CN103954750B - A kind of method of immunomagnetic beads and fast detection Oneida Shewanella - Google Patents
A kind of method of immunomagnetic beads and fast detection Oneida Shewanella Download PDFInfo
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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
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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
- G01N33/543—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
- G01N33/54326—Magnetic particles
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2446/00—Magnetic particle immunoreagent carriers
- G01N2446/80—Magnetic particle immunoreagent carriers characterised by the agent used to coat the magnetic particles, e.g. lipids
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Abstract
The invention discloses a kind of immunomagnetic beads and the quick method detecting Oneida Shewanella thereof.The carboxylic polystyrene magnetic microsphere of Oneida Shewanella specific antibody and activation carries out hatching and can obtain by being prepared as of immunomagnetic beads.Method for quick, for immunomagnetic beads is put into sample solution, makes the antibody on immunomagnetic beads fully be combined with Oneida Shewanella isoantigen; Remove sample substrate and obtain immunomagnetic beads-Oneida Shewanella immunocomplex, washes clean, add nitrite ion generation chromogenic reaction, then add the reaction of sulfuric acid solution color development stopping, detect its OD
450 nmvalue, if OD
450 nmvalue more than 0.029, containing Oneida Shewanella in interpret sample.The inventive method has higher sensitivity, detects and is limited to 5.0 × 10
3cfu/ml, easy to operate, rapid, can be used for Site Detection on the spot; And there is good specificity, testing result is hardly by the impact of other common microbiological.
Description
Technical field
The invention belongs to microorganism detection field, relate to a kind of immunomagnetic beads and the quick method detecting Oneida Shewanella thereof.
Background technology
Oneida Shewanella (
shewanella oneidensismR-1,
s. oneidensis) belong to the Gram-negative facultative anaerobe of γ-distortion Gammaproteobacteria, be extensively present in seawater, oceanic sediment and fresh water environment.Under anaerobic, Oneida Shewanella can while degradation of organic substances reducing heavy metal ion.It is with a wide range of applications in environmental pollution field of repairing, and such as it directly can by water miscible U (VI)) be reduced into U (IV).In addition it can also produce sulphion by reduced sulphur thiosulfate, is then combined with heavy metal ion and forms sulfide precipitation.In view of Oneida Shewanella is in the application prospect of environmental area, the method for quick of developing low-cost is significant.At present, for the detection method of Oneida Shewanella, there are Surface enhanced raman spectroscopy method and microarray hybridization method.But, these methods not only complicated operation and also detection sensitivity lower.
Immunity magnetic separation technique is the novel concentration and separation technology of one that immunology and magnetic carrier technology combine and grow up.The magnetic microsphere that this technology adopts is coated with specific antibody, can form immunomagnetic beads antigenic compound with corresponding target material specific binding, by externally-applied magnetic field effect, and immunomagnetic beads antigenic compound and solution quick separating.Therefore, have that detection time is short, sensitivity is higher and not by advantages such as sample substrate affect.In recent years, the principle adopting immune magnetic separation technique to combine with modern signal amplification system, develops numerous novel detection method.Such as, immuno magnetic cell separation technology combines with PCR, flow cytometry and ELISA etc.But these methods have the instrument etc. that detection time is long, cost is high and need complexity.There are some researches show, the membrane-bound c type cytochrome of Oneida Shewanella 80% are positioned at epicyte, and these C type cytochrome have similar catalase activity to horseradish peroxidase (HRP).Existing technology retrieval is found, at present also not for the immune Magneto separate detection method of Oneida Shewanella.Therefore, utilize Oneida Shewanella epicyte catalase activity, binding immunoassay Beads enrichment technology, a kind of method for quick of novel Oneida Shewanella can be developed.
Summary of the invention
In order to solve above-mentioned Problems existing, the present invention can cross research, signal amplification based on the outer catalase activity of Oneida Shewanella born of the same parents is combined with immuno magnetic cell separation technology and is creatively combined, build the method detecting Oneida Shewanella fast, it is high that the method has specificity, simple to operate, detect rapidly.
The object of the present invention is to provide a kind of immunomagnetic beads.
Another object of the present invention is to the method that fast a kind of quick detection Oneida Shewanella is provided.
The technical solution used in the present invention is:
A kind of immunomagnetic beads, its preparation method comprises the following steps:
1) Oneida Shewanella specific antibody is prepared;
2) carboxylic polystyrene magnetic microsphere is activated, washes clean, then carry out oscillation incubation with the Oneida Shewanella specific antibody prepared in step 1), antibody is fully combined with magnetic bead;
3) the magnetic bead washes clean of will hatch, then closes, cleans up, 4 DEG C of preservations, can adaptive immune magnetic bead.
A method for quick detection Oneida Shewanella, comprises the following steps:
1) above-mentioned immunomagnetic beads is added in testing sample solution, oscillating reactions at 18 ~ 27 DEG C, the antibody on immunomagnetic beads is fully combined with Oneida Shewanella isoantigen;
2) after association reaction completes in step 1), with magnet, immunomagnetic beads and sample substrate are separated, remove sample substrate, obtain immunomagnetic beads-Oneida Shewanella immunocomplex;
3) by 2) in immunomagnetic beads-Oneida Shewanella immunocomplex washes clean of obtaining, add nitrite ion, chromogenic reaction occur, make blue product generate completely;
4) in blue product, add the reaction of sulfuric acid solution color development stopping, blue product is converted into yellow product, detects it in 450 nanometers OD values, if OD value is more than 0.029, illustrates in this sample containing Oneida Shewanella.
Further, in step 1), immunomagnetic beads and sample solution volume ratio are (1 ~ 5): 50.
Further, the time of oscillating reactions described in step 1) is 10 ~ 70min.
Further, the detailed process of washing described in step 3) is first with containing 4 ~ 6%(v/v) the PBS buffer solution of polysorbas20 3 ~ 4 times, cleaner with pure water.
Further, the volume of nitrite ion described in step 3) and the ratio of sample solution volume are (0.8 ~ 1.2): 5.
Further, 0.1 ~ 5mM hydrogen peroxide that nitrite ion described in step 3) contains, 90 ~ 110mM sodium citrate, 190 ~ 210mM sodium hydrogen phosphate, 0.3 ~ 0.35mM tetramethyl benzidine, pH value is 4 ~ 5.
Further, the time of chromogenic reaction described in step 3) is 5 ~ 60min.
The invention has the beneficial effects as follows:
(1) immunomagnetic beads of the present invention has complete superparamagnetism, monodispersity, and suspension is good;
(2) Oneida Shewanella has the outer catalase activity of born of the same parents, in the presence of hydrogen peroxide can catalytic oxidation tetramethyl benzidine, produces blue reaction product, reaches the object detected fast;
(3) detection method of Oneida Shewanella of the present invention has higher sensitivity, is limited to 5.0 × 10 to the detection of Oneida Shewanella
3cfu/ml, testing process is convenient, rapid, can be used for Site Detection on the spot;
(4) Oneida Shewanella detection method of the present invention has good specificity, and other common microorganisms have an impact hardly to detection.
Accompanying drawing explanation
Fig. 1 is that immunomagnetic beads prepares schematic diagram;
Fig. 2 is the schematic diagram detecting Oneida Shewanella fast;
Fig. 3 is optical microphotograph camera lens and the scanning electron microscope (SEM) photograph of Oneida Shewanella and immunomagnetic beads complex;
Fig. 4 is that the class peroxidase activity of Oneida Shewanella and immunomagnetic beads detects;
Fig. 5 is the optimization of the condition of work detecting Oneida Shewanella fast;
Fig. 6 is the regression curve that Oneida Shewanella detects;
Fig. 7 is specificity experiments result figure;
Fig. 8 is the impact of sample substrate composition on detection method.
Embodiment
the preparation of immunomagnetic beads
(1) immunizing antigen is prepared: described immunizing antigen adopts 1% Formalin inactivation Oneida Shewanella liquid and makes;
(2) Oneida Shewanella specific antibody is prepared: described Oneida Shewanella specific antibody adopts deactivation immunizing antigen immunize New Zealand white rabbits method to obtain antiserum, and adopt two step ammonium sulfate salting-out process purifying antiserums, obtain Oneida Shewanella specific antibody;
(3) immunomagnetic beads is prepared: described immunomagnetic beads adopts the Oneida Shewanella specific antibody of purifying and the carboxylic polystyrene magnetic microsphere oscillation incubation coupling of activation to be prepared from;
the method of quick detection Oneida Shewanella
1) immunomagnetic beads of above-mentioned preparation is added in analyte sample fluid, room temperature (18 ~ 27 DEG C) oscillating reactions 10 ~ 70min, the antibody on immunomagnetic beads is fully combined with Oneida Shewanella isoantigen; Wherein immunomagnetic beads and sample solution volume are than being (1 ~ 5): 50;
2) with magnet, immunomagnetic beads and sample substrate are separated, remove sample substrate, obtain immunomagnetic beads-Oneida Shewanella immunocomplex;
3) by 2) in immunomagnetic beads-Oneida Shewanella immunocomplex washes clean of obtaining, add nitrite ion, the volume of nitrite ion and the ratio of sample solution volume are (0.8 ~ 1.2): 5, and chromogenic reaction 5 ~ 60min occurs, and make blue product generate completely;
4) in blue product, add the reaction of sulfuric acid solution color development stopping, blue product is converted into yellow product, detects it in 450 nanometers OD values, if OD value is more than 0.029, illustrates in this sample containing Oneida Shewanella.
Preferably, in step 1), immunomagnetic beads and sample solution volume ratio are 4:50.
Preferably, the time of oscillating reactions described in step 1) is 50min.
Preferably, the volume of nitrite ion described in step 3) and the ratio of sample solution volume are 1:5
Preferably, the detailed process of washing described in step 3) is first with containing 4 ~ 6%(v/v) the PBS buffer solution of polysorbas20 3 ~ 4 times, cleaner with pure water.
Preferably, 0.1 ~ 5mM hydrogen peroxide that nitrite ion described in step 3) contains, 90 ~ 110mM sodium citrate, 190 ~ 210mM sodium hydrogen phosphate, 0.3 ~ 0.35mM tetramethyl benzidine, pH value is 4 ~ 5.
Most preferred, nitrite ion described in step 3) contains 2mM hydrogen peroxide, 100mM sodium citrate, 200mM sodium hydrogen phosphate, 0.32mM tetramethyl benzidine, and pH is 4.3.
Preferably, the time of chromogenic reaction described in step 3) is 30min.
Briefly introduce the know-why of the inventive method below:
Under having Oneida Shewanella to exist, immunomagnetic beads is combined with Oneida Shewanella generation antigen-antibody and forms immunocomplex; Under outside magnetic field effect, immunomagnetic beads-Oneida Shewanella immunocomplex is separated with sample substrate, with pipettor pipette samples matrix, obtains immunomagnetic beads-Oneida Shewanella immunocomplex; The Oneida Shewanella of non-specific binding is washed away with the PBST containing 1%BSA; When there being hydrogen peroxide to exist, Oneida Shewanella adventitia c type cytochrome catalytic oxidation tetramethyl benzidine (TMB), creates blue reaction product; This blue product and sulfuric acid reaction change into yellow, and available microplate reader detects OD value in 450 nanometers.Detection obtains OD value and Oneida Shewanella quantity in certain limit positive correlation.
Below in conjunction with specific embodiment, the present invention is further illustrated, but be not limited thereto.
embodiment 1: the preparation of immunomagnetic beads
(1) Oneida Shewanella specific antibody preparation
Being inoculated in 500ml triangular flask by Oneida Shewanella glycerine conserving liquid with 1:100 ratio, is 30 DEG C in temperature, shaken cultivation 16h in the constant-temperature table of rotating speed 160rpm.Bacterium liquid will be cultivated with the centrifugal 20min of 5000g, collecting precipitation, after washing 3 times with PBS, with PBS, bacterial concentration will be adjusted to 10
9cfu/mL.After 1% Formalin inactivation bacteria suspension 24h, then wash 3 times with PBS, bacterial concentration is adjusted to 10
8cfu/mL, and with Freund's adjuvant according to 1:1(V/V) fully emulsified obtained immunizing antigen.To prepare antigen immune New Zealand white rabbit, first immunisation is after 30 days, every immunity in 10 days once, and immunity 4 times altogether.Immune latter 10 days the last time, collect ear venous blood, be separated and obtain Oneida Shewanella specific antisera.The antiserum of preparation is respectively saltoutd once with 50%, 33% saturation degree ammonium sulfate, and then with PBS dialysis 72h, after dislysate is concentrated with PEG 20000, packing is stored in-20 DEG C.
(2) immunomagnetic beads preparation
As shown in Figure 1, get the carboxylic polystyrene magnetic microsphere that 1ml diameter is 1-2 μm, with 2-(N-morpholine) ethanesulfonic acid buffer (MES) (0.05 M, pH 5.5) wash 3 times, add 200 microlitre 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride (EDC) solution (100mg/ml), N-hydroxy-succinamide (NHS) solution (100mg/ml) and 600 microlitre MES, shaken at room temperature 30min respectively.After the immunomagnetic beads of activation is washed 3 times with MES, add 1.4mg/ml purifying Oneida Shewanella antibody, after shaken at room temperature 3h, wash 3 times with PBS, be resuspended in 1% bovine serum albumin solution (W/V) and close, 4 DEG C of preservations.
embodiment 2: the method detecting Oneida Shewanella fast
1) in 1.5ml centrifuge tube, immunomagnetic beads and the 500 μ L testing samples of preparation in 10 ~ 50 embodiments 1 are added, room temperature (18 ~ 27 DEG C) vibration 50 ~ 60min;
2) centrifuge tube is placed in 5min on magnet, magnetic bead is separated completely with solution, bottom centrifuge tube, suck sample substrate with pipettor, obtain immunomagnetic beads-Oneida Shewanella immunocomplex;
3) by 2) in the complex that obtains with containing 4 ~ 6%(v/v) the PBS cleansing solution of polysorbas20 washs 3 times, 96 orifice plates are transferred to after pure water 1 time, 100 μ L nitrite ion (0.1 ~ 5mM hydrogen peroxide are added in 96 orifice plates, 90 ~ 110mM sodium citrate, 190 ~ 210mM sodium hydrogen phosphate, 0.3 ~ 0.35mM tetramethyl benzidine, pH is 4 ~ 5), room temperature (18 ~ 27 DEG C) chromogenic reaction 5 ~ 60min;
4) add 50 μ L 2M sulfuric acid solution cessation reactions, by Magneto separate, get supernatant, detect it in 450nm place OD value by microplate reader, if OD value is more than 0.029, illustrate in this sample containing Oneida Shewanella.
embodiment 3: the method detecting Oneida Shewanella fast
As shown in Figure 2, concrete operation step is the schematic diagram of the method for quick detection Oneida Shewanella:
1) in 1.5ml centrifuge tube, add immunomagnetic beads and the 500 μ L 5.0 × 10 of preparation in 40 μ L embodiments 1
6the Oneida Shewanella liquid of CFU, room temperature (18 ~ 27 DEG C) vibration 60min;
2) centrifuge tube is placed in 5min on magnet, magnetic bead is separated completely with solution, bottom centrifuge tube, suck sample substrate with pipettor, obtain immunomagnetic beads-Oneida Shewanella immunocomplex;
3) by 2) in the complex that obtains with containing 5%(v/v) the PBS cleansing solution of polysorbas20 washs 3 times, 96 orifice plates are transferred to after pure water 1 time, 100 μ L nitrite ion (100mM sodium citrates are added in 96 orifice plates, 200mM sodium hydrogen phosphate, 0.32mM tetramethyl benzidine and 2mM hydrogen peroxide, pH 4.3), room temperature (18 ~ 27 DEG C) chromogenic reaction 30min;
4) add 50 μ L 2M sulfuric acid solution cessation reactions, by Magneto separate, get supernatant, detect it 450nm place OD value (OD value is 0.60) by microplate reader.
In order to confirm further immunomagnetic beads with
s. oneidensiswhether the combination of (Oneida Shewanella) is relevant with the antibody on immunomagnetic beads surface, goes to catch respectively with magnetic bead and immunomagnetic beads
s. oneidensis, detect catching result, result display magnetic bead fails to capture
s. oneidensis, as shown in Figure 3 a, wherein in optical microscope figure and scanning electron microscope (SEM) photograph all without shaft-like thalline, and in immunomagnetic ca pture figure (as shown in Figure 3 b), in its optical microscope figure and scanning electron microscope (SEM) photograph, all can see that is shaft-like
s. oneidensisthalline, illustrate immunomagnetic beads of the present invention be by the antibody on its surface with
s. oneidensisthe specific recognition of somatic surface antigen and carrying out combines.
In addition, some some magnetic bead of research report itself has the activity (Wei, H. & Wang, the E. Fe that are similar to peroxidase
3o
4magnetic nanoparticles as peroxidase mimetics and their applications in H
2o
2and glucose detection.
anal. Chem.80,2250-2254 (2008) .), in order to confirm further in method for quick of the present invention adopt magnetic bead whether also to have the activity being similar to peroxidase? does is chromogenic reaction wherein subject to the impact of adopted magnetic bead? respectively will
s. oneidensis, cytochrome c (positive control),
s. oneidensisadd containing H with the complex of immunomagnetic beads, immunomagnetic beads
2o
2with in the solution of TMB, carry out chromogenic reaction, and scanning reactant liquor UV-Vis spectra, result as shown in Figure 4, wherein
s. oneidensis(Fig. 4 a), cytochrome c (Fig. 4 b),
s. oneidensisall can produce blue reaction product with the complex (Fig. 4 c) of immunomagnetic beads, and at 650nm place, there is obvious extinction peak, and immunomagnetic beads group (Fig. 4 d) and only containing H
2o
2with in the negative control group (Fig. 4 e) of TMB all without blue product, also there is not corresponding extinction peak, illustrate that the magnetic bead that the present invention selects does not have the activity being similar to peroxidase, interference is not existed to detection method of the present invention.
embodiment 4: the method detecting Oneida Shewanella fast
In order to obtain best Detection results, the present embodiment is further on the parameter (immunomagnetic beads volumetric concentration, the H that affect Detection results
2o
2concentration, immune response time, chromogenic reaction time) further optimize.
Immunomagnetic beads volumetric concentration is optimized: experimental implementation process, with embodiment 2, is 5.0 × 10 in Oneida Shewanella liquid concentration
6cfu/mL, volume is 500 μ L, H
2o
2concentration is 3mM, study best immunomagnetic beads concentration under the condition of immune response time 60 min, chromogenic reaction time 30min, the volume that immunomagnetic beads adds is chosen for 10,20,30,40,50 μ L respectively, finally detect the OD value of reactant liquor at 450nm place adding different volumes immunomagnetic beads, testing result as shown in Figure 5 a, therefrom can find out when the volume that immunomagnetic beads adds is 40 μ L, end reaction liquid is the highest in the OD value at 450nm place, Detection results is the sensitiveest, therefore the immunomagnetic beads volumetric concentration of the best is 8%(40 μ L/500 μ L).
H
2o
2concentration optimization: method for quick of the present invention depends on the peroxidase activity of Oneida Shewanella itself, and the sensitivity of peroxidase activity should be able to be subject to H
2o
2the impact of concentration, therefore be necessary to visit the H grinding the best in the inventive method
2o
2concentration.
Be 5.0 × 10 in Oneida Shewanella liquid concentration
6cfu/mL, volume is 500 μ L, and immunomagnetic beads volumetric concentration is 8%, study best H under the condition of immune response time 60 min, chromogenic reaction time 30min
2o
2concentration, H
2o
2the volume of concentration is chosen for 0.1,1,2,3,4,5 mM respectively, finally detects and adds variable concentrations H
2o
2the OD value of reactant liquor at 450nm place, testing result as shown in Figure 5 b, therefrom can be found out and work as H
2o
2when concentration is 2mM, end reaction liquid is the highest in the OD value at 450nm place, and Detection results is the sensitiveest, therefore the H of the best
2o
2concentration is 2mM.
Immune response is time-optimized: be 5.0 × 10 in Oneida Shewanella liquid concentration
6cfu/mL, volume is 500 μ L, and immunomagnetic beads volumetric concentration is 8%, H
2o
2concentration is 2mM, study the optimal immune response time under the condition of chromogenic reaction time 30min, the immune response time elects 10,20,30,40,50,60 min respectively as, finally detect the OD value of reactant liquor at 450nm place after adding the different immune response time, testing result as shown in Figure 5 c, therefrom can find out when immunity inverse time between be 10 ~ 50min time, OD value increases with the growth of immune response time, when the immune response time is 50 ~ 60min, OD value tends towards stability, almost no longer increase, so the most preferred immune response time is 50min.
Chromogenic reaction is time-optimized: be 5.0 × 10 in Oneida Shewanella liquid concentration
6cfu/mL, volume is 500 μ L, immunomagnetic beads volumetric concentration is 8%, the immune response time 50 min, H
2o
2concentration is study the best chromogenic reaction time under the condition of 2mM, the chromogenic reaction time elects 5,10,20,30,40 min respectively as, finally detect the OD value of the reactant liquor after the different chromogenic reaction time at 450nm place, testing result as fig 5d, therefrom can find out when the chromogenic reaction time is 30min, end reaction liquid is the highest in the OD value at 450nm place, and Detection results is the sensitiveest, therefore the chromogenic reaction time of the best is 30min.
According to the above-mentioned optimization to each parameter, the method for optimum quick detection Oneida Shewanella can be obtained, comprise following operation steps:
1) in 1.5ml centrifuge tube, immunomagnetic beads and the 500 μ L bacterium liquid samples of preparation in 40 μ L embodiments 1 are added, room temperature (18 ~ 27 DEG C) vibration 50min;
2) centrifuge tube is placed in 5min on magnet, magnetic bead is separated completely with solution, bottom centrifuge tube, suck sample substrate with pipettor, obtain immunomagnetic beads-Oneida Shewanella immunocomplex;
3) by 2) in the complex that obtains with containing 5%(v/v) the PBS cleansing solution of polysorbas20 washs 3 times, 96 orifice plates are transferred to after pure water 1 time, 100 μ L nitrite ion (100mM sodium citrates are added in 96 orifice plates, 200mM sodium hydrogen phosphate, 0.32mM tetramethyl benzidine and 2mM hydrogen peroxide, pH 4.3), room temperature (18 ~ 27 DEG C) chromogenic reaction 30min;
4) add 50 μ L 2M sulfuric acid solution cessation reactions, by Magneto separate, get supernatant, detect it in 450nm place OD value by microplate reader, OD value, more than 0.029, can illustrate in this sample containing Oneida Shewanella.
Further recruitment evaluation is done to this bright immunomagnetic beads and the quick method detecting Oneida Shewanella thereof below.
one, sensitivity
By 10
8cfu/mL Oneida Shewanella suspension dilutes successively for concentration is respectively 5.0 × 10
7, 5.0 × 10
6, 2.5 × 10
6, 1.25 × 10
6, 6.3 × 10
5, 3.2 × 10
5, 1.6 × 10
5, 8.0 × 10
4, 4.0 × 10
4, 2.0 × 10
4, 1.0 × 10
4, 5.0 × 10
3the bacteria suspension of cfu/mL, the optimum detection methodology set up with embodiment 4 detects the Oneida Shewanella liquid of these variable concentrations, take cell concentration as horizontal ordinate, OD value is as ordinate, and curve plotting, is shown in Fig. 6, analyze this curve known, its regression equation is Y=1.133
-7x+0.027, (R
2=0.9988), equal 3 judgement the inventive method according to signal noise ratio and can detect that to cause concentration be 5.0 × 10
3cfu/mL, linear detection range is 5.0 × 10
3~ 5.0 × 10
6cfu/mL.
two, specificity
Compound concentration is 5.0 × 10 respectively
6the Oneida Shewanella bacterium liquid of cfu/ml, Escherichia coli bacteria liquid and bacillus subtilis bacterium liquid, and blank, the optimum detection methodology determined in embodiment 4 is adopted to detect the bacterium liquid of these different strains, its OD value as shown in Figure 7, therefrom can find out, immunomagnetic beads of the present invention and method for quick thereof only have Detection results to going out how to reach Shewanella.Illustrate that the inventive method has specificity very and accuracy.
In order to determine that the inventive method is applicable to real testing process further, gather real river respectively compound concentration be 5.0 × 10
6the Oneida Shewanella bacterium liquid of cfu/ml (River water+
s. oneidensis), Escherichia coli bacteria liquid (River water+E.
coli) and bacillus subtilis bacterium liquid (River water+B.
subtilis), and containing the mixed bacteria liquid of these three kinds of bacterium, (Mixture, the concentration of often kind of bacterium is 5.0 × 10 simultaneously
6cfu/ml), with river (River water) in contrast (as shown in Figure 8), testing result shows, Oneida Shewanella bacterium liquid and mixed bacteria liquid is only had to occur very strong light absorption value, and the light absorption value of the two is without significant difference (as shown in Figure 8), this illustrates that composition in river and other mushrooms do not affect immunomagnetic beads of the present invention and method for quick thereof to the specific detection of Oneida Shewanella, the method of immunomagnetic beads of the present invention and fast detection Oneida Shewanella has good specificity, and is applicable to the detection to this bacterium in environment.
three, repeatable
Be 5.0 × 10 to concentration
6the Oneida Shewanella bacterium liquid of cfu/ml carries out 5 secondary pollutant duplicate detection, and testing result all demonstrates similar light absorption value, and aligning standby difference is 5.41%, illustrates that the inventive method has good repeatability.
four, with the comparing of its Oneida Shewanella detection method
With other Oneida Shewanella detection method of bibliographical information, as Surface enhanced raman spectroscopy method (Yang, X., Gu, C., Qian, F., Li, Y. & Zhang, J. Z. Highly sensitive detection of proteins and bacteria in aqueous solution using surface-enhanced Raman scattering and optical fibers. Anal. Chem. 83, 5888-5894 (2011) .) and microarray hybridization method (Wu, L. et al. Development and evaluation of microarray-based whole-genome hybridization for detection of microorganisms within the context of environmental applications. Environ. Sci. Technol. 38, 6775-6782 (2004) .), its lowest detectable limit is respectively 10
6cells/mL and 2.5 × 10
5cells/mL compares, and (lowest detection is limited to 5.0 × 10 to the inventive method
3cells/mL) there is better detection perform.In addition, according to bibliographical information, the bacterial concentration of the low biomass water sample in biological prosthetic site is up to 3 × 10
6cells/mL, therefore, the inventive method can meet the monitoring of biological prosthetic process function micro organism quantity.
In sum, the method for immunomagnetic beads of the present invention and fast detection Oneida Shewanella has higher sensitivity, is limited to 5.0 × 10 to the detection of Oneida Shewanella
3cfu/ml, testing process is convenient, rapid, can be used for the Site Detection on the spot of Oneida Shewanella; In addition, detection method of the present invention has good specificity, and other common microorganisms and environment composition do not produce interference to detection.
Above embodiment is only introduces preferred case of the present invention, to those skilled in the art, not deviating from any apparent changes and improvements of carrying out in the scope of spirit of the present invention, all should be regarded as a part of the present invention.
Claims (8)
1. detect a method for Oneida Shewanella fast, it is characterized in that: comprise the following steps:
1) immunomagnetic beads is added in testing sample solution, oscillating reactions at 18 ~ 27 DEG C, the antibody on immunomagnetic beads is fully combined with Oneida Shewanella isoantigen;
2) after association reaction completes in step 1), with magnet, immunomagnetic beads and sample substrate are separated, remove sample substrate, obtain immunomagnetic beads-Oneida Shewanella immunocomplex;
3) by 2) in immunomagnetic beads-Oneida Shewanella immunocomplex washes clean of obtaining, add nitrite ion, chromogenic reaction occur, make blue product generate completely;
4) in blue product, add the reaction of sulfuric acid solution color development stopping, blue product is converted into yellow product, detects it in 450 nanometers OD values, if OD value is more than 0.029, illustrates in this sample containing Oneida Shewanella.
2. the method for a kind of quick detection Oneida Shewanella according to claim 1, is characterized in that: in step 1), immunomagnetic beads and sample solution volume are than being (1 ~ 5): 50.
3. the method for a kind of quick detection Oneida Shewanella according to claim 1, is characterized in that: the time of oscillating reactions described in step 1) is 10 ~ 70min.
4. the method for a kind of quick detection Oneida Shewanella according to claim 1, is characterized in that: the detailed process of washing described in step 3) is first with containing 4 ~ 6%(v/v) the PBS buffer solution of polysorbas20 3 ~ 4 times, cleaner with pure water.
5. the method for a kind of quick detection Oneida Shewanella according to claim 1, is characterized in that: the volume of nitrite ion described in step 3) and the ratio of sample solution volume are (0.8 ~ 1.2): 5.
6. the method for a kind of quick detection Oneida Shewanella according to claim 1, it is characterized in that: 0.1 ~ 5mM hydrogen peroxide that nitrite ion described in step 3) contains, 90 ~ 110mM sodium citrate, 190 ~ 210mM sodium hydrogen phosphate, 0.3 ~ 0.35mM tetramethyl benzidine, pH value is 4 ~ 5.
7. the method for a kind of quick detection Oneida Shewanella according to claim 1, is characterized in that: the time of chromogenic reaction described in step 3) is 5 ~ 60min.
8. the method for a kind of quick detection Oneida Shewanella according to claim 1, is characterized in that: the preparation method of described immunomagnetic beads comprises the following steps:
1) Oneida Shewanella specific antibody is prepared;
2) carboxylic polystyrene magnetic microsphere is activated, washes clean, then carry out oscillation incubation with the Oneida Shewanella specific antibody prepared in step 1), antibody is fully combined with magnetic bead;
3) the magnetic bead washes clean of will hatch, then closes, cleans up, 4 DEG C of preservations, can adaptive immune magnetic bead.
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