CN108362872A - A kind of quantitative detecting method of the white diarrhea and avian infectious bronchitis nephritis virus of non-diagnostic purpose - Google Patents
A kind of quantitative detecting method of the white diarrhea and avian infectious bronchitis nephritis virus of non-diagnostic purpose Download PDFInfo
- Publication number
- CN108362872A CN108362872A CN201810022503.7A CN201810022503A CN108362872A CN 108362872 A CN108362872 A CN 108362872A CN 201810022503 A CN201810022503 A CN 201810022503A CN 108362872 A CN108362872 A CN 108362872A
- Authority
- CN
- China
- Prior art keywords
- added
- infectious bronchitis
- avian infectious
- magnetic
- nephritis virus
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- 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
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
- G01N33/54346—Nanoparticles
-
- 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
Abstract
The invention discloses a kind of rapid detection methods of the white diarrhea and avian infectious bronchitis nephritis virus of non-diagnostic purpose.The detection method of the present invention prepares the coupled to Nano magnetic bead (IgG MNPs) for being marked with S. pullonum and avian infectious bronchitis nephritis virus antibody and antibody and the silicon dioxide nanosphere (GOx IgG SiNPs) of enzyme double labelling first, when there are purpose bacterium to be measured, form the immune complex IgG MNPs/S.pullorum and S.gallinarum GOx IgG SiNPs of sandwich " sandwich " structure, then pass through enzymic catalytic reaction, reaction product forms complexing product with color developing agent, the concentration of white diarrhea and avian infectious bronchitis nephritis virus is obtained finally by the absorbance value of detection architecture, to realize the quantitative detection of S. pullonum and avian infectious bronchitis nephritis virus.The cascade reaction combination Fe that the present invention is triggered with glucose oxidase (GOx)3+‑SCN‑Color Appearance System realizes the quantitative detection of S. pullonum and avian infectious bronchitis nephritis virus, and the detection method is convenient and efficient, easy to operate, has good sensitivity, specificity and stability.
Description
Technical field
The invention belongs to food and raw-food material safety detection technology field, more particularly to the one of food and raw-food material
The quantitative detecting method of the white diarrhea and avian infectious bronchitis nephritis virus of the non-diagnostic purpose of kind.
Background technology
S. pullonum (Salmonella pullorum, S.pullorum) and avian infectious bronchitis nephritis virus
(Salmonella gallinarum, S.gallinarum) is white diarrhea (Pullorum Disease, PD) and fowl typhoid
The pathogen of (Fowl typhoid, FT), brings about great losses, and seriously threaten the safety of food to poultry farming.By
In S. pullonum and avian infectious bronchitis nephritis virus O1 having the same, O9, O12 antigens, it is stringent both distinguish it is highly difficult and
Also it is not necessarily in practice examining, thus the two detects typically together.To S. pullonum and avian infectious bronchitis nephritis virus
Detection it is meaningful for prevention and control food safety risk.
Detection method to pathogenic bacteria includes standard method and a variety of rapid detection methods, such as digital category judgement method, base
In immunolabelling technique and multiclass detection method based on molecular biology.Existing method has their own advantages and deficiency, therefore, uses
Multidisciplinary new knowledge, new material and new technology mixing together build the certainty that better Fast Detection Technique is development and application
Trend.
Chen etc. reports a kind of glucose oxidase catalysis glucose solution generation hydrogen peroxide, with PSS-GN-Pt
Analogies of the nanocomposite as peroxidase are catalyzed tetramethyl biphenyl colour developing with this, and then realize to glucose
Colorimetric estimation.This provides new approaches for the new colorimetric system of our design constructions, and glucose oxidase is catalyzed glucose and generates
H2O2, and H2O2Catalysis oxidation and chromogenic reaction can be carried out under the assistance of horseradish peroxidase (HRP).Ferric ion
(Fe3+) classical reaction be exactly itself and thiocyanate anions (SCN-) chromogenic reaction, generate macroscopic red.Cause
This, this reaction is usually applied to Fe3+Measurement.In addition, in acidic environment, Fe2+It can be oxidized to Fe3+.Therefore,
The cascade reaction combination Fe of glucose oxidase triggering can be utilized3+-SCN-Color Appearance System establishes novel immune analysis method.
To sum up, the present invention is directed to the defect of existing S. pullonum and avian infectious bronchitis nephritis virus detection technique, exploitation
Go out a kind of cascade reaction system combination Fe triggered based on glucose oxidase3+-SCN-Colorimetric method is immunized in the magnetic control of Color Appearance System,
Object bacteria S. pullonum to be measured and avian infectious bronchitis nephritis virus are detached and be enriched with IgG-MNPs, GOx-IgG-
SiNPs is as signal designation object；Build IgG-MNPs-S. pullorum and S.gallinarum-GOx-IgG-SiNPs folders
Core structure can be catalyzed glucose with GOx and generate H2O2, H2O2Oxidation of Fe2+Become Fe3+, Fe3+It can quickly and SCN-It is complexed
Reaction generates red product, by measuring the absorbance of red product, to realize white diarrhea Salmonella in quantitative determination food
The concentration of bacterium and avian infectious bronchitis nephritis virus.
Invention content
The purpose of the present invention is to provide the quantitative detections of a kind of white diarrhea of non-diagnostic purpose and avian infectious bronchitis nephritis virus
Method.The cascade reaction combination Fe that the present invention is triggered with GOx3+-SCN-Color Appearance System is realized to white diarrhea Salmonella in food
The quantitative detection of bacterium and avian infectious bronchitis nephritis virus, the detection method is convenient and efficient, easy to operate, with good sensitivity, specifically
Property and stability.
To achieve the above object, the present invention takes following technical scheme：
A kind of quantitative detecting method of the white diarrhea and avian infectious bronchitis nephritis virus of non-diagnostic purpose, includes the following steps：
(1) by white diarrhea and avian infectious bronchitis nephritis virus antibody (Anti-S.pullorum and S.gallinarum) and
Glucose oxidase (GOx) is covalently bound to the surface of silicon dioxide nanosphere (SiNPs), forms enzyme marker (GOx-
IgG-SiNPs), spare；
(2) use white diarrhea and avian infectious bronchitis nephritis virus antibody (Anti-S.pullorum and S.gallinarum) right
Fe3O4/SiO2The magnetic nanoparticle surface of nucleocapsid is modified, and antibody coupling nanometer magnetic bead (IgG-MNPs) is obtained,
It is spare；
(3) the antibody coupling nanometer magnetic bead (IgG-MNPs) that step (2) is prepared is added to sample to be tested suspension
In in 37 DEG C be incubated certain times, washed, Magnetic Isolation obtains having magnetic immune complex A (IgG-MNPs-
S.pullorum and S.gallinarum)；
(4) immune complex (the IgG-MNPs-S.pullorum and being prepared in step (3)
S.gallinarum the enzyme marker (GOx-IgG-SiNPs) that step (1) is prepared is added in) and is incubated in 37 DEG C certain
Time, washed, Magnetic Isolation obtain having magnetic immune complex B (IgG-MNPs-S.pullorum and
S.gallinarum-GOx-IgG-SiNPs)；
(5) glucose is added in the immune complex B that step (4) obtains and carries out enzymic catalytic reaction；
(6) after the completion of enzymic catalytic reaction, colour reagent KSCN solution is added, using microplate reader determination sample in wavelength
Absorbance value at 466nm is ordinate according to absorbance value, and the logarithm of white diarrhea and avian infectious bronchitis nephritis virus concentration is cross
White diarrhea and avian infectious bronchitis nephritis virus content can be calculated in the standard curve of coordinate.
In the above-mentioned methods, the silicon dioxide nanosphere in the step (1) is prepared via a method which to obtain：
Triton X-100, n-hexyl alcohol, hexamethylene are mixed evenly, ultra-pure water is added later and stirs evenly to form W/
O-shaped microemulsion, adds tetraethyl orthosilicate, after being stirred to react, ammonium hydroxide is added, hydrolysis-condensation reaction occurs as catalyst, then
Through demulsification, centrifugation, washing step, product silicon dioxide nanosphere is obtained.
In the above-mentioned methods, the specific preparation process of enzyme marker is as follows in the step (1)：
(1) silane coupling agent 3- aminopropyls three will in silicon dioxide nanosphere ultrasonic disperse to absolute ethyl alcohol, be added
Ethoxysilane (APTES), reacts 12h at ambient temperature, with absolute ethyl alcohol and phosphate buffer (PBS) wash respectively to
Less three times, the silicon dioxide nanosphere of amino chemical modification is obtained；
(2) by the silicon dioxide nanosphere ultrasonic disperse to phosphate buffer of amino chemical modification, penta 2 are added
Aldehyde solution, is protected from light and is stirred to react 3h, is washed repeatedly with phosphate buffer, obtains aldehyde radical improved silica nanoparticle；Again
White diarrhea and avian infectious bronchitis nephritis virus antibody is added, is stirred to react 3h under room temperature, extremely with phosphate buffer centrifuge washing
It is resuspended in phosphate buffer after 3 times few, glucose oxidase (GOx) is added and is stirred to react, obtains enzyme marker (GOx-
IgG-SiNPs it) precipitates, bovine serum albumin(BSA) (BSA) is added and is stirred to react with sealase mark with GOx-IgG-SiNPs precipitation room temperatures
The nonspecific binding site for remembering object (GOx-IgG-SiNPs) surface residual, obtains enzyme marker.
In the above-mentioned methods, the Fe in the step (2)3O4/SiO2The magnetic nanoparticle of nucleocapsid passes through such as lower section
Method is prepared：
(1) Fe will be contained3+Solution be added in the deionized water of advance deoxygenation, be added FeSO4·7H2O is waited for
State FeSO4·7H2Ammonium hydroxide is rapidly joined after the dissolving of O solids and is vigorously stirred, N2Under protective condition, 80 DEG C are vigorously stirred reaction
60min is fully washed with deionized water after liquid cooling, obtains Fe3O4Magnetic nanoparticle；
(2) Fe is taken3O4Magnetic nanoparticle is added to absolute ethyl alcohol, deionized water and ammonium hydroxide in the mixed solvent, is vigorously stirred
Afterwards, tetraethyl orthosilicate is added, is stirred to react for 24 hours in 40 DEG C of waters bath with thermostatic control, obtains magnetic nanoparticle (MNPs).
In the above-mentioned methods, the antibody coupling nanometer magnetic bead (IgG-MNPs) in the step (2) is made by the following method
It is standby to obtain：
(1) magnetic nanoparticle (MNPs) is distributed in absolute ethyl alcohol, adds three second of silane coupling agent 3- aminopropyls
Oxysilane is washed three times or more with absolute ethyl alcohol and phosphate buffer, is obtained after reacting 12h at ambient temperature respectively
Amino modified MNPs；
(2) amino modified magnetic nanoparticle is resuspended into phosphate buffer, glutaraldehyde solution is added, is protected from light and stirs
It after mixing reaction 6h, is fully washed with phosphate buffer repeatedly, obtains aldehyde radical and be modified MNPs；Add S. pullonum and
Avian infectious bronchitis nephritis virus antibody is stirred to react 3h under room temperature, then bovine serum albumin(BSA) (BSA) and antibody is added in washing
Coupled to Nano magnetic bead precipitation room temperature is stirred to react to close the surfaces IgG-MiNPs nonspecific binding site, obtains antibody coupling
Nanometer magnetic bead (IgG-MNPs).
In the above-mentioned methods, the concentration of the antibody coupling nanometer magnetic bead in the step (3) is preferably 6 mg/mL, is incubated
Time is preferably 45min.
In the above-mentioned methods, the concentration of the enzyme marker in the step (4) is preferably 20mg/mL, and incubation time is preferred
For 30min.
In the above-mentioned methods, the glucose in the step (5) dissolves to obtain a concentration of with the tris-HCl of pH=7.0
The solution form of 50mM is added.
In the above-mentioned methods, the colour reagent in the step (6) is to contain 4.5 mM with what 0.2M HCl were prepared
FeSO4With the solution of 0.3mM KSCN.
In the above-mentioned methods, the linear equation of step (6) standard curve is y=0.1992x-0.31102, x
It is S. pullonum and avian infectious bronchitis nephritis virus concentration denary logarithm value, unit CFU/mL, y are absorbances
Value.
The cascade reaction combination Fe that the present invention is triggered with glucose oxidase (GOx)3+-SCN-It is white that Color Appearance System realizes chicken
The quantitative detection of dysentery salmonella and avian infectious bronchitis nephritis virus.To build this detection platform, it is prepared for two kinds of nanometers first and visits
Needle includes the antibody coupling nanometer magnetic bead (IgG-MNPs) and label for being marked with S. pullonum and avian infectious bronchitis nephritis virus
There are enzyme marker (GOx-IgG-SiNPs), wherein IgG-MNPs to be used for purpose bacterium S. pullonum as capture probe
It is enriched with the fast Acquisition of avian infectious bronchitis nephritis virus, GOx-IgG-SiNPs is used for signal transduction as detection probe.It is waited for when existing
When surveying purpose bacterium, the immune of IgG-MNPs S.pullorum S.gallinarum-GOx-IgG-SiNPs interlayer structures is formed
Compound B, the GOx that the GOx-IgG-SiNPs in interlayer structure is carried can be catalyzed glucose solution generate gluconic acid and
H2O2, the H of generation2O2And then it can be with Oxidation of Fe2+As Fe3+, and Fe3+Can be quickly and SCN-Complex reaction occurs, is formed red
The complexing product of color.The concentration of the absorbance value and purpose bacterium S. pullonum and avian infectious bronchitis nephritis virus of the color products
Logarithm present linearly related, can quantitatively obtain S. pullonum and chicken in food by measuring absorbance value whereby and hinder
The concentration of cold salmonella.
The present invention has following technical characterstic：
1) the cascade reaction combination Fe that the present invention is triggered with glucose oxidase3+-SCN-Color Appearance System realizes in food
The quantitative detection of S. pullonum and avian infectious bronchitis nephritis virus, the detection method are convenient, fast, economical, easy to operate.
2) present invention has good sensitivity, specificity and stability.
3) of the invention based on Fe3+-SCN-The immune sensing system of Color Appearance System can be by changing target antibody into one
Expanded application is walked in other pathogenic bacteria of detection.
Description of the drawings
Fig. 1 a antibody coupling nanometer magnetic bead preparation process schematic diagrames；
Fig. 1 b enzyme marker preparation process schematic diagrames；
The cascade reaction combination Fe of Fig. 1 c GOx triggerings3+-SCN-Color Appearance System quantitative determines white diarrhea fowl typhoid Salmonella
Bacterium Method And Principle schematic diagram；
The relational graph of 2 absorbance value of Fig. 2 embodiments and antibody coupling nanometer magnetic bead concentration；
The relational graph of Fig. 3 embodiments 2 absorbance value and enzyme marker concentration；
The relational graph of Fig. 4 embodiments 3 absorbance value and white diarrhea and avian infectious bronchitis nephritis virus concentration；
4 specificity experiments of Fig. 5 embodiments：The relational graph of absorbance value and bacterial species；
5 stability experiment of Fig. 6 embodiments：The relational graph of absorbance value and storage time.
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) synthesis of silica nanometer ball (SiNPs)：
SiNPs is synthesized using reverse microemulsion process.Specific building-up process is as follows：2mL Triton X-100 (are lived on surface
Property agent), 2mL n-hexyl alcohols (cosurfactant), 8mL hexamethylenes (organic solvent) stir 20min to clear shape, are added
For 480 μ L ultra-pure waters as dispersed phase, stirring 20min makes water droplet is fully dispersed in form W/ that is uniform and stablizing in above-mentioned oil phase
O-shaped microemulsion adds precursor substance of the 100 μ L tetraethyl orthosilicates (TEOS) as synthesis SiNPs, is stirred to react 30min
100 μ L ammonium hydroxide (catalyst) are added in above-mentioned system liquid afterwards to promote TEOS to form nanometer by hydrolysis-condensation reaction faster
Microballoon.10mL acetone is added after magnetic agitation reaction 48h to be demulsified to microemulsion, collect and sink after 10000 rpm centrifugations 5min
It forms sediment, then distinguishes centrifuge washing 3 times with acetone, absolute ethyl alcohol and ultra-pure water.Gained SiNPs is scattered in ultra-pure water, room temperature condition
Under save backup.
(2) synthesis of enzyme marker (GOx-IgG-SiNPs)：
The preparation principle of GOx-IgG-SiNPs is as shown in Figure 1 b.The 30 mg SiNPs for taking above-mentioned reverse microemulsion process to prepare
With absolute ethyl alcohol centrifuge washing three times, then it will be made to keep its fully dispersed into 10mL absolute ethyl alcohols after precipitation ultrasound 30min, then
200 μ L silane coupling agent 3- aminopropyl triethoxysilanes (APTES) are added, after reacting 12h at ambient temperature, APTES is logical
Crossing hydrolysis-condensation reaction makes amino be introduced into the surfaces SiNPs, with absolute ethyl alcohol and 0.01M phosphate buffers (PBS, pH
7.4) centrifuge washing three times respectively, obtain the SiNPs (SiNPs-NH of amino chemical modification2)。
By SiNPs-NH2In ultrasonic disperse to 10mL PBS, 2.5% glutaraldehyde solutions of 5mL are added, is protected from light and is stirred to react
After 3h, be modified SiNPs with aldehyde radical is obtained after PBS centrifuge washings 6 times, then be resuspended with 4mL PBS, be added 40 μ L white diarrhea and
Avian infectious bronchitis nephritis virus antibody (anti-S.pullorum and S. gallinarum), is stirred to react 3h under room temperature, with
PBS 10000rpm centrifuge washings are resuspended in 4mL PBS for 3 times, and 500 μ L 6mg/mL GOx are added and are stirred to react 6h in 4 DEG C, with
PBS washings obtain GOx-IgG-SiNPs after removing unbonded GOx molecules for several times.GOx-IgG-SiNPs is resuspended with 4mL PBS
It precipitates and 1mL 1%BSA is added and reaction 1.5h is stirred at room temperature to close the non-specific binding of GOx-IgG-SiNPs surface residuals
Site.To be resuspended into 2mL PBS after PBS centrifuge washings 3 times, saved backup in 4 DEG C of refrigerators.
(3) Fe3O4The synthesis of magnetic nanoparticle：
Method according to the literature is modified slightly, and utilizes coprecipitation synthesis Fe3O4Magnetic nanoparticle[.It will
2.7mL 1M Fe3+Solution is added to 70mL in the deionized water of advance deoxygenation, and 0.375g FeSO are added4·7H2O is waited for
Above-mentioned FeSO4·7H25mL ammonium hydroxide is rapidly joined after the dissolving of O solids and is vigorously stirred, oxygen is extracted with vacuum pump and is passed through high-purity
Nitrogen creates oxygen-free environment, N2Under protective condition, 80 DEG C are vigorously stirred reaction 60min, after liquid cooling, are filled with deionized water
Divide washing, obtains Fe3O4Magnetic nanoparticle, and be resuspended into the deionized water of 60mL deoxygenations, 4 DEG C of refrigerators save backup.
(4) synthesis of magnetic nanoparticle (MNPs)：
SiO2It coats to Fe3O4The process on magnetic nanoparticle surface is with reference to classicalMethod, and carried out a little improvement.
Specific cladding process is as follows：Take the above-mentioned Fe of 1mL3O4Magnetic nanoparticle is added to 60mL absolute ethyl alcohols, 10mL deionized waters and
In 9mL ammonium hydroxide, after being vigorously stirred 20min, 1mL TEOS is added, is stirred to react for 24 hours in 40 DEG C of waters bath with thermostatic control, is washed with deionization
It washs for several times, obtains MNPs, be resuspended into 500 μ L deionized waters, saved backup in 4 DEG C of refrigerators.
(5) synthesis of antibody coupling nanometer magnetic bead (IgG-MNPs)：
IgG-MNPs preparation principles are as shown in Figure 1a.The MNPs of the 500 above-mentioned synthesis of μ L is taken to be washed three times with absolute ethyl alcohol, then
Keep its fully dispersed into 10mL absolute ethyl alcohols, adds 200 μ L silane coupling A PTES, react 12h at ambient temperature
Afterwards, APTES makes amino be introduced into the surfaces MNPs by hydrolysis-condensation reaction, with absolute ethyl alcohol and 0.01M phosphate-buffereds
Liquid (PBS, pH 7.4) washs three times respectively, obtains amino modified MNPs (MNPs-NH2)。
By MNPs-NH2It is resuspended into 10mL PBS, 2.5% glutaraldehyde solutions of 5mL is added, are protected from light after being stirred to react 6h,
Aldehyde radical is obtained after fully washing 6 times with PBS and is modified MNPs, then sediment is resuspended with 4mL PBS, and the anti-of 40 μ L is added
S.pullorum and S.gallinarum are stirred to react 3h under room temperature, are washed 3 times with PBS, obtain IgG-MNPs.With
4mL PBS, which are resuspended IgG-MNPs and 1mL 1%BSA are added, is stirred at room temperature reaction 1.5h to close the surfaces IgG-MNPs non-specificity
Binding site.It is resuspended into 500 μ L PBS after being washed 3 times with PBS, is saved backup in 4 DEG C of refrigerators.
(6) GOx triggers enzymatic cascade reaction combination Fe3+-SCN-Color Appearance System is to white diarrhea and avian infectious bronchitis nephritis virus
Detecting step：
Utilize Fe3+-SCN-Color Appearance System detects white diarrhea and avian infectious bronchitis nephritis virus (S.pullorum and S.
Gallinarum) testing principle is as illustrated in figure 1 c.Specific detecting step is as follows：The antibody coupling of a concentration of 6mg/mL of 20 μ L
The solution of nanometer magnetic bead IgG-MNPs is added in 1mL white diarrheas and avian infectious bronchitis nephritis virus suspension and is incubated 45min in 37 DEG C
Afterwards, 3 times are washed with PBS and removes unbonded white diarrhea and avian infectious bronchitis nephritis virus and other substances, Magnetic Isolation obtains IgG-
MNPs-S.pullorum-and- S.gallinarum immune complexs A.Immune complex A after 20 μ L PBS are resuspended, then
The enzyme marker GOx-IgG-SiNPs that 20 a concentration of 20mg/mL of μ L are added is incubated 30min in 37 DEG C, then is thoroughly washed with PBS
Unbonded GOx-IgG-SiNPs is removed, Magnetic Isolation obtains IgG-MNPs-S. pullorum-and-S.gallinarum-
50 μ L 50mM glucose are added (with pH7.0tris- in the immune complex B of sandwich " sandwich " structures of GOx-IgG-SiNPs
HCl dissolves) after 37 DEG C carry out enzymic catalytic reaction 1min, isometric colour reagent is successively added and (is prepared with 0.2M HCl
4.5mM FeSO4, 3%KSCN solution), in measuring its absorbance value at wavelength 466nm in microplate reader within 5min.It is all
Measurement room temperature carry out (25 ± 1.0 DEG C).
Embodiment 2：GOx triggers enzymatic cascade reaction combination Fe3+-SCN-Color Appearance System is to white diarrhea and fowl typhoid Salmonella
The foundation of bacterium quantitative detecting method
(1) optimization of antibody coupling nanometer magnetic bead concentration
The concentration for changing antibody coupling nanometer magnetic bead IgG-MNPs is respectively：2.4mg/mL, 3mg/mL, 4mg/mL, 6mg/
ML, 12mg/mL, 24mg/mL, 48mg/mL, other experiment conditions are same as Example 1, quantitatively detect 1mL 107CFU·mL-1S.pullorum and S.gallinarum bacterium samples to be measured.As shown in Figure 2, as IgG-MNPs concentration increases, extinction
Downward trend is presented after keeping one section of plateau after progressivelying reach maximum value in angle value, is accordingly used in the IgG-MNPs of analysis test
Optium concentration is 6mg/mL.
(2) optimization of enzyme marker concentration
The concentration for changing enzyme marker GOx-IgG-SiNPs is respectively：2mg/mL, 2.5mg/mL, 3.3 mg/mL, 5mg/
ML, 10mg/mL, 20mg/mL, 40mg/mL, other experiment conditions are same as Example 1, quantitatively detect 1mL 107CFU·mL-1S.pullorum and S.gallinarum bacterium samples to be measured.From the figure 3, it may be seen that as enzyme marker concentration increases, extinction
Angle value kept after progressivelying reach maximum value stablize, therefore select for analyze test GOx-IgG-SiNPs optium concentrations for
20mg/mL。
Embodiment 3：Sensitivity technique
8.4 × 10 are detected under conditions of embodiment 10CFU·mL-1To 8.4 × 107CFU·mL-1White diarrhea and chicken wound
Salmonella (S.pullorum and S.gallinarum) tremble with fear to determine sensitivity.Absorbance value and white diarrhea and fowl typhoid
The concentration relationship of salmonella as shown in figure 4, when S.pullorum and S. gallinarum concentration 8.4 ×
103CFU·mL-1To 8.4 × 107CFU·mL-1When variation, absorbance value and S.pullorum and S.gallinarum concentration
Logarithm good linear relationship is presented.Linear equation is y=0.1992x-0.31102, coefficient of variation R2=0.98706.
This method is limited to 2.36 × 10 to the detection of purpose bacterium S.pullorum and S.gallinarum3CFU·mL-1。
Embodiment 4：Specific detection
Good specificity is particularly important in immunoassay, therefore has investigated the new method for building up and detected chicken for analysis
The specific effect of dysentery characterized by white mucous stool and avian infectious bronchitis nephritis virus (S.pullorum and S.gallinarum).In the condition of embodiment 1
It is lower to detect 10 respectively7CFU·mL-1S.pullorum and S.gallinarum), bacillus ceylonensis A
(Sh.sonnei), escherichia coli (E.coli), salmonella typhimurium (S. typhimurium), takes formula lemon lactic acid
Bacterium (C.freundii) and PBS are as blank control.Experimental result such as Fig. 5 shows, only S.pullorum and
S.gallinarum causes apparent color change and absorbance value to be significantly larger than other control groups, this shows that this method can be special
Property effectively detection is realized to S.pullorum and S. gallinarum.
Embodiment 5：Stability experiment
By GOx-IgG-SiNPs after preserving 1,7,30,60,90,120d respectively in 4 DEG C of refrigerators, the probe is recycled to examine
Survey 106CFU·mL-1S.pullorum and S.gallinarum.The experimental results showed that 7 are stored in GOx-IgG-SiNPs,
After 30,60,90,120d, signal strength values become the 94.29% of initial signal value respectively, and 97.77%, 99.98%,
62.32% and 61.37%, this shows that the bioactivity of enzyme marker is not decreased obviously, and also still preserves higher reaction
Activity stores and still can be used for analyzing detection S.pullorum and S.gallinarum within 60d, without having obviously
Signal weaken.
Embodiment 6：White diarrhea and avian infectious bronchitis nephritis virus example are detected in food
Actual sample is simulated with chicken gizzard.Chicken gizzard is purchased from supermarket, and machine homogeneous is patted into homogenate with sterile homogeneous.It will
S.pullorum and S.gallinarum are added in the chicken gizzard suspension of preparation, and S.pullorum and are respectively prepared
S.gallinarum final concentration of 8.4 × 103CFU·mL-1,8.4×105CFU·mL-1With 8.4 × 107 CFU·mL-1Reality
Border sample, every part of three groups of sample setting are parallel.Testing result is as shown in table 1, and newly-established method is detected with National Standard Method
S.pullorum and S.gallinarum result relative errors are no more than 17.14%, therefore the method that this research is established is steady
Fixed, accuracy is good, can be used for detecting actual sample detection S.pullorum and S.gallinarum.
The result of the method detection mark-on chicken gizzard sample of 1 present invention of table is compared with National Standard Method
The average value of a detections three times
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 claim protection domain of the present invention.
Claims (10)
1. a kind of quantitative detecting method of the white diarrhea and avian infectious bronchitis nephritis virus of non-diagnostic purpose, which is characterized in that including with
Lower step：
(1) white diarrhea and avian infectious bronchitis nephritis virus antibody and glucose oxidase are covalently bound to silicon dioxide nanosphere
Surface forms enzyme marker, spare；
(2) use white diarrhea and avian infectious bronchitis nephritis virus antibody to Fe3O4/SiO2The magnetic nanoparticle surface of nucleocapsid carries out
Modification, obtains antibody coupling nanometer magnetic bead, spare；
(3) the antibody coupling nanometer magnetic bead that step (2) is prepared is added in sample to be tested suspension and is incubated one in 37 DEG C
It fixes time, washed, Magnetic Isolation obtains having magnetic immune complex A；
(4) enzyme marker that step (1) is prepared is added in the immune complex that step (3) is prepared and is incubated in 37 DEG C
It educates certain time, washed, Magnetic Isolation, obtains having magnetic immune complex B；
(5) glucose is added in the immune complex B that step (4) obtains and carries out enzymic catalytic reaction；
(6) after the completion of enzymic catalytic reaction, colour reagent KSCN solution is added, using microplate reader determination sample at wavelength 466nm
Absorbance value, be ordinate according to absorbance value, the logarithm of white diarrhea and avian infectious bronchitis nephritis virus concentration is abscissa
White diarrhea and avian infectious bronchitis nephritis virus content can be calculated in standard curve.
2. quantitative detecting method as described in claim 1, which is characterized in that the silica nanometer in the step (1)
Microballoon is prepared via a method which to obtain：
Triton X-100, n-hexyl alcohol, hexamethylene are mixed evenly, ultra-pure water is added later and stirs evenly to form w/o type
Microemulsion adds tetraethyl orthosilicate, after being stirred to react, ammonium hydroxide is added, hydrolysis-condensation reaction occurs as catalyst, then pass through
Demulsification, centrifugation, washing step, obtain product silicon dioxide nanosphere.
3. quantitative detecting method as described in claim 1, which is characterized in that the specific system of enzyme marker in the step (1)
It is standby that steps are as follows：
(1) three ethoxy of silane coupling agent 3- aminopropyls will in silicon dioxide nanosphere ultrasonic disperse to absolute ethyl alcohol, be added
Base silane reacts 12h at ambient temperature, is washed respectively at least three times with absolute ethyl alcohol and phosphate buffer, obtains amino
The silicon dioxide nanosphere of chemical modification；
(2) by the silicon dioxide nanosphere ultrasonic disperse to phosphate buffer of amino chemical modification, it is molten that glutaraldehyde is added
Liquid is protected from light and is stirred to react 3h, is washed repeatedly with phosphate buffer, obtains aldehyde radical improved silica nanoparticle；It adds
White diarrhea and avian infectious bronchitis nephritis virus antibody, are stirred to react 3h under room temperature, with phosphate buffer centrifuge washing at least 3 times
After be resuspended in phosphate buffer, be added glucose oxidase be stirred to react, obtain enzyme marker precipitation, be added ox blood it is pure
Albumen is stirred to react with enzyme marker precipitation room temperature to close the nonspecific binding site of enzyme marker surface residual, obtains enzyme
Marker.
4. quantitative detecting method as described in claim 1, which is characterized in that the Fe in the step (2)3O4/SiO2Nucleocapsid knot
The magnetic nanoparticle of structure is prepared via a method which to obtain：
(1) Fe will be contained3+Solution be added in the deionized water of advance deoxygenation, be added FeSO4·7H2O is waited for above-mentioned
FeSO4·7H2Ammonium hydroxide is rapidly joined after the dissolving of O solids and is vigorously stirred, N2Under protective condition, 80 DEG C are vigorously stirred reaction
60min is fully washed with deionized water after liquid cooling, obtains Fe3O4Magnetic nanoparticle；
(2) Fe is taken3O4Magnetic nanoparticle is added to absolute ethyl alcohol, deionized water and ammonium hydroxide in the mixed solvent, after being vigorously stirred,
Tetraethyl orthosilicate is added, is stirred to react for 24 hours in 40 DEG C of waters bath with thermostatic control, obtains magnetic nanoparticle.
5. quantitative detecting method as described in claim 1, which is characterized in that the antibody coupling nano magnetic in the step (2)
Pearl is prepared via a method which to obtain：
(1) magnetic nanoparticle is distributed in absolute ethyl alcohol, adds silane coupling agent 3- aminopropyl triethoxysilanes,
After reacting 12h under room temperature, is washed respectively three times or more with absolute ethyl alcohol and phosphate buffer, obtain amino modified magnetic
Property nano particle；
(2) amino modified magnetic nanoparticle is resuspended into phosphate buffer, glutaraldehyde solution is added, it is anti-to be protected from light stirring
After answering 6h, is fully washed with phosphate buffer repeatedly, obtain aldehyde radical modified magnetic nano particle；Add white diarrhea Salmonella
Bacterium and avian infectious bronchitis nephritis virus antibody, are stirred to react 3h under room temperature, washing, and it is even with antibody that bovine serum albumin(BSA) is then added
Connection nanometer magnetic bead precipitation room temperature is stirred to react with blocking antibody coupled to Nano magnetic bead surfaces nonspecific binding site, obtains antibody
Coupled to Nano magnetic bead.
6. quantitative detecting method as described in claim 1, which is characterized in that the antibody coupling nano magnetic in the step (3)
The concentration of pearl is preferably 6mg/mL, and incubation time is preferably 45min.
7. quantitative detecting method as described in claim 1, which is characterized in that the concentration of the enzyme marker in the step (4)
Preferably 20mg/mL, incubation time are preferably 30min.
8. quantitative detecting method as described in claim 1, which is characterized in that the glucose in the step (5) is with pH=7.0
Tris-HCl dissolve to obtain the solution form of a concentration of 50mM and be added.
9. quantitative detecting method as described in claim 1, which is characterized in that colour reagent in the step (6) be with
What 0.2M HCl were prepared contains 4.5mM FeSO4With the solution of 3wt%KSCN.
10. quantitative detecting method as described in claim 1, which is characterized in that the linear side of step (6) standard curve
Formula is y=0.1992x-0.31102, and x is S. pullonum and avian infectious bronchitis nephritis virus concentration denary logarithm
Value, unit CFU/mL, y are absorbance values.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810022503.7A CN108362872A (en) | 2018-01-10 | 2018-01-10 | A kind of quantitative detecting method of the white diarrhea and avian infectious bronchitis nephritis virus of non-diagnostic purpose |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810022503.7A CN108362872A (en) | 2018-01-10 | 2018-01-10 | A kind of quantitative detecting method of the white diarrhea and avian infectious bronchitis nephritis virus of non-diagnostic purpose |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN108362872A true CN108362872A (en) | 2018-08-03 |
Family
ID=63011326
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810022503.7A Pending CN108362872A (en) | 2018-01-10 | 2018-01-10 | A kind of quantitative detecting method of the white diarrhea and avian infectious bronchitis nephritis virus of non-diagnostic purpose |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN108362872A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109988810A (en) * | 2019-03-11 | 2019-07-09 | 上海交通大学 | A kind of fast-bacteria-detection method and its application |
| CN111024943A (en) * | 2019-12-20 | 2020-04-17 | 吉林农业大学 | Switch-on/off type composite fluorescent nano probe for rapid detection of salmonella and preparation method thereof |
| CN113049651A (en) * | 2021-03-15 | 2021-06-29 | 重庆大学 | In-situ electrochemical immunosensor for simultaneously detecting four breast cancer markers |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101566567A (en) * | 2009-05-31 | 2009-10-28 | 武汉理工大学 | Thin-film material for optical fiber biosensor probe and preparation method thereof |
| CN106526197A (en) * | 2016-10-10 | 2017-03-22 | 中南大学 | Method for detecting human IgG concentration |
| CN107478823A (en) * | 2017-08-01 | 2017-12-15 | 长沙理工大学 | A kind of method based on acetylcholine ester enzyme signal amplification principle detection bisphenol-A |
-
2018
- 2018-01-10 CN CN201810022503.7A patent/CN108362872A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101566567A (en) * | 2009-05-31 | 2009-10-28 | 武汉理工大学 | Thin-film material for optical fiber biosensor probe and preparation method thereof |
| CN106526197A (en) * | 2016-10-10 | 2017-03-22 | 中南大学 | Method for detecting human IgG concentration |
| CN107478823A (en) * | 2017-08-01 | 2017-12-15 | 长沙理工大学 | A kind of method based on acetylcholine ester enzyme signal amplification principle detection bisphenol-A |
Non-Patent Citations (2)
| Title |
|---|
| EMILIA WITKOWSKA NERY: "《Analysis of Samples of Clinical and Alimentary interest with paper based devices》", 31 December 2016 * |
| YIHENG LUO等: "Rapid electrochemical quantification of Salmonella Pullorum and Salmonella Gallinarum based on glucose oxidase and antibody-modified silica nanoparticles", 《ANAL BIOANAL CHEM》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109988810A (en) * | 2019-03-11 | 2019-07-09 | 上海交通大学 | A kind of fast-bacteria-detection method and its application |
| CN111024943A (en) * | 2019-12-20 | 2020-04-17 | 吉林农业大学 | Switch-on/off type composite fluorescent nano probe for rapid detection of salmonella and preparation method thereof |
| CN113049651A (en) * | 2021-03-15 | 2021-06-29 | 重庆大学 | In-situ electrochemical immunosensor for simultaneously detecting four breast cancer markers |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Wu et al. | Application of nano-ELISA in food analysis: Recent advances and challenges | |
| CN104280542B (en) | Double; two enhanced chemiluminescence immunoassays that and nanometer particle to mark luminous based on Reinforced by Metal amplifies | |
| CN108362872A (en) | A kind of quantitative detecting method of the white diarrhea and avian infectious bronchitis nephritis virus of non-diagnostic purpose | |
| CN103926405B (en) | Creatine kinase isozyme detection kit and preparation thereof | |
| Weidemaier et al. | Real-time pathogen monitoring during enrichment: a novel nanotechnology-based approach to food safety testing | |
| CN108362879A (en) | A kind of histamine immunoassay method based on platinum-gold duplex metal nano granule class peroxidase activity | |
| CN109765361A (en) | A kind of Detection Method of Pathogenic Microorganism | |
| CN108303532A (en) | A kind of escherichia coli O157 of non-diagnostic purpose:The rapid detection method of H7 | |
| Luo et al. | Highly sensitive homogenous chemiluminescence immunoassay using gold nanoparticles as label | |
| Won et al. | Signal enhancement of a micro-arrayed polydiacetylene (PDA) biosensor using gold nanoparticles | |
| CN103995130A (en) | Alpha 1-microglobulin detection kit and preparation | |
| CN109061207A (en) | A kind of biochip for heart infarction marker detection, detection method | |
| Sun et al. | A nonenzymatic optical immunoassay strategy for detection of Salmonella infection based on blue silica nanoparticles | |
| CN102135541A (en) | Detection method of enterobacter sakazakii and gold-labeled rapid diagnosis kit for same and preparation method thereof | |
| Sun et al. | Blue silica nanoparticle-based colorimetric immunoassay for detection of Salmonella pullorum | |
| Kaur et al. | Novel screening test for celiac disease using peptide functionalised gold nanoparticles | |
| Chen et al. | Sensitive and hook effect–free lateral flow assay integrated with cascade signal transduction system | |
| Zhao et al. | Antibiotic-enzyme-inorganic nanoflowers based immunoassay for the ultrasensitive detection of Staphylococcus aureus | |
| Ge et al. | Lateral flow immunoassay for visible detection of human brucellosis based on blue silica nanoparticles | |
| CN107084976A (en) | A kind of method recognized based on aptamers with Mimetic Peroxidase Visual retrieval salmonella | |
| Duan et al. | Preparation of immunomagnetic iron-dextran nanoparticles and application in rapid isolation of E. coli O157: H7 from foods | |
| CN106290326A (en) | The detection colorimetric sensor of lipopolysaccharide, its preparation method and application | |
| CN110907442B (en) | Colorimetric detection kit and detection method for milk allergen | |
| CN108828231A (en) | A kind of cardio-pulmonary function marker magnetic particle microflow controlled biochip, detection method | |
| CN111474340A (en) | Enzyme-labeled antigen for novel coronavirus detection, preparation method, kit and application |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20180803 |