CN108663343A - Fluorimetry based on aptamer-quantum dot combination B. thuringiensis spores - Google Patents
Fluorimetry based on aptamer-quantum dot combination B. thuringiensis spores Download PDFInfo
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- CN108663343A CN108663343A CN201810415673.1A CN201810415673A CN108663343A CN 108663343 A CN108663343 A CN 108663343A CN 201810415673 A CN201810415673 A CN 201810415673A CN 108663343 A CN108663343 A CN 108663343A
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- quantum dot
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
Abstract
The invention discloses a kind of fluorimetries based on aptamer quantum dot combination B. thuringiensis spores, including Cry1Ab albumen, that is, Bt albumen, aptamer quantum azeotropic mixture preparation, aptamer quantum dot is combined with subtilis spore.The present invention is detected Bt albumen result in transgenic paddy rice by the method that aptamer quantum dot is combined with subtilis spore, testing result is objective, easy judgement, quantitative analysis can be carried out to the content of transgenic product, while detection speed is fast, detection sensitivity is high.
Description
Technical field
The invention belongs to the detection technique fields of transgene component, and in particular to one kind being based on aptamer-quantum dot
In conjunction with the fluorimetry of B. thuringiensis spores.
Background technology
The safety evaluation of genetically modified organism is the premise of genetically modified organism and products thereof marketization and commercialization, is commented in safety
The detection of valence transgenic ingredient is an important content.
China is in world lead level to the research of transgenic paddy rice, wherein some of research and development are disease-resistant, insect-resistant transgenic
Rice strain has been applied producing plantation bio-safety certificate, but some are former due to their GMO bio-safety problem etc.
Cause, there has been no strain approvals to commercially produce application so far.Maturation in view of transgenic paddy rice technology and its product marketization
Trend, therefore establish it is a kind of quickly, the method for easy detection Bt albumen, there is necessity and urgency.
The detection of transgene component can be carried out from two levels of nucleic acid and protein of foreign gene at present.Nucleic acid water
Flat detection, nucleotide sequence mainly for the foreign gene being transferred to simultaneously are set according to the homology of foreign gene and endogenous gene
Detection method is counted, the method mainly used is PCR amplification, i.e., according to being transferred to the promoter of foreign gene, target gene, terminator
And expression (recombination) carrier information, specific primer is designed, PCR is carried out, according to the presence or absence of product or how much is sentenced
Whether disconnected is transgenic product.PCR reactions have the characteristics that high sensitivity, high specific, high efficiency, are transgenic product primary dcreening operations
Stage predominantly detects method.
The horizontal detection of albumen inspection, is mainly based upon the immunological method of antigen and antibody interaction, for transgenosis
The specific proteins of external source destination gene expression carry out qualitative and quantitative detection in plants and plant product.Sweden in 1971
Engvall et al. using cellulose and polystyrene test tube as solid phase carrier adsorption antigen/antibody, establishes enzyme linked immunological respectively
Absorption method (Enzyme Linked ImmunosrbentAssay, abbreviation ELISA method).Voller in 1974 et al. uses instead poly-
Styrene micro-reaction plate enables ELISA method to promote and apply as solid-phase immunity absorption carrier so that is positioned for antigen
Enzyme-labelled antibody technique develop into the assay method of micro substance in liquid sample, and be increasingly becoming in antigen and antibody
A kind of the most commonly used method.It is by the catalytic amplification phase of the immune response of enzyme marker synantigen antibody complex and enzyme
In conjunction with, the sensibility of enzymic catalytic reaction was not only maintained, but also maintain the specificity of antigen-antibody reaction, thus greatly improve
Sensitivity.It is a kind of heterogeneous immunoassay method again simultaneously, i.e., has washing process in each step of reaction, to remove
Unreacted reactant and interfering substance.Since ELISA method has, high sensitivity, high specificity, easy to operate, detection is rapid and non-
Radioactivity and many advantages, such as can measure in batches so that ELISA method is more and more widely used.
But it is higher and higher with the requirement of detection sensitivity, pass through two levels of detection and protein of transgene component
Detection cannot meet the needs developed at present completely.
Invention content
It is an object of the invention to overcome the deficiencies of existing technologies, a kind of Gao Ling of the Bt suitable for transgenic paddy rice is provided
Sensitivity rapid detection method realizes quickly detection using aptamer-quantum dot combination B. thuringiensis spores.
To achieve the above object, the present invention provides the following technical solutions：
A kind of fluorimetry based on aptamer-quantum dot combination B. thuringiensis spores, including it is following
Step：
(1) preparation of aptamer-quantum dot：
1.1,13.9 μ L 10mM SMCC (succinimide -4- (N- maleimides) thiacyclohexane -1-1 carboxylic esters) are added
It is added in 125 μ L quantum dots and places one hour at room temperature to activate quantum dot；
1.2,5 ' mercapto-modified oligonucleotides are prepared in the PBS solution of 300 μ L mono- times of working concentration of 1mg/ml, to
The dithiothreitol (DTT) (DTT) of 6.1 μ l 1m is added in solution and places 30 minutes at room temperature to reduce aptamers；
1.3, the solution of activation quantum dot and the solution of reduction aptamers pass through a nap-5 desalting column respectively
(Sephadex G-25 desalting columns), each solution collect 500 μ l；
1.4 and then quantum dot and reduction aptamer are reacted a hour at room temperature.By 10.1 μ L beta -mercaptoethanols (100
μM ultimate density) be added in mixture to quench conjugation reaction, and place 30 minutes at room temperature, by column purification, altogether
It is collected into 250 μ L aptamers-quantum dot；
1.5,10 μ l aptamers-quantum dot is dissolved in 1ml reverse-osmosis pure waters.With a uv-2100 points
Light photometer weighs the absorbance in 260nm；
1.6, the actual concentrations of aptamer-quantum dot by with initial aptamer concentration (1.08mg/ml) into
Row relatively determines.
(2) aptamer-quantum dot is combined with subtilis spore：
2.1,30 μ L aptamers-quantum dot is placed in a test tube and is diluted with 600 μ L PBS.
2.2, a series of reaction of repetitions is carried out with same program：Aptamer-quantum dot is uncombined without spore
Quantum dot and Bt spores, aptamer-quantum dot are combined with the Bt spores of purifying, aptamer-quantum dot and BG spores
In conjunction with；
2.3, the spore collected before is washed 13mm microporous membrane filters (PVDF, hydrophily, 0.45 μm) twice with PBS,
Spore, which is exposed to after any one of aptamer-quantum dot or uncombined quantum dot to collect in the filter, to be used in combination
Three times, washed spore is collected in 1ml PBS for the PBS solution washing of mono- times of working concentration of 1ml.
As the preferred of the embodiment of the present invention, in the step 1.4, reaction product is concentrated using 0.5ml concentrators, so
After be added to 200 column Superdex desalting columns and eluted with PBS..
As the preferred of the embodiment of the present invention, in the step 1.6, the percentage that aptamer is conjugated quantum dot is true
It is set to 98%.
As the preferred of the embodiment of the present invention, the diluted 100 μ L aptamers-quantum dots of PBS are separately added into 900 μ L
In the Bt spores of 107,106,105,104,103CFU.Mixture is with the of short duration vortex (speed 8) of 3 vortex mixers of VSM and puts
It sets and reacts 20min at room temperature.
Compared with prior art, the beneficial effects of the invention are as follows：
1. the present invention is economical and practical, reaction is carried out under conditions of constant temperature, so do not need expensive PCR instrument, and
Detection cycle is shorter.
2. present invention detection is simple：This method detects that Bt albumen results in transgenic paddy rice are objective, easy judgement, can be to turning base
Because the content of product carries out quantitative analysis.
Description of the drawings
Fig. 1 is the secondary circuit structural schematic diagram of the aptamer of BT；
Fig. 2 is the fluorescence spectra of bt spores；
Fig. 3 is free quantum dot and Bt spore combination fluorescence spectras；
Fig. 4 is conjugation aptamer-quantum dot combination Bt spore fluorescence spectras；
Fig. 5 is the Bt spore fluorescence spectras for being conjugated aptamer-quantum dot and combining purifying；
Fig. 6 is conjugation aptamer-quantum dot combination BG spore fluorescence spectras；
Specific implementation mode
Following will be combined with the drawings in the embodiments of the present invention, and technical solution in the embodiment of the present invention carries out clear, complete
Site preparation describes, it is clear that described embodiments are only a part of the embodiments of the present invention, instead of all the embodiments.It is based on
Embodiment in the present invention, it is obtained by those of ordinary skill in the art without making creative efforts every other
Embodiment shall fall within the protection scope of the present invention.
One, prepared by Cry1Ab albumen
The preparation process of Cry1Ab albumen is as follows：Shown in Fig. 1 to 2, the Su Yun gold buds that will be stored in -70 DEG C of refrigerators
Born of the same parents' bacillus strain is crossed to 30 DEG C of growths on LB tablets and overnight, chooses single bacterium and fall on the LB cultures of 10mL based on 30 DEG C of shaken cultivation mistakes
Night.The bacterium solution being incubated overnight is added in the PGSM culture mediums of 200mL after 30 DEG C of shaken cultivations 3-4 days, under the microscope again
Observation cell can see the cell of cracking.Then 4 DEG C, 4000rpm centrifuges 15min and collects culture, with ice-cold 0.1mol/
L NaCl washing precipitations are primary, 4 DEG C, and 4000rpm centrifuges 15min and collects culture, then washs precipitation one with ice-cold distilled water
Secondary, at 4 DEG C, 4000rpm centrifuges 15min and collects culture, and precipitation is suspended in the ice-cold pure water of 15mL.Again by solution in 4
DEG C, 4000rpm centrifuges 15min and collects precipitation, and precipitation is Cry1Ab crystalline proteins.Precipitation is suspended in 0.1mol/L NaOH
Middle 15min makes Cry1Ab crystalline proteins be completely dissolved, and 1mol/LTris buffer solutions is used to adjust the pH to 7.0 of solution at once, in
After 12000rpm centrifuges 10min, the PBS solution dialysis of 100 times of volumes of supernatant is taken three times.Its concentration uses Bradford methods
It measures.
Wherein：
The PCR Molecular Detections of Cry 1Ab genes, upstream and downstream primer sequence are respectively
5'-GAGAACTTCGGTACGTAGC-3’
5'-AACACATGAGCGGTAAGG-3’
The reagent component and its proportioning is as follows：
LB culture mediums：Tryptone (Tryptone) 10g/L, yeast extract (Yeast extract) 5g/L, sodium chloride
(NaCl) 10g/L, NaOH adjust the pH of the culture medium, reach 7.4；
PGSM culture mediums：Bacto peptone 7.5g, glucose 1g, KH2PO43.4g, K2HPO44.35g add distilled water fixed
Hold to 1L, adjust pH to 7.2, sterilize 30min under 121 DEG C of high-temperature steams；
PBS buffer solution：KH2PO40.2g, NaCl 8.0g, KCl 0.2g, Na2HPO42.9g, Tween-200.5mL, add
Distilled water is settled to 1L, adjusts pH to 7.4；
Tris buffer solutions：50ml 0.1mol/L trishydroxymethylaminomethanes (Tris) solution 0.1mol/L hydrochloric acid tune
PH to 7.0 is saved, 100ml is diluted with water to
Two, the preparation of water-soluble CdSe/ZnS quantum dot
The preparation of water-soluble CdSe/ZnS quantum dot comprises the concrete steps that：Weigh 0.0127g (0.1mmo l) CdO and
0.1140g stearic acid is put into the three-necked bottle of 50mL, under protection of argon gas, is heated to 130 DEG C, CdO is made to be completely dissolved in tristearin
In acid.After cooling the temperature to room temperature, weighs trioctylphosphine oxide (TOPO) and hexadecylamine (HDA) each 1.5g is put into three-necked bottle
In, 230 DEG C are stirred and heated to, 0.032g (1mmo l) S powder is separately weighed, under protection of argon gas, is allowed to be dissolved in 2mL trioctylphosphine phosphorus
(TOP) it in, is pumped into the needle tubing of 5mL and is used as storing solution.It is when Cd predecessor temperature reaches 230 DEG C, Se powder storing solutions is quick
After injecting three-necked bottle, temperature is down to 180 DEG C, and 10min, collection CdSe samples is kept to be dissolved in spare in chloroform.
The sulphur powder of the zinc stearate and 0.032g that weigh 0.632g is separately added into the octadecene (ODE) of 2mL and is heated to
120 DEG C of fully dissolvings, cool to 70 DEG C, two kinds of solution are mixed and are saved backup at 70 DEG C.It will be scattered in chloroform
CdSe samples are added in bottle, are heated to 120 DEG C of holding 30m in, are evaporated chloroform, are warming up to 200 DEG C.Extract zinc stearate and sulphur
The octadecene solution of powder injects ZnS shells with 0.2mL/m in, after the completion of injection, is warming up to 230 DEG C of holding 1h, cools to
60 DEG C, reaction product methanol precipitation, eccentric cleaning three times, obtains CdSe/ZnS chloroformic solutions.Take CdSe/ZnS chloroformic solutions,
Mercaptopropionic acid and each 1mL of deionized water are put into bottle and mix, and stir 3h strongly, quantum dot in initial chloroformic solution i.e. by shifting
Into in aqueous solution, extract upper layer aqueous solution, add methanol eccentric cleaning three times, after be scattered in 1mL deionized waters both
CdSe/ZnS quantum dot mother liquors.It is shown in Figure 3, carry out uv-visible absorption spectra, fluorescence spectrum, transmission electron microscope
Characterization.
Three, aptamer-quantum dot is conjugated to prepare
13.9 μ L 10mM SMCC (succinimide -4- (N- maleimides) thiacyclohexane -1-1 carboxylic esters) are added to
One hour is placed in 125 μ L quantum dots and at room temperature to activate quantum dot.In the PBS of 300 μ L mono- times of working concentration of 1mg/ml
5 ' mercapto-modified oligonucleotides are prepared in solution, the dithiothreitol (DTT) (DTT) of 6.1 μ l 1m are added into solution and in room temperature
It is lower to place 30 minutes to reduce aptamers.The solution for activating quantum dot and the solution for reducing aptamers pass through a nap- respectively
5 desalting columns (Sephadex G-25 desalting columns), each solution collect 500 μ l.Then at room temperature by quantum dot and reduction aptamer
React a hour.10.1 μ L beta -mercaptoethanols (100 μM of ultimate densities) are added in mixture to quench conjugation reaction,
And it places 30 minutes at room temperature.Reaction product concentrates (7000 turns, 3000 × g, 15min) using 0.5ml concentrators, then adds
It is added to 200 column Superdex desalting columns and is eluted with PBS.By column purification, it is collected into 250 μ L aptamers-amount altogether
Sub- point.10 μ l aptamers-quantum dot is dissolved in 1ml reverse-osmosis pure waters.With a uv-2100 spectrophotometer
To weigh the absorbance in 260nm.The actual concentrations of aptamer-quantum dot by with initial aptamer concentration
(1.08mg/ml) is compared to determine.The percentage of aptamer conjugation quantum dot is confirmed as 98%.
Four, aptamer-quantum dot is combined with subtilis spore
Shown in Fig. 4 to 6, by 30 μ L aptamers-quantum dot be placed in a test tube and with 600 μ L PBS into
Row dilution.In this typical reaction, the diluted 100 μ L aptamers-quantum dots of PBS are separately added into 900 μ L 107,
In the Bt spores of 106,105,104,103CFU.Mixture is with the of short duration vortex (speed 8) of 3 vortex mixers of VSM and is placed on
20min is reacted at room temperature.A series of reaction of repetitions is carried out with same program：Aptamer-quantum dot is without spore, non-knot
The quantum dot of conjunction and Bt spores, aptamer-quantum dot are combined with the Bt spores of purifying, aptamer-quantum dot and BG
Spore combines.The spore collected before is washed 13mm microporous membrane filters (PVDF, hydrophily, 0.45 μm) twice with PBS.Spore
Son is exposed to be collected after any one of aptamer-quantum dot or uncombined quantum dot and uses 1ml in the filter
The PBS solution washing of one times of working concentration is three times.Washed spore is collected in 1ml PBS.All experiments are duplicate.
Reagent source and its component and its proportioning described in this patent is as follows：
1, reagent source
The special reagent of Primary Chemical used in the present invention (omits the canal of common agents from following commercially available channel
Road)：
CdO (Sinopharm Chemical Reagent Co., Ltd.)；Argon gas (Sinopharm Chemical Reagent Co., Ltd.)；Cd (traditional Chinese medicines
Chemical reagent Co., Ltd of group)；Carbodiimide (EDC) (Sinopharm Chemical Reagent Co., Ltd.)；N- hydroxysuccinimidyls acyl is sub-
Amine (NHS) (Shanghai Chinese medicines group)；；Trishydroxymethylaminomethane Tris (Sinopharm Chemical Reagent Co., Ltd.)；Cadmium oxide
(CdO, content 99.99%), hexadecylamine (HDA, content 90%), trioctylphosphine oxide (TOPO, content 90%), trioctylphosphine
Phosphorus (TOP, content 90%), sulphur powder (S, 99%), mercaptopropionic acid (MPA, 98%).
2, instrument source
Current chart level instruments and meters Co., Ltd on FA1004 type electronic balances
Ying Yu Yu Hua instrument plants of Gongyi City of the type universal mixer Henan Province DC-2
300 type ultraviolet-visible spectrophotometer Thermo Electron Corporation of Evolution
Perkin Elmer companies of the LS-55 sepectrophotofluorometer type sepectrophotofluorometers U.S.
TEM-H-70000FA transmission electron microscope Hitachi, Japan
Millipore companies of the Milli-Q ultrapure water machines U.S.
G25 desalting and purifying columns Beijing Bo Erxi Science and Technology Ltd.s
It although an embodiment of the present invention has been shown and described, for the ordinary skill in the art, can be with
Understanding without departing from the principles and spirit of the present invention can carry out these embodiments a variety of variations, modification, replace
And modification, the scope of the present invention is defined by the appended.
Claims (4)
1. a kind of fluorimetry based on aptamer-quantum dot combination B. thuringiensis spores, which is characterized in that
Include the following steps：
(1) preparation of aptamer-quantum dot：
1.1,13.9 μ L 10mM SMCC (succinimide -4- (N- maleimides) thiacyclohexane -1-1 carboxylic esters) are added to
One hour is placed in 125 μ L quantum dots and at room temperature to activate quantum dot；
1.2,5 ' mercapto-modified oligonucleotides are prepared in the PBS solution of 300 μ L mono- times of working concentration of 1mg/ml, to solution
The middle dithiothreitol (DTT) (DTT) that 6.1 μ l 1m are added simultaneously places 30 minutes to reduce aptamers at room temperature；
1.3, the solution of activation quantum dot and the solution of reduction aptamers pass through a nap-5 desalting columns (Sephadex respectively
G-25 desalting columns), each solution collects 500 μ l；
1.4 then by quantum dot and reduction aptamer react a hour at room temperature.By 10.1 μ L beta -mercaptoethanols (100 μM
Ultimate density) it is added in mixture to quench conjugation reaction, and place 30 minutes at room temperature, by column purification, collect altogether
To 250 μ L aptamers-quantum dot；
1.5,10 μ l aptamers-quantum dot is dissolved in 1ml reverse-osmosis pure waters.It is divided light with a uv-2100
Degree is counted to weigh the absorbance in 260nm；
1.6, the actual concentrations of aptamer-quantum dot with initial aptamer concentration (1.08mg/ml) by being compared
Relatively determine；
(2) aptamer-quantum dot is combined with subtilis spore：
2.1,30 μ L aptamers-quantum dot is placed in a test tube and is diluted with 600 μ L PBS；
2.2, a series of reaction of repetitions is carried out with same program：Aptamer-quantum dot is without spore, uncombined quantum
Point and Bt spores, aptamer-quantum dot are combined with the Bt spores of purifying, and aptamer-quantum dot is combined with BG spores；
2.3, the spore collected before is washed 13mm microporous membrane filters (PVDF, hydrophily, 0.45 μm) twice, spore with PBS
It is exposed to after any one of aptamer-quantum dot or uncombined quantum dot and collects in the filter and with 1ml mono-
Three times, washed spore is collected in 1ml PBS for the PBS solution washing of times working concentration.
2. the fluorescence according to claim 1 based on aptamer-quantum dot combination B. thuringiensis spores is surveyed
Determine method, it is characterised in that：In the step 1.4, reaction product is concentrated using 0.5ml concentrators, is then added to 200 columns
Superdex desalting columns are simultaneously eluted with PBS.
3. the fluorescence according to claim 1 based on aptamer-quantum dot combination B. thuringiensis spores is surveyed
Determine method, it is characterised in that：In the step 1.6, the percentage of aptamer conjugation quantum dot is confirmed as 98%.
4. ultrasonic emulsification device according to claim 1, it is characterised in that：It is in the step (2.2), PBS is dilute
The 100 μ L aptamers-quantum dot released is separately added into the Bt spores of 900 μ L 107,106,105,104,103CFU.Mixing
The object of short duration vortex (speed 8) of 3 vortex mixers of VSM and place react 20min at room temperature.
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| CN201810415673.1A CN108663343A (en) | 2018-05-03 | 2018-05-03 | Fluorimetry based on aptamer-quantum dot combination B. thuringiensis spores |
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| CN201810415673.1A CN108663343A (en) | 2018-05-03 | 2018-05-03 | Fluorimetry based on aptamer-quantum dot combination B. thuringiensis spores |
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101113979A (en) * | 2007-07-25 | 2008-01-30 | 中山大学肿瘤防治中心 | CBA reagent kit for detecting EB virus gp78 antibody and method for making same |
| US20120288852A1 (en) * | 2010-01-15 | 2012-11-15 | Richard Willson | Force Mediated Assays |
| CN103197075A (en) * | 2012-01-16 | 2013-07-10 | 华中农业大学 | Method for detecting Bt protein in transgenic rice by quantum dot |
-
2018
- 2018-05-03 CN CN201810415673.1A patent/CN108663343A/en not_active Withdrawn
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101113979A (en) * | 2007-07-25 | 2008-01-30 | 中山大学肿瘤防治中心 | CBA reagent kit for detecting EB virus gp78 antibody and method for making same |
| US20120288852A1 (en) * | 2010-01-15 | 2012-11-15 | Richard Willson | Force Mediated Assays |
| CN103197075A (en) * | 2012-01-16 | 2013-07-10 | 华中农业大学 | Method for detecting Bt protein in transgenic rice by quantum dot |
Non-Patent Citations (1)
| Title |
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| MILADA IKANOVIC 等: "Fluorescence Assay Based on Aptamer-Quantum Dot Binding to Bacillus thuringiensis Spores", 《JOURNAL OF FLUORESCENCE》 * |
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