CN105137061B - A kind of soil remains the original position ELISA quantitative determination method of bt albumen - Google Patents
A kind of soil remains the original position ELISA quantitative determination method of bt albumen Download PDFInfo
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- 239000002689 soil Substances 0.000 title claims abstract description 104
- 238000002965 ELISA Methods 0.000 title claims abstract description 10
- 238000004445 quantitative analysis Methods 0.000 title claims abstract description 9
- 238000000034 method Methods 0.000 claims abstract description 24
- 238000001514 detection method Methods 0.000 claims abstract description 14
- 239000012535 impurity Substances 0.000 claims abstract description 10
- 239000002245 particle Substances 0.000 claims abstract description 9
- 230000001954 sterilising effect Effects 0.000 claims abstract description 8
- 238000000227 grinding Methods 0.000 claims abstract description 3
- 239000006228 supernatant Substances 0.000 claims description 31
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 30
- 238000004140 cleaning Methods 0.000 claims description 21
- 239000000203 mixture Substances 0.000 claims description 21
- 229910000397 disodium phosphate Inorganic materials 0.000 claims description 16
- 239000007836 KH2PO4 Substances 0.000 claims description 15
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 15
- 239000011780 sodium chloride Substances 0.000 claims description 15
- 239000000376 reactant Substances 0.000 claims description 12
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 claims description 9
- 239000007788 liquid Substances 0.000 claims description 8
- 239000012153 distilled water Substances 0.000 claims description 6
- 229940005654 nitrite ion Drugs 0.000 claims description 6
- 239000000758 substrate Substances 0.000 claims description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 6
- IOVCWXUNBOPUCH-UHFFFAOYSA-M Nitrite anion Chemical compound [O-]N=O IOVCWXUNBOPUCH-UHFFFAOYSA-M 0.000 claims description 5
- 238000006243 chemical reaction Methods 0.000 claims description 5
- 102000004169 proteins and genes Human genes 0.000 claims description 5
- 108090000623 proteins and genes Proteins 0.000 claims description 5
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 4
- 230000012447 hatching Effects 0.000 claims description 4
- YASYEJJMZJALEJ-UHFFFAOYSA-N Citric acid monohydrate Chemical compound O.OC(=O)CC(O)(C(O)=O)CC(O)=O YASYEJJMZJALEJ-UHFFFAOYSA-N 0.000 claims description 3
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 claims description 3
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 claims description 3
- 239000006260 foam Substances 0.000 claims description 3
- 238000002372 labelling Methods 0.000 claims description 3
- 238000002156 mixing Methods 0.000 claims description 3
- 239000001117 sulphuric acid Substances 0.000 claims description 3
- 235000011149 sulphuric acid Nutrition 0.000 claims description 3
- AQLJVWUFPCUVLO-UHFFFAOYSA-N urea hydrogen peroxide Chemical compound OO.NC(N)=O AQLJVWUFPCUVLO-UHFFFAOYSA-N 0.000 claims description 3
- 238000005406 washing Methods 0.000 claims description 3
- 229960002303 citric acid monohydrate Drugs 0.000 claims description 2
- 150000001875 compounds Chemical class 0.000 claims description 2
- 229960000935 dehydrated alcohol Drugs 0.000 claims description 2
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 claims 5
- 235000019800 disodium phosphate Nutrition 0.000 claims 3
- 235000019796 monopotassium phosphate Nutrition 0.000 claims 3
- 238000013459 approach Methods 0.000 abstract description 3
- 238000012544 monitoring process Methods 0.000 abstract description 3
- 229920000742 Cotton Polymers 0.000 description 9
- UAIUNKRWKOVEES-UHFFFAOYSA-N 3,3',5,5'-tetramethylbenzidine Chemical compound CC1=C(N)C(C)=CC(C=2C=C(C)C(N)=C(C)C=2)=C1 UAIUNKRWKOVEES-UHFFFAOYSA-N 0.000 description 7
- 238000010521 absorption reaction Methods 0.000 description 4
- 230000000694 effects Effects 0.000 description 3
- 239000000284 extract Substances 0.000 description 3
- 238000000605 extraction Methods 0.000 description 3
- 238000002331 protein detection Methods 0.000 description 3
- 239000003053 toxin Substances 0.000 description 3
- 231100000765 toxin Toxicity 0.000 description 3
- 102000004190 Enzymes Human genes 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- 230000015556 catabolic process Effects 0.000 description 2
- 238000006731 degradation reaction Methods 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 230000001900 immune effect Effects 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 231100000614 poison Toxicity 0.000 description 2
- 239000002574 poison Substances 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 238000005070 sampling Methods 0.000 description 2
- 230000028327 secretion Effects 0.000 description 2
- BVKZGUZCCUSVTD-UHFFFAOYSA-L Carbonate Chemical compound [O-]C([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-L 0.000 description 1
- 238000008157 ELISA kit Methods 0.000 description 1
- UUWCNZDJRQCXKW-UHFFFAOYSA-N N(=O)O.CC=1C=C(C=C(C1N)C)C1=CC(=C(N)C(=C1)C)C Chemical compound N(=O)O.CC=1C=C(C=C(C1N)C)C1=CC(=C(N)C(=C1)C)C UUWCNZDJRQCXKW-UHFFFAOYSA-N 0.000 description 1
- 229920001213 Polysorbate 20 Polymers 0.000 description 1
- 238000010802 RNA extraction kit Methods 0.000 description 1
- 230000000507 anthelmentic effect Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000002734 clay mineral Substances 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
- 230000009514 concussion Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- UQLDLKMNUJERMK-UHFFFAOYSA-L di(octadecanoyloxy)lead Chemical compound [Pb+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O UQLDLKMNUJERMK-UHFFFAOYSA-L 0.000 description 1
- 229960004756 ethanol Drugs 0.000 description 1
- 239000008187 granular material Substances 0.000 description 1
- 239000003864 humus Substances 0.000 description 1
- 238000011065 in-situ storage Methods 0.000 description 1
- 230000000749 insecticidal effect Effects 0.000 description 1
- 230000000968 intestinal effect Effects 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 239000004570 mortar (masonry) Substances 0.000 description 1
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 1
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 1
- 231100000683 possible toxicity Toxicity 0.000 description 1
- 230000001681 protective effect Effects 0.000 description 1
- 238000000751 protein extraction Methods 0.000 description 1
- 239000002002 slurry Substances 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 239000004094 surface-active agent Substances 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 230000009261 transgenic effect Effects 0.000 description 1
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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
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/535—Production of labelled immunochemicals with enzyme label or co-enzymes, co-factors, enzyme inhibitors or enzyme substrates
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- Health & Medical Sciences (AREA)
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- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Chemical & Material Sciences (AREA)
- Hematology (AREA)
- Urology & Nephrology (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Cell Biology (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
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Abstract
The invention discloses the original position ELISA quantitative determination method remaining Bt albumen in a kind of soil, comprise the following steps: 1) remove the plant and animal residues in soil sample to be measured, it is fully ground, makes soil particle diameter at below 0.2mm by 50 60 mesh sieve；2) soil sample sterilizing 15 20 minutes under high pressure wet heat condition after grinding；3) weigh soil sample 1 2g after sterilizing, put into and 10ml centrifuge tube is carried out remove soil impurity；4) the soil soil sample surface removing impurity is closed；5) the soil soil sample after closing is carried out antibodies detection；The present invention is monitoring Bt albumen residual in soil and impact on soil ecosystem provides a new approach, and this method not only may be used for the detection of soil bt albumen, after changing different antibody, more may be used for the residual protein content detecting in various soil.
Description
Technical field
The present invention relates to protein detection technology field, be specifically related in a kind of soil remain the original position enzyme linked immunological of Bt albumen
Quantitative detecting method.
Background technology
In recent years, along with the continuous expansion of converted Bt crops cultivated area, the potential risk of ecological environment is also increasingly becoming by it
Expert and the focus of scholar's research both at home and abroad.Converted Bt crops, after plantation, in its whole trophophase, can pass through root system
Secretions, pollen, plant stubble discharge Bt toxin to soil, and the Bt toxin of these secretions can be by the clay mineral in soil
With the absorption of the surfactant granules such as humus, and this combined state toxin still has stronger insecticidal activity, in soil environment
Can retain for a long time.Soil is the important place that in ecosystem, material circulation and energy convert, animal in soil and micro-
Biotic population plays very important effect for mass degradation and the energy conversion of soil ecosystem.The poison of these residuals
Element may produce potential toxicity to invertebrates, microbial population in soil, affects soil animal and microbial species
The Diversity structure of group, thus destroy material circulation and the energy conversion of soil.Therefore, research transgenic Bt crops poison egg
Residual in soil and degradation dynamic are of great significance for safety tool of preserving the ecological environment in vain.
Currently for the detection method of residual Bt albumen in soil, the main extracting solution that passes through extracts Bt from soil
ELISA method detection is carried out after albumen.The method of residual Bt albumen in soil of extracting at present mainly has SDS method, carbonate
The methods such as method, artificial anthelmintic intestinal protein extracting solution method, PBST RNA isolation kit.These methods above-mentioned are at pedo relict Bt
Protein Detection plays certain effect, but the most all there is certain defect.These detection soil bt albumen existing
Method, be and first extract after bt albumen with extracting solution, then detect, the method is simple to operate, but for soil
The when of this absorption affinity the strongest medium, often effect will not be highly desirable, some firmly absorption on soil particle surface
Bt albumen is difficult to be eluted, and therefore can not reflect the true residual content of Bt albumen in soil.
Summary of the invention
Goal of the invention: the present invention is directed to the drawback of current existing soil Bt method of protein detection, have developed a kind of new straight
Being connected on the method that soil particle surface carries out enzyme linked immunological detection by quantitative bt albumen in situ, the method can be straight without extracting
Connect and be accurately detected the difficult extraction bt albumen being adsorbed in soil particle surface, truly reflect the residual feelings of Bt albumen in soil
Condition.Meanwhile, in conjunction with the method for bt albumen in existing Detection and Extraction liquid, both data are added, can be complete obtain
The real content of bt albumen in soil.The present invention is monitoring Bt albumen remaining and to soil ecosystem in soil
Impact provides a new approach, and this method not only may be used for the detection of soil bt albumen, is changing difference
Antibody after more may be used for the residual protein content that detects in various soil.
Technical scheme: in order to solve above-mentioned technical problem, the invention provides the original position remaining Bt albumen in a kind of soil
ELISA quantitative determination method, comprises the following steps:
1) remove the plant and animal residues in soil sample to be measured, be fully ground, by 50-60 mesh sieve, soil particle diameter is existed
Below 0.2mm；
2) soil sample sterilizing 15-20 minute under high pressure wet heat condition after grinding；
3) weigh soil sample 1-2g after sterilizing, put into and 10ml centrifuge tube is carried out remove soil impurity；
4) the soil soil sample surface removing impurity is closed；
5) the soil soil sample after closing is carried out antibodies detection:
5.1) soil sample after closing adds 3ml reactant liquor I, after fully shaking up, be horizontally placed at shaking table, room temperature,
50rpm/min, hatches 2h, and period overturns centrifuge tube for several times every 20min, and 10000rpm/min is centrifuged 5min afterwards,
Abandon supernatant；
5.2) add cleaning mixture II, after fully shaking up, be horizontally placed at shaking table, room temperature, 100rpm/min, shake 5min；
10000rpm/min is centrifuged 5min afterwards, abandons supernatant；
5.3) step 5.2 is repeated) three to five times；
5.4) add 3ml reactant liquor II, after fully shaking up, be horizontally placed at shaking table, room temperature, 50rpm/min, hatch 1h,
Period overturns centrifuge tube for several times every 20min, and 10000rpm/min is centrifuged 5min afterwards, abandons supernatant；
5.5) add cleaning mixture II, after fully shaking up, be horizontally placed at shaking table, room temperature, 100rpm/min, shake 5min；
10000rpm/min is centrifuged 5min afterwards, abandons supernatant；
5.6) step 5.5 three to five times are repeated；
5.7) add the 3ml TMB nitrite ion of fresh configuration, after fully shaking up, be horizontally placed at shaking table, room temperature,
100rpm/min, hatches 30min, and period overturns centrifuge tube for several times every 10min, adds 2M sulphuric acid after hatching end
1ml terminates reaction, and 10000rpm/min is centrifuged 5min afterwards, takes supernatant and detects in microplate reader 450nm.
Wherein, above-mentioned steps 3) in remove soil impurity step as follows:
3.1) add cleaning mixture I 3ml, after fully shaking up, be horizontally placed at shaking table, room temperature, 200rpm/min, shake 10min,
10000rpm/min is centrifuged 5min afterwards, abandons supernatant；
3.2) step 3.1 is repeated) once；
3.3) add cleaning mixture II 3ml, after fully shaking up, be horizontally placed at shaking table, 37 DEG C, 200rpm/min, shake 5min,
10000rpm/min is centrifuged 5min afterwards, abandons supernatant；
3.4) step 3.3 is repeated), until supernatant no longer has foam.
Wherein, above-mentioned steps 4) in soil sample surface carry out close step as follows:
4.1) soil sample after washing adds 3ml confining liquid, after fully shaking up, be horizontally placed at shaking table, room temperature,
50rpm/min, hatches 1h, and period overturns centrifuge tube for several times every 10min, and 10000rpm/min is centrifuged 5min afterwards,
Abandon supernatant；
4.2) add cleaning mixture II, after fully shaking up, be horizontally placed at shaking table, room temperature, 100rpm/min, shake 5min；
10000rpm/min is centrifuged 5min afterwards, abandons supernatant；
4.3) step 4.2 is repeated) three to five times.
Wherein, above-mentioned cleaning mixture I formula is: NaCl 137mmol/L, KCl 2.7mmol/L, Na2HPO410mmol/L,
KH2PO42mmol/L, 2% (m/v) SDS, 0.2mmol/L EDTA.
Wherein, above-mentioned cleaning mixture II formula is: NaCl 137mmol/L, KCl 2.7mmol/L, Na2HPO410mmol/L,
KH2PO42mmol/L。
Wherein, above-mentioned confining liquid formula is: NaCl 137mmol/L, KCl 2.7mmol/L, Na2HPO410mmol/L,
KH2PO42mmol/L, 8%m/vBSA, 0.5%v/vTween-20.
Wherein, above-mentioned reactant liquor I formula: NaCl 137mmol/L, KCl 2.7mmol/L, Na2HPO410mmol/L,
KH2PO42mmol/L, 5%m/vBSA, 0.5%v/vTween-20,0.5%v/vBt protein antibodies.
Wherein, above-mentioned reactant liquor II formula: NaCl 137mmol/L, KCl 2.7mmol/L, Na2HPO410mmol/L,
KH2PO42mmol/L, 5%m/vBSA, 0.5%v/vTween-20,0.05%v/vHRP labelling two resists.
Wherein, the compound method of above-mentioned TMB (3,3', 5,5'-tetramethyl benzidine) nitrite ion is: weigh TMB (3,3', 5,5'-
Tetramethyl benzidine) 20mg, dissolve with dehydrated alcohol 10ml, fully add distilled water constant volume after vibration to 100ml, this
For substrate A；Take the hydrogen peroxide urea 0.64ml of citric acid monohydrate 2.1g, anhydrous Na 2HPO4 2.82g, 0.75%,
Distilled water is settled to 100ml, and this is substrate B；During use, A:B respectively takes 1.5ml mixing.
Beneficial effect: the present invention is monitoring Bt albumen residual in soil and impact on soil ecosystem provides
Article one, new approach, and this method not only may be used for the detection of soil bt albumen, after changing different antibody more
May be used for the residual protein content detecting in various soil.
Detailed description of the invention
Below technical solution of the present invention is described in detail, but protection scope of the present invention is not limited to described embodiment.
Embodiment 1
Pedotheque employed in this programme is taken respectively from In Nanjing continuous 1 year, and plantation in 2 years and 5 years turns Bt Cotton Gossypii
With the cotton field of normal conon, sampling position is the Nanjing six directions.
At the florescence of Cotton Gossypii, turning Bt cotton field and conventional cotton field random acquisition soil sample, use 5 point sampling methods, every piece of cotton field sets
Put 5 sampled points.Each sample point, at a distance of 30～50m, randomly selects 5 strain Cotton Gossypiis, in every strain around each sampled point
(in main root 5cm, away from earth's surface 5～20cm), take about 500g soil around the rhizosphere of Cotton Gossypii altogether, load and seal bag, make
It it is a test specimen.Laboratory taken back by the sample gathered, and-20 DEG C of Refrigerator stores are to be measured.
Take 1-2g soil, carry out as follows:
1. remove the plant and animal residues in soil sample to be measured, be fully ground so that soil particle diameter (can pass through 50-60 at below 0.2mm
Mesh sieve)；
2. soil sample (103.4 kPas of vapour pressures, 121.3 DEG C) sterilizing 15-20 minute under high pressure wet heat condition；
3. take the soil sample after sterilizing and put in 10ml centrifuge tube, in the steps below removal soil impurity:
3.1 add cleaning mixture I (cleaning mixture I formula: NaCl 137mmol/L, KCl 2.7mmol/L, Na2HPO4
10mmol/L, KH2PO42mmol/L, 2% (m/v) SDS, 0.2mmol/L EDTA) 3ml, fully shakes
After even, it be horizontally placed at shaking table, room temperature, 200rpm/min, shake 10min.10000rpm/min is centrifuged afterwards
5min, abandons supernatant；
3.2 repeat step 3.1 once；
3.3 add cleaning mixture II (cleaning mixture II formula: NaCl 137mmol/L, KCl 2.7mmol/L, Na2HPO4
10mmol/L, KH2PO42mmol/L) 3ml, after fully shaking up, is horizontally placed at shaking table, 37 DEG C, 200rpm/min,
Concussion 5min.10000rpm/min is centrifuged 5min afterwards, abandons supernatant；
3.4 repeat step 3.3, until supernatant no longer has foam.
The most in the steps below soil sample surface is closed:
4.1 to washing after soil samples in add 3ml confining liquid (confining liquid formula: NaCl 137mmol/L, KCl 2.7mmol/L,
Na2HPO410mmol/L, KH2PO42mmol/L, 8% (m/v) BSA, 0.5% (v/v) Tween-20),
After fully shaking up, being horizontally placed at shaking table, room temperature, 50rpm/min, hatch 1h, period overturns every 10min
Centrifuge tube is for several times.10000rpm/min is centrifuged 5min afterwards, abandons supernatant；
4.2 add cleaning mixture II, after fully shaking up, are horizontally placed at shaking table, room temperature, 100rpm/min, shake 5min；It
Rear 10000rpm/min is centrifuged 5min, abandons supernatant；
4.3 repeat step 4.2 three to five times.
Carry out antibodies detection the most in the steps below:
5.1 soil samples after closing add 3ml reactant liquor I (reactant liquor I formula: NaCl 137mmol/L, KCl
2.7mmol/L, Na2HPO410mmol/L, KH2PO42mmol/L, 5% (m/v) BSA, 0.5% (v/v)
Tween-20,0.5% (v/v) Bt protein antibodies), after fully shaking up, it is horizontally placed at shaking table, room temperature, 50rpm/min,
Hatching 2h, period overturns centrifuge tube for several times every 20min.10000rpm/min is centrifuged 5min afterwards, abandons
Clear liquid；
5.2 add cleaning mixture II, after fully shaking up, are horizontally placed at shaking table, room temperature, 100rpm/min, shake 5min；It
Rear 10000rpm/min is centrifuged 5min, abandons supernatant；
5.3 repeat step 5.2 three to five times；
5.4 add 3ml reactant liquor II (reactant liquor II formula: NaCl 137mmol/L, KCl 2.7mmol/L, Na2HPO4
10mmol/L, KH2PO42mmol/L, 5% (m/v) BSA, 0.5% (v/v) Tween-20,0.05% (v/v)
HRP labelling two resists), after fully shaking up, it is horizontally placed at shaking table, room temperature, 50rpm/min, hatches 1h, phase
Between overturn centrifuge tube for several times every 20min.10000rpm/min is centrifuged 5min afterwards, abandons supernatant；
5.5 add cleaning mixture II, after fully shaking up, are horizontally placed at shaking table, room temperature, 100rpm/min, shake 5min；It
Rear 10000rpm/min is centrifuged 5min, abandons supernatant；
5.6 repeat step 5.5 three to five times；
(TMB nitrite ion formula: weigh TMB 20mg, with anhydrous for the 3ml TMB nitrite ion of the 5.7 fresh configurations of addition
Ethanol 10ml dissolves, and fully adds distilled water constant volume after vibration to 100ml, and this is substrate A；Substrate B, takes one
The hydrogen peroxide urea 0.64ml of water citric acid 2.1g, anhydrous Na 2HPO4 2.82g, 0.75%, distilled water constant volume
To 100ml.During use, A:B respectively takes 1.5ml mixing), after fully shaking up, it is horizontally placed at shaking table, room temperature,
100rpm/min, hatches 30min, and period overturns centrifuge tube for several times every 10min.2M is added after hatching end
Sulphuric acid 1ml terminates reaction.10000rpm/min is centrifuged 5min afterwards, takes supernatant and enters in microplate reader 450nm
Row detection；
Comparative example 1
Take 1-2g soil (soil sample is in the same manner as in Example 1), take SDS method [ZL201010137168.9] to carry out
Bt protein extraction detects, as follows:
1. taking 100g pedotheque, the mesh screen using aperture to be 48 μm sieves the impurity such as removal root system of plant residuum, claims
Take the pedotheque after 5g sieves to be placed in mortar, grind 1min and make soil particle fine uniform；
2. the pedotheque after being ground by 0.5g is placed in 2mL centrifuge tube, adds 1.5mL SDS extracting solution, is placed in
Vibrate on vortex mixed instrument 1min so that it is is sufficiently mixed uniformly；
3. above-mentioned Soil Slurry is placed in controllable temperature shaking table, 200r min at 50 DEG C-1Vibration 4～16h；
4. the centrifuge tube after vibration is taken out, be immediately placed in centrifuge, 12000r min-1Centrifugal 1～2min；
5., after centrifugal end, carefully draw the supernatant in centrifuge tube with micropipettor, proceed in new centrifuge tube, should
Supernatant is the solution containing Bt albumen；
6. use ELISA kit that the solution containing Bt albumen is carried out quantitatively.
Experimental result:
The soil of the present invention remains original position ELISA quantitative determination method and the SDS method of Bt albumen
[ZL201010137168.9] extracts the comparision contents result of soil Bt albumen
1 two kinds of methods extraction Cotton Soil Bt albumen results of table:
As seen from the above table, one of the soil Bt albumen percentage only accounting for soil total Bt albumen that employing SDS method an elutes left side
The right side, illustrates that the Bt albumen major part in soil is to adsorb combination to be present in soil, is difficult to be eluted,
And use the present invention can detect the Bt albumen that this part and soil particle are combined closely, thus measure accurately and be unearthed
The real content of Bt albumen in earth.
It addition, by detection plantation different years turn Bt Cotton Gossypii Cotton Soil after it was found that the Bt egg of adsorption by soil
Bai Hanliang does not increase along with the growth of Planting Years, explanation soil may have absorption and the protective effect of Bt albumen
Certain saturation capacity, Bt albumen will not be accumulated.
Claims (4)
1. a soil remains the original position ELISA quantitative determination method of Bt albumen, it is characterised in that comprise the following steps:
1) remove the plant and animal residues in soil sample to be measured, be fully ground, make soil particle diameter at below 0.2mm by 50-60 mesh sieve；
2) soil sample sterilizing 15-20 minute under high pressure wet heat condition after grinding；
3) weigh soil sample 1-2g after sterilizing, put into and 10ml centrifuge tube is carried out remove soil impurity；
4) the soil soil sample surface removing impurity is closed；
5) the soil soil sample after closing is carried out antibodies detection:
5.1) adding 3ml reactant liquor I in the soil sample after closing, after fully shaking up, be horizontally placed at shaking table, room temperature, 50rpm/min, hatch 2h, period overturns centrifuge tube for several times every 20min, and 10000rpm/min is centrifuged 5min afterwards, abandons supernatant；
5.2) add cleaning mixture II, after fully shaking up, be horizontally placed at shaking table, room temperature, 100rpm/min, shake 5min；10000rpm/min is centrifuged 5min afterwards, abandons supernatant；
5.3) step 5.2 is repeated) three to five times；
5.4) adding 3ml reactant liquor II, after fully shaking up, be horizontally placed at shaking table, room temperature, 50rpm/min, hatch 1h, period overturns centrifuge tube for several times every 20min, and 10000rpm/min is centrifuged 5min afterwards, abandons supernatant；
5.5) add cleaning mixture II, after fully shaking up, be horizontally placed at shaking table, room temperature, 100rpm/min, shake 5min；10000rpm/min is centrifuged 5min afterwards, abandons supernatant；
5.6) step 5.5 three to five times are repeated；
5.7) the 3ml TMB nitrite ion of fresh configuration is added, after fully shaking up, it is horizontally placed at shaking table, room temperature, 100rpm/min, hatch 30min, period overturns centrifuge tube for several times every 10min, adding 2M sulphuric acid 1ml after hatching end and terminate reaction, 10000rpm/min is centrifuged 5min afterwards, takes supernatant and detects in microplate reader 450nm；
Described cleaning mixture II formula is: NaCl 137mmol/L, KCl 2.7mmol/L, Na2HPO4 10mmol/L, KH2PO4 2mmol/L；
Described reactant liquor I formula: NaCl 137mmol/L, KCl 2.7mmol/L, Na2HPO4 10mmol/L, KH2PO4 2mmol/L, 5%m/vBSA, 0.5%v/vTween-20,0.5%v/vBt protein antibodies；
Described reactant liquor II formula: NaCl 137mmol/L, KCl 2.7mmol/L, Na2HPO4 10mmol/L, KH2PO4 2mmol/L, 5%m/vBSA, 0.5%v/vTween-20,
0.05%v/vHRP labelling anti-Bt protein antibodies.
A kind of soil the most according to claim 1 remains the original position ELISA quantitative determination method of Bt albumen, it is characterised in that described step 3) is removed soil impurity step as follows:
3.1) adding cleaning mixture I 3ml, after fully shaking up, be horizontally placed at shaking table, room temperature, 200rpm/min, shake 10min, 10000rpm/min is centrifuged 5min afterwards, abandons supernatant；
3.2) step 3.1 is repeated) once；
3.3) add cleaning mixture II 3ml, after fully shaking up, be horizontally placed at shaking table, 37 DEG C, 200rpm/min, shake 5min, 10000rpm/min is centrifuged 5min afterwards, abandons supernatant；
3.4) step 3.3 is repeated), until supernatant no longer has foam；
Described cleaning mixture I formula is: NaCl 137mmol/L, KCl 2.7mmol/L, Na2HPO4
10mmol/L, KH2PO4
2mmol/L, 2%m/vSDS, 0.2mmol/L EDTA.
In a kind of soil the most according to claim 1 remain Bt albumen original position ELISA quantitative determination method, it is characterised in that in described step 4) soil sample surface carry out close step as follows:
4.1) adding 3ml confining liquid in the soil sample after washing, after fully shaking up, be horizontally placed at shaking table, room temperature, 50rpm/min, hatch 1h, period overturns centrifuge tube for several times every 10min, and 10000rpm/min is centrifuged 5min afterwards, abandons supernatant；
4.2) add cleaning mixture II, after fully shaking up, be horizontally placed at shaking table, room temperature, 100rpm/min, shake 5min；10000rpm/min is centrifuged 5min afterwards, abandons supernatant；
4.3) step 4.2 is repeated) three to five times；
Described confining liquid formula is: NaCl 137mmol/L, KCl 2.7mmol/L, Na2HPO4 10mmol/L, KH2PO4 2mmol/L, 8%m/vBSA, 0.5%v/vTween-20.
A kind of soil the most according to claim 1 remains the original position ELISA quantitative determination method of Bt albumen, it is characterized in that, the compound method of described TMB nitrite ion is: weigh TMB 20mg, dissolves with dehydrated alcohol 10ml, fully adding distilled water constant volume after vibration to 100 ml, this is substrate A；Take citric acid monohydrate 2.1g, anhydrous Na2HPO4 2.82g, the hydrogen peroxide urea 0.64ml of 0.75%, distilled water is settled to 100ml, and this is substrate B；During use, A:B respectively takes 1.5ml mixing.
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| CN101812121A (en) * | 2010-04-01 | 2010-08-25 | 环境保护部南京环境科学研究所 | Method for efficiently extracting soil residual BT protein based on SDS buffer solution |
| CN102095855A (en) * | 2010-12-23 | 2011-06-15 | 浙江大学 | Insecticidal crystal protein CrylAc enzyme linked immunosorbent detection kit |
| CN102590523A (en) * | 2011-01-06 | 2012-07-18 | 宋敦伦 | Method for testing trace Bt (bacillus thuringiensis) toxic protein in bodies of insects in transgenic Bt crop habitat |
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| CN101812121A (en) * | 2010-04-01 | 2010-08-25 | 环境保护部南京环境科学研究所 | Method for efficiently extracting soil residual BT protein based on SDS buffer solution |
| CN102095855A (en) * | 2010-12-23 | 2011-06-15 | 浙江大学 | Insecticidal crystal protein CrylAc enzyme linked immunosorbent detection kit |
| CN102590523A (en) * | 2011-01-06 | 2012-07-18 | 宋敦伦 | Method for testing trace Bt (bacillus thuringiensis) toxic protein in bodies of insects in transgenic Bt crop habitat |
Non-Patent Citations (4)
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| Bt 蛋白在几种土壤中的降解与残留;黄威等;《华中农业大学学报》;20090430;第28卷(第2期);第169-173页 * |
| Determination of insecticidal Cry1Ab protein in soil collected in the final growing seasons of a nine-year field trial of Bt-maize MON810;Helga Gruber,et al.;《Transgenic Res》;20110416;第21卷;第77-88页 * |
| Effects of physicochemical interactions and microbial activity on the persistence of Cry1Aa Bt (Bacillus thuringiensis) toxin in soil;Nordine Helassa，et al.;《Soil Biology & Biochemistry》;20110212;第43卷;第1089-1097页 * |
| 苏云金杆菌HBF-1 菌株在土壤中毒蛋白含量的ELISA检测;吴瑞华等;《中国生物防治》;20070831;第23卷(第3期);第263-268页 * |
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