CN108303532A - A kind of escherichia coli O157 of non-diagnostic purpose:The rapid detection method of H7 - Google Patents
A kind of escherichia coli O157 of non-diagnostic purpose:The rapid detection method of H7 Download PDFInfo
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Abstract
The invention discloses a kind of escherichia coli O157 of non-diagnostic purpose:The rapid detection method of H7.The rapid detection method of the present invention is with Au Pt SiO2Composite nano-microsphere is that carrier forms signal transduction probe by binding antibody and enzyme；Enrichment and separation is carried out to object in sample with the superparamagnetism of immune magnetic probe；By detected purpose bacterium escherichia coli O157:The signal of H7 is converted to the signal of glucose molecule, is read with portable glucose meter.The present invention is based on blood glucose meter and two kinds of nano-complex capture probes and signal transduction probe, build a kind of novel economy, simplicity, quickly, escherichia coli O157 in sensitive detection food:The new method of H7.
Description
Technical field
The invention belongs to Biological Detection technical fields, more particularly to a kind of escherichia coli of non-diagnostic purpose
O157:The rapid detection method of H7.
Background technology
Escherichia coli O157:H7(Escherichia coli O157:H7, E.Coli O157:H7), it is a kind of heavy
The food-borne pathogens wanted belong to one of first five position of main food-borne pathogens.E.coli O157:H7 mainly passes through pollution
Beef, milk, chicken and its product, the foods such as vegetables are propagated.When people has infected E.Coli O157:After H7, it may appear that stomach and intestine
Tract disease more has severe patient even to develop into thrombocytopenic purpura (TTP)[8-10].At present to E.Coli O157:H7 is detected
Method mainly has National Standard Method (Zengjing Granule method) and various PCR methods etc., respectively there is advantage and disadvantage.Therefore, it establishes a kind of better
E.Coli O157:The novel Fast Detection Technique of H7 qualitative, quantitatives has larger realistic meaning.
In recent years, with high-specific surface area, high conductivity, catalytic, hypotoxicity the golden platinum nanoparticle of many advantages, such as
(Au-Pt NPs) and have many advantages, such as that high-specific surface area, chemical property be stable, Nano particles of silicon dioxide of good biocompatibility
(SiNPs) as the research hotspot of field of nanometer material technology, the researchs such as bioanalysis, chemical detection, food inspection have been applied to
Field.Au-Pt NPs are proven to have excellent biocompatibility, can be with the amino and cysteine residues phase in protein
Interaction hand over hand adsorbed proteins and keep its activity, the large biological molecules such as its binding antibody can be used.However, individually
Au-Pt NPs Chang Yinwei unstable be easier to occur to reunite and influence the activity of protein marker.Studies have found that, incite somebody to action in the recent period
SiNPs carries out amination or sulfhydrylation modification, in combination with the metals such as Au, Ag, Pt and Pb, be compounded to form SiNPs/Au,
SiNPs/Ag, SiNPs/Pt reduce aggtegation, moreover it is possible to simultaneously in conjunction with not only can effectively improve respective dispersibility later
Gather around the advantages of there are two types of nano materials.Currently, golden platinum silicon nanoparticle (APS NPs) while labelled antibody and enzyme are prepared letter
Number transduction probe Ab2/ APS NPs/Invertase are applied to the research that food-borne pathogens and other determinands detect and go back
It has not been reported.
To sum up, the present invention is directed to existing escherichia coli O157:The defect of H7 detection techniques develops a kind of large intestine angstrom
Uncommon Salmonella O157:The rapid detection method of H7.The rapid detection method of the present invention passes through binding antibody using APS NPs as carrier
Signal transduction probe (Ab is formed with enzyme2/APS NPs/Invertase)；With the superparamagnetic of immune magnetic probe (MNPs-IgG)
Property enrichment and separation is carried out to object in sample, purpose bacterium escherichia coli O157 that will be detected:The signal of H7 is converted
For the signal of glucose molecule, read with portable glucose meter (Personal Glucose Meter, PGM).The wound of this research
New point is to be based on blood glucose meter and two kinds of nano-complexes MNPs-IgG and Ab2/ APS NPs/Invertase are respectively as capture
Probe and signal transduction probe, a kind of novel economy of structure, simplicity, escherichia coli quickly, in sensitive detection food
Bacterium O157:The new method of H7.
Invention content
The purpose of the present invention is to provide a kind of escherichia coli O157 of non-diagnostic purpose:The quick detection side of H7
Method.The present invention is to be based on blood glucose meter and two kinds of nano-complexes MNPs-IgG and Ab2/ APS NPs/Invertase respectively as
Capture probe and signal transduction probe are realized to the escherichia coli O157 in food:The quick detection of H7 qualitative, quantitatives,
The detection method economy, simplicity, quickly, have good sensitivity, specificity, stability and accuracy.
To achieve the above object, the present invention takes following technical scheme：
A kind of escherichia coli O157 of non-diagnostic purpose:The rapid detection method of H7, includes the following steps：
(1) with the anti-escherichia coli O157 of mouse:H7 antibody is to Fe3O4/SiO2The magnetic nanoparticle surface of nucleocapsid
It is modified, obtains immune magnetic probe, it is spare；
(2) Au-Pt nanoparticles are modified to the surface nano SiO 2 particle (SiNPs), obtains Au-Pt-SiO2It is multiple
Close nanoparticle (APS NPs)；
(3) Au-Pt-SiO being prepared with step (2)2Composite nano-microsphere (APS NPs) is carrier combination large intestine angstrom
Uncommon Salmonella O157:H7 antibody and invertase obtain signal transduction probe, spare；
(4) sample to be tested is taken, the glucose initial concentration in sample to be tested is detected with blood glucose meter, then makes step (1)
Standby obtained immune magnetic probe, which is added in the bacteria suspension of sample to be tested, is incubated certain time in 37 DEG C, washed, magnetic point
From magnetic composite A is separated；
(5) step (3) signal transduction probe for being prepared is added in the isolated magnetisable material A of step (4) simultaneously
It is incubated certain time in 37 DEG C, washed, Magnetic Isolation separates magnetic composite B；
(6) sucrose solution is added in the magnetic composite B that step (5) obtains, reaction certain time is stood at 55 DEG C
The concentration of glucose C2 of blood glucose meter detection architecture is used afterwards；It is ordinate according to the variation difference DELTA C of concentration of glucose, large intestine angstrom is uncommon
Salmonella O157:The logarithm of H7 concentration is the standard curve of abscissa, can calculate escherichia coli O157 in sample:H7
Content, wherein Δ C=C2-C1.
In the above-mentioned methods, the magnetic nanoparticle in the step (1) is prepared via a method which and obtains：
(1) poly(4-styrene sulfonic acid-co-maleic acid) sodium salt (PSSMA) is dissolved in ethylene glycol, magnetic agitation obtains
Uniform orange solution；Add Iron(III) chloride hexahydrate and anhydrous sodium acetate；Then, the uniform liquid of obtained rufous is fallen
Enter into hydrothermal reaction kettle, 200 DEG C of reaction 10h；It is to be cooled to after room temperature, by gained black Fe3O4Sediment is in external magnetic field
It is lower with milli-Q water at least three times；
(2) Fe is taken3O4Solution is placed in conical flask, and ethyl alcohol, deionized water and ammonium hydroxide are added thereto, after mixing, then
Tetraethyl orthosilicate (TEOS) is added, reaction 18h is stirred at room temperature；Then ethyl alcohol and distillation are used successively under external magnetic field
Water washing is multiple, and the sediment being finally separating to obtain is magnetic nanoparticle.
In the above-mentioned methods, the specific preparation process of immune magnetic probe is as follows in the step (1)：Magnetism is taken to receive first
Rice grain is simultaneously washed with absolute ethyl alcohol, and the magnetic nanoparticle after washing is resuspended in ethyl alcohol, and three ethoxy of 3- aminopropyls is added
Base silane (APTES), after being stirred to react 12h at room temperature, Magnetic Isolation simultaneously uses ethyl alcohol and phosphate buffer washing more respectively
It is secondary, obtain amido modified magnetic nanoparticle；Meanwhile by 1- (3- dimethylamino-propyls) -3- ethyl-carbodiimide hydrochlorides
(EDC) it is added to the anti-escherichia coli O157 of the diluted mouse of phosphate buffer with n-hydroxysuccinimide (NHS):
H7 monoclonals capture in antibody, and 4 DEG C are incubated overnight；Add the amido modified magnetic Nano for being dissolved in phosphate buffer
Grain, room temperature concussion reaction 4h, Magnetic Isolation are simultaneously washed repeatedly with phosphate buffer, and obtaining surface modification has the anti-large intestine angstrom of mouse uncommon
Salmonella O157:The magnetic nanoparticle of H7 antibody.
In the above-mentioned methods, the nano SiO 2 particle in the step (2) is prepared via a method which and obtains：
Tetraethyl orthosilicate is added in ethyl alcohol and obtains A liquid, ammonium hydroxide and ethyl alcohol are added to the water to obtain B liquid, then will
A liquid is promptly added in B liquid, reacts 5h under room temperature；After, it centrifuges, is distinguished with absolute ethyl alcohol and distilled water
Washing is multiple.
In the above-mentioned methods, the Au-Pt-SiO in the step (2)2Composite nano-microsphere be prepared via a method which and
：
(1) chlorauric acid solution being added in ultra-pure water, oil bath heating is to boiling, then rapidly joins sodium citrate solution,
After a few minutes, solution reddens, and shows to have generated colloidal gold；Then, platinum acid chloride solution is added, then is rapidly added anti-bad
Hematic acid solution, is stirred continuously, and reaction 20min postcoolings obtain Au-Pt nanoparticles to room temperature；
(2) it takes nano SiO 2 particle to be distributed to absolute ethyl alcohol after ultrasound, 3- aminopropyl triethoxysilanes is added,
Reaction is stirred at room temperature overnight, centrifuges after reaction, uses ethyl alcohol and milli-Q water respectively, obtains amidized two
Silica nano particle；Amidized nano SiO 2 particle is distributed in the aqueous solution of Au-Pt nanoparticles, after ultrasound
Be added poly(4-styrene sulfonic acid-co-maleic acid) sodium salt solution, be stirred at room temperature reaction 12h, after use ultra-pure water and phosphoric acid
Salt buffer distinguishes centrifuge washing 3 times, obtains the sediment of kermesinus, as Au-Pt-SiO2Composite nano-microsphere.
In the above-mentioned methods, the specific preparation process of signal transduction probe is as follows in the step (3)：By escherichia coli
Bacterium O157:H7 labelled antibodies are added to Au-Pt-SiO2In composite nano-microsphere solution, gentle agitation 4h, adds under 4 DEG C of environment
Enter to use the diluted sucrose enzyme solutions of phosphate buffer, continue gentle agitation 12h, after reaction, centrifuge, washing obtains letter
Number transduction probe.
In the above-mentioned methods, in the step (4) immune magnetic probe a concentration of 10mg/mL, volume be 50 μ L, be incubated
Time is 45min.
In the above-mentioned methods, signal transduction concentration and probe concentration is 5mg/mL in the step (5), and volume is 100 μ L, is incubated
Time is preferably 30min.
In the above-mentioned methods, sucrose solution is dissolved in HAc-NaAc buffer solutions by sucrose and is prepared in the step (6),
The concentration of sucrose solution is preferably 0.5M.
The linear equation of step (6) standard curve is y=5.0724x-6.6421, and x is that large intestine angstrom is uncommon in sample
The O157 of Salmonella:H7 concentration denary logarithm values, unit CFU/mL, y are the variation difference DELTA C of concentration of glucose, single
Position is mmol/L.
The present invention has following technical characterstic：
1) present invention is using sodium citrate and ascorbic acid restores gold chloride as reducing agent and chloroplatinic acid prepares Au-Pt
Nanoparticle, then amido modified silica nanoparticle surface is attached to by Electrostatic Absorption and the effect of Pt-NH keys, not only
Simple and convenient easy preparation, and building-up process is safe and non-toxic, it is often more important that Au-Pt-SiO2Composite nano-microsphere is in sessile antibody
With the reagent and step for avoiding glutaraldehyde method complexity when enzyme, enzyme and antibody caused by also avoiding in modification due to crosslinking
The influence of loss of activity improves modification efficiency.
2) for the present invention using immune magnetic probe as capture probe, the enrichment and separation object bacteria from sample improves detection
Sensitivity, interlayer structure is formed with signal transduction probe and object bacteria and immune magnetic probe, due to band in sandwich complex
There is invertase, in the presence of having sucrose, its energy catalyzing hydrolysis, which generates glucose, makes glucose from scratch, and concentration increases, then
The concentration that glucose is measured using blood glucose meter, in this way can be by escherichia coli O157:H7 concentration is contacted with concentration of glucose
Get up, reaches qualitative and quantitative detection escherichia coli O157:The purpose of H7；If there is no purpose bacterium in sample, just there is no enzyme
Catalysis generates glucose.
3) the escherichia coli O157 that the present invention develops:The rapid detection method of H7, can be to the large intestine angstrom in food
Uncommon Salmonella O157:H7 carry out qualitative and quantitative detection, detection process economy, simplicity, quickly, and can store at 4 DEG C 90d without mistake
It is living, with good stability and good sensitivity, specificity and accuracy.
Description of the drawings
The Fe that Fig. 1 embodiments 1 are prepared3O4The transmission electron microscope picture of nano-particle；
The Fe that Fig. 2 embodiments 1 are prepared3O4/SiO2The projection electron microscope of the magnetic nanoparticle of nucleocapsid；
The transmission electron microscope picture for the nano SiO 2 particle that Fig. 3 embodiments 1 are prepared；
The transmission electron microscope picture for the Au-Pt nano-particles that Fig. 4 embodiments 1 are prepared；
The Au-Pt-SiO that Fig. 5 embodiments 1 are prepared2The transmission electron microscope picture of composite nano-microsphere；
Fig. 6 signal transduction probe set immune magnetic probes quickly detect escherichia coli O157:H7 Method And Principles show
It is intended to；
2 concentration of glucose of Fig. 7 embodiments changes the relational graph of (Δ C) and the amount of capture probe；
2 concentration of glucose of Fig. 8 embodiments changes (Δ C) and invertase and E.Coli O157:The ratio of H7 labelled antibodies
Relational graph；
2 concentration of glucose of Fig. 9 embodiments changes the relational graph of (Δ C) and the concentration of signal transduction probe；
3 concentration of glucose of Figure 10 embodiments changes (Δ C) and E.Coli O157:The relational graph of H7 log concentrations.
4 specific test of Figure 11 embodiments：The relational graph of concentration of glucose variation (Δ C) and bacterial species is (from left to right
It is followed successively by Cronobacter Enterobacter sakazakii (C.sakazakii), Bacterium enteritidis (S.enteritidis), Staphylococcus aureus
Bacterium (S.aureus), vibrio parahemolyticus (V.parahaemolyticus) take formula lemon lactic acid bacteria (C.freundii), and chicken is white
Dysentery avian infectious bronchitis nephritis virus (S.pullorum and S.gallinarum), PBS, escherichia coli O157:H7(E.coli
O157:H7)；All bacteria concentrations are 108CFU/mL)；
5 stability experiment of Figure 12 embodiments：Concentration of glucose changes (Δ C) and storage time relational graph.
Specific implementation mode
Following specific examples is the further explanation to method provided by the invention and technical solution, but is not construed as
Limitation of the present invention.
Embodiment 1：
(1) Fe3O4/SiO2The synthesis of the magnetic nanoparticle of nucleocapsid
With solvent structure Fe3O4Nano-particle.Specific building-up process is as follows：By 0.5g it is poly- (4- styrene sulfonic acids-altogether
Poly- maleic acid) sodium salt PSSMA is dissolved in 20mL ethylene glycol, and magnetic agitation obtains uniform orange solution.Add six water of 0.54g
Close ferric trichloride and 1.5g anhydrous sodium acetates.Then, the uniform liquid of obtained rufous is poured into 100mL hydrothermal reaction kettles
In, 200 DEG C of reaction 10h.It is to be cooled to after room temperature, by gained black precipitate under external magnetic field with milli-Q water at least
It three times and is settled in 6 mL ultra-pure waters, for use.
Take the Fe of the above-mentioned preparations of 1mL3O4Solution is placed in conical flask, and 60mL ethyl alcohol, 10mL deionized waters are added thereto
1mL tetraethyl orthosilicate TEOS are added after 10min is mixed with 9mL ammonium hydroxide, reaction 18h is stirred at room temperature.Then
Use ethyl alcohol and distillation water washing multiple successively under external magnetic field, the sediment being finally separating to obtain is magnetic nanoparticle
(magnetic nanoparticles, MNPs), constant volume is for use to 1mL.
As shown in Figure 1, Fe3O4Nano-particle has preferable monodispersity, and all particles are typical spherical solid
Structure, it is about 350nm to refer to scale to measure the average diameter of nano particle by Nano ZS laser particle analyzers and comparison.Such as Fig. 2
It is shown, it utilizesMethod is to Fe3O4After nano-particle is modified, it can be seen that a kind of apparent nucleocapsid, SiO2Shell
Thickness be about 70nm, illustrate SiO2Successfully it has been coated to Fe3O4The surface of nano-particle.
(2) synthesis of immune magnetic probe
The above-mentioned prepared magnetic nanoparticles (MNPs) of 150 μ L are taken first and are washed 2 times with absolute ethyl alcohol, by MNPs
It is resuspended in 20mL ethyl alcohol, 200 μ L3- aminopropyl triethoxysilanes (APTES), after being stirred to react 12h at room temperature, magnetic is added
Property detach and to use ethyl alcohol and phosphate buffer solution (PBS) (pH 7.4) to wash respectively multiple, obtain MNPs-NH2.Meanwhile
48.3mg 1- (3- dimethylamino-propyls) -3- ethyl-carbodiimide hydrochlorides (EDC) and 60.4mg N- hydroxysuccinimidyls acyl is sub-
Amine (NHS) is added to the 1.2mL anti-E.Coli O157 of the diluted mouse of PBS:H7 monoclonals capture in antibody (145 μ g/mL), and 4
It DEG C is incubated overnight.Add the 2.4mg MNPs-NH for being dissolved in 120 μ L PBS2, room temperature concussion reaction 4h, Magnetic Isolation simultaneously uses PBS
Washing is multiple, and for last constant volume in 200 μ L PBS, obtaining surface modification has the anti-E.Coli O157 of mouse:The Fe of H7 antibody3O4/
SiO2The magnetic nanoparticle of nucleocapsid, i.e. immune magnetic probe.It is preserved at 4 DEG C for use.
(3) synthesis of nano SiO 2 particle
It utilizesMethod synthetic silica nano particle (SiNPs).Specific building-up process is as follows：Prepare first A liquid and
B liquid.A liquid：0.9mL TEOS are added in 9.1mL ethyl alcohol；B liquid：0.6mL ammonium hydroxide and 3.4mL ethyl alcohol are added to 6.0mL
In water.A liquid is promptly added in B liquid again, reacts 5h under room temperature.After, by gained sediment in 10000rpm
Lower centrifugation washs 3 times and constant volume respectively with absolute ethyl alcohol and distilled water.
It can be obtained by Fig. 3, the Nanoparticle Size of synthesis is uniform, and grain grain separates, good dispersion, and pattern is typical and light
Sliding is spherical.It is 200nm by the hydration grain size that Nano ZS laser particle analyzers measure.
(4) Au-Pt-SiO2The synthesis of composite nano-microsphere
0.5mL 1wt% chlorauric acid solutions are added in 50mL ultra-pure waters first, oil bath heating is to boiling.Quickly add again
Enter 0.75mL 1wt% sodium citrate solutions, after a few minutes, solution reddens, and shows to have generated colloidal gold.Then, add
Enter 0.625mL 1wt% platinum acid chloride solutions, then be rapidly added 1mL 0.1M ascorbic acid solutions, is stirred continuously.After reacting 20min
Stop heating.To be cooled to after room temperature, constant volume obtains Au-Pt NPs solution, is preserved at 4 DEG C for use.
It takes above-mentioned 0.05g SiNPs to be distributed to the absolute ethyl alcohol of 20mL after ultrasound, 200 μ L APTES is added, in room temperature
Under be stirred to react overnight, amino is introduced on the surfaces SiNPs after hydrolysis.It centrifuges and divides under the conditions of 10000rpm
Not Yong ethyl alcohol and milli-Q water 3 times, obtain amidized SiNPs.The amidized SiNPs of 4mg are distributed to 1mL water and 4mL
In Au-Pt NPs solution, 1mL 3%PSSMA solution (w/v) is added after ultrasound.Reaction 12h is stirred at room temperature, after with ultrapure
Water and 0.01M pH 7.2PB buffer solutions distinguish centrifuge washing 3 times, obtain the sediment of kermesinus, as Au-Pt-SiO2It is compound
Nanoparticle, last constant volume is in 5mL PBS.
As shown in figure 4, the Au-Pt nano-particle patterns of synthesis exactly like " sea urchin shape ", Pt uniform particles it is coated on Au
Rough spherical shape is all presented in sublist face, and uniform in size, good dispersion, all particles.It is surveyed by Nano ZS laser particle analyzers
The hydration grain size obtained is about 30nm.
The Au-Pt-SiO synthesized as shown in Figure 52Composite nano-microsphere good dispersion, and the uniform surfaces SiNPs distributed
Many Au-Pt NPs, Percentage bound are higher.The hydration grain size measured by Nano ZS laser particle analyzers is about 250nm.
(5) synthesis of signal transduction probe
By 100 μ L E.Coli O157:H7 labelled antibodies (80 μ g/mL) are added to above-mentioned 5 mLAu-Pt-SiO2It is compound to receive
In rice microspheres solution, 500 μ L are added with the diluted sucrose enzyme solutions (12mg/mL) of PBS in the gentle agitation 4h under 4 DEG C of environment,
Continue gentle agitation 12h.In this process, antibody and enzyme can be attached to by the Pt-S/Pt-NH keys between albumen and golden platinum
The surfaces APS NPs.After reaction, under 4 DEG C of environment centrifuge with 10000r/min centrifuge 5min, with PB buffer solutions wash 3 times with
On.Obtain Ab2/APS NPs/Invertase.It is finally stored in the 7.2 PB buffer solutions of pH containing 0.5wt%BSA.4
It is preserved under DEG C refrigerator for use.
(6) blood glucose meter combination immune magnetic probe is to escherichia coli O157:The detection method of H7
With blood glucose meter binding signal transduction probe Ab2/ APS NPs/Invertase quickly detect E.coli O157:H7's
Principle is as shown in Figure 6.Specific detecting step is as follows：50 μ L immune magnetics probes (MNPs-IgG) (10mg/mL) are added to 1mL
E.coli O157:In H7 bacteria suspensions after 37 DEG C are incubated 45min, washs 3 times with PBS and remove unbonded E.coli
O157:H7 and other substances, obtain magnetic composite A (MNPs-IgG/E.coli O157:H7 immune complexs).Magnetic Isolation
After add 100 μ L 5mg/mL signal transduction probes Ab2/ APS NPs/Invertase are incubated 30min in 37 DEG C, then use PBS
Thoroughly washing removes unbonded Ab2/ APS NPs/Invertase obtain magnetic composite B (MNPs-IgG/E.coli
O157:H7/Ab2The immune complex of sandwich " sandwich " structures of/APS NPs/Invertase).After Magnetic Isolation, then to body
50 μ L 0.5M sucrose solutions (using 4.5 HAc-NaAc buffer solutions of pH) are added in system, after standing reaction 90min at 55 DEG C
The concentration of glucose is detected with blood glucose meter.
Embodiment 2：Signal transduction probe combination immune magnetic probe immuno-sandwich method quickly detects escherichia coli
O157:The foundation of H7 methods
(1) optimization of immune magnetic probe usage amount
It is respectively 30 μ L, 35 μ L, 40 μ L, 50 μ L and 55 μ L, other experiment conditions and reality to change immune magnetic probe dosage
It is identical to apply example 1.As shown in fig. 7, the concentration for the glucose that sucrose enzyme hydrolysis generates is continuous with the increase of the amount of capture probe
Become larger, signal constantly enhances, and when the amount of addition reaches 50 μ L, concentration of glucose reaches maximum, and the signal of PGM is also most strong.
As amount continues growing, concentration of glucose reduces instead.Therefore, it is 50 μ L to select the amount for the capture probe being added.
(2) invertase and E.Coli O157:H7 labelled antibodies are modified in Au-Pt-SiO2The ratio of composite nano-microsphere
Optimization
The ratio for changing invertase and antibody is respectively 3:1,2:1,1:1,1:2,1:3, other experiment conditions and embodiment 1
It is identical.As shown in figure 8, with the reduction of ratio, sucrose is first increased by the concentration for the glucose that sucrose enzyme hydrolysis generates, and illustrates anti-
Body combines more and more, and the interlayer structure of formation is more secured, and the signal value of generation is also bigger, when ratio is 2:Signal when 1
It is maximum.As the ratio of enzyme and antibody continues to reduce, the concentration for hydrolyzing the glucose of generation is begun to decline, this illustrates signal transduction
The enzyme combined on probe is fewer and fewer, so the glucose generated tails off, signal value is lower, and thereby reduces the sensitive of reaction
Degree.Therefore, it is 2 to select the ratio of enzyme and antibody:1.
(3) optimization of the concentration of signal transduction probe
Under conditions of keeping other factors consistent, it is respectively provided with 40 μ L, 60 μ L, 80 μ L, 100 μ L, the amount of 120 μ L
To detect 108CFU/mL E.Coli O157:H7.The results are shown in Figure 9, increases with the amount of addition, hydrolyzes the Portugal of generation
Grape sugar is more and more, and when the amount of addition is 100 μ L, PGM signal values reach maximum.When the amount of addition is 120 μ L, production
Raw signal value is similar with before, illustrates that the signal transduction probe being added has made sandwich complex reach saturation state.
Therefore, it is cost-effective and reduction blank value, it is 100 μ L to select the amount for the signal transduction probe being added.
Embodiment 3：Sensitivity technique
Under the testing conditions of embodiment 1, the detection method pair 3.5 × 10 is had studied1To 3.5 × 108CFU/mL ranges
Interior E.Coli O157:The testing result of H7.PGM signal values and E.Coli O157:The concentration relationship of H7 is as shown in Figure 10, when
E.Coli O157:The concentration of H7 is 3.5 × 102CFU/mL to 3.5 × 106When CFU/mL changes, concentration of glucose changes difference
With E.Coli O157:Good linear relationship is presented in the logarithm of the concentration of H7.Linear equation is y=5.0724x-
6.6421 coefficient of variation R2=0.9863, and method is to object bacteria E.Coli O157:The detection of H7 is limited to 1.83 × 102 CFU/
ML, the detection are limited than traditional ELISA method (1.0 × 105CFU/mL) want much lower.E.Coli O157 in sample:H7 concentration
It can quantitatively be obtained by equation of linear regression.
Embodiment 4：Specificity experiments
Select E.Coli O157:H7 is as object bacteria, Cronobacter Enterobacter sakazakii (C.sakazakii), Salmonella
Bacterium (S.enteritidis), staphylococcus aureus (S.aureus), vibrio parahemolyticus (V. parahaemolyticus),
Take formula lemon lactic acid bacteria (C.freundii), white diarrhea avian infectious bronchitis nephritis virus (S. pullorum and S.gallinarum)
Bacterium carries out specificity experiments to several frequently seen food-borne pathogens as a contrast, and PBS is as blank control.Experimental result such as Figure 11
It is shown, E.coli O157:H7 groups hydrolysis generate glucose be far longer than other food-borne pathogens groups and PBS groups generation
Glucose.These results prove that this method can effectively distinguish E.Coli O157:H7 and other food-borne pathogens and PBS are right
E.Coli O157:The detection of H7 has good specificity.
Embodiment 5：Stability experiment
By capture probe MNP-IgG and signal transduction probe Ab2/ APS NPs/Invertase are respectively in 4 DEG C of refrigerators
After preserving 1,7,30,60,90d respectively, both probe in detecting 10 are recycled8CFU/mL E. coli O157:H7.Such as Figure 12
It is shown, during trimestral experiment, although the glucose amount that enzymatic hydrolysis generates is declined, but keep relatively stablizing
The state and glucose amount of generation is more.Show MNP-IgG and Ab2Two kinds of nano-probes of/APS NPs/Invertase are at 4 DEG C
It remains to keep good reactivity after lower storage 90d.
The explanation of above example is only intended to help to understand the method for the present invention and its core concept.It should be pointed out that for
For those skilled in the art, without departing from the principle of the present invention, if can also be carried out to the present invention
Dry improvement and modification, these improvement and modification are also fallen into the claims in the present invention protection domain.
Claims (10)
1. a kind of escherichia coli O157 of non-diagnostic purpose:The rapid detection method of H7, which is characterized in that including following step
Suddenly：
(1) with the anti-escherichia coli O157 of mouse:H7 antibody is to Fe3O4/SiO2The magnetic nanoparticle surface of nucleocapsid carries out
Modification, obtains immune magnetic probe；
(2) Au-Pt nanoparticles are modified to the surface nano SiO 2 particle (SiNPs), obtains Au-Pt-SiO2Composite Nano
Microballoon (APS NPs)；
(3) Au-Pt-SiO being prepared with step (2)2Composite nano-microsphere (APS NPs) is carrier combination escherichia coli
Bacterium O157:H7 antibody and invertase obtain signal transduction probe；
(4) sample to be tested is taken, the glucose initial concentration C1 in sample to be tested is detected with blood glucose meter, then prepares step (1)
Obtained immune magnetic probe, which is added in the bacteria suspension of sample to be tested, is incubated certain time in 37 DEG C, washed, Magnetic Isolation,
Magnetic composite A is separated；
(5) be added in the isolated magnetic composite A of step (4) step (3) the signal transduction probe that is prepared and in
37 DEG C of incubation certain times, washed, Magnetic Isolation separate magnetic composite B；
(6) sucrose solution is added in the magnetic composite B that step (5) obtains, reaction is stood at 55 DEG C and is used after a certain period of time
The concentration of glucose C2 of blood glucose meter detection architecture；It is ordinate, escherichia coli according to the variation difference DELTA C of concentration of glucose
O157:The logarithm of H7 concentration is the standard curve of abscissa, can calculate escherichia coli O157 in sample:H7's contains
Amount, wherein Δ C=C2-C1.
2. rapid detection method as described in claim 1, which is characterized in that the magnetic nanoparticle in the step (1)
It is prepared via a method which and obtains：
(1) poly(4-styrene sulfonic acid-co-maleic acid) sodium salt (PSSMA) is dissolved in ethylene glycol, magnetic agitation obtains uniformly
Orange solution；Add Iron(III) chloride hexahydrate and anhydrous sodium acetate；Then, the uniform liquid of obtained rufous is poured into
In hydrothermal reaction kettle, 200 DEG C of reaction 10h；It is to be cooled to after room temperature, by gained black Fe3O4Sediment is used under external magnetic field
Milli-Q water is at least three times；
(2) Fe is taken3O4Solution is placed in conical flask, and ethyl alcohol, deionized water and ammonium hydroxide are added thereto, after mixing, is added
Reaction 18h is stirred at room temperature in tetraethyl orthosilicate (TEOS)；Then ethyl alcohol and distillation is used to wash successively under external magnetic field
It washs repeatedly, the sediment being finally separating to obtain is magnetic nanoparticle.
3. rapid detection method as described in claim 1, which is characterized in that the tool of immune magnetic probe in the step (1)
Preparation step is as follows：Magnetic nanoparticle is taken first and is washed with absolute ethyl alcohol, and the magnetic nanoparticle after washing is resuspended
In ethyl alcohol, 3- aminopropyl triethoxysilanes (APTES) are added, after being stirred to react 12h at room temperature, Magnetic Isolation simultaneously makes respectively
It is washed with ethyl alcohol and phosphate buffer and repeatedly obtains amido modified magnetic nanoparticle；Meanwhile by 1- (3- dimethylaminos
Propyl) -3- ethyl-carbodiimide hydrochlorides (EDC) and n-hydroxysuccinimide (NHS) be added to it is dilute with phosphate buffer
The anti-escherichia coli O157 of mouse released:H7 monoclonals capture in antibody, and 4 DEG C are incubated overnight；It adds and is dissolved in phosphate-buffered
The amido modified magnetic nanoparticle of liquid, room temperature concussion reaction 4h, Magnetic Isolation are simultaneously washed repeatedly with phosphate buffer, are obtained
There is the anti-escherichia coli O157 of mouse to surface modification:The magnetic nanoparticle of H7 antibody.
4. rapid detection method as described in claim 1, which is characterized in that the silica nanometer in the step (2)
Grain is prepared via a method which and obtains：
Tetraethyl orthosilicate is added in ethyl alcohol and obtains A liquid, ammonium hydroxide and ethyl alcohol are added to the water to obtain B liquid, then A liquid is fast
It is added to fastly in B liquid, reacts 5h under room temperature；After, it centrifuges, is washed respectively with absolute ethyl alcohol and distilled water more
It is secondary.
5. rapid detection method as described in claim 1, which is characterized in that the Au-Pt-SiO in the step (2)2It is compound to receive
Meter Wei Qiu is prepared via a method which and obtains：
(1) chlorauric acid solution is added in ultra-pure water, oil bath heating is to boiling, then rapidly joins sodium citrate solution, passes through
After a few minutes, solution reddens, and shows to have generated colloidal gold；Then, platinum acid chloride solution is added, then is rapidly added ascorbic acid
Solution is stirred continuously, and reaction 20min postcoolings obtain Au-Pt nanoparticles to room temperature；
(2) it takes nano SiO 2 particle to be distributed to absolute ethyl alcohol after ultrasound, 3- aminopropyl triethoxysilanes is added, in room
It is stirred to react under temperature overnight, centrifuges after reaction, use ethyl alcohol and milli-Q water respectively, obtain amidized titanium dioxide
Nano silicon particles；Amidized nano SiO 2 particle is distributed in the aqueous solution of Au-Pt nanoparticles, is added after ultrasound
Reaction 12h is stirred at room temperature in poly(4-styrene sulfonic acid-co-maleic acid) sodium salt solution, after it is slow with ultra-pure water and phosphate
Fliud flushing distinguishes centrifuge washing 3 times, obtains the sediment of kermesinus, as Au-Pt-SiO2Composite nano-microsphere.
6. rapid detection method as described in claim 1, which is characterized in that in the above-mentioned methods, sucrose in the step (3)
Enzyme and escherichia coli O157:The ratio of H7 antibody is preferably 2:1.
7. rapid detection method as described in claim 1, which is characterized in that the tool of signal transduction probe in the step (3)
Preparation step is as follows：By escherichia coli O157:H7 labelled antibodies are added to Au-Pt-SiO2Composite nano-microsphere solution
In, the gentle agitation 4h under 4 DEG C of environment, the diluted sucrose enzyme solutions of addition phosphate buffer continue gentle agitation 12h,
After reaction, it centrifuges, washing obtains signal transduction probe：
8. rapid detection method as described in claim 1, which is characterized in that immune magnetic probe is dense in the step (4)
Degree is 10mg/mL, and volume is 50 μ L, incubation time 45min.
9. rapid detection method as described in claim 1, which is characterized in that signal transduction concentration and probe concentration in the step (5)
For 5mg/mL, volume is 100 μ L, and incubation time is preferably 30min.
10. rapid detection method as described in claim 1, which is characterized in that the linear side of step (6) standard curve
Journey is y=5.0724x-6.6421, and x is the O157 of escherichia coli in sample:H7 concentration denary logarithm values, unit
It is variation the difference DELTA C, unit mmol/L of concentration of glucose for CFU/mL, y.
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