CN104569432A - Method and device for detecting transgenic ingredients in food - Google Patents
Method and device for detecting transgenic ingredients in food Download PDFInfo
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- CN104569432A CN104569432A CN201510005351.6A CN201510005351A CN104569432A CN 104569432 A CN104569432 A CN 104569432A CN 201510005351 A CN201510005351 A CN 201510005351A CN 104569432 A CN104569432 A CN 104569432A
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- hollow fiber
- plate body
- porous plate
- fiber polypropylene
- protein
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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
- G01N33/68—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
Abstract
The invention discloses a method for detecting transgenic ingredients in food. The method comprises the steps of step I. extracting total proteins of a raw material; step II. injecting the total protein obtained in the step I into a detection device, and enabling the total protein to pass through the detection device, wherein the detection device comprises a sample feeding device, a plurality of hollow fiber polypropylene films and a first porous plate body, each hollow fiber polypropylene film is provided with a first antibody which is specifically combined with the transgenic protein in an attaching manner, the sample feeding device comprises a second porous plate body and a cone, and two ends of each hollow fiber polypropylene film are respectively fixed on the first porous plate body and the second porous plate body and are respectively communicated with holes in the first and second porous plate bodies; step III. washing; step IV. injecting a second antibody which is specifically combined with the transgenic protein and provided with marks into sample feeding holes of the detection device, and enabling the second antibody to pass through the detection device; step V. washing; and step VI, detecting whether the hollow fiber polypropylene film has the mark or not, and correlating the detection result with whether the transgenic ingredient exists in the raw material.
Description
Technical field
The present invention relates to a kind of detection method and device of transgene component in food.
Background technology
Transferred in certain specific biosome by one or more allogenic genes by technique for gene engineering, and make 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 enetically modified food.Difference according to enetically modified food source can be divided into vegetalitas enetically modified food, animality enetically modified food and microbes enetically modified food.Enetically modified food has more advantage: can increase crop yield; Production cost can be reduced; Crop insect pest, antiviral etc. ability can be strengthened; Improve agricultural product storage property.The safety evaluation of genetically modified organism is genetically modified organism and products thereof marketization and commercial prerequisite, in safety evaluation, the detection of transgene component is an important content, set up genetically modified organism quick, easy, accurately detection method be that safety evaluation and management lay the foundation.The detection of vegetalitas enetically modified food mainly adopts following two technology paths, and one is the foreign gene that the technology for detection such as application PCR, Southern are inserted, and two is the albumen using Elisa, Western etc. to detect transgene expression.The detection of protein level, mainly based on antigen and the interactional immunological method of antibody, quantitative and qualitative analysis detection is carried out for the specific proteins of external source destination gene expression in genetically modified plants and products thereof, its result is relatively more accurate, reliability is also better, but the means of the detection albumen that Elisa and Western etc. are current are all more loaded down with trivial details, go out result also slower.Especially at the position that customs etc. imports and exports, the testing of transgene component is very loaded down with trivial details and efficiency is lower.
Summary of the invention
An object of the present invention is to provide a kind of detection method of transgene component in food, and protein level detects transgene component in food fast;
Another object of the present invention is to provide a kind of pick-up unit of transgene component in food, and this device can binding specificity ground albumen quickly and easily, and use simple, and efficiency is high.
Technical scheme provided by the invention is:
A detection method for transgene component in food, comprising:
Step one, extract raw-material total protein;
Step 2, gross protein step one obtained is expelled to the well of a pick-up unit, make gross protein by described pick-up unit, this pick-up unit comprises sample adding device, many hollow fiber polypropylene screens, with the first porous plate body, described hollow fiber polypropylene screen is attached with the first antibody with the transgene protein specific binding in described total protein, described sample adding device comprise the second porous plate body and to be arranged on described second porous plate body and with the cone of its formation one accommodation space, the diameter of described cone reduces gradually from down to up and its free end forms described well, one end of described many hollow fiber polypropylene screens is fixed on described second porous plate body and is communicated with the hole on described second porous plate body, the other end of described many hollow fiber polypropylene screens is fixed on described first porous plate body and is communicated with the hole on described first porous plate body, when described total protein is by described pick-up unit, close the hole on described first porous plate body, occur over just between one end of described many hollow fiber polypropylene screens and the fenestra of described hollow fiber polypropylene screen to make the flowing of total protein, this transgenic protein and described first antibody is made to be combined and then to be attached on described hollow fiber polypropylene screen, carry out step 3 afterwards,
Step 3, opens the hole on described first porous plate body, washs the residue in the chamber of described hollow fiber polypropylene screen; Carry out step 4 afterwards;
Step 4, closes the hole on described first porous plate body again, by with described transgenic protein specific binding and be expelled to the well of described pick-up unit with markd second antibody, make second antibody by described pick-up unit; Carry out step 5 afterwards;
Step 5, again opens the hole on described first porous plate body, washs the residue in the chamber of described hollow fiber polypropylene screen; Carry out step 6 afterwards; With
Step 6, detects the mark whether described hollow fiber polypropylene screen existing described second antibody, if there is described mark, then thinks to there is this transgene protein in this total protein, and judges to there is transgene component in these starting material.
Preferably, in the detection method of described transgene component in food, the detailed process extracting raw-material total protein in described step one comprises:
(1.1) starting material are placed in a sealing bag, in liquid nitrogen after quick-frozen 30s, are placed in mortar liquid nitrogen grinding and become powder, backward described sealing bag in add dissociating buffer than 6: 1 to starting material according to volume mass, in 0 ~ 4 DEG C of fully mixing;
(1.2) potpourri obtained in step (1.1) is drawn in centrifuge tube, in temperature 4 DEG C, after the centrifugal 10min of 12000rpm, abandon supernatant, afterwards according to being add lysate resuspended precipitation at 1: 1 with raw-material volume mass ratio, leave standstill 10min; And,
(1.3) by the potpourri in step (1.2) in temperature 4 DEG C, the centrifugal 20min of 12000rpm, gets supernatant and namely obtains 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 of described mortar are fitted, described sealing bag comprises the skin and internal layer that are connected to each other from outside to inside, be provided with several beaded glasses between described skin and described internal layer, described sealing bag is made up of polysulfone material.
Preferably, in the detection method of described transgene component in food, close and open the hole on described first porous plate body by use diaphragm seal.
Preferably, in the detection method of described transgene component in food, wherein saidly be labeled as horseradish peroxidase, in described step 6, DAB is utilized to detect the mark whether described hollow fiber polypropylene screen existing described second antibody, if produce brown precipitate when being joined by DAB on described hollow fiber polypropylene screen, then judge that described hollow fiber polypropylene screen exists described mark.
Preferably, in the detection method of described transgene component in food, in described step 2, gross protein step one obtained is drawn in a syringe, afterwards by be inserted into by described syringe in described well thus to be expelled in this pick-up unit by this total protein.
Preferably, in the detection method of described transgene component in food, described starting material comprise corn, paddy rice, soybean and wheat.
A kind of pick-up unit of transgene component in food, this pick-up unit comprises sample adding device, many hollow fiber polypropylene screens, with the first porous plate body, described hollow fiber polypropylene screen is attached with the first antibody with the transgenic protein specific binding in described total protein, described sample adding device comprise the second porous plate body and to be arranged on described second porous plate body and with the cone of its formation one accommodation space, the diameter of described cone reduces gradually from down to up and its free end forms well, one end of described many hollow fiber polypropylene screens is fixed on described second porous plate body and is communicated with the hole on described second porous plate body, the other end of described many hollow fiber polypropylene screens is fixed on described first porous plate body and is communicated with the hole on described first porous plate body, when described total protein is by described pick-up unit, close the hole on described first porous plate body, occur over just between one end of described many hollow fiber polypropylene screens and the fenestra of described hollow fiber polypropylene screen to make the flowing of total protein, this transgenic protein is made to be combined with described first antibody and to be attached on described hollow fiber polypropylene screen.
Beneficial effect of the present invention: in pick-up unit provided by the invention, the first antibody of transgenic protein is attached on hollow fiber polypropylene screen, thus detect in starting material whether there is transgenic protein by making raw-material total protein to be detected flow through this pick-up unit, and by syringe, protein fluid is joined in pick-up unit of the present invention, be easy to operation, device of the present invention can detect whether there is transgene protein effectively rapidly.Transgene protein can be detected fast according to method of the present invention.In Protein Extraction, starting material are loaded in a sealing bag, the shape of described sealing bag and the inwall of described mortar are fitted, like this, when grinding in mortar, sealing bag can not be moved, described sealing bag comprises the skin and internal layer that are connected to each other from outside to inside, is provided with several beaded glasses between described skin and described internal layer.Like this, when grinding, stone pestle contacts with beaded glass, and starting material number of times polished in sealing bag and area are increased all greatly, and described sealing bag is made up of polysulfone material simultaneously, wear-resisting, low temperature resistant, can not damage.And, after using dissociating buffer that the starting material of pulverizing are resuspended, be easy to all draw out from sealing bag, and can operate at low ambient temperatures, avoid in common mortar grinder process, due to liquid nitrogen rapid evaporation, and have to from mortar, take out raw-material powder fast.Sometimes, owing to taking out not in time, cause raw-material waste, also result in the waste of time and manpower and materials.Present method avoids this kind of situation.Method of the present invention is quick for the detection of transgene protein, accurately, efficiency is high.
Accompanying drawing explanation
Fig. 1 is the structural representation of the hole of the first porous plate body of pick-up unit of the present invention when closing;
Fig. 2 is the structural representation of the hole of the first porous plate body of pick-up unit of the present invention when opening.
Embodiment
Below in conjunction with accompanying drawing, the present invention is described in further detail, can implement according to this with reference to instructions word to make those skilled in the art.
Nuclear isolation buffer: 0.25M sucrose, 18mM PIPES (pH6.8), 5mM MgCl
2, 60mM KCl, 15mM NaCl, 1mM CaCl
2, the Triton X-100 of 1.0%, 1mM PMSF (protease inhibitors is now with now adding).Fresh configuration 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 is now with now adding).Fresh configuration in 4 DEG C of preservations.
TBST damping fluid: 12.114gTris alkali, adjusts pH to 7.5,16.332gNaCl, 1M1Tween-20, adds water to 1L.
DAB horseradish peroxidase chromogenic reagent box is bought from the green skies, and production code member is P0202.
The invention provides a kind of detection method of transgene component in food, the sample that the method detects is liquid, comprising:
Step one, extract raw-material total protein, the detailed process extracting raw-material total protein comprises:
(1.1) starting material are placed in a sealing bag, in liquid nitrogen after quick-frozen 30s, be placed in mortar liquid nitrogen grinding and become powder, backward described sealing bag according to volume mass than 6: 1 to starting material add dissociating buffer (as needs extract cytoplasmic protein, then add common dissociating buffer, if desired Nuclear extract matter is extracted, then use nuclear isolation buffer), in the present embodiment, the nuclear isolation buffer as above herein added, then in 0 ~ 4 DEG C of fully mixing; The shape of described sealing bag and the inwall of described mortar are fitted, and described sealing bag comprises the skin and internal layer that are connected to each other from outside to inside, is provided with several beaded glasses between described skin and described internal layer, and described sealing bag is made up of polysulfone material.
(1.2) potpourri obtained in step (1.1) is drawn in centrifuge tube, in temperature 4 DEG C, after the centrifugal 10min of 12000rpm, abandon supernatant, afterwards according to being add lysate resuspended precipitation at 1: 1 with raw-material volume mass ratio, what add is Nuclei lysis buffer herein, leaves standstill 10min; And,
(1.3) by the potpourri in step (1.2) in temperature 4 DEG C, the centrifugal 20min of 12000rpm, gets supernatant and namely obtains described total protein.A syringe is utilized to be drawn in this syringe by gross protein.Certainly, after other purification measures also can be taked to carry out further purifying to this total protein, then step 2 is carried out.
Step 2, be expelled in pick-up unit by described syringe being inserted into this total protein obtained in described well thus by step one, make gross protein by described pick-up unit, (in testing process, need to apply certain pressure on this syringe, flowed out by the fenestra of hollow fiber polypropylene screen to enable the liquid in gross protein sample.This pick-up unit comprises sample adding device, many hollow fiber polypropylene screens, with the first porous plate body, described hollow fiber polypropylene screen is attached with the first antibody with the transgene protein specific binding in described total protein, described sample adding device comprise the second porous plate body and to be arranged on described second porous plate body and with the cone of its formation one accommodation space, the diameter of described cone reduces gradually from down to up and its free end forms described well, one end of described many hollow fiber polypropylene screens is fixed on described second porous plate body and is communicated with the hole on described second porous plate body, the other end of described many hollow fiber polypropylene screens is fixed on described first porous plate body and is communicated with the hole on described first porous plate body, when described total protein is by described pick-up unit, diaphragm seal is sealed up to close the hole on described first porous plate body under the first porous plate body, occur over just between one end of described many hollow fiber polypropylene screens and the fenestra of described hollow fiber polypropylene screen to make the flowing of total protein, this transgenic protein is combined with described first antibody and is attached on described hollow fiber polypropylene screen, carry out step 3 afterwards, there is a lot of fold on the surface of this hollow fiber polypropylene screen, to increase its surface area, and with the contact area of total protein, in a pick-up unit, the hollow fiber polypropylene screen that 20 ~ 30 5 ~ 8cm are long is set.Every root hollow fiber polypropylene screen oneself is formed with a chamber, and the diameter of every root hollow fiber polypropylene screen is 5 ~ 10mm.
Step 3, removes diaphragm seal to open the hole on described first porous plate body, the residue in the chamber of hollow fiber polypropylene screen described in use TBST buffer solution 3 times, each 20mL, 10min; Carry out step 4 afterwards;
Step 4, closes the hole on described first porous plate body again with new diaphragm seal, by with described transgenic protein specific binding and be expelled to the well of described pick-up unit with markd second antibody, make second antibody by described pick-up unit; Carry out step 5 afterwards; Wherein saidly be labeled as horseradish peroxidase, second antibody and this horseradish peroxidase.
Step 5, removes current diaphragm seal and again opens hole on described first porous plate body, uses residue in the chamber of hollow fiber polypropylene screen described in TBST buffer solution 3 times, each 20mL, 10min; Carry out step 6 afterwards; With
Step 6, DAB horseradish peroxidase chromogenic reagent box (the green skies) is utilized to detect the mark whether described hollow fiber polypropylene screen existing described second antibody, if produce brown precipitate when being joined by DAB on described hollow fiber polypropylene screen, then judge that described hollow fiber polypropylene screen exists described mark.If there is described mark, then think to there is this transgene protein in this total protein, and judge to there is transgene component in these starting material.Method of the present invention both can be detected on the transgenic protein of expressing in tenuigenin, also can be detected on the transgenic protein of expressing in nucleus.
In one embodiment of the invention, described starting material are any one in corn, paddy rice, soybean and wheat.
The present invention also provides a kind of pick-up unit of transgene component in food, as depicted in figs. 1 and 2, this pick-up unit comprises sample adding device, many hollow fiber polypropylene screens 3, with the first porous plate body 2, on described hollow fiber polypropylene screen, 3 are attached with the first antibody with the transgenic protein specific binding in described total protein, described sample adding device comprise the second porous plate body 4 and to be arranged on described second porous plate body and with the cone 5 of its formation one accommodation space, the diameter of described cone 5 reduces gradually from down to up and its free end forms well 1, one end of described many hollow fiber polypropylene screens 3 is fixed on described second porous plate body 4 and is communicated with the hole on described second porous plate body 4, the other end of described many hollow fiber polypropylene screens 3 is fixed on described first porous plate body 2 and is communicated with the hole on described first porous plate body 2, when described total protein is by described pick-up unit, as shown in Figure 1, close the hole on described first porous plate body 2, occur over just between one end of described many hollow fiber polypropylene screens 3 and the fenestra of described hollow fiber polypropylene screen to make the flowing of total protein, this transgenic protein is combined with described first antibody and is attached on described hollow fiber polypropylene screen, other waste liquids have just flowed out this pick-up unit, a waste liquid pool can be placed in below and catch waste liquid.When needs wash, as shown in Figure 2, open the hole on this first porous plate body 2, cleansing solution is flowed out.
Although embodiment of the present invention are open as above, but it is not restricted to listed in instructions and embodiment utilization, it can be applied to various applicable the field of the invention completely, for those skilled in the art, can easily realize other amendment, therefore do not deviating under the universal that claim and equivalency range limit, the present invention is not limited to specific details and illustrates here and the legend described.
Claims (8)
1. a detection method for transgene component in food, comprising:
Step one, extract raw-material total protein;
Step 2, gross protein step one obtained is expelled to the well of a pick-up unit, make gross protein by described pick-up unit, this pick-up unit comprises sample adding device, many hollow fiber polypropylene screens, with the first porous plate body, described hollow fiber polypropylene screen is attached with the first antibody with the transgene protein specific binding in described total protein, described sample adding device comprise the second porous plate body and to be arranged on described second porous plate body and with the cone of its formation one accommodation space, the diameter of described cone reduces gradually from down to up and its free end forms described well, one end of described many hollow fiber polypropylene screens is fixed on described second porous plate body and is communicated with the hole on described second porous plate body, the other end of described many hollow fiber polypropylene screens is fixed on described first porous plate body and is communicated with the hole on described first porous plate body, when described total protein is by described pick-up unit, close the hole on described first porous plate body, occur over just between one end of described many hollow fiber polypropylene screens and the fenestra of described hollow fiber polypropylene screen to make the flowing of total protein, this transgenic protein and described first antibody is made to be combined and then to be attached on described hollow fiber polypropylene screen, carry out step 3 afterwards,
Step 3, opens the hole on described first porous plate body, washs the residue in the chamber of described hollow fiber polypropylene screen; Carry out step 4 afterwards;
Step 4, closes the hole on described first porous plate body again, by with described transgenic protein specific binding and be expelled to the well of described pick-up unit with markd second antibody, make second antibody by described pick-up unit; Carry out step 5 afterwards;
Step 5, again opens the hole on described first porous plate body, washs the residue in the chamber of described hollow fiber polypropylene screen; Carry out step 6 afterwards; With
Step 6, detects the mark whether described hollow fiber polypropylene screen existing described second antibody, if there is described mark, then thinks to there is this transgene protein in this total protein, and judges to there is transgene component in these starting material.
2. the detection method of transgene component in food as claimed in claim 1, the detailed process extracting raw-material total protein in wherein said step one comprises:
(1.1) starting material are placed in a sealing bag, in liquid nitrogen after quick-frozen 30s, are placed in mortar liquid nitrogen grinding and become powder, backward described sealing bag in add dissociating buffer than 6: 1 to starting material according to volume mass, in 0 ~ 4 DEG C of fully mixing;
(1.2) potpourri obtained in step (1.1) is drawn in centrifuge tube, in temperature 4 DEG C, after the centrifugal 10min of 12000rpm, abandon supernatant, afterwards according to being add lysate resuspended precipitation at 1: 1 with raw-material volume mass ratio, leave standstill 10min; And,
(1.3) by the potpourri in step (1.2) in temperature 4 DEG C, the centrifugal 20min of 12000rpm, gets supernatant and namely obtains described total protein.
3. the detection method of transgene component in food as claimed in claim 2, in wherein said step (1.1), the shape of described sealing bag and the inwall of described mortar are fitted, described sealing bag comprises the skin and internal layer that are connected to each other from outside to inside, be provided with several beaded glasses between described skin and described internal layer, described sealing bag is made up of polysulfone material.
4. the detection method of transgene component in food as claimed in claim 1, wherein closes and opens the hole on described first porous plate body by use diaphragm seal.
5. the detection method of transgene component in food as claimed in claim 1, wherein saidly be labeled as horseradish peroxidase, in described step 6, DAB is utilized to detect the mark whether described hollow fiber polypropylene screen existing described second antibody, if produce brown precipitate when being joined by DAB on described hollow fiber polypropylene screen, then judge that described hollow fiber polypropylene screen exists described mark.
6. the detection method of transgene component in food as claimed in claim 1, in wherein said step 2, gross protein step one obtained is drawn in a syringe, afterwards by be inserted into by described syringe in described well thus to be expelled in this pick-up unit by this total protein.
7. the detection method of transgene component in food as claimed in claim 1, wherein said starting material comprise corn, paddy rice, soybean and wheat.
8. the pick-up unit of a transgene component in food, this pick-up unit comprises sample adding device, many hollow fiber polypropylene screens, with the first porous plate body, described hollow fiber polypropylene screen is attached with the first antibody with the transgenic protein specific binding in described total protein, described sample adding device comprise the second porous plate body and to be arranged on described second porous plate body and with the cone of its formation one accommodation space, the diameter of described cone reduces gradually from down to up and its free end forms well, one end of described many hollow fiber polypropylene screens is fixed on described second porous plate body and is communicated with the hole on described second porous plate body, the other end of described many hollow fiber polypropylene screens is fixed on described first porous plate body and is communicated with the hole on described first porous plate body, when described total protein is by described pick-up unit, close the hole on described first porous plate body, occur over just between one end of described many hollow fiber polypropylene screens and the fenestra of described hollow fiber polypropylene screen to make the flowing of total protein, this transgenic protein is made to be combined with described first antibody and to be attached on described hollow fiber polypropylene screen.
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| CN106324258A (en) * | 2016-08-29 | 2017-01-11 | 黎志春 | Auxiliary method for detecting whether genetically modified protein exists in food |
| CN106405102A (en) * | 2016-08-29 | 2017-02-15 | 黎志春 | Apparatus for aided detection of existence of transgenic protein 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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| Publication number | Publication date |
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| CN104569432B (en) | 2016-10-05 |
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