CN102495213A - Kit used for detecting phytopathogenic fungi and detection method thereof - Google Patents
Kit used for detecting phytopathogenic fungi and detection method thereof Download PDFInfo
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- CN102495213A CN102495213A CN2011103895397A CN201110389539A CN102495213A CN 102495213 A CN102495213 A CN 102495213A CN 2011103895397 A CN2011103895397 A CN 2011103895397A CN 201110389539 A CN201110389539 A CN 201110389539A CN 102495213 A CN102495213 A CN 102495213A
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Abstract
The invention discloses a kit used for detecting phytopathogenic fungi and a detection method thereof. The kit comprises a microfluidic chip, antibody to phytopathogenic fungi, alkaline phosphatase labeled secondary antibody specifically binding to the antibody to phytopathogenic fungi, positive control of phytopathogenic fungi and a chromogenic substrate, wherein the microfluidic chip comprises a chip base plate, a microfluidic chip substrate provided with a pipeline and a track etching polycarbonate filter membrane. The kit can rapidly finish detection of a plurality of phytopathogenic fungi and uses a tiny amount of reagents and samples for detection, thereby saving detection cost.
Description
Technical field
The invention belongs to the detection technique field, be specifically related to a kind of kit and detection method that detects phytopathogen.
Background technology
Entry and exit vegetable seeds nursery stock detects very important, and plant disease becomes the port and detects emphasis with hiding property, allocation and transportation propagation risk height again.But all the time, the detection of port vegetable seeds nursery stock disease is difficult problem, and reason has: 1. the target that detects of port is the circulation commodity, and the requirement that detects the time limit is arranged, but the hiding property of plant disease, characteristics decision detection such as reveal any symptoms does not need the time long; 2. plant disease (poison) amount of carrying disease germs is low, thus require the detection technique level high, detection method highly sensitive; 3. disease detects a project needs the long time, and a general sample also requires to detect simultaneously several disease projects, and labour intensity and workload are big; 4. plant disease different detection project needs different operation; Testing staff's technical merit is required high (needing deeper professional knowledge background and higher technical operation level), and 5. the plant disease test item is various; Different test items need be bought a large amount of different detection reagent, and the reagent cost is high.
In recent years, the detection difficult problem of vegetable seeds nursery stock disease has the trend of aggravation.The dramatic growth of port vegetable seeds nursery stock foreign trade amount; Test sample batch increase in enormous quantities year by year; And at the beginning of 2009; Country has strengthened the quarantine dynamics to the seed of high economic value added, the method for the examining inspection project of nursery stock import, and a sample is increased to tens kinds even tens of kinds by the several legal test items of script.Spend more time, more reagent, more complex manual operation like this, improved working strength and strengthened workload greatly.In the present detection method, in the immune detection to vegetative bacteria, comprise conventional sense means such as ELISA kit and test strips, all can only detect a project of a sample simultaneously, can't improve flux.
Under the requirement that " speeds passage through customs " at the port; Face these problems; A kind of high flux be must seek, personnel labour and reagent spent less, practice thrift; And can detect a plurality of legal test item of a sample simultaneously,, improve the detection level at port, the more effectively prevention and control adventive invasion of venereal disease evil of especially hiding to quicken the speed of being open to the custom at port.
Microflow control technique is because its volume is little, and amount of samples is little, and can the high-throughout characteristics of simple realization, can be applied to easily and fast in the immune detection, and a plurality of parameters of using microflow control technique can high flux to accomplish a plurality of samples fast detect simultaneously.Micro-fluidic chip has a plurality of parallelpipeds, can feed different albumen in each pipeline, and the albumen that on second direction, feeds so just can react with multiple different albumen simultaneously, thereby reaches high flux, the purpose that high information quantity detects.
Nanometer technology is being advanced develop rapidly in 20 years, and the nanometer technology of China maintains the leading position in the world, but the relevant research of nanometer technology focuses mostly in basic scientific research, and the application in the actual production life is still very not enough.Reason is because the relevant research in nanosecond science and technology field is directly cooperated with practical application and exchanged deficiency.Can't advanced technology be converted into practical application, obtain higher social economic value.
The film that the present invention will have nanostructured is attached in the micro-fluidic chip; Utilize the nanostructured surface of film to have bigger specific surface area; Adhesion protein and generation immune response in the above; Utilize micro-fluidic high integration again, realize high-throughout immune detection analysis, Preliminary Applications is in the research that the port phytopathogen detects.At present, both at home and abroad also not about using the report that detects phytopathogen with the film of nanostructured as the micro-fluidic chip of substrate.
Summary of the invention
Technical matters to be solved by this invention provides a kind of kit that detects phytopathogen, and this kit can be accomplished the detection to the various plants germ fast, and the amount of reagent that uses and sample is all considerably less, practices thrift and detects cost.
Second technical matters to be solved by this invention provides a kind of method of using kit according to the invention to detect phytopathogen.
For solving the problems of the technologies described above, the technical scheme that the present invention adopted is:
A kind of kit that detects phytopathogen, this kit comprise that micro-fluidic chip, phytopathogen antibody, specificity combine two anti-, the phytopathogen positive control and the chromogenic substrate of the alkali phosphatase enzyme mark of phytopathogen antibody; Wherein, said micro-fluidic chip comprises the chip base plate, is provided with the micro-fluidic chip substrate and the track etching polycarbonate leaching film of pipeline.
Further, said phytopathogen is one or more in corn bacterial wilt, universal melon bacterial fruit blotch, bacterial ring rot o potato bacterium, tomato bacterial canker germ or the onion rotten pathogenic bacteria.
Further, said chromogenic substrate is NBT/5-bromo-4-chloro-3-indolylphosphate salt.
Further, the material of said chip base plate and micro-fluidic chip substrate is dimethyl silicone polymer.
Also a kind of detection method of using the said kit of claim 1 to detect phytopathogen of the present invention is characterized in that this method may further comprise the steps:
(1) chip assembling
Be placed on the chip base plate track etching polycarbonate leaching film is smooth; There is not bubble between said filter membrane and the chip base plate; The micro-fluidic chip substrate that will be provided with pipeline then is placed on the said track etching polycarbonate leaching film, does not have bubble between said micro-fluidic chip substrate and the filter membrane;
(2) use PBS solution to dilute the positive control and the phytopathogen sample of phytopathogen respectively; Solution after will diluting then feeds the pipeline of micro-fluidic chip substrate respectively; Hatched 5-60 minute; Form phytopathogen positive control and sample strip, hatch and finish back extraction solution, upper strata micro-fluidic chip substrate is taken off;
(3) dripping lock solution on the said track etching polycarbonate leaching film, sealed 10-60 minute, then with the lock solution flush away, said lock solution is 5%BSA;
(4) on perpendicular to described phytopathogen positive control of step (2) and sample strip, place another micro-fluidic chip substrate; In pipeline, feed corresponding phytopathogen antibody then; Hatched 5-60 minute, and hatched and finish back extraction antibody-solutions, upper strata micro-fluidic chip substrate is taken off;
(5) anti-at the alkali phosphatase enzyme mark two that drips specificity combination phytopathogen antibody on the said track etching polycarbonate leaching film, hatched 5-60 minute, with two anti-solution flush awaies, use the chromogenic substrate colour developing at last;
(6) examine under a microscope and photographic images.
Further, said phytopathogen is one or more in corn bacterial wilt, universal melon bacterial fruit blotch, bacterial ring rot o potato bacterium, tomato bacterial canker germ or the onion rotten pathogenic bacteria.
Further, said chromogenic substrate is NBT/5-bromo-4-chloro-3-indolylphosphate salt.
Beneficial effect of the present invention:
1. detection time: detecting each step incubation time in original ELISA kit test method needs 30 minutes to 1 hour at least, and chip detection can shorten to each 5 minutes to 10 minutes step with this process.
2. detectable consumption: using needed sample of micro-fluidic chip and amount of reagent is 1 microlitre (it is 100 microlitres that conventional ELISA tests); Reagent (detection antibody) concentration is 10 times of conventional ELISA experimental concentration, uses 1/10 of conventional amount of reagent can carry out the detection of a plurality of bacteria samples.Cost is reduced to 1/50 of conventional ELISA.The few advantage for sample of reagent dosage is that some precious sample can be only obtain the result with seldom an amount, thus for the advantage of antibody reagent be can be fewer the reagent dosage use amount that can reduce expensive reagent reduce cost.
3. detection integrated level: pipeline can be designed to quantity (1 to 100 or more) arbitrarily; Because the dot matrix that intersects reacts; The chip that adds for the second time can be confirmed the amount (1 to 100 or more) of detected parameters, so can make a plurality of parameters that detect a plurality of samples simultaneously according to the detection demand.
4. kit according to the invention can carry out the detection of various plants germ simultaneously, also can be directed against a kind of phytopathogen, measures the sample of separate sources.
Description of drawings
Fig. 1 micro-fluidic chip piping plan;
The testing result of Fig. 2 five kind of plant germs on micro-fluidic chip.
The specific embodiment mode
Below in conjunction with specific embodiment, advance-go on foot to set forth the present invention.But these embodiment only limit to the present invention is described and are not used in restriction protection scope of the present invention.Method therefor is conventional method if no special instructions among the following embodiment.
Reagent:
1. corn bacterial wilt (erwinia stewartii) antibody kit comprises corn bacterial wilt antibody, and AP (alkaline phosphatase) mark two is anti-, corn bacterial wilt positive control;
Universal melon bacterial fruit blotch (watermelon fruit spot, Acidovorax avenae subsp. Citrulli) antibody kit comprises universal melon bacterial fruit blotch antibody, and AP mark two is anti-, universal melon bacterial fruit blotch positive control;
Bacterial ring rot o potato bacterium (clavibacter michiganensis subsp. sepidonicus) antibody kit comprises the bacterial ring rot o potato bacteria antibody, and AP mark two is anti-, bacterial ring rot o potato bacterium positive control;
Tomato bacterial canker germ (Clavibacter michiganensis subsp.michiganensis) antibody kit comprises tomato bacterial canker bacteria antibody, and AP mark two is anti-, tomato bacterial canker germ positive control;
Onion rotten pathogenic bacteria (Burkholderia gladioli pv.alliicola) antibody kit comprises onion rotten pathogenic bacteria antibody, and AP mark two is anti-, onion rotten pathogenic bacteria positive control;
The mentioned reagent box is all purchased in An Dezhen Bioisystech Co., Ltd (www.adgen.com);
2. onion rotten pathogenic bacteria (Burkholderia gladioli pv.alliicola) positive control; Corn bacterial wilt (erwinia stewartii) positive control; Universal melon bacterial fruit blotch (watermelon fruit spot; Acidovorax avenae subsp. Citrulli) positive control; Bacterial ring rot o potato bacterium (clavibacter michiganensis subsp. sepidonicus) positive control, tomato bacterial canker germ (Clavibacter michiganensis subsp.michiganensis) positive control, Beijing Entry-Exit Inspection and Quarantine Bureau; Can purchase in An Dezhen Bioisystech Co., Ltd (www.adgen.com)
3. phosphate buffer (PBS; PH=7.2-7.4), added phosphate buffer (PBST, the pH=7.2-7.4 of 0.1%Tween as the protein solution dilution; 0.1%Tween 20) as the protein solution dilution, 5% BSA solution (being dissolved in the PBS the inside).
4. chromogenic substrate: NBT/BCIP (NBT/5-bromo-4-chloro-3-indolylphosphate salt) buys the source: Sigma company
Preparation of embodiment 1 chip and assembled part
1. the design of micro-fluidic chip substrate structure and mask print
The material of said micro-fluidic chip substrate is PDMS (polydimethylsiloxane, a dimethyl silicone polymer).
Draw required pipeline configuration in the chip substrate with software L-edit.Micro-fluidic chip substrate specification such as following table that this experiment is used, chip pipeline configuration figure sees Fig. 1:
Table 1 micro-fluidic chip pipeline specifications table
| Specification number | Duct width (μ m) | Pipeline center's spacing (μ m) | Pipeline maximum length (cm) |
| 1 | 150 | 1100 | 2.0 |
Send to professional film printing company to the figure that designs and be printed as film, as the mask of next step experiment usefulness.Because photoetching process need use SU8 to bear photoresist, pipeline configuration partly needs printing opacity.Therefore on the film mask of being printed, pipeline is transparent, and non-conduit region is that black is lighttight.
2. the preparation of micro-fluidic chip template
The main process of preparation is photoetching, promptly utilizes photoresist in the characteristics making of convertibility matter under the ultraviolet ray irradiation and the on all four photoresist template of figure on the mask.Photoetching need be carried out in specific ultra-clean chamber.
2.1 instrument and reagent articles for use
Instrument: uv-exposure machine, photoresist spinner, heating plate, baking oven (DHG-9030A type electric heating constant temperature air dry oven)
Reagent and articles for use: SU-8 2025 photoresists and supporting developer solution thereof (USA Clariant Corporation), 5 cun or 3.5 cun silicon chips (Luoyang Monocrystal Silicon Company)
2.2 experimental procedure
(1) take out 5 cun new silicon chips of a slice, with photoresist spinner on silicon chip, throw away the photoresist that thickness is about 40 μ m (SU-8 2025,2000rpm, 30s).At this moment, silicon chip surface photoresist thickness is even, surface smoothing.
(2) preceding baking is put into behind the baking oven and is risen to 95 ℃ by 65 ℃, keeps 10min.
(3) with the silicon slice placed that one deck photoresist is arranged on the surface in the uv-exposure machine, put before ready film, graphic printing faces down.With quartz glass it is pressed on photoresist layer, the adjustment position is positioned in the middle of the silicon chip figure, fixes.
(4) open instrument and make public, the time shutter is 120s.
(5) take off film mask, take off silicon chip, the back baking, step is with (2).
(6) develop: silicon chip is put into developer solution, and the responseless photoresist of flush away after rinsing well repeatedly, dries up with nitrogen.The photoresist of flush away promptly is not the structure that is designed, and is projected on the silicon chip, forms template.After the silanization processing, masterplate can use in long period of time repeatedly.
3. the preparation of micro-fluidic detection pipeline and chip base plate
3.1 soft etching obtains micro-fluidic pipeline and chip base plate
PDMS (polydimethylsiloxane; Dimethyl silicone polymer) is a kind of transparent silicon rubber; Under common state, be transparent and liquid thickness, curable after also heating with hardening agent reaction (184 silicone elastomer curing agent).Utilize PDMS can the projection figure on the silicon chip template be converted into corresponding pipeline figure, thereby obtain micro-fluidic pipeline.
3.2 instrument and reagent articles for use
Instrument: electronic balance (YP-6001 type), vacuum pump (2XZ-4 type sliding vane rotary vacuum pump), baking oven (DHG-9030A type electric heating constant temperature air dry oven)
Reagent and articles for use: PDMS monomer (184 silicone elastomer, SYLGARD company), hardening agent (184 silicone elastomer curing agent; SYLGARD company), glass rod, disposable cup; Scotch white glues band, syringe (the band syringe needle polishes)
3.3 experimental procedure
(1) take by weighing the PDMS monomer according to required amount, take by weighing hardening agent and mixing with mass ratio 10:1, stir about 10min is to even.Decompression pumps bubble in vacuum pump.
(2) having the potpourri of pouring PDMS and hardening agent on the silicon chip template of miniflow pipeline into, pump bubble.
(3) put into 70 ℃ in baking oven, two hours, to solidifying; Perhaps 80 ℃ of extremely curing in a hour.Outstanding photoresist on the silicon chip can stamp groove shape pipeline with PDMS.
(4) PDMS with this curing downcuts from template, and punches with the syringe of band syringe needle at the injection port place that designs.With Scotch white glues band cleaning PDMS pipe surface.
(5) in smooth double dish, pour PDMS and the curing agent mixture that mixes into, pump bubble, it is dull and stereotyped to obtain smooth PDMS after the heat curing, as the base plate use of chip.
4. has nano structure membrane
The surface has the track etching polycarbonate leaching film of 200 nano-pores and buys the company in Whatman; Model is: 110606. 25 mm diameters; 100/box; The track etching polycarbonate of being buied is ganoid white polycarbonate leaching film, and the surface has the cylindrical bore that 200 nanometers use the method for track etching to obtain.
5. chip assemble method
Smooth being placed on a slice PDMS flat board of track etching polycarbonate leaching film with buying do not have bubble between filter membrane and the PDMS flat board, and the five pipeline PDMS chip substrates that prepare are placed on the polycarbonate leaching film, do not have bubble between PDMS chip substrate and the filter membrane.Next step use of another five pipeline PDMS chip substrate.
The composition of embodiment 2 kits
Adopt and implement 1 said method preparation and assemble micro-fluidic chip.
Detect the kit of onion rotten pathogenic bacteria and form, comprising: micro-fluidic chip, phytopathogen antibody, specificity combine two anti-, the phytopathogen positive control and the chromogenic substrate of the alkali phosphatase enzyme mark of phytopathogen antibody; Wherein, said micro-fluidic chip comprises the chip base plate, is provided with the micro-fluidic chip substrate and the track etching polycarbonate leaching film of pipeline.
Kit described in the present embodiment is to following 5 kind of plant germs: corn bacterial wilt, universal melon bacterial fruit blotch, bacterial ring rot o potato bacterium, tomato bacterial canker germ and onion rotten pathogenic bacteria.
Said chromogenic substrate is NBT/5-bromo-4-chloro-3-indolylphosphate salt.
Two of the alkali phosphatase enzyme mark of above-mentioned said phytopathogen antibody, specificity combination phytopathogen antibody resists and the phytopathogen positive control can obtain through buying, and also can utilize method well known in the prior art to prepare.
Embodiment 3 five kind of plant germ detection methods are set up
1. the kind of the phytopathogen that will detect
Onion rotten pathogenic bacteria (Burkholderia gladioli pv.alliicola); Corn bacterial wilt (erwinia stewartii); Universal melon bacterial fruit blotch (watermelon fruit spot; Acidovorax avenae subsp. Citrulli), bacterial ring rot o potato bacterium (clavibacter michiganensis subsp. sepidonicus), tomato bacterial canker germ (Clavibacter michiganensis subsp.michiganensis).
2. phytopathogen is cultivated
The culture medium prescription that uses is (1L): beef peptone: 1.0 g, yeast extract: 2.0 g, peptone: 5.0 g, NaCl:5.0 g, agar (joining solid medium uses): 15 g, KH
2PO
4: 0.45 g, Na
2HPO
412H
2O:2.39 g regulates pH to 6.8, optimum growth temp: 28 ℃.
3. the preparation of bacterium liquid sample
With 1ml shake bacterium liquid (OD value be 0.7-0.8) that training spends the night carry out centrifugal, 5000r/min, 20 minutes; Supernatant liquor is discarded (being mainly nutrient culture media), bacterium liquid is used the PBS washed twice, each 1ml; 5000r/min, 20 minutes. bacterium liquid is dispersed among the 100 microlitre PBS.At this moment, the concentration of bacterium is roughly 10
8Individual/ml, 4 ℃ of preservations.
The bacterium liquid preparation of deactivation: the bacterium liquid that will wash boiled 20 minutes at 100 ℃.All at last bacterium liquid same 121 ℃ (0.1MPa), 30 min deactivations.The element sterilization that bubble such as associated vessel is strong.
4. two-dimensional detection model dot matrix test experience
It is the high flux of a kind of uniqueness in the microflow control technique, the detection method of high information quantity that dot matrix detects.This is tested so-called dot matrix and detects, and promptly refers to utilize the micro-fluidic chip substrate on a direction of polycarbonate leaching film, to feed a kind of protein, is adsorbed in and forms band on the film; Change vertical direction again and feed another kind of protein equally, thereby react, and form dot matrix at the infall of both direction protein band.Micro-fluidic chip has a plurality of parallelpipeds, can feed different albumen in each pipeline, and the albumen that on second direction, feeds so just can react with multiple different albumen simultaneously, thereby reaches high flux, the purpose that high information quantity detects.
5. use embodiment 2 described kits to set up the detection method of 5 kind of plant germs, as follows:
A. the positive control with five kinds of germs uses PBS solution to dilute in advance; Respectively five kinds of bacterium are fed five pipelines to working concentration; The sample that each pipeline uses is 2 microlitres/pipeline; Method is to use pipettor that 2 microlitre samples are put into an injection port of pipeline, uses syringe to vacuumize at another mouthful, and fluid sample can be inhaled into pipeline and be full of pipeline; Two chips detect as parallel simultaneously, and the bacterium liquid positive that consumes each concentration is totally 4 microlitres.Hatch 10 minutes as fixed form.Hatch and finish back extraction solution, upper strata PDMS chip is taken off.
B. 200 microlitre 5%BSA solution are used for sealing in the polycarbonate leaching film substrate.Flush away after 30 minutes (PBST flushing one time, PBS flushing one time).
C. put into 5 pipeline chip substrates in vertical direction, feed the antibody of corresponding phytopathogen successively at each pipeline, a kind of bacterium of each pipeline, liquor capacity is 1 microlitre then; Two chips detect simultaneously, and the antibody that consumes each concentration is totally 2 microlitres.Hatch and carried out immune response in 10 minutes.Hatch and finish back extraction antibody-solutions, upper strata PDMS chip is taken off.
D. anti-at the goat anti-rabbit igg that drips 100 microlitre AP marks in the polycarbonate leaching film substrate two, concentration is 1:400, and the PBS dilution was hatched 10 minutes.With two anti-solution flush awaies (PBST flushing one time, PBS flushing one time).(reagent consumption: 0.5 microlitre AP mark, the two anti-1:400 that are diluted to, two parallel 200 microlitres two that use altogether are anti-), use AP chromogenic substrate NBI/BCIP colour developing at last.
E. examine under a microscope and photographic images.
5. experimental result
After the testing conditions of five kinds of bacterium is optimized; Select the optimal conditions of each bacterium: for universal melon bacterial fruit blotch (watermelon fruit spot; Acidovorax avenae subsp. Citrulli), detecting AC uses PBS to dilute for antibody stoste to be 1:160; Bacterial ring rot o potato bacterium (clavibacter michiganensis subsp. sepidonicus), detection AC are used the PBS dilution for antibody stoste and are 1:40; For onion rotten pathogenic bacteria (Burkholderia gladioli pv.alliicola), the detection AC uses the PBS dilution for antibody stoste and is 1:320; For corn bacterial wilt (erwinia stewartii), the detection AC uses the PBS dilution for antibody stoste and is 1:20; Tomato bacterial canker germ (Clavibacter michiganensis subsp.michiganensis), detection AC are used the PBS dilution for antibody stoste and are 1:20) on same chip, carry out again detecting in five kinds of bacterium.Testing result is seen Fig. 2.
Five kinds of bacterium are carried out immune detection according to the condition of optimizing in same chip, can produce in the zone that bacterium band and antibody intersect and combine square, and can observed atropurpureus square by producing after the two anti-colour developings.By finding out among Fig. 2; Five kinds of bacterium testing results are consistent with monoxenic effect; Bga bacterium and Aac bacterium that antibody response is good obtain square clearly, and antibody response signal in single kind bacterial classification chip is also more weak in the integrated chip of five kinds of bacterium with regard to more weak kind.We can draw, and micro-fluidic chip can success detects when carrying out multiple bacterium, but detecting effect antagonist reactivity has requirement, and good antibody can obtain effect preferably.
Obviously, the above embodiment of the present invention only be for clearly the present invention is described and is done for example, and be not to be qualification to embodiment of the present invention.For the those of ordinary skill in affiliated field, on the basis of above-mentioned explanation, can also make other multi-form variation or change.Here can't give exhaustive to all embodiments.Everyly belong to the row that conspicuous variation that technical scheme of the present invention extends out or change still are in protection scope of the present invention.
Claims (7)
1. a kit that detects phytopathogen is characterized in that, this kit comprises that micro-fluidic chip, phytopathogen antibody, specificity combine two anti-, the phytopathogen positive control and the chromogenic substrate of the alkali phosphatase enzyme mark of phytopathogen antibody; Wherein, said micro-fluidic chip comprises the chip base plate, is provided with the micro-fluidic chip substrate and the track etching polycarbonate leaching film of pipeline.
2. the kit of detection phytopathogen according to claim 1; It is characterized in that said phytopathogen is one or more in corn bacterial wilt, universal melon bacterial fruit blotch, bacterial ring rot o potato bacterium, tomato bacterial canker germ or the onion rotten pathogenic bacteria.
3. the kit of detection phytopathogen according to claim 1 is characterized in that, said chromogenic substrate is NBT/5-bromo-4-chloro-3-indolylphosphate salt.
4. the kit of detection phytopathogen according to claim 1 is characterized in that, the material of said chip base plate and micro-fluidic chip substrate is dimethyl silicone polymer.
5. detection method of using the said kit of claim 1 to detect phytopathogen is characterized in that this method may further comprise the steps:
(1) chip assembling
Be placed on the chip base plate track etching polycarbonate leaching film is smooth; There is not bubble between said filter membrane and the chip base plate; The micro-fluidic chip substrate that will be provided with pipeline then is placed on the said track etching polycarbonate leaching film, does not have bubble between said micro-fluidic chip substrate and the filter membrane;
(2) use PBS solution to dilute the positive control and the phytopathogen sample of phytopathogen respectively; Solution after will diluting then feeds the pipeline of micro-fluidic chip substrate respectively; Hatched 5-60 minute; Form phytopathogen positive control and sample strip, hatch and finish back extraction solution, upper strata micro-fluidic chip substrate is taken off;
(3) dripping lock solution on the said track etching polycarbonate leaching film, sealed 10-60 minute, then with the lock solution flush away, said lock solution is 5%BSA;
(4) on perpendicular to described phytopathogen positive control of step (2) and sample strip, place another micro-fluidic chip substrate; In pipeline, feed corresponding phytopathogen antibody then; Hatched 5-60 minute, and hatched and finish back extraction antibody-solutions, upper strata micro-fluidic chip substrate is taken off;
(5) anti-at the alkali phosphatase enzyme mark two that drips specificity combination phytopathogen antibody on the said track etching polycarbonate leaching film, hatched 5-60 minute, with two anti-solution flush awaies, use the chromogenic substrate colour developing at last;
(6) examine under a microscope and photographic images.
6. detection method according to claim 5; It is characterized in that said phytopathogen is one or more in corn bacterial wilt, universal melon bacterial fruit blotch, bacterial ring rot o potato bacterium, tomato bacterial canker germ or the onion rotten pathogenic bacteria.
7. detection method according to claim 5 is characterized in that, said chromogenic substrate is NBT/5-bromo-4-chloro-3-indolylphosphate salt.
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| CN107907535A (en) * | 2017-10-20 | 2018-04-13 | 浙江省柑桔研究所 | A kind of method for diagnosing red bayberry dead-arm |
| CN107918012A (en) * | 2016-10-11 | 2018-04-17 | 上海市农业科学院 | A kind of immunity detection reagent of muskmelon macula lutea virus and its application |
| CN109580506A (en) * | 2018-11-28 | 2019-04-05 | 天津瑞生物科技股份有限公司 | A kind of fungal detection system and two Methods for Fungi Detection based on digital microfluidic technology |
| CN112725494A (en) * | 2020-12-31 | 2021-04-30 | 中国农业科学院蔬菜花卉研究所 | Method and primer for detecting 17 tomato pathogens by using micro-fluidic chip |
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| CN104237519A (en) * | 2014-09-29 | 2014-12-24 | 江南大学 | Double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) method for detecting erwinia stewartii dye |
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| CN107907535A (en) * | 2017-10-20 | 2018-04-13 | 浙江省柑桔研究所 | A kind of method for diagnosing red bayberry dead-arm |
| CN109580506A (en) * | 2018-11-28 | 2019-04-05 | 天津瑞生物科技股份有限公司 | A kind of fungal detection system and two Methods for Fungi Detection based on digital microfluidic technology |
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