CN105974113B - For detecting Escherichia coli O 157:H7 interlayer type immune chromatography test paper - Google Patents
For detecting Escherichia coli O 157:H7 interlayer type immune chromatography test paper Download PDFInfo
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- CN105974113B CN105974113B CN201610288500.9A CN201610288500A CN105974113B CN 105974113 B CN105974113 B CN 105974113B CN 201610288500 A CN201610288500 A CN 201610288500A CN 105974113 B CN105974113 B CN 105974113B
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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
It is of the invention a kind of for detecting Escherichia coli O 157:H7 interlayer type immune chromatography test paper, including a supporting layer, adsorption layer is provided with the upper side of supporting layer, adsorption layer is followed successively by the absorbent material layer of adsorbing fiber layer, fluorescence antibody fibrous layer, cellulose film layer and handle end from test lead, detection line and nature controlling line are provided with cellulose film layer, detection line is by anti-Escherichia coli O 157:H7 monoclonal antibody or polyclonal antibody is constituted, and nature controlling line is made up of goat-anti or rabbit anti-mouse IgG antibody or goat anti-rabbit igg antibody, and fluorescence antibody, the anti-Escherichia coli O 157 that fluorescence antibody is marked by FITC are adsorbed with fluorescence antibody fibrous layer:H7 monoclonal antibody or polyclonal antibody is constituted.The invention also discloses the preparation method of above-mentioned interlayer type immune chromatography test paper.The test paper detecting method of the present invention is simple, high specificity, and sensitivity is high, intuitively, accurately, minimum detectable 1CFU/mL trace contamination.
Description
Technical field
The invention belongs to bioengineering field, it is related to a kind of immune chromatography test paper, it is particularly a kind of to be used to detect large intestine bar
Bacterium O157:H7 interlayer type immune chromatography test paper.
Background technology
Escherichia coli O 157:H7 types (Escherichia coli O157:H7) it is a kind of intestinal bleeding Escherichia coli,
It is one of main food source pathogenic bacteria.By Escherichia coli O 157:Enteric infection disease has become global concern caused by H7
Public health problem.Escherichia coli O 157:H7 infection can causing bleeding property colitis (HC), appendicitis and perforation of colon etc. are serious
Gastrointestinal complication, serious causes thrombotic thrombocytic purpura (TTP) and hemolytic urinary tract syndrome (HUS) etc. systemic
Complication, wherein 3-5% patient deaths, there are about 12% patient has serious sequelae.And the infective dose of people is extremely low, intake
10-100 viable bacteria can cause morbidity.In recent years, Escherichia coli O 157:H7 is in China's food samples, especially animal flesh system
Recall rate is higher in product.The bacterium is all detected in the numerous food of the provinces and cities such as Fujian, Beijing, Guangdong.Escherichia coli O 157:
Environment has compared with strong adaptability H7 to external world, has certain resistance to temperature, pH and drying, can be in food, sandy soil, water, fertilizer
Survival is up to the several months in material and artificial microenvironment.
Current Escherichia coli O 157:H7 quick determination methods mainly include biochemical method, molecular biology method, biological biography
Sensor, metabolism method and immunological method etc..Biochemical method takes time and effort, and easily missing inspection natural mutant.Molecule is given birth to
Thing method is high to equipment dependency degree due to its complex operation, the reason such as the accuracy and repeatability detected in addition has much room for improvement,
Therefore the detection, diagnostic field in pathogenic bacteria are not yet used widely.Biology sensor detection technique and traditional detection method
Compared to having the advantages that the good, sensitivity of selectivity is high, analyze speed is fast, cost is low, can on-line checking, but it generally requires optics
Or chemical original paper, detecting needs instrument to aid in, and testing cost is higher.Metabolism group method is convenient, sensitive, with great application
Potentiality, but it is only used for total plate count identification, it is impossible to be used in strain idenfication.Immunological method is based on the special of Ag-Ab
Property association reaction development protein level on serial detection method, mainly including enzyme linked immunosorbent assay (ELISA),
Latex agglutination test, immunochromatography technique and protein chip technology.Immune chromatography test paper technology is in detection speed and convenience
Immunological detection method the most superior.
The current main development direction of immune chromatography test paper is to improve sensitivity, quantitative detection and multivariate detection.For spirit
The currently used method of sensitivity problem is classified as two classes：Using the monoclonal antibody that purity is high, affinity is strong and using new mark
Remember thing.Upper conversion fluorescent nano particle, quantum dot and fluorescent microsphere etc. are the novel emission spectral type label of representative, and optics is special
Property it is excellent, nano material luminous intensity is high, by the amino coupled after their functionalization with bacteria antibody, forms stable luminous
Complex, by specific light source by detecting that sensitivity just can be able to a certain degree of raising by light signal strength.But these are new
The preparation technology of material is more complicated, and cost is higher, and raw material are difficult to obtain, very remote from a distance from practical application.
Fluorescent marker FITC is the fluorescein being most widely used at present, its a length of 490- of absorption maximum light wave
495nm, a length of 525-530nm of emission maximum light wave, are presented bright yellow-green fluorescence, can under conditions of pH is 9.0-9.5
Reaction is carried out with amino and forms thiocarbamide, and FITC fluorescent labelling techniques are more ripe, and labeling method is simple and easy to apply, but it is used as mark
Thing is not studied applied to immune chromatography test paper.
The content of the invention
For above-mentioned technical problem of the prior art, it is used to detect Escherichia coli O 157 the invention provides one kind:H7
Interlayer type immune chromatography test paper, it is described this to be used to detect Escherichia coli O 157:H7 interlayer type immune chromatography test paper will
Solve Escherichia coli O 157 in detection food in the prior art:H7 method sensitivity is not high, detection time length, detection process are multiple
Miscellaneous technical problem.
It is used to detect Escherichia coli O 157 the invention provides one kind:H7 interlayer type immune chromatography test paper, including one
Adsorption layer is provided with supporting layer, the upper side of described supporting layer, described adsorption layer is followed successively by adsorbing fiber from test lead
The absorbent material layer of layer, fluorescence antibody fibrous layer, cellulose film layer and handle end, described adsorbing fiber layer and should antibody it is fine
Dimension layer is closely connected, and described fluorescence antibody fibrous layer and described cellulose film layer are closely connected, described cellulose film layer
Closely connected with described absorbent material layer, detection line and nature controlling line are provided with cellulose film layer, described detection line is by resisting
Escherichia coli O 157:H7 monoclonal antibody or polyclonal antibody is constituted, and described nature controlling line is resisted by goat-anti or rabbit anti-mouse IgG
Body or goat anti-rabbit igg antibody are constituted, and are adsorbed with fluorescence antibody on the fluorescence antibody fibrous layer, described fluorescence antibody by
The anti-Escherichia coli O 157 of FITC marks:H7 monoclonal antibody or polyclonal antibody is constituted.
Further, the upper end of described adsorption layer sets matcoveredn,
Further, the anti-Escherichia coli O 157 of described FITC marks:H7 monoclonal antibody or polyclonal antibody
Preparation method comprises the following steps：
The step of (1) configuration cross-linking reaction liquid, 7.56g NaHCO are added in every liter of reaction solution3, 1.06g
Na2CO3With 7.36g NaCl, surplus is ultra-pure water, by anti-Escherichia coli O 157 after purification:H7 monoclonal antibody is right in 4 DEG C
Dialysed 2~4 times in cross-linking reaction liquid, to pH=9.0；
(2) reaction solution after dialysis is transferred in reaction vessel, be put on magnetic stirring apparatus, according to anti-Escherichia coli
O157:H7 monoclonal antibody：FITC amount is 1mg:FITC DMSO solution, lucifuge, 4 DEG C of reactions are added dropwise in 150 μ g ratio
Overnight, 5mol/L NH is added44 DEG C of Cl extremely final concentration of 5mmol/L, terminating reaction 2h；
(3) cross-linking agent is dialysed more than 8 times in PBS, limpid to dialyzate, 4 DEG C of dark place preservations are standby.
Further, the material of described adsorbing fiber layer is glass fibre cotton, nylon membrane, PVDF membrane or poly-
Any one in ester film；The material of the fluorescence antibody fibrous layer be glass fibre cotton, nylon membrane, PVDF membrane or
Any one in polyester film；The material of described absorbent material layer is absorbent filter, and the material of described supporting layer is not inhale
The toughness material of water；The material of described cellulose film layer is in nitrocellulose filter, pure cellulose film or carboxylated cellulose film
Any one.
Further, the stealthy control trace in the stealthy detection trace and nature controlling line in the detection line is arranged in parallel
" the linear traces of ︱ ︱ ", or trace is arranged for " 10 " font, or for " ┬ ┬ " fonts arrange trace, or are " ┴ ┴ " fonts
Arrange trace, or for " ├ ├ " fonts arrange trace, or for " ┤ ┤ " fonts arrange trace, or are " ● ● " point-like arrangement print
Mark.
Further, on adsorbing fiber layer, fluorescence antibody fibrous layer and absorbent material layer covered with diaphragm,
Sample mark line is printed with adsorbing fiber layer diaphragm corresponding with fluorescence antibody fibrous layer intersection, mark line deviation is inhaled
Attached fibrous layer side 0.3-0.7cm.
It is used to detect Escherichia coli O 157 present invention also offers above-mentioned one kind:H7 interlayer type immune chromatography test paper
Preparation method, comprises the following steps：
1) one prepares Escherichia coli O 157:The step of H7 monoclonal antibodies or polyclonal antibody；
2) one the step of prepare fluorescence antibody；
3) one the step of prepare adsorbing fiber layer, described adsorbing fiber layer is using glass fibre cotton, nylon membrane, poly- inclined
Difluoride membranes or polyester film are made；
4) one the step of prepare fluorescence antibody fibrous layer, fluorescence is adsorbed with described fluorescence antibody fibrous layer and is resisted
Body；
5) one the step of prepare cellulose film layer, described cellulose film layer uses nitrocellulose filter, pure cellulose
Film or carboxylated cellulose film, with point sample instrument, specking detects trace on cellulose membrane, standby after drying；
6) the step of assembling test paper, by adsorbing fiber layer, fluorescence antibody fibrous layer, cellulose film layer, absorbent material
Layer is attached on the supporting layer with adhesive successively from left to right, and supporting layer, adsorption layer and protective layer are assembled into test paper successively.
Further, the preparation method of described adsorbing fiber layer is as follows：By glass fibre cotton, nylon membrane, gather inclined difluoro
In vinyl film or polyester film immersion TBS cushioning liquid, the concentration of described cushioning liquid is 0.01M, and pH is 7.8, and described is slow
Rush in solution, also containing PVP, sucrose, Tw-20, BSA, in described cushioning liquid, PVP mass percent concentration is
0.5%, the mass percent concentration of sucrose is that 1%, Tw-20 mass percent concentration is 0.5%, BSA mass percent
Concentration is 1%, is defined by soaking, and is freezed, standby.
Present invention also offers detect Escherichia coli O 157 using above-mentioned interlayer type immune chromatography test paper:H7 method,
Comprise the following steps：
1) bacterium is increased before sample：FITC is dissolved in DMSO, FITC solution is added in enriched medium, sample progress is added
Zengjing Granule；
2) test paper is detected：Test paper is inserted into sample solution, takes out and keeps flat after 10~20 seconds, is examined after 5-10min by observing
The detection line of test paper has in unstressed configuration, judgement sample whether contain Escherichia coli O 157:H7, or the progress of bar instrument is read by fluorescence
Semi-quantitative analysis.
The test paper of the present invention has advantages below：
(1) making is simple, cost is low, high specificity, and sensitivity is high.Test paper of the present invention and detection method are by immunofluorescence skill
Art is combined with immunochromatography technique, the target Escherichia coli O 157 in measuring samples:H7 passes through the enriched medium with FITC
Just it can be fluoresced after culture, it is strong without any mark flow, low manufacture cost, photostability, FITC labelled antibodies are introduced and tried
After paper, the effect of antigen-antibody dual signal amplification is reached, the sensitivity higher than common colloid gold test paper can be obtained.And
FITC mark costs are low compared with collaurum.It is high, specific that the test strips maintain the easy quick, sensitivity of traditional colloidal gold strip
Good advantage, minimum detectable 1CFU/mL trace contamination.
(2) it is easy, quick.The test paper is directly detected after food samples being increased with bacterium processing, is directly observed under ultraviolet light
As a result, also half-quantitative detection can be realized by the direct readings of phosphor reader.Only test paper need to be inserted test sample 10 during detection
~20 seconds, 5-10min is interior to can determine that testing result, time saving and energy saving, and easy to operate, a step is completed.
(3) result display is vivid, directly perceived, accurate.Test strips with show yellow green " ︱ " and " ︱ ︱ " (or " ten ", " ┬ ",
" ┴ ", " ├ ", " ┤ " "●") feminine gender and positive mark of the trace as detection, i.e., a yellow green is shown on cellulose membrane
" ︱ " trace, represents to be free of Escherichia coli O 157 in test sample:H7；" ︱ ︱ " traces represent test sample to two yellow greens of display
In contain Escherichia coli O 157:H7.This result with the naked eye can be observed directly, judge vivid, directly perceived, accurate, simple and clear, be difficult
There is false positive and false negative etc. artificially to judge by accident.
(4) expense is saved.Without separately matching somebody with somebody instrument and equipment and other reagents during the ELISA test strip, it can be examined whenever and wherever possible
Survey, can qualitative detection again can quantify detection；Testing cost is cheap, can save a large amount of expensive instruments and the Meteorological of equipment.
(5) it is applied widely, it is easy to utilize.The need for the test paper can meet different levels personnel, including specialization
Test, customs quarantine control, health quarantine, quality-monitoring, livestock products processing, raiser and consumer individual etc., both suitable for single sample
The detection of product, is suitable to the examination of a large amount of samples, can apply to the fields such as medical diagnosis on disease, Bacteria Detection and environment measuring again.This hair
It is bright that there is meaning of crucial importance in terms of ensuring food safety, protecting consumer health, with obvious economic benefit and society
Benefit.
The present invention is compared with prior art, and its technological progress is significant.The test paper and its detection method of the present invention is simple,
High specificity, sensitivity is high, intuitively, accurately, can carry out quantitative and semi-quantitative detection, and minimum detectable 1CFU/mL trace is dirty
Dye, applied widely, cost is low, it is easy to popularization and application.
Brief description of the drawings
Fig. 1 interlayer type test paper schematic diagrams.
The structural representation of Fig. 2 interlayer type test paper.
The vertical view figure structure schematic representation of Fig. 3 interlayer type test paper.
The sensitivity technique result of Fig. 4 interlayer type test paper.
Fig. 5 interlayer type test paper semi-quantitative analysis results.
Fig. 6 interlayer type test paper cross reaction experimental results.
Fig. 7 interlayer types test paper simulates experimental result of carrying disease germs.
Embodiment
Embodiment 1
The preparation process of test paper of the present invention includes：Escherichia coli O 157:Preparation, the absorption of H7 monoclonals or polyclonal antibody
The steps such as preparation, the preparation of cellulose film layer and the assembling of test strips of fibrous layer.
(1) anti-Escherichia coli O 157:The preparation of H7 monoclonal antibodies or polyclonal antibody
It is prepared by monoclonal antibody：Using the concentration of inactivation as 108Cfu/mL Escherichia coli O 157:The full week old of bacterial immunity 6~8 of H7
Balb/C mouse 3~4 times, each 3~5 weeks immunization interval time, determine that antibody titer is superpower immune after meeting the requirements, afterwards 3
~4 days, sinus under immune mouse socket of the eye is taken a blood sample, separate positive serum；De- neck is lethal, with 75% alcohol-pickled 5~10min of mouse
Body surface is sterilized, it is sterile to take its spleen, spleen is shredded and ground, is filtered through 120 mesh nylon gauzes, 1000rpm centrifugation 10min,
Collect splenocyte.By 1 × 108Splenocyte and NS0 myeloma cell press 10:1 ratio mixing, 1000rpm centrifugation 10min,
Supernatant is abandoned, cell pellet is slowly added into 0.7~1.0mL Escherichia coli O 157s in 37 DEG C of water-baths:H7 50%PEG4000,
1min is acted on, the culture medium 15mL of serum-free 1640 is then slowly added into, to terminate PEG effect, 37 DEG C of 5~10min of water-bath,
1000rpm centrifuges 10min, abandons supernatant, cell pellet is resuspended in HAT Selective agar mediums, and adds 96 hole cell culture
Plate hole (100 μ L~200 μ L/ holes), is placed in 37 DEG C, 5%CO2Cultivated 10~14 days in incubator, sun is carried out with indirect elisa method
Property hole sizer choosing, selection strong positive, the hole that inhibiting rate is high, cell growth is vigorous carry out 3 limited dilution clonings, then expand training
Support, set up the hybridoma prepared by hybridoma cell strain, the monoclonal antibody of secretion 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 epitopes,
Printing for T lines.(the present embodiment is intended merely to describe the preparation process of monoclonal antibody, any commercially available and Escherichia coli
O157:The monoclonal antibody of H7 reactions can be used in the present invention)
It is more anti-to prepare：With the Escherichia coli O 157 of inactivation:NZw is immunized in H7, and it is 10 that original content, which is immunized,8Cfu/mL,
4~6 points of injections of dorsal sc point.Head exempts from, and not formula Freund's complete adjuvant is added in immunogene, fully emulsified；In booster immunization, immunogene
Incomplete Freund's adjuvant is added, it is fully emulsified, carry out continuous immunity 4~5 times within 2~3 weeks after head exempts from, every minor tick 2~3 weeks, most
Afterwards 10~15 days after primary immune response, it is surveyed with indirect ELISA determine potency and reach 105During the above, take a blood sample and separate and collect height and exempt from blood
Clearly.IgG antibody is extracted with saturated ammonium sulfate salting out method, that is, takes 1 portion of hyper-immune serum plus 2 parts of PBS (pH7.2) to mix, plus in equal volume
Saturated ammonium sulfate solution is mixed, and is put 4 DEG C of refrigerators 12h, 4 DEG C, 2500rpm centrifugation 15min, is abandoned supernatant；Again with appropriate PBS
(pH7.2) dissolving precipitation, plus saturated ammonium sulfate solution is to final concentration 33%, puts 4 DEG C of refrigerator 2h, 4 DEG C, 2500rpm centrifugations
15min, abandons supernatant；Dissolved and precipitated with appropriate PBS (pH7.2), put with 48~72h of PBS (pH7.2) dialysis in 4 DEG C of refrigerators, it is middle
Change liquid for several times, 4 DEG C, 12000rpm centrifugation 15min collect supernatant, obtain the anti-Escherichia coli O 157 of purifying:H7 polyclonal antibodies ,-
20 DEG C freeze, the printing for T lines.
(2) preparation of fluorescence antibody fibrous layer (pad)
The preparation of fluorescence antibody fibrous layer, it is necessary first to prepare FITC fluorescence antibodies, concretely comprise the following steps：
1) cross-linking reaction liquid is configured：7.56g NaHCO3, 1.06g Na2CO3, 7.36g NaCl, plus ultra-pure water are settled to
1L.Monoclonal antibody after purification is dialysed 3 times in 4 DEG C to cross-linking reaction liquid, to pH=9.0.
2) reaction solution after dialysis is transferred into vial to be put on magnetic stirring apparatus, according to antibody：FITC amount is
1mg:FITC DMSO solution is added dropwise in 150 μ g ratio, and lucifuge, 4 DEG C of reactions are stayed overnight.Add 5mol/L NH44 DEG C of Cl is to end
Concentration is 5mmol/L, terminating reaction 2h.
3) cross-linking agent is dialysed more than 8 times in PBS, it is limpid to dialyzate.4 DEG C of dark place preservations are standby.
Secondly, with point sample instrument on fiber tunic specking FTIC antibody, make fluorescence mark band, it is standby in the drying of 37 DEG C of lucifuges
With.
(3) preparation of adsorbing fiber layer (sample pad)
Test lead adsorbing fiber layer glass fibre cotton, nylon membrane, polyvinylidene fluoride pvdf membrane or polyester film preparation, will
Fibrous material is cut into wide 1.5cm band, puts it into sample pad treatment fluid and soaks 30min, standby in 37 DEG C of drying.
(4) preparation of cellulose film layer (analysis pad)
Cellulose film layer nitrocellulose filter, pure cellulose film or carboxylated cellulose film, cut into wide 1.5cm specifications
Band, with point sample instrument on cellulose membrane specking Escherichia coli O 157:H7 monoclonal antibodies or polyclonal antibody and goat-anti or rabbit
Anti-mouse IgG antibody or goat anti-rabbit igg antibody, make stealthy detection trace band and control trace band, in 37 DEG C of dry for standby.
(5) assembling of interlayer type test paper：By adsorbing fiber layer (sample pad), fluorescence antibody fibrous layer (pad), fiber
Plain film layer (analyzing film), absorbent material layer are attached on the supporting layer with adhesive (bottom plate), and are cut into 3- successively from right to left
Test strips wide 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 test paper test lead insertion testing sample solution, solution to be measured is made by siphon
With drive luminous Escherichia coli O 157 to be measured:H7 spreads to cellulose film layer, and eventually penetrates the absorbent material layer of handle end.
In diffusion process, Escherichia coli O 157 to be measured:H7 can be with FITC fluorescence antibodies formation FITC- bacterium-antibody-FITC on pad
Compound, again can be by Escherichia coli O 157 thereon when flowing to T lines:H7 antibody capture is so as to show detection trace, ultraviolet
Line excites down (or using the direct readings of phosphor reader) to form yellow green detection trace band " ︱ ", is positive represent；Otherwise sample
Without Escherichia coli O 157 in product solution:During H7, then can not be combined with the fluorescence antibody on pad, and with it is big on cellulose membrane
Enterobacteria O157:H7 antibody detection trace is combined, and does not show that yellow green detects trace band " ︱ ", is negative represent；No matter sample
Whether Escherichia coli O 157 is contained:H7, FITC fluorescence antibody all can be with the goat-anti on cellulose membrane or rabbit anti-mouse IgG (or sheep
Anti-rabbit IgG) antibody capture, form a yellow green control trace band " ︱ " (as shown in Figure 1 C).
The detection method of the interlayer type immune chromatography test paper of the present invention is as follows：
(1) bacterium is increased before sample：It should be examined as early as possible after sample collection.Even if if can not detect, 18h can be preserved at 2-4 DEG C.Nothing
Bacterium operation sampling 25g (mL) is added in 225mLEC the or NB meat soups containing FTIC solution, and above-mentioned solution is put into homogenizer
In, the continuous homogenizing 1-2min on slap type homogenizer, or it is put into the homogeneous cup for filling 225mLEC or NB meat soups (containing FITC)
In, 8000-10000r/min homogeneous 1-2min, in 37 DEG C of some hours (as shown in Figure 1A) of culture.Do positive and negative simultaneously
Control.
(2) test paper is detected：Test paper is inserted into sample solution, takes out and keeps flat after 10~20 seconds, is examined after 5-10min by observing
The detection line of test paper has in unstressed configuration, judgement sample whether contain Escherichia coli O 157:H7, or the progress of bar instrument is read by fluorescence
Semi-quantitative analysis.
Following examples illustrate the structure and detection method of test strips.
Embodiment 2
Referring to Fig. 2, Fig. 3.
1 is supporting layer in figure, is made of plastic slice bar, and 2 be adsorbing fiber layer, is made of glass fibre cotton, fluorescence resists
FITC fluorescent monoclonal antibodies are adsorbed with body fibrous layer 3, cellulose film layer 4 uses nitrocellulose filter, the water suction of handle end
Material layer 5 is made of absorbent filter, and adsorbing fiber layer 2, fluorescence antibody fibrous layer 3, cellulose film layer 4, absorbent material layer 5 is each
Layer is pasted and fixed on supporting layer 1 successively from right to left, and intersection fiber crosses one another infiltration each other.In cellulose film layer
4, provided with stealthy detection trace 6, use Escherichia coli O 157:H7 monoclonal antibodies are made；Stealth control trace 7 uses sheep anti-Mouse
IgG antibody solution trace on cellulose membrane is made " ︱ ", and two trace bands are arranged in parallel, forms combination trace band " ︱ ︱ ".
8-1 is to be covered in the white diaphragm of sample end above adsorbing fiber layer 2 and fluorescence antibody fibrous layer 3, and 8-2 is to cover
The other color diaphragms (such as yellow) covered on absorbent material layer 5,9 be sample mark line, and it is fine that the mark line is located at absorption
Tie up at the white diaphragm deviation adsorbing fiber layer 2 sides about 0.5cm corresponding with the intersection of fluorescence antibody fibrous layer 3 of layer 2, in mark
On the right side of note line arrow and max printed words are printed on diaphragm.
(1) test paper sensitivity technique of the present invention：By Escherichia coli O 157：H7 single bacterium colonies are inoculated in containing 0.4mg/mL's
Cultivated in FITC culture mediums, using plate count method, bacterium solution is diluted to 10 respectively8-104CFU/mL, uses interlayer type test paper
Testing result is observed after detection, 5-10min under uviol lamp, range estimation minimum detection limit (LOD) is that minimum bacterium when T lines are visible is dense
Degree, as a result as shown in figure 4, the LOD of test paper is 106CFU/mL.Bar instrument is read with fluorescence semi-quantitative analysis is carried out to test paper, as a result such as
Shown in Fig. 5, test paper is 10 in bacteria concentration5-108It is linearly related during CFU/mL, and LOD is 105CFU/mL。
(2) test paper specific detection of the present invention：Using its specificity of cross reaction test for identification.It is 10 by concentration8CFU/
ML common food-borne pathogens, as shown in table 1, detect, if positive have intersection, testing result is as schemed with interlayer type test paper
Shown in 6, Escherichia coli interlayer type test paper can only detect three plants of Escherichia coli, and other common food-borne pathogens can not be detected,
I.e. the test paper has preferable specificity.
Common food-borne pathogens and source used in the cross reaction of table 1
(3) measure of test paper stability of the present invention：To adding drier, lucifuge after being sealed with a batch of interlayer type test paper
It is positioned over 4 DEG C.Preserving 1 day, 1,5,10, after 15 and 20 weeks detect its sensitivity respectively, as a result show the examination after preserving 20 weeks
The sensitivity of paper with preserve 1 day after it is unchanged, therefore the test paper at least can preserve 20 weeks and retention property at 4 DEG C stably.
(4) experiment of carrying disease germs is simulated：From local supermarket purchase bread, milk, jelly sample, three kinds of samples are identified with PCR methods
Without Escherichia coli O 157：H7 pollutes.Every kind of sample 25g is taken to be cultivated as in the 225mL culture mediums with FITC with sterile working,
Viable bacteria solution is added in culture medium makes bacteria concentration be 1CFU/mL, and blake bottle is put in 37 DEG C of shaking table cultures, samples and detects every 2h,
As a result it is as shown in Figure 7.As a result it can be detected after showing bread, milk sample culture 10h, jelly culture 8h can be detected；Interlayer type is tried
Paper can detect the sample that initial bacteria concentration is 1CFU/mL.
Embodiment 3
Test strips structure and embodiment 1 are identical.Difference is：Fluorescence antibody fibrous layer 3 is adsorbed with FITC marks
Anti- Escherichia coli O 157：H7 polyclonal antibodies, adsorbing fiber layer 2 is made of nylon membrane, and cellulose film layer 4 uses pure cellulose
Film, stealth is imprinted as goat anti-rabbit igg antibody.It is " ten " that stealthy detection trace band and stealth, which compare trace band, and 8-2 is to be covered in
Handle end blue diaphragm above absorbent material layer 5.
The preparation of testing sample and detection operating procedure：
Detect bread：Sterile working sampling 25g is added in 225mLEC the or NB meat soups containing FTIC solution, will be above-mentioned
Solution is put into homogenizer, the continuous homogenizing 1-2min on slap type homogenizer, in 37 DEG C of culture some hours.
Operating method：Test paper is inserted into sample solution, takes out and keeps flat after 10~20 seconds, is detected after 5-10min by observing
The detection line of test paper has in unstressed configuration, judgement sample whether contain Escherichia coli O 157:H7, or bar instrument progress half is read by fluorescence
Quantitative analysis.
Result judgement：If (a) positive show two yellow green trace bands " ten " on cellulose membrane, detection knot is represented
Fruit is the positive, illustrates to contain Escherichia coli O 157 in testing sample:H7；If (b) feminine gender shows one on cellulose membrane
Bar yellow green trace band " ten ", it is feminine gender to represent testing result, illustrates to be free of Escherichia coli O 157 in testing sample:H7；(c)
If failure does not have yellow green band to show on cellulose membrane, show that test strips have failed.
Embodiment 4
Test paper structure and embodiment 2 are essentially identical, and difference is：Cellulose film layer 3 is adsorbed with anti-Escherichia coli
O157:H7 polyclonal antibody, adsorbing fiber layer 2 is made of nylon membrane, and cellulose film layer 3 uses pure cellulose film, stealth inspection
It is to be covered in the handle end greenism film above absorbent material layer 4 that trace, which is surveyed, for " ┬ ", 6-2.
For detecting milk sample：Sterile working sampling 25mL is added in 225mLEC the or NB meat soups containing FTIC solution,
Above-mentioned solution is put into homogenizer, is put into the homogeneous cup for filling 225mLEC or NB meat soups (containing FITC), 8000-10000r/
Min homogeneous 1-2min, in 37 DEG C of culture some hours.
Operating method：Test paper is inserted into sample solution, takes out and keeps flat after 10~20 seconds, read after 5-10min by fluorescence
The direct readings of device, numerical value is the fluorescence intensity of T lines, draws standard curve according to peak value or peak area, calculates actual content.
Embodiment 5
Test paper structure and embodiment 2 are essentially identical, and difference is：Adsorbing fiber layer 2 uses polyvinylidene fluoride PVDF
Film is made, and cellulose film layer 3 uses carboxylated cellulose film, and 6-2 is to be covered in the handle end greenism above absorbent material layer 4
Film, stealth detection trace band is " ┴ ".
For detecting jelly：Increase bacterium, result judgement and the equal be the same as Example two of operating method before sample, do not repeat.
Embodiment 6
Test paper structure and embodiment 2 are essentially identical, and difference is：Adsorbing fiber layer 2 is made of polyester film, cellulose
Film layer 3 uses carboxylated cellulose film, and stealth detection trace band is " ├ ".
Increase bacterium, result judgement and the equal be the same as Example two of operating method before meat sample, sample for detecting, do not repeat.
Embodiment 7
Test paper structure and embodiment 2 are essentially identical, and difference is：Adsorbing fiber layer 2 is made of nylon membrane.Detection print
Mark band is " ┤ ".Detect sample, result judgement and operating method with example two.
Embodiment 8
Essentially identical with embodiment 2, difference is：Cellulose film layer 3 is adsorbed with anti-Escherichia coli O 157:H7's is more
Clonal antibody, detection trace band is "●".Detection sample is bread sample.
Embodiment 9
Essentially identical with embodiment 2, difference is：Cellulose film layer 3 is adsorbed with anti-Escherichia coli O 157:H7's is more
Clonal antibody, detection sample is milk sample.
Embodiment 8
Essentially identical with embodiment 2, difference is：Cellulose film layer 3 is adsorbed with anti-Escherichia coli O 157:H7's is more
Clonal antibody, detection sample is jelly sample.
Embodiment 10
Essentially identical with embodiment 2, difference is：Cellulose film layer 3 is adsorbed with anti-Escherichia coli O 157:H7's is more
Clonal antibody, detection sample is meat sample.
Claims (1)
1. a kind of detection Escherichia coli O 157 of non-diseases diagnostic purpose:H7 method, it is characterised in that comprise the following steps：
1) bacterium is increased before sample：FITC is dissolved in DMSO, FITC solution is added in enriched medium, sample is added and carries out increasing bacterium
Culture；
2) test paper is detected：Test paper is inserted into sample solution, takes out and keeps flat after 10~20 seconds, by observing detection examination after 5-10min
The detection line of paper has in unstressed configuration, judgement sample whether contain Escherichia coli O 157:H7, or bar instrument progress semidefinite is read by fluorescence
Amount analysis；Described test paper includes supporting layer, and adsorption layer, described absorption are provided with the upper side of described supporting layer
Layer is followed successively by the absorbent material layer of adsorbing fiber layer, fluorescence antibody fibrous layer, cellulose film layer and handle end from test lead, described
Adsorbing fiber layer and described fluorescence antibody fibrous layer closely connect, described fluorescence antibody fibrous layer and described cellulose
Film layer is closely connected, and described cellulose film layer and described absorbent material layer are closely connected, provided with inspection in cellulose film layer
Survey line and nature controlling line, described detection line is by anti-Escherichia coli O 157:H7 monoclonal antibody or polyclonal antibody is constituted, described
Nature controlling line be made up of goat-anti or rabbit anti-mouse IgG antibody or goat anti-rabbit igg antibody, on the fluorescence antibody fibrous layer adsorb
There are fluorescence antibody, the anti-Escherichia coli O 157 that described fluorescence antibody is marked by FITC:H7 monoclonal antibody or Anti-TNF-α
Body is constituted.
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| CN111521827A (en) * | 2020-05-21 | 2020-08-11 | 上海理工大学 | Immunofluorescence test strip for rapidly determining titer of monoclonal antibody based on antigen tracing |
| 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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| US4863875A (en) * | 1985-08-26 | 1989-09-05 | Gia Research Company, L.P. | Dye labelled antibodies as reagents for use in immunoassay systems |
| CN101526534A (en) * | 2009-03-10 | 2009-09-09 | 沈鹤柏 | Fluorescence immune chromatography test paper and preparing method and application thereof |
| CN101539580A (en) * | 2009-03-18 | 2009-09-23 | 侯巍 | Protein fluorescence detection card and detection method thereof |
| CN202814987U (en) * | 2012-08-23 | 2013-03-20 | 南京基蛋生物科技有限公司 | In-vitro diagnostic test strip for ferritin |
| CN104007265B (en) * | 2014-05-30 | 2016-02-24 | 山东出入境检验检疫局检验检疫技术中心 | Enterohemorrhagic Escherichia coli O 157: H7 quantum dot fluorescence immunochromatographiassay assay quantitative detection test paper and detection method thereof |
| CN104316686A (en) * | 2014-10-11 | 2015-01-28 | 南昌大学 | Test paper box capable of detecting escherichia coli O157:H7 |
| CN104237537A (en) * | 2014-10-21 | 2014-12-24 | 南京基蛋生物科技有限公司 | Immune chromatography fluorescence reagent strip for detecting cardiac troponin and preparation method thereof |
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