are coated with protecting film; the protecting film that adsorbing fiber layer is corresponding with fluorescent antibody fibrous layer intersection is printed with sample mark line, this mark line deflection adsorbing fiber layer side 0.3-0.7cm.
Present invention also offers above-mentioned one for detecting Escherichia coli O 157: the preparation method of the sandwich type immune chromatography test paper of H7, comprise the following steps:
1) Escherichia coli O 157 is prepared for one: H7 monoclonal antibody or the step of polyclonal antibody；
2) step of a preparation fluorescent antibody；
3) step preparing adsorbing fiber layer, described adsorbing fiber layer uses glass fibre cotton, nylon membrane, PVDF membrane or polyester film to make；
4) a preparation fibrolaminar step of fluorescent antibody, is adsorbed with fluorescent antibody on described fluorescent antibody fibrous layer；
5) step preparing cellulose membrane layer, described cellulose membrane layer uses nitrocellulose filter, pure cellulose film or carboxylated cellulose film, with point sample instrument specking detection trace on cellulose membrane, standby after drying；
6) step assembling reagent paper, is attached to adsorbing fiber layer, fluorescent antibody fibrous layer, cellulose membrane layer, absorbent material layer the most successively on the supporting layer with binding agent, successively supporting layer, adsorption layer and protective layer is assembled into reagent paper.
Further, the preparation method of described adsorbing fiber layer is as follows: glass fibre cotton, nylon membrane, PVDF membrane or polyester film are immersed in TBS buffer solution, the concentration of described buffer solution is 0.01M, pH is 7.8, in described buffer solution, possibly together with PVP, sucrose, Tw-20, BSA, in described buffer solution, the mass percent concentration of PVP is 0.5%, and the mass percent concentration of sucrose is 1%, and the mass percent concentration of Tw-20 is 0.5%, the mass percent concentration of BSA is 1%, it is as the criterion to soak, lyophilizing, standby.
Present invention also offers the method using above-mentioned sandwich type immune chromatography test paper detection Escherichia coli O 157: H7, comprise the steps:
1) increase bacterium before sample: FITC is dissolved in DMSO, enriched medium adds FITC solution, adds sample and carry out Zengjing Granule；
2) detection paper: reagent paper is inserted sample solution, take out after 10～20 seconds and keep flat, there is unstressed configuration by the detection line of observation Test paper after 5-10min, it is judged that whether sample contains Escherichia coli O 157: H7, or carry out semi-quantitative analysis by fluorescence reading bar instrument.
The reagent paper of the present invention has the advantage that
(1) simple, low cost, high specificity are made, highly sensitive.Immunofluorescence technique is combined by reagent paper of the present invention and detection method with immunochromatography technique, target Escherichia coli O 157 in measuring samples: H7 just can fluoresce after cultivating with the enriched medium of FITC, without any labelling flow process, cost of manufacture is low, light stability is strong, after FITC traget antibody is introduced reagent paper, reach the effect that antigen-antibody dual signal amplifies, sensitivity more higher than common colloid gold test paper can have been obtained.And FITC labelling cost is low compared with gold colloidal.The advantage that this test strips maintains tradition, and colloidal gold strip simplicity is quick, highly sensitive, specificity is good, the minimum trace contamination detecting 1CFU/mL.
(2) easy, quickly.This reagent paper directly can detect after food samples is increased bacterium process, direct observed result under ultraviolet light, it is possible to by the direct readings of phosphor reader, it is achieved half-quantitative detection.Reagent paper only need to insert during detection test sample 10～20 seconds, i.e. can determine that testing result in 5-10min, time saving and energy saving, easy and simple to handle, a step completes.
(3) result display is vivid, directly perceived, accurately.Test strips is using display yellow green " " and " " (or " ten ", " ┬ ", " ┴ ", " ├ ", " ┤ " "●") trace as the feminine gender of detection and positive mark, on cellulose membrane, i.e. show yellow green " " trace, represent in test sample without Escherichia coli O 157: H7；Show two yellow green " " traces, represent in test sample containing Escherichia coli O 157: H7.This result can the most directly be observed, it is determined that vivid, directly perceived, accurate, simple and clear, is difficult to the artificially erroneous judgement such as false positive and false negative occur.
(4) expense is saved.Without separately joining instrument and equipment and other reagent during this ELISA test strip, can detect whenever and wherever possible, can qualitative detection again can detection by quantitative；Testing cost is cheap, can save the Meteorological of a large amount of expensive instrument and equipment.
(5) applied widely, it is simple to popularization and application.This reagent paper can meet the needs of different levels personnel, including specialty chemical examination, customs quarantine control, health quarantine, quality-monitoring, livestock products processing, raiser and consumer individual etc., both the detection of single sample had been suitable to, be suitable to again the examination of a large amount of sample, can apply to the fields such as medical diagnosis on disease, Bacteria Detection and environment measuring.The present invention has meaning of crucial importance in terms of ensuring food safety, protecting consumer health, has obvious economic benefit and social benefit.
The present invention compares with prior art, and its technological progress is significant.The reagent paper of the present invention and detection method thereof are simple, and high specificity is highly sensitive, intuitively, accurately, can carry out quantitative and semi-quantitative detection, and the minimum trace contamination detecting 1CFU/mL is applied widely, low cost, it is easy to popularization and application.
Accompanying drawing explanation
Fig. 1 sandwich type reagent paper schematic diagram.
The structural representation of Fig. 2 sandwich type reagent paper.
The top view structural representation of Fig. 3 sandwich type reagent paper.
The sensitivity technique result of Fig. 4 sandwich type reagent paper.
Fig. 5 sandwich type reagent paper semi-quantitative analysis result.
Fig. 6 sandwich type reagent paper cross reaction experimental result.
Experimental result of carrying disease germs simulated by Fig. 7 sandwich type reagent paper.
Detailed description of the invention
Embodiment 1
The preparation process of reagent paper of the present invention includes: Escherichia coli O 157: the steps such as the assembling of H7 monoclonal or the preparation of polyclonal antibody, the preparation of adsorbing fiber layer, the preparation of cellulose membrane layer and test strips.
(1) anti-Escherichia coli O 157: H7 monoclonal antibody or the preparation of polyclonal antibody
Prepared by monoclonal antibody: with inactivation concentration for 108The full bacterial immunity of the Escherichia coli O 157 of cfu/mL: H7 6～8 week old Balb/C mice 3～4 times, each immunization interval time 3～5 weeks, determine antibody titer meet the requirements after superpower immunity, 3～4 days afterwards, hole under immune mouse socket of the eye is taken a blood sample, separates positive serum；De-neck is lethal, and the alcohol-pickled mice 5～10min with 75% is sterilized body surface, aseptic takes its spleen, is shredded by spleen and grinds, filtering through 120 mesh nylon gauzes, and 1000rpm is centrifuged 10min, collection splenocyte.By 1 × 108Splenocyte mix in the ratio of 10:1 with NS0 myeloma cell, 1000rpm is centrifuged 10min, abandon supernatant, cell precipitate is slowly added into the 50%PEG4000 of 0.7～1.0mL Escherichia coli O 157: H7 in 37 DEG C of water-baths, effect 1min, then serum-free 1640 culture medium 15mL it is slowly added into, to terminate the effect of PEG, 37 DEG C of water-baths 5～10min, 1000rpm is centrifuged 10min, abandons supernatant, is resuspended in HAT Selective agar medium by cell precipitate, and add 96 porocytes cultivation plate hole (100 μ L～200 μ L/ holes), it is placed in 37 DEG C, 5%CO2Incubator is cultivated 10～14 days, positive hole sizer choosing is carried out with indirect elisa method, strong positive, suppression ratio height, the eugonic hole of cell is selected to carry out 3 limited dilution clonings, then amplification culture, set up the hybridoma prepared by hybridoma cell strain, secretion monoclonal antibody can specifically with Escherichia coli O 157: H7 reacts, and affinity constant reaches 1010～1012, for Escherichia coli O 157: the monoclonal antibody of H7 specific epitope, for the printing of T line.(the present embodiment is intended merely to describe the preparation process of monoclonal antibody, and any commercially available monoclonal antibody reacted with Escherichia coli O 157: H7 can use in the present invention)
Prepared by multi-resistance: with the Escherichia coli O 157 of inactivation: H7 immunity New Zealand white rabbit, immunogen concentration is 108Cfu/mL, dorsal sc divides 4～6 injections.Head exempts from, and adds not formula Freund's complete adjuvant in immunogen, fully emulsified；Booster immunization, adds incomplete Freund's adjuvant in immunogen, fully emulsified, and head carries out continuous immunity 4～5 times for 2～3 weeks after exempting from, every minor tick 2～3 weeks, after last immunity 10～15 days, surveys it with indirect ELISA and determines titer and reach 105Time above, take a blood sample and separate and collect hyper-immune serum.Extract IgG antibody with saturated ammonium sulfate salting out method, i.e. take 1 portion of hyper-immune serum and add the mixing of 2 parts of PBS (pH7.2), add the mixing of equal-volume saturated ammonium sulfate solution, put 4 DEG C of refrigerator 12h, 4 DEG C, 2500rpm be centrifuged 15min, abandon supernatant；Again with appropriate PBS (pH7.2) dissolution precipitation, add saturated ammonium sulfate solution to final concentration 33%, put 4 DEG C of refrigerator 2h, 4 DEG C, 2500rpm be centrifuged 15min, abandon supernatant；With appropriate PBS (pH7.2) dissolution precipitation, put the 4 DEG C of interior PBS of refrigerators (pH7.2) and dialyse 48～72h, liquid is changed for several times in centre, 4 DEG C, 12000rpm be centrifuged 15min, collect supernatant, obtain the anti-Escherichia coli O 157 of purification: H7 polyclonal antibody ,-20 DEG C frozen, for the printing of T line.
(2) preparation of fluorescent antibody fibrous layer (pad)
The fibrolaminar preparation of fluorescent antibody, it is necessary first to preparation FITC fluorescent antibody, concretely comprises the following steps:
1) configuration cross-linking reaction liquid: 7.56g NaHCO3, 1.06g Na2CO3, 7.36g NaCl, add ultra-pure water and be settled to 1L.Cross-linking reaction liquid is dialysed 3 times by monoclonal antibody after purification in 4 DEG C, to pH=9.0.
2) reactant liquor after dialysis is transferred to vial be put on magnetic stirring apparatus, according to antibody: the amount of FITC is the DMSO solution of the ratio dropping FITC of 1mg:150 μ g, and lucifuge, 4 DEG C of reactions are overnight.Add the NH of 5mol/L4Cl 4 DEG C to final concentration of 5mmol/L, terminates reaction 2h.
3) cross-linking agent is dialysed more than 8 times in PBS, limpid to dialysis solution.4 DEG C of dark place preservations are standby.
Secondly, with point sample instrument specking FTIC antibody on fiber tunic, fluorescence mark band is made, in 37 DEG C of lucifuge dry for standby.
(3) preparation of adsorbing fiber layer (sample pad)
Prepared by test lead adsorbing fiber layer glass fibre cotton, nylon membrane, polyvinylidene fluoride pvdf membrane or polyester film, fibrous material is cut into the band of wide 1.5cm, puts it into immersion 30min in sample pad treatment fluid, in 37 DEG C of drying, standby.
(4) preparation of cellulose membrane layer (analyzing pad)
Cellulose membrane layer nitrocellulose filter, pure cellulose film or carboxylated cellulose film, cut into the band of wide 1.5cm specification, with point sample instrument specking Escherichia coli O 157 on cellulose membrane: H7 monoclonal antibody or polyclonal antibody and goat-anti or rabbit anti-mouse IgG antibody or goat anti-rabbit igg antibody, make stealthy detection trace band and comparison trace band, in 37 DEG C of dry for standby.
(5) assembling of sandwich type reagent paper: adsorbing fiber layer (sample pad), fluorescent antibody fibrous layer (pad), cellulose membrane layer (analyzing film), absorbent material layer are attached to the most successively on the supporting layer (base plate) with binding agent, and are cut into the wide test strips of 3-4cm.(as shown in Figure 1B).
(6) the detection reaction principle of test strips of the present invention:
When detection Escherichia coli O 157: after H7 reagent paper test lead inserts testing sample solution, solution to be measured drives luminous Escherichia coli O 157: H7 to be measured to spread to cellulose membrane layer by siphonage, and eventually penetrates the absorbent material layer of handle end.In diffusion process, Escherichia coli O 157 to be measured: H7 can be with the complex of the FITC fluorescent antibody formation FITC-bacterium-antibody-FITC on pad, again can be by Escherichia coli O 157 thereon when flowing to T line: H7 antibody capture thus show detection trace, under ultraviolet excites, (or using the direct readings of phosphor reader) forms yellow green detection trace band " ", is positive expression；Otherwise without Escherichia coli O 157 in sample solution: during H7, then can not the fluorescent antibody on pad be combined, and the Escherichia coli O 157 on cellulose membrane: H7 antibody test trace is combined, and does not show yellow green detection trace band " ", is negative expression；No matter whether sample contains Escherichia coli O 157: H7, FITC fluorescent antibody all can form yellow green comparison trace band " " (as shown in Figure 1 C) with the goat-anti on cellulose membrane or rabbit anti-mouse IgG (or goat anti-rabbit igg) antibody capture.
The detection method of the sandwich type immune chromatography test paper of the present invention is as follows:
(1) bacterium is increased before sample: should check as early as possible after sample collecting.Even if if can not detect, 18h can be preserved at 2-4 DEG C.Sterile working's sampling 25g (mL) joins in 225mLEC or the NB meat soup containing FTIC solution, above-mentioned solution is put in homogenizer, continuous homogenizing 1-2min on slap type homogenizer, or put in the homogenizing cup filling 225mLEC or NB meat soup (containing FITC), 8000-10000r/min homogenizing 1-2min, cultivates some hours (as shown in Figure 1A) in 37 DEG C.Do the positive and negative control simultaneously.
(2) detection paper: reagent paper is inserted sample solution, take out after 10～20 seconds and keep flat, there is unstressed configuration by the detection line of observation Test paper after 5-10min, it is judged that whether sample contains Escherichia coli O 157: H7, or carry out semi-quantitative analysis by fluorescence reading bar instrument.
Following example illustrate structure and the detection method of test strips.
Embodiment 2
See Fig. 2, Fig. 3.
In figure, 1 is supporting layer, make with plastic slice bar, 2 is adsorbing fiber layer, make with glass fibre cotton, being adsorbed with FITC fluorescent monoclonal antibody on fluorescent antibody fibrous layer 3, cellulose membrane layer 4 uses nitrocellulose filter, and the absorbent material layer 5 of handle end is made with absorbent filter, adsorbing fiber layer 2, fluorescent antibody fibrous layer 3, cellulose membrane layer 4, each layer of absorbent material layer 5 being pasted and fixed on the most successively on supporting layer 1, intersection fiber crosses one another infiltration each other.Cellulose membrane layer 4 is provided with stealthy detection trace 6, with Escherichia coli O 157: H7 monoclonal antibody is made；Stealthy comparison trace 7 goat anti-mouse igg antibody solution trace on cellulose membrane makes " ", and two trace bands are arranged in parallel, form combination trace band " ".
8-1 is to cover the sample end white protecting film on adsorbing fiber layer 2 and fluorescent antibody fibrous layer 3; 8-2 is to cover other color protecting film (such as yellow) on absorbent material layer 5; 9 is sample mark line; this mark line is positioned at the white protecting film deflection adsorbing fiber layer 2 side about 0.5cm that adsorbing fiber layer 2 is corresponding with fluorescent antibody fibrous layer 3 intersection, is printed on arrow and max printed words on the right side of mark line on protecting film.
(1) reagent paper sensitivity technique of the present invention: by Escherichia coli O 157: the mono-colony inoculation of H7 is cultivated in the FITC culture medium containing 0.4mg/mL, uses plate count method, bacterium solution is diluted to 10 respectively8-104CFU/mL, uses sandwich type detection paper, observes testing result after 5-10min under uviol lamp, and minimum bacteria concentration when range estimation lowest detectable limit (LOD) is seen from T line, as shown in Figure 4, the LOD of reagent paper is 10 to result6CFU/mL.Reading bar instrument with fluorescence and reagent paper is carried out semi-quantitative analysis, result is as it is shown in figure 5, reagent paper is 10 at bacteria concentration5-108Linearly it is correlated with during CFU/mL, and LOD is 105CFU/mL。
(2) reagent paper specific detection of the present invention: use its specificity of cross reaction test for identification.It is 10 by concentration8The common food-borne pathogens of CFU/mL, as shown in table 1, use sandwich type detection paper, if the positive i.e. has intersection, testing result is as shown in Figure 6, escherichia coli sandwich type reagent paper can only detect three strain escherichia coli, and other common food-borne pathogens all can not detect, i.e. this reagent paper has preferable specificity.
Common food-borne pathogens used by table 1 cross reaction and source
(3) mensuration of reagent paper stability of the present invention: to adding desiccant with a batch of sandwich type reagent paper after sealing, lucifuge is positioned over 4 DEG C.Preserving 1 day, after 1,5,10,15 and 20 weeks, detecting its sensitivity respectively, result show the sensitivity of the reagent paper after preserving 20 weeks and preserve 1 day after unchanged, therefore this reagent paper at least can keep stable performance 4 DEG C of preservations 20 weeks.
(4) simulate experiment of carrying disease germs: buy bread, milk, fruit jelly sample from local supermarket, identify that three kinds of samples are all without Escherichia coli O 157 by PCR method: H7 pollutes.Taking every kind of sample 25g with sterile working to cultivate as in the 225mL culture medium of band FITC, add viable bacteria solution and make bacteria concentration be 1CFU/mL in culture medium, culture bottle is put in 37 DEG C of shaking tables and cultivates, and every 2h sampling detection, result is as shown in Figure 7.Result shows that bread, milk sample can detect after cultivating 10h, and fruit jelly is cultivated 8h and can be detected；Sandwich type reagent paper can detect the sample that initial bacteria concentration is 1CFU/mL.
Embodiment 3
Test strips structure is identical with embodiment 1.Difference is: fluorescent antibody fibrous layer 3 is adsorbed with the anti-Escherichia coli O 157 of FITC labelling: H7 polyclonal antibody, and adsorbing fiber layer 2 is made with nylon membrane, and cellulose membrane layer 4 uses pure cellulose film, and stealth is imprinted as goat anti-rabbit igg antibody.Stealthy detection trace band and stealthy comparison trace band are " ten ", and 8-2 is to cover the handle end blue protecting film on absorbent material layer 5.
The preparation of testing sample and detection operating procedure:
Detection bread: sterile working sampling 25g joins in 225mLEC or the NB meat soup containing FTIC solution, puts in homogenizer by above-mentioned solution, continuous homogenizing 1-2min on slap type homogenizer cultivates some hours in 37 DEG C.
Operational approach: reagent paper is inserted sample solution, takes out after 10～20 seconds and keeps flat, and has unstressed configuration by the detection line of observation Test paper, it is judged that whether contain Escherichia coli O 157 in sample: H7 after 5-10min, or carries out semi-quantitative analysis by fluorescence reading bar instrument.
Result judges: if (a) positive shows two yellow greens trace band " ten " on cellulose membrane, represents that testing result is positive, illustrates in testing sample containing Escherichia coli O 157: H7；If b () feminine gender shows yellow green trace band " ten " on cellulose membrane, represent that testing result is negative, illustrate in testing sample without Escherichia coli O 157: H7；Do not had yellow green band to show on cellulose membrane if c () is lost efficacy, then showed that test strips lost efficacy.
Embodiment 4
Test paper structure and embodiment 2 are essentially identical; difference is: cellulose membrane layer 3 is adsorbed with the polyclonal antibody of anti-Escherichia coli O 157: H7; adsorbing fiber layer 2 is made with nylon membrane; cellulose membrane layer 3 uses pure cellulose film, stealthy detection trace be " ┬ ", 6-2 be covering handle end greenism film on absorbent material layer 4.
For detecting milk sample: sterile working sampling 25mL joins in 225mLEC or the NB meat soup containing FTIC solution, above-mentioned solution is put in homogenizer, put in the homogenizing cup filling 225mLEC or NB meat soup (containing FITC), 8000-10000r/min homogenizing 1-2min, cultivates some hours in 37 DEG C.
Operational approach: reagent paper is inserted sample solution, takes out after 10～20 seconds and keeps flat, and by the direct readings of phosphor reader after 5-10min, numerical value is the fluorescence intensity of T line, draws standard curve according to peak value or peak area, calculates actual content.
Embodiment 5
Test paper structure and embodiment 2 are essentially identical; difference is: adsorbing fiber layer 2 polyvinylidene fluoride pvdf membrane is made; cellulose membrane layer 3 uses carboxylated cellulose film, and 6-2 is to cover the handle end greenism film on absorbent material layer 4, and stealthy detection trace band is " ┴ ".
For detecting fruit jelly: increase bacterium before sample, result judges and operational approach is all with embodiment two, does not repeats.
Embodiment 6
Test paper structure and embodiment 2 are essentially identical, and difference is: adsorbing fiber layer 2 is made with polyester film, and cellulose membrane layer 3 uses carboxylated cellulose film, and stealthy detection trace band is " ├ ".
For detecting meat sample, before sample, increasing bacterium, result judgement and operational approach are all with embodiment two, do not repeat.
Embodiment 7
Test paper structure and embodiment 2 are essentially identical, and difference is: adsorbing fiber layer 2 is made with nylon membrane.Detection trace band is " ┤ ".Detection sample, result judge and operational approach is with example two.
Embodiment 8
Essentially identical with embodiment 2, difference is: cellulose membrane layer 3 is adsorbed with the polyclonal antibody of anti-Escherichia coli O 157: H7, and detection trace band is "●".Detection sample is bread sample.
Embodiment 9
Essentially identical with embodiment 2, difference is: cellulose membrane layer 3 is adsorbed with the polyclonal antibody of anti-Escherichia coli O 157: H7, and detection sample is milk sample.
Embodiment 8
Essentially identical with embodiment 2, difference is: cellulose membrane layer 3 is adsorbed with the polyclonal antibody of anti-Escherichia coli O 157: H7, and detection sample is fruit jelly sample.
Embodiment 10
Essentially identical with embodiment 2, difference is: cellulose membrane layer 3 is adsorbed with the polyclonal antibody of anti-Escherichia coli O 157: H7, and detection sample is meat sample.
Claims (9)
1. it is used for detecting a sandwich type immune chromatography test paper of Escherichia coli O 157: H7, including a supporting layer, described supporting layer
Upper side on be provided with adsorption layer, described adsorption layer from test lead be followed successively by adsorbing fiber layer, fluorescent antibody fibrous layer,
Cellulose membrane layer and the absorbent material layer of handle end, described adsorbing fiber layer and should antibody fibrous layer compact siro spinning technology, described
Fluorescent antibody fibrous layer and described cellulose membrane layer compact siro spinning technology, described cellulose membrane layer and described absorbent material layer
Compact siro spinning technology, is provided with detection line and nature controlling line on cellulose membrane layer, it is characterised in that: described detection line is by anti-large intestine bar
The monoclonal antibody of bacterium O157:H7 or polyclonal antibody are constituted, described nature controlling line by goat-anti or rabbit anti-mouse IgG antibody,
Or goat anti-rabbit igg antibody is constituted, described fluorescent antibody fibrous layer being adsorbed with fluorescent antibody, described fluorescent antibody is by FITC
The anti-Escherichia coli O 157 of labelling: the monoclonal antibody of H7 or polyclonal antibody are constituted.
It is the most according to claim 1 for detecting Escherichia coli O 157: the sandwich type immune chromatography test paper of H7, it is characterised in that:
The upper end of described adsorption layer is provided with protective layer.
It is the most according to claim 1 for detecting Escherichia coli O 157: the sandwich type immune chromatography test paper of H7, it is characterised in that:
The anti-Escherichia coli O 157 of described FITC labelling: the monoclonal antibody of H7 or the preparation method of polyclonal antibody include as follows
Step,
The step of (1) configuration cross-linking reaction liquid, adds 7.56g NaHCO in every liter of reactant liquor3, 1.06g Na2CO3With
7.36g NaCl, surplus is ultra-pure water, the anti-Escherichia coli O 157 by after purification: the monoclonal antibody of H7 in 4 DEG C to crosslinking
Reactant liquor is dialysed 2～4 times, to pH=9.0；
(2) reactant liquor after dialysis is transferred in reaction vessel, is put on magnetic stirring apparatus, according to anti-Escherichia coli O 157: H7
Monoclonal antibody: the amount of FITC be 1mg:150 μ g ratio dropping FITC DMSO solution, lucifuge, 4 DEG C were reacted
At night, add the NH of 5mol/L4Cl 4 DEG C to final concentration of 5mmol/L, terminates reaction 2h；
(3) being dialysed in PBS more than 8 times by cross-linking agent, limpid to dialysis solution, 4 DEG C of dark place preservations are standby.
It is the most according to claim 1 for detecting Escherichia coli O 157: the sandwich type immune chromatography test paper of H7, it is characterised in that:
The material of described adsorbing fiber layer is any one in glass fibre cotton, nylon membrane, PVDF membrane or polyester film；
The fibrolaminar material of described fluorescent antibody is any one in glass fibre cotton, nylon membrane, PVDF membrane or polyester film
Kind；The material of described absorbent material layer is absorbent filter, and the material of described supporting layer is the toughness material not absorbed water；Institute
The material of the cellulose membrane layer stated is any one in nitrocellulose filter, pure cellulose film or carboxylated cellulose film.
It is the most according to claim 1 for detecting Escherichia coli O 157: the sandwich type immune chromatography test paper of H7, it is characterised in that:
Stealthy detection trace in described detection line and the stealthy comparison trace in nature controlling line are " | " orthoscopic print arranged in parallel
Mark, or be that " 10 " font arranges trace, or beFont arrangement trace, or beFont arranges
Trace, or beFont arrangement trace, or beFont arrangement trace, or be " ● ● " point-like arrangement
Trace.
It is the most according to claim 1 for detecting Escherichia coli O 157: the sandwich type immune chromatography test paper of H7, it is characterised in that:
Described adsorbing fiber layer, fluorescent antibody fibrous layer and absorbent material layer are coated with protecting film, at adsorbing fiber layer and fluorescence
Sample mark line it is printed with, this mark line deflection adsorbing fiber layer side on the protecting film that antibody fibrous layer intersection is corresponding
0.3-0.7cm。
7. being used for described in claim 1 detects the preparation method of sandwich type immune chromatography test paper of Escherichia coli O 157: H7, its feature
It is to comprise the following steps:
1) Escherichia coli O 157 is prepared for one: H7 monoclonal antibody or the step of polyclonal antibody；
2) step of a preparation fluorescent antibody；
3) step preparing adsorbing fiber layer, described adsorbing fiber layer uses glass fibre cotton, nylon membrane, polyvinylidene fluoride
Film or polyester film are made；
4) a preparation fibrolaminar step of fluorescent antibody, is adsorbed with fluorescent antibody on described fluorescent antibody fibrous layer；
5) step preparing cellulose membrane layer, described cellulose membrane layer uses nitrocellulose filter, pure cellulose film or carboxylation fine
Dimension element film, with point sample instrument specking detection trace on cellulose membrane, standby after drying；
6) one assemble reagent paper step, by adsorbing fiber layer, fluorescent antibody fibrous layer, cellulose membrane layer, absorbent material layer from a left side to
The right side is attached on the supporting layer with binding agent successively, successively supporting layer, adsorption layer and protective layer is assembled into reagent paper.
Preparation method the most according to claim 7, it is characterised in that the preparation method of described adsorbing fiber layer is as follows: by glass
Cellucotton, nylon membrane, PVDF membrane or polyester film immerse in TBS buffer solution, the concentration of described buffer solution
It is 7.8 for 0.01M, pH, in described buffer solution, possibly together with PVP, sucrose, Tw-20, BSA, delays described
In dissolved liquid, the mass percent concentration of PVP is 0.5%, and the mass percent concentration of sucrose is 1%, the quality hundred of Tw-20
Proportion by subtraction concentration is 0.5%, and the mass percent concentration of BSA is 1%, is as the criterion to soak, and lyophilizing is standby.
9. use the method that the sandwich type immune chromatography test paper described in claim 1 detects Escherichia coli O 157: H7, it is characterised in that bag
Include following steps:
1) increase bacterium before sample: FITC is dissolved in DMSO, enriched medium adds FITC solution, adds sample and increase
Bacterium is cultivated；
2) detection paper: reagent paper is inserted sample solution, takes out after 10～20 seconds and keeps flat, by observing detection examination after 5-10min
The detection line of paper has unstressed configuration, it is judged that whether contain Escherichia coli O 157 in sample: H7, or is entered by fluorescence reading bar instrument
Row semi-quantitative analysis.
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| CN113238047A (en) * | 2021-05-10 | 2021-08-10 | 深圳市光明区疾病预防控制中心 | Graphene quantum dot immunochromatography test strip for rapidly detecting Escherichia coli O157H 7 |
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| CN112034155A (en) * | 2020-08-26 | 2020-12-04 | 广东石油化工学院 | Methamidophos rapid detection test paper based on aptamer molecule competitive binding and preparation method thereof |
| CN113238047A (en) * | 2021-05-10 | 2021-08-10 | 深圳市光明区疾病预防控制中心 | Graphene quantum dot immunochromatography test strip for rapidly detecting Escherichia coli O157H 7 |
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