CN107015014A - Crop fungal spore liquefaction resistance device and detection method - Google Patents
Crop fungal spore liquefaction resistance device and detection method Download PDFInfo
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- CN107015014A CN107015014A CN201710155750.XA CN201710155750A CN107015014A CN 107015014 A CN107015014 A CN 107015014A CN 201710155750 A CN201710155750 A CN 201710155750A CN 107015014 A CN107015014 A CN 107015014A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
Abstract
The invention discloses a kind of crop fungal spore liquefaction resistance device, including syringe pump, micro-fluidic chip, temperature control device, cmos image sensor, reaction zone and reaction detection area are provided with micro-fluidic chip, it is combined using microflow controlled biochip and cmos image sensor, improves the accuracy of spore detection；The invention also discloses a kind of crop fungal spore liquefaction resistance method, it is only necessary to which the amount of reagent and spore that will be detected wait observed result from the entrance injection set.Crop fungal spore liquefaction resistance device and detection method in the present invention, accuracy that is simple to operate and can improving testing result are easy to avoid repetitive operation, improve detection efficiency.
Description
Technical field
The present invention relates to microorganism field, more particularly to a kind of crop fungal spore liquefaction resistance device and detection side
Method.
Background technology
At present, not yet occur for spore resistance to the action of a drug diagnostic techniques, disease disposal has larger hysteresis quality, to farming
High yield brings serious strike.Research finds that, with a large amount of, extensive adaptations of agricultural chemicals, resistance problem is also produced therewith.To mesh
Before untill, there are various crop fungies to be respectively provided with drug-fast report to agricultural chemicals.Because disease mainly passes through the sexual spore of germ
Propagated in the modes such as air-flow, therefore, fungal spore is detected in atmosphere, whether crops are judged by disease infestation, to realizing
Crops early diagnosis, prevention are significant.
The content of the invention
Goal of the invention：In order to solve the deficiencies in the prior art, the invention provides a kind of inspection of crop fungal spore resistance to the action of a drug
Device and detection method are surveyed, simple, intuitive the resistance to medicine characteristic of spore can be detected, and improve accuracy.
Technical scheme：In order to solve the above technical problems, crop fungal spore liquefaction resistance device in the present invention, the device
Including syringe pump, micro-fluidic chip, temperature control device, cmos image sensor, unidirectional response is provided with the micro-fluidic chip
Passage, is disposed with sample inlet, buffer inlet, coloring agent solution inlet and reactant row on the unidirectional response passage
Outlet, the first chip reaction zone, the second chip reaction zone, chip are divided into according to the traveling stage of reactant by half-duplex channel
Detection zone, the sample inlet and buffer inlet access the first chip reaction zone, the first chip reaction zone by micro-mixer
The second chip reaction zone is accessed by micro-mixer with coloring agent solution inlet, the second chip reaction zone connects with chip detection zone
Connect, reactant outlet is arranged on the end of chip detection zone；The sample inlet, buffer inlet and coloring agent solution inlet
It is connected respectively with the syringe pump equipped with respective substance；At least two groups of the sample inlet, one of which enters as spores solution
Mouthful, remaining is used as the pesticide solution entrance；The temperature control device is laid in around the micro-fluidic chip supplies warm, described CMOS for it
Imaging sensor carries out IMAQ to chip detection zone.
The position relationship of each group is in the sample inlet：In the axle centered on the position of spores solution entrance, remaining group
Sample inlet be even number, it is and substantially symmetrical about its central axis on this.
Correspondingly, present invention also offers a kind of crop fungal spore liquefaction resistance method, using said apparatus to true
The resistance to the action of a drug of bacterium spore is detected that this method comprises the following steps：
(1) spores solution and the pesticide solution are injected separately into using sample inlet, buffer solution, profit is injected using buffer inlet
Coloring agent is injected with coloring agent solution inlet；
(2) spores solution and the pesticide solution are continued after micro-mixer is mixed into the progress of the first chip reaction zone pre-
If the reaction of time；
(3) the second chip reaction zone is entered after the spores solution for dyeing agent solution and being infected is mixed by micro-mixer
It is middle to be dyed；
(5) when the mixed liquor for dyeing agent solution and the spores solution infected flow to chip detection zone, schemed using CMOS
As sensor carries out IMAQ to micro-fluidic chip.
Further, during for detection spore to the resistances to the action of a drug of different pesticide concentrations, different groups of sample inlets difference are utilized
Inject the pesticide solution of various concentrations；And isolated each the pesticide solution with the mixed liquor of spores solution using buffer solution.
Further, during for detection spore to the resistance to the action of a drug of agricultural chemicals not of the same race, noted respectively using different groups of sample inlets
Enter the pesticide solution not of the same race；And isolated each the pesticide solution with the mixed liquor of spores solution using buffer solution.
Beneficial effect：Crop fungal spore liquefaction resistance device in the present invention is provided using micro-fluidic chip for spore
Reaction zone and reaction detection area, it is simple to operate, it is only necessary to which that the amount of reagent and spore that will be detected are waited from the entrance injection set
Observed result；The device possesses sample pretreatment function, reduces the complexity of whole detection process, is not required to professional
Operation and fixed-site；It is combined using microflow controlled biochip and cmos image sensor, improves the accuracy of spore detection；
By setting multigroup sample inlet to realize the work in the resistance to the action of a drug of the detection spore to single medicine various concentrations, the present invention
Thing fungal spore liquefaction resistance method, it is simple to operate using said apparatus, it can also detect that spore resists to a variety of medicines simultaneously
The property of medicine, it is to avoid repeated operation, saves the time, and improve detection efficiency.
Brief description of the drawings
Fig. 1 is the structural representation of crop fungal spore liquefaction resistance device in the present invention；
Fig. 2 is the structural representation of micro-fluidic chip；
Fig. 3 is the structural representation of the micro-mixer of micro-fluidic chip；
Fig. 4 is that the chip detection zone of micro-fluidic chip detects target display schematic diagram in cmos image sensor；
Fig. 5 is the flow chart of crop fungal spore liquefaction resistance method in the present invention；
In figure, 1 is syringe pump, and 2 be micro-fluidic chip, and 3 be single-chip microcomputer, and 4 be temperature control device, and 5 be cmos image sensor,
6 be host computer, and 7 be power supply, and 201 be sample inlet, and 202 be buffer inlet, and 203 be coloring agent solution inlet, and 204 be micro- mixed
Clutch, 205 be chip reaction zone, and 206 be chip detection zone, and 207 be reactant outlet.
Embodiment
With reference to embodiment, the present invention is described in further detail.
Crop fungal spore liquefaction resistance device in Fig. 1, including syringe pump 1, micro-fluidic chip 2, single-chip microcomputer 3, temperature
Equipment 4, cmos image sensor 5, host computer 6 and power supply 7 are controlled, syringe pump 1 is connected with micro-fluidic chip 2, for completing to institute
Need the sample introduction task of sample；Temperature control device 4, cmos image sensor 5 are respectively positioned on around micro-fluidic chip 2, and single-chip microcomputer 3 is distinguished
It is connected with temperature control device 4, cmos image sensor 5 and host computer 6, single-chip microcomputer 3 is used for the work feelings for controlling temperature control device 4
Condition, it is ensured that the optimum temperature needed for detection environment, the control cmos image of single-chip microcomputer 3 can be reached during spore liquefaction resistance
Sensor 5 works and received and store the picture that cmos image sensor 5 is transmitted；Single-chip microcomputer 3 is by the picture collected
Shown in real time on data transfer to host computer 6, be easy to the observation of tester；Power supply 7 and single-chip microcomputer 3, host computer 6 and note
Penetrate pump 1 to be connected, power supply 7 is powered to this three, it is ensured that its normal operation.
As illustrated in fig. 1 and 2, unidirectional response passage is provided with micro-fluidic chip 2, is set successively on the unidirectional response passage
Sample inlet 201, buffer inlet 202, coloring agent solution inlet 203 and reactant outlet 207 are equipped with, according to reactant
Half-duplex channel is divided into the first chip reaction zone, the second chip reaction zone, chip detection zone 206, the first core by the traveling stage
Piece reaction zone and the second chip reaction zone are referred to as chip reaction zone 205, and sample inlet 201, buffer inlet 202 pass through micro- mixed
Clutch 204 accesses the first chip reaction zone, and the first chip reaction zone and coloring agent solution inlet 203 are connect by micro-mixer 204
Enter the second chip reaction zone；Second chip reaction zone is connected with chip detection zone 206, and reactant outlet 207 is arranged on chip
The end of detection zone 206.Wherein, sample inlet 201 uses Y type passages, and Y types passage connects corresponding syringe pump, passes through Y respectively
The reactant that type passage enters accesses unidirectional response passage also by micro-mixer 204, experimental to need sample inlet 201
N+1 groups can be divided into, No. 1-1, No. 1-2,1-2 ' numbers, No. 1-3,1-3 ' numbers No. 1-N, 1-N ' numbers, No. 1-1 work are designated as respectively
For spores solution entrance, remaining each group is as the pesticide solution entrance, and sample inlet number in remaining each group is even number, such as
Shown in Fig. 2, No. 1-2 and 1-2 ' numbers entrance be it is symmetrical on No. 1-1, the like, No. 1-3,1-3 ' numbers entrance be on 1-1
It is number symmetrical, and No. 1-N, 1-N ' numbers entrance it is symmetrical on-No. 1.Sample number entrance 201 is connected with syringe pump 1, if according to reality
Testing needs to be provided with N groups sample inlet 201, then each entrance is corresponding with the syringe pump 1 equipped with different material respectively is connected, and temperature control is set
Standby 4 are placed on the lower section of the chip reaction zone 205 of micro-fluidic chip 2, and cmos image sensor 5 is positioned over micro-fluidic chip 2
The lower section of chip detection zone 206.
The chip can not only realize that detection spore, to the resistance to the action of a drug of single medicine various concentrations, detection spore can be also realized simultaneously
Son is to the resistance to the action of a drug of a variety of medicines, so as to realize detection object diversification, automaticity is high.
As shown in figure 3, in order to increase the micro-mixer 204 on chaos degree, raising mixing efficiency, micro-fluidic chip 2
The rectangular flat plate obstruction arranged according to certain rules is placed in diffusion admittance increases convection current with this.
As shown in figure 4, the chip detection zone 206 on micro-fluidic chip 2 uses bending channel shape, bending channel is added
The length of chip detection zone 206 so that testing result can be showed preferably；The present invention uses cmos image sensor 5
To observe the color after sporocyst infects, the existing state of spore is judged with this, facilitates the shooting of testing result.
As shown in figure 5, when detecting spore to the resistance to the action of a drug of medicine using the device in Fig. 1, comprising the following steps：
(1) spore stoste and water are injected from the 1-1 Y type passages two ends of sample inlet 201 respectively, by agricultural chemicals and moisture
Two ends not from other group of Y type passage of sample inlet 201 are injected, such as 1-2 and 1-2 ' numbers mouth, complete early stage solution and prepare mixing
Pretreatment work；
(2) solution (the pesticide solution and spores solution) prepared enters first after micro-mixer 204 is mixed fully
Chip reaction zone continue the reaction of preset time；
(3) coloring agent stoste and water are injected from the Y type passages two ends of coloring agent solution inlet 203 respectively, and filled
Divide mixing, complete the preparation of dyeing agent solution, mixed afterwards with the spores solution infected by agricultural chemicals；
(4) mixed liquor (mixed liquor of dyeing agent solution and the spores solution infected by agricultural chemicals) passes through micro-mixer 204
Diffusion admittance is thoroughly mixed, and is dyed in the second chip reaction zone, hybrid channel in the second chip reaction zone
Length, which is set, ensures that dyeing is complete；
(5) when mixed liquor mixing is complete and flow to chip detection zone 206, detected using cmos image sensor 5
The spore of dyeing is completed, and sends picture to single-chip microcomputer 3, single-chip microcomputer 3 transmits the image data collected to host computer 6
Show in real time.
With above-mentioned principle, when detecting spore to the resistances to the action of a drug of medicine various concentrations a kind of using the device in Fig. 1, accordingly
Crop fungal spore liquefaction resistance method comprises the following steps：
(1) preparation of various concentrations the pesticide solution and the mixing with the pesticide solution and spore
1) water and spore of corresponding dosage are separately added into the corresponding syringe pump of 1-1 mouth Y type passages of sample inlet 201
Son, in 1-2 and 1-2 ' water and agricultural chemicals of corresponding dosage are separately added into number corresponding syringe pump of mouth Y type passages, in 1-3 and 1-3 '
It is separately added into the water and agricultural chemicals of corresponding dosage in number corresponding syringe pump of mouth Y type passages, the ratio of its reclaimed water and agricultural chemicals is according to reality
Needs are tested to be configured, the like, until completing the concentration group number for needing to detect；
Buffer solution is added in the corresponding syringe pump of buffer inlet 202；
It is proportionally added into respectively in the corresponding syringe pump of Y type passages both sides sample inlet of coloring agent solution inlet 203
FDA and PI solution (both are mixed to form dyeing agent solution)；
2) while promoting 2 (N+1)+1+2 being connected with sample inlet 201, buffer inlet 202 and coloring agent entrance 203
Individual syringe pump, the spores solution that the 1-1 mouths of sample inlet 201 flow out after the mixing of micro-mixer 204, other group of entrance warp
The agricultural chemicals of the various concentrations flowed out after micro-mixer 204 is mixed is crossed, what buffer inlet 202 injected is buffer solution, because of passage
Length is isometric, so the mixed liquor and buffer solution of each passage can flow to sample inlet 201, buffer inlet 202 simultaneously and connect
Contact forward part, stops promoting；
3) syringe pump being connected with 1-1 mouths is first promoted, injecting fluid volume is set as 1ul, stops other syringe pumps
Injection, stops injection, spores solution now is the sample infected without decoction, it detects knot after the syringe pump completes to inject
Fruit can judge the infection ability of which kind of concentration of this kind of medicine most with the sporogenesis blank control infected below by medicine with this
By force；
Start the ejection of syringe pump buffer solution of buffer inlet 202, set injecting fluid volume as 0.1ul, work as syringe pump
Complete to stop injection after injection；
The syringe pump being connected with 1-1 mouths, 1-2 mouths and 1-2 ' numbers mouth is promoted simultaneously, sets this injecting fluid volume
For 1ul, after the completion of injection task, stop injection, be again started up connecting the ejection of syringe pump buffer solution of No. 2 mouths, set parenteral solution
Body volume is 0.1ul, after the completion of injection task, stops injection, by that analogy, and until N kinds, (N size is set according to experiment needs
Put) medicine of concentration injects to finish and obtains various concentrations medicine and spore mixed solution.
(2) spores solution and the pesticide solution enter the first chip reaction zone after micro-mixer 204 is mixed fully and carried out
Continue the reaction of preset time.
1) micro-mixer 204 is entered by the spores solution that agricultural chemicals infects, the process ensures that spores solution and medicine are sufficient
Mixing, obtains the spores solution infected by medicine.
2) mixed solution of the pesticide solution and spores solution reacts into the first chip reaction zone, the first chip reaction zone
Length ensure that spore contaminates required duration and (if the time is not enough, can integrally slow down the sample introduction speed of syringe pump to coordinate medicine
Infect the time span needed for spore).
(3) dyeing agent solution by the spore that agricultural chemicals infects to being dyed.
When the liquid in chip is flow to before coloring agent solution inlet 203, the syringe pump of connection coloring agent solution inlet 203 is opened
Begin to inject the FDA-PI solution mixed.
(4) spores solution and the mixed liquor of FDA-PI solution infected by agricultural chemicals is again introduced into micro- under the push of syringe pump
Blender 204, afterwards into the second chip reaction zone, FDA-PI solution gives the spore infected by the medicament of the same race of various concentrations
Sub- solution is dyed.
Spore suspension is after the mono- dyes of FDA, in 5 times observations of the cmos image sensor allowed with fluorescence detection function spore living
Son is in bright green fluorescence；Fusarinm solani heats lethal spore suspension after the mono- dyes of PI, and dead spore sends red fluorescence.Will be new
Fresh spore is mixed with heating lethal spore, after the double dyes of FDA-PI, visible green and red fluorescence difference under fluorescence microscope
Clearly, illustrate to judge spore anyway with the double fluorescent stainings of FDA-PI, wherein spore living is in green fluorescence, dead spore
Son takes on a red color fluorescence.
(5) after spores solution is contaminated completely by FDA-PI solution, liquid flow to chip detection zone 206, is schemed by CMOS
As sensor 5 is shot to viewing area, red fluorescence is presented in dead spore, and green fluorescence is presented in spore living,
Gained picture reaches host computer 6 and shown, is easy to observe and judges the activity of spore, and it is dense to this kind of medicine difference to complete spore with this
The liquefaction resistance of degree.
With above-mentioned principle, when detecting spore to the resistance to the action of a drug of medicine not of the same race using the device in Fig. 1, corresponding crop is true
The step of bacterium spore liquefaction resistance method is with the above method is identical, unique the difference is that being accomplished that difference in step (1)
The preparation of the pesticide solution and the mixing with spore.When being detected to several samples decoction, in sample inlet 2011-1
Mouthful Y type passages both sides sample inlet be separately added into the water and spore of corresponding dosage in proportion, in 1-2 and 1-2 ' the Y types of number mouth
Passage both sides sample inlet is proportionally added into the water and the first medicine of corresponding dosage respectively, in 1-3 and 1-3 ' number mouth addition point
The water and second of medicine of corresponding dosage, by that analogy, the experimental group number needed are not proportionally added into.
It the above is only the preferred embodiment of the present invention, it should be pointed out that implement row above and restriction, phase are not constituted to the present invention
Close staff in the range of without departing from the technology of the present invention thought, carried out it is various change and modifications, all fall within the present invention
Protection domain in.
Claims (8)
1. a kind of crop fungal spore liquefaction resistance device, it is characterised in that the device include syringe pump, micro-fluidic chip,
Unidirectional response passage is provided with temperature control device, cmos image sensor, the micro-fluidic chip, on the unidirectional response passage
Sample inlet, buffer inlet, coloring agent solution inlet and reactant outlet are disposed with, according to the traveling rank of reactant
Half-duplex channel is divided into the first chip reaction zone, the second chip reaction zone, chip detection zone by section, and the sample inlet eases up
Fliud flushing entrance accesses the first chip reaction zone by micro-mixer, and the first chip reaction zone and coloring agent solution inlet pass through micro- mixed
Clutch accesses the second chip reaction zone, and the second chip reaction zone is connected with chip detection zone, and reactant outlet is arranged on chip
The end of detection zone；The sample inlet, buffer inlet and coloring agent solution inlet respectively with the injection equipped with respective substance
Pump is connected；At least two groups of the sample inlet, one of which is as spores solution entrance, and remaining is used as the pesticide solution entrance；
The temperature control device is laid in around the micro-fluidic chip to be entered for it for warm, described cmos image sensor to chip detection zone
Row IMAQ.
2. crop fungal spore liquefaction resistance device according to claim 1, it is characterised in that in the sample inlet
The position relationship of each group is：Sample inlet in the axle centered on the position of spores solution entrance, remaining group is even number, and is closed
It is substantially symmetrical about its central axis in this.
3. crop fungal spore liquefaction resistance device according to claim 2, it is characterised in that the sample inlet and
Coloring agent solution inlet uses Y type passages.
4. crop fungal spore liquefaction resistance device according to claim 2, it is characterised in that the micro-mixer
Obstruction is provided with diffusion admittance.
5. crop fungal spore liquefaction resistance device according to claim 2, it is characterised in that the cmos image is passed
Sensor is located at the lower section of the chip detection zone.
6. a kind of crop fungal spore liquefaction resistance method, is examined using the crop fungal spore resistance to the action of a drug described in claim 1
Survey device, it is characterised in that this method comprises the following steps：
（1）Spores solution and the pesticide solution are injected separately into using sample inlet, buffer solution is injected using buffer inlet, utilizes dye
Toner solution inlet injects coloring agent；
（2）When spores solution and the pesticide solution into the first chip reaction zone persistently preset after micro-mixer is mixed
Between reaction；
（3）Enter to enter in the second chip reaction zone after the spores solution for dyeing agent solution and being infected is mixed by micro-mixer
Row dyeing；
（5）When the mixed liquor for dyeing agent solution and the spores solution infected flow to chip detection zone, passed using cmos image
Sensor carries out IMAQ to micro-fluidic chip.
7. crop fungal spore liquefaction resistance method according to claim 6, it is characterised in that for detection spore pair
During the resistance to the action of a drug of different pesticide concentrations, the pesticide solution of various concentrations is injected separately into using different groups of sample inlets；And utilize slow
Fliud flushing is isolated each the pesticide solution with the mixed liquor of spores solution.
8. crop fungal spore liquefaction resistance method according to claim 7, it is characterised in that for detection spore pair
During the resistance to the action of a drug of agricultural chemicals not of the same race, the pesticide solution not of the same race is injected separately into using different groups of sample inlets；And utilize buffer solution will
Each the pesticide solution is isolated with the mixed liquor of spores solution.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201710155750.XA CN107015014B (en) | 2017-03-16 | 2017-03-16 | Detection device and detection method for drug resistance of crop fungal spores |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710155750.XA CN107015014B (en) | 2017-03-16 | 2017-03-16 | Detection device and detection method for drug resistance of crop fungal spores |
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| CN107015014A true CN107015014A (en) | 2017-08-04 |
| CN107015014B CN107015014B (en) | 2020-04-21 |
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| CN112023989A (en) * | 2019-06-03 | 2020-12-04 | 利多(香港)有限公司 | Microfluidic detection integrated chip and sample detection method |
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