CN104569432B - The detection method of a kind of transgene component in food and device - Google Patents
The detection method of a kind of transgene component in food and device Download PDFInfo
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- CN104569432B CN104569432B CN201510005351.6A CN201510005351A CN104569432B CN 104569432 B CN104569432 B CN 104569432B CN 201510005351 A CN201510005351 A CN 201510005351A CN 104569432 B CN104569432 B CN 104569432B
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- G—PHYSICS
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- 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
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
- G01N33/6827—Total protein determination, e.g. albumin in urine
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Abstract
The invention discloses the detection method of a kind of transgene component in food, comprising: step one, extract raw-material total protein；Step 2, the gross protein obtaining step one are expelled in detection device, make total protein by detection device, detection device includes sample adding device, many doughnut polypropylene screens and the first porous plate body, it is attached with the first antibody specific binding with transgene protein on doughnut polypropylene screen, sample adding device includes the second porous plate body and cone, and the two ends of doughnut polypropylene screen are separately fixed on the first and second porous plate bodys and through with the hole on it respectively；Step 3, washing；Step 4, by specific binding with transgenic protein and be expelled to detection device with markd SA well, makes SA by detection device；Step 5, washing；Step 6, whether detection doughnut polypropylene screen exists mark, and by whether there is transgene component in testing result and raw material connects.
Description
Technical field
The present invention relates to detection method and the device of a kind of transgene component in food.
Background technology
By technique for gene engineering, one or more allogenic genes are transferred to certain specific organism
In, and making it effectively give expression to corresponding product (polypeptide or protein), this process is transgenosis.With
Genetically modified organism be Raw material processing produce food be exactly GM food.Originate according to GM food
Difference can be divided into vegetalitas GM food, animality GM food and microbes GM food.
GM food has more advantage: can increase crop yield；Production cost can be reduced；Work can be strengthened
Thing insect pest, antiviral etc. ability；Improve agricultural product storage property.The safety evaluation of genetically modified organism is
Genetically modified organism and products thereof marketization and the premise of commercialization, the inspection of transgene component in safety evaluation
Survey is an important content, set up genetically modified organism quick, easy, accurately detection method comment for safety
Valency and management lay the foundation.The detection of vegetalitas GM food mainly uses following two technology paths,
One is the foreign gene that the technology for detection such as application PCR, Southern are inserted, and two is to use Elisa, Western
Albumen Deng detection transgene expression.The detection of protein level, is mainly based upon antigen and antibody is mutual
The immunological method of effect, for external source destination gene expression specific in genetically modified plants and products thereof
Albumen carries out qualitative and quantitative detection, and its result is relatively more accurate, and reliability is also more preferable, but Elisa
The means of the detection albumen current with Western etc. are all comparatively laborious, go out result also slower.Especially exist
The position that customs etc. import and export, the detection work of transgene component is very loaded down with trivial details and inefficient.
Content of the invention
An object of the present invention is to provide the detection method of a kind of transgene component in food, at protein
Transgene component in quick detection food in level；
It is a further object of the present invention to provide the detection device of a kind of transgene component in food, this device energy
The enough ground of binding specificity quickly and easily albumen, and use simple, efficiency is high.
The technical scheme that the present invention provides is:
A kind of detection method of transgene component in food, comprising:
Step one, extract raw-material total protein；
Step 2, the gross protein obtaining step one are expelled to the well of a detection device, make total egg
White matter by described detection device, this detection device include sample adding device, many doughnut polypropylene screens,
With the first porous plate body, described doughnut polypropylene screen is attached with and the transgenosis in described total protein
The first antibody that protein-specific combines, described sample adding device includes the second porous plate body and is arranged at described
On second porous plate body and the cone of a formed accommodation space, the diameter of described cone is arrived by down
On be gradually reduced and its free end forms described well, one end of described many doughnut polypropylene screens
It is fixed on described second porous plate body and connect with the hole on described second porous plate body, in described many
The other end of hollow fiber polypropylene screen be fixed on described first porous plate body and with on described first porous plate body
Hole connection, when described total protein by described detection device when, close on described first porous plate body
Hole so that the flowing of total protein occurs over just one end and the institute of described many doughnut polypropylene screens
State between the fenestra of doughnut polypropylene screen, make this transgenic protein be incorporated into described first antibody
And be attached on described doughnut polypropylene screen；Carry out step 3 afterwards；
Step 3, opens the hole on described first porous plate body, washs described doughnut polypropylene screen
Chamber in residue；Carry out step 4 afterwards；
Step 4, closes the hole on described first porous plate body again, will be with described transgenic protein
Specific binding and be expelled to described detection device with markd SA well, makes second
Antibody is by described detection device；Carry out step 5 afterwards；
Step 5, is again turned on the hole on described first porous plate body, washs described doughnut poly-third
Residue in the chamber of alkene film；Carry out step 6 afterwards；With
Step 6, detects the mark that whether there is described SA on described doughnut polypropylene screen,
If there is described mark, then it is assumed that this total protein exists this transgene protein, and judges in this raw material
There is transgene component.
Preferably, in the detection method of described transgene component in food, described step one is extracted former
The detailed process of the total protein of material includes:
(1.1) raw material are placed in a sealing bag, in liquid nitrogen after quick-frozen 30s, are placed in mortar use
Liquid nitrogen grinding becomes powder, backward described sealing bag according to volume mass than 6: 1 to raw material add separation
Buffer solution, in 0～4 DEG C of abundant mixing；
(1.2) it is drawn into the mixture obtaining in step (1.1) in centrifuge tube, in temperature 4 DEG C,
After 12000rpm centrifuges 10min, abandon supernatant, afterwards according to being to add at 1: 1 with raw-material volume mass ratio
Enter the resuspended precipitation of lysate, stand 10min；And,
(1.3) by the mixture in step (1.2) in temperature 4 DEG C, 12000rpm centrifuges 20min,
Take supernatant and i.e. obtain described total protein.
Preferably, in the detection method of described transgene component in food, in described step (1.1),
The shape of described sealing bag and the inwall laminating of described mortar, described sealing bag includes connecting each other from outside to inside
The outer layer connecing and internal layer, be provided with several beades, described sealing between described outer layer and described internal layer
Bag is made up of polysulfone material.
Preferably, in the detection method of described transgene component in food, closed by using diaphragm seal
With the hole opened on described first porous plate body.
Preferably, it, in the detection method of described transgene component in food, wherein said is labeled as horseradish
Whether peroxidase, in described step 6, utilize and deposit on the described doughnut polypropylene screen of DAB detection
At the mark of described SA, if producing palm fibre when DAB being joined on described doughnut polypropylene screen
Look precipitates, then judge there is described mark on described doughnut polypropylene screen.
Preferably, in the detection method of described transgene component in food, in described step 2, will step
Rapid one gross protein obtaining is drawn in a syringe, described by being inserted into described syringe afterwards
It in well thus is expelled to this total protein in this detection device.
Preferably, in the detection method of described transgene component in food, described raw material include corn,
Paddy rice, soybean and wheat.
The detection device of a kind of transgene component in food, this detection device includes sample adding device, in many
Hollow fiber polypropylene screen and the first porous plate body, described doughnut polypropylene screen is attached with described
The specific binding first antibody of transgenic protein in total protein, described sample adding device includes more than second
Orifice plate body and being arranged on described second porous plate body and the cone of a formed accommodation space, described
The diameter of cone is gradually reduced from down to up and its free end forms well, described many doughnuts
One end of polypropylene screen is fixed on described second porous plate body and connects with the hole on described second porous plate body
Logical, the other end of described many doughnut polypropylene screens be fixed on described first porous plate body and with described
Hole connection on first porous plate body, when described total protein is by described detection device, closes described
Hole on first porous plate body, so that the flowing of total protein occurs over just described many doughnuts poly-third
Between one end of alkene film and the fenestra of described doughnut polypropylene screen, make this transgenic protein with described
First antibody combines and is attached on described doughnut polypropylene screen.
Beneficial effects of the present invention: in the detection device that the present invention provides, by the first of transgenic protein
Antibody is attached on doughnut polypropylene screen, thus flows through this by making raw-material total protein to be detected
Detection device detects whether there is transgenic protein in raw material, and by syringe by protein
Liquid joins in the detection device of the present invention, it is easy to operation, assembly of the invention can be effectively and rapidly
Detect whether there is transgene protein.Can quickly detect transgene protein according to the method for the present invention.At egg
During white matter is extracted, loading raw material in one sealing bag, the shape of described sealing bag is interior with described mortar
Wall is fitted, and so, sealing bag when grinding in mortar will not be moved, and described sealing bag includes from outward
It to the interior outer layer being connected to each other and internal layer, between described outer layer and described internal layer, is provided with several beades.
So, when grinding, stone pestle contacts with bead so that the number of times that raw material are ground in sealing bag
All being greatly increased with area, simultaneously described sealing bag is made up of polysulfone material, wear-resisting, low temperature resistant, will not
Damage.And, use dissociating buffer by after resuspended for the raw material pulverized, it is easy to from sealing bag
In all draw out, and can operate at low ambient temperatures, it is to avoid during common mortar grinder, due to
Liquid nitrogen rapid evaporation, and have to quickly from mortar, take out raw-material powder.Sometimes, owing to taking out
Not in time, cause raw-material waste, also result in the waste of time and manpower and materials.The present invention's
Method avoids this kind of situation.The method of the present invention is quick, accurate for the detection of transgene protein, effect
Rate is high.
Brief description
Fig. 1 is the structural representation during hole closing of the first porous plate body of detection device of the present invention
Figure；
Fig. 2 is structural representation when opening of the hole of the first porous plate body of detection device of the present invention
Figure.
Detailed description of the invention
The present invention is described in further detail below in conjunction with the accompanying drawings, to make those skilled in the art's reference
Specification word can be implemented according to this.
Nuclear isolation buffer: 0.25M sucrose, 18mM PIPES (pH6.8), 5mM MgCl2,
60mM KCl, 15mM NaCl, 1mM CaCl2, the Triton X-100 of 1.0%, 1mM PMSF (egg
White enzyme inhibitor is now with now adding).Fresh configuration and in 4 DEG C of preservations.
Nuclei lysis buffer: 50mM HEPES (pH7.5), 150mM NaCl, 1mM EDTA,
1.0%SDS, 0.1% courage oxycholic acid sodium, the TritonX-100 of 1.1%, 1 μ g/mLpestatinA and 1 μ
G/mL aprotinin (protease inhibitors now with now add).Fresh configuration and in 4 DEG C of preservations.
TBST buffer solution: 12.114gTris alkali, adjusts pH to 7.5,16.332gNaCl, 1M1Tween-20,
Add water to 1L.
DAB horseradish peroxidase colour reagent box is bought from the green skies, and production code member is P0202.
The present invention provides the detection method of a kind of transgene component in food, and the sample of the method detection is liquid
Body, comprising:
Step one, extracting raw-material total protein, the detailed process extracting raw-material total protein includes:
(1.1) raw material are placed in a sealing bag, in liquid nitrogen after quick-frozen 30s, are placed in mortar use
Liquid nitrogen grinding becomes powder, backward described sealing bag according to volume mass than 6: 1 to raw material add separation
Buffer solution (if desired for extracting cytoplasmic protein, then adds common dissociating buffer, if desired extracts
Nuclear extract matter, then use nuclear isolation buffer), in the present embodiment, the as above institute that herein adds
The nuclear isolation buffer stated, then at 0～4 DEG C of abundant mixing；The shape of described sealing bag is ground with described
The inwall laminating of alms bowl, described sealing bag includes outer layer and the internal layer being connected to each other from outside to inside, described outer layer
And it is provided with several beades between described internal layer, described sealing bag is made up of polysulfone material.
(1.2) it is drawn into the mixture obtaining in step (1.1) in centrifuge tube, in temperature 4 DEG C,
After 12000rpm centrifuges 10min, abandon supernatant, afterwards according to being to add at 1: 1 with raw-material volume mass ratio
Enter the resuspended precipitation of lysate, add herein for Nuclei lysis buffer, stand 10min；And,
(1.3) by the mixture in step (1.2) in temperature 4 DEG C, 12000rpm centrifuges 20min,
Take supernatant and i.e. obtain described total protein.A syringe is utilized to be drawn to gross protein in this syringe.When
So, it is possible to take other purification measures to carry out this total protein further after purification, then carry out step 2.
Step 2, by described syringe is inserted into obtain in described well thus by step one should
Total protein is expelled in detection device, makes gross protein pass through described detection device, (during detection,
Need on this syringe, apply certain pressure, so that during the liquid in gross protein sample can pass through
The fenestra of hollow fiber polypropylene screen flows out.This detection device includes sample adding device, many doughnuts poly-third
Alkene film and the first porous plate body, described doughnut polypropylene screen is attached with in described total protein
The specific binding first antibody of transgene protein, described sample adding device includes the second porous plate body and setting
On described second porous plate body and the cone of a formed accommodation space, the diameter of described cone
It is gradually reduced from down to up and its free end forms described well, described many doughnut polypropylene screens
One end be fixed on described second porous plate body and connect with the hole on described second porous plate body, described
The other end of many doughnut polypropylene screens be fixed on described first porous plate body and with described first porous
Hole connection on plate body, when described total protein is by described detection device, under the first porous plate body
Seal up diaphragm seal to close the hole on described first porous plate body, so that the flowing of total protein occurs over just
Between one end of described many doughnut polypropylene screens and the fenestra of described doughnut polypropylene screen, make
This transgenic protein is combined and is attached to described first antibody on described doughnut polypropylene screen；It
After carry out step 3；There is a lot of fold on the surface of this doughnut polypropylene screen, to increase its surface area,
And the contact area with total protein, in a detection device, arrange in 20～30 5～8cm length
Hollow fiber polypropylene screen.Every doughnut polypropylene screen oneself is formed with a chamber, every doughnut
A diameter of 5～10mm of polypropylene screen.
Step 3, removes diaphragm seal opening the hole on described first porous plate body, uses TBST to delay
Rush the residue 3 times in the chamber of the liquid described doughnut polypropylene screen of washing, each 20mL, 10min；It
After carry out step 4；
Step 4, closes the hole on described first porous plate body again with new diaphragm seal, will with described
Transgenic protein specific binding and be expelled to described detection the adding of device with markd SA
Sample hole, makes SA pass through described detection device；Carry out step 5 afterwards；Wherein said be labeled as peppery
Root peroxidase, SA and this horseradish peroxidase.
Step 5, removes the hole that current diaphragm seal is again turned on described first porous plate body, uses
Residue in the chamber of doughnut polypropylene screen described in TBST buffer solution 3 times, each 20mL,
10min；Carry out step 6 afterwards；With
Step 6, utilizes DAB horseradish peroxidase colour reagent box (the green skies) to detect described hollow fine
Whether there is the mark of described SA, if DAB is joined described doughnut on dimension polypropylene screen
Produce brown precipitate when on polypropylene screen, then judge there is described mark on described doughnut polypropylene screen.
If there is described mark, then it is assumed that this total protein exists this transgene protein, and judges in this raw material
There is transgene component.The method of the present invention both can be detected in cytoplasm the transgenic protein expressed,
Also the transgenic protein expressed can be detected in nucleus.
In one embodiment of the invention, described raw material are in corn, paddy rice, soybean and wheat
Any one.
The present invention also provides the detection device of a kind of transgene component in food, as depicted in figs. 1 and 2,
This detection device includes sample adding device, many doughnut polypropylene screens 3 and the first porous plate body 2, institute
State on doughnut polypropylene screen 3 be attached with specific binding with the transgenic protein in described total protein
First antibody, described sample adding device includes the second porous plate body 4 and is arranged at described second porous plate body
The cone 5 of a upper and formed accommodation space, the diameter of described cone 5 gradually subtracts from down to up
Little and its free end forms well 1, and one end of described many doughnut polypropylene screens 3 is fixed on institute
State the second porous plate body 4 and connect with the hole on described second porous plate body 4, described many hollow fibres
Dimension polypropylene screen 3 the other end be fixed on described first porous plate body 2 and with described first porous plate body 2
On hole connection, when described total protein is by described detection device, as it is shown in figure 1, close described
Hole on first porous plate body 2, so that the flowing of total protein occurs over just described many doughnuts and gathers
Between one end of propylene film 3 and the fenestra of described doughnut polypropylene screen, make this transgenic protein with
Described first antibody combines and is attached on described doughnut polypropylene screen, and other waste liquids have just flowed out this
Detection device, can place a waste liquid pool and catch waste liquid in lower section.When needs wash, such as Fig. 2 institute
Show, open the hole on this first porous plate body 2, make cleaning solution flow out.
Although embodiment of the present invention are disclosed as above, but it is not restricted to specification and embodiment party
Listed utilization in formula, it can be applied to various applicable the field of the invention completely, for being familiar with ability
For the personnel in territory, be easily achieved other modification, therefore without departing substantially from claim and etc. homotype
Enclosing under limited universal, the present invention is not limited to specific details and shown here as the figure with description
Example.
Claims (8)
1. the detection method of a transgene component in food, comprising:
Step one, extract raw-material total protein；
Step 2, the gross protein obtaining step one are expelled to the well of a detection device, make total egg
White matter by described detection device, this detection device include sample adding device, many doughnut polypropylene screens,
With the first porous plate body, described doughnut polypropylene screen is attached with and the transgenosis in described total protein
The first antibody that protein-specific combines, described sample adding device includes the second porous plate body and is arranged at described
On second porous plate body and the cone of a formed accommodation space, the diameter of described cone is arrived by down
On be gradually reduced and its free end forms described well, one end of described many doughnut polypropylene screens
It is fixed on described second porous plate body and connect with the hole on described second porous plate body, in described many
The other end of hollow fiber polypropylene screen be fixed on described first porous plate body and with on described first porous plate body
Hole connection, when described total protein by described detection device when, close on described first porous plate body
Hole so that the flowing of total protein occurs over just one end and the institute of described many doughnut polypropylene screens
State between the fenestra of doughnut polypropylene screen, make this transgenic protein be incorporated into described first antibody
And be attached on described doughnut polypropylene screen；Carry out step 3 afterwards；
Step 3, opens the hole on described first porous plate body, washs described doughnut polypropylene screen
Chamber in residue；Carry out step 4 afterwards；
Step 4, closes the hole on described first porous plate body again, will be with described transgenic protein
Specific binding and be expelled to described detection device with markd SA well, makes second
Antibody is by described detection device；Carry out step 5 afterwards；
Step 5, is again turned on the hole on described first porous plate body, washs described doughnut poly-third
Residue in the chamber of alkene film；Carry out step 6 afterwards；With
Step 6, detects the mark that whether there is described SA on described doughnut polypropylene screen,
If there is described mark, then it is assumed that this total protein exists this transgene protein, and judges in this raw material
There is transgene component.
2. the detection method of transgene component in food as claimed in claim 1, wherein said step one
The middle detailed process extracting raw-material total protein includes:
(1.1) raw material are placed in a sealing bag, in liquid nitrogen after quick-frozen 30s, are placed in mortar use
Liquid nitrogen grinding becomes powder, backward described sealing bag according to volume mass than 6:1 to raw material add separate
Buffer solution, in 0～4 DEG C of abundant mixing；
(1.2) it is drawn into the mixture obtaining in step (1.1) in centrifuge tube, in temperature 4 DEG C,
After 12000rpm centrifuges 10min, abandon supernatant, add than for 1:1 according to raw-material volume mass afterwards
Enter the resuspended precipitation of lysate, stand 10min；And,
(1.3) by the mixture in step (1.2) in temperature 4 DEG C, 12000rpm centrifuges 20min,
Take supernatant and i.e. obtain described total protein.
3. the detection method of transgene component in food as claimed in claim 2, wherein said step
(1.1), in, the shape of described sealing bag and the inwall laminating of described mortar, described sealing bag includes from outward
It to the interior outer layer being connected to each other and internal layer, between described outer layer and described internal layer, is provided with several beades,
Described sealing bag is made up of polysulfone material.
4. the detection method of transgene component in food as claimed in claim 1, wherein close by using
The hole that sealer is closed and opened on described first porous plate body.
5. the detection method of transgene component in food as claimed in claim 1, wherein said is labeled as
Horseradish peroxidase, in described step 6, utilizes and on the DAB described doughnut polypropylene screen of detection is
The no mark that there is described SA, if producing when joining DAB on described doughnut polypropylene screen
Raw brown precipitate, then judge there is described mark on described doughnut polypropylene screen.
6. the detection method of transgene component in food as claimed in claim 1, wherein said step 2
In, the gross protein obtaining step one is drawn in a syringe, afterwards by inserting described syringe
Enter in described well thus be expelled to this total protein in this detection device.
7. the detection method of transgene component in food as claimed in claim 1, wherein said raw material
Including corn, paddy rice, soybean and wheat.
8. a detection device for transgene component in food, this detection device include sample adding device, many
Doughnut polypropylene screen and the first porous plate body, described doughnut polypropylene screen is attached with always
The specific binding first antibody of transgenic protein in albumen, described sample adding device includes the second porous
Plate body and being arranged on described second porous plate body and the cone of a formed accommodation space, described circle
The diameter of cone is gradually reduced from down to up and its free end forms well, and described many doughnuts gather
One end of propylene film is fixed on described second porous plate body and connects with the hole on described second porous plate body
Logical, the other end of described many doughnut polypropylene screens be fixed on described first porous plate body and with described
Hole connection on first porous plate body, when total protein is by described detection device, closes described first
Hole on porous plate body, so that the flowing of total protein occurs over just described many doughnut polypropylene screens
One end and the fenestra of described doughnut polypropylene screen between, make this transgenic protein and described first
Antibody combines and is attached on described doughnut polypropylene screen.
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| CN106405102A (en) * | 2016-08-29 | 2017-02-15 | 黎志春 | Apparatus for aided detection of existence of transgenic protein in food |
| CN106324258A (en) * | 2016-08-29 | 2017-01-11 | 黎志春 | Auxiliary method for detecting whether genetically modified protein exists in food |
| CN106645702A (en) * | 2017-02-04 | 2017-05-10 | 上海为然环保科技有限公司 | Rapid detection device for genetically modified ingredients in grain and oils |
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| CA2559943A1 (en) * | 2004-03-15 | 2005-10-06 | Purdue Research Foundation | Cell concentration and pathogen recovery |
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| CN85104030A (en) * | 1984-05-11 | 1986-11-19 | 海布利德克公司 | Method of immunity and device |
| EP0310406A3 (en) * | 1987-10-01 | 1990-11-22 | E-Y Laboratories, Inc. | Directed flow diagnostic device and method |
| US5022411A (en) * | 1989-09-18 | 1991-06-11 | La Mina Ltd. | Modular fluid testing device |
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