CN209669229U - A kind of canine virus fluorescence quantitative PCR detection micro-fluidic chip - Google Patents
A kind of canine virus fluorescence quantitative PCR detection micro-fluidic chip Download PDFInfo
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- CN209669229U CN209669229U CN201920129800.1U CN201920129800U CN209669229U CN 209669229 U CN209669229 U CN 209669229U CN 201920129800 U CN201920129800 U CN 201920129800U CN 209669229 U CN209669229 U CN 209669229U
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- fluid channel
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Abstract
The utility model relates to a kind of canine virus fluorescence quantitative PCR detection micro-fluidic chips, are made of substrate, upper cover plate and lower cover plate；Sample holes A, fluid channel, DNA separation chamber, triangle vavle, waste liquid chamber, sample holes B, mixing chamber, PCR reaction zone and fluorescence detection room are provided on substrate；Upper cover plate reserves the position of corresponding sample holes A, sample holes B and triangle vavle, and the opening chamber of corresponding waste liquid chamber position setting protrusion is bonded to the upper surface of substrate；Lower cover plate is bonded to the lower surface of substrate, and base lower surface is closed.The utility model can realize DNA separation and fluorescence quantitative PCR detection on a chip, convenient for users to from latter step operation is sampled you can learn that testing result；The waste liquid chamber of autonomous closure is set, is convenient for manipulator motorized operation, avoids the pollution to instrument；The utility model had both been applicable to large automatic detecting instrument, and also applicable small device is operated manually, and operating process is simple and efficient, strong flexibility.
Description
Technical field
The utility model relates to animal epidemic detection technique fields, and in particular to a kind of canine virus quantitative fluorescent PCR inspection
The micro-fluidic chip of survey.
Background technique
Virus with other microbial ratios, structure, chemical composition, modes of reproduction and to drug susceptibility in terms of have it is aobvious
Write difference.Animal virosis has the characteristics that infectiousness is strong, prevalence is extensive, harm is serious and disease incidence is high, especially pet disease
Viral disease, it is also possible to which pet-breeder is impacted.Therefore how quick diagnosis, quantitative Diagnosis is at control and to treat these dynamic
The key factor of object virosis.
Under present case, each veterinary laboratories, pets hospital detection animal virosis mainly utilize serological test, molecule
Biological test and the test paper card or ELISA designed based on colloidal gold principle.But there is drawback, serology in these methods
Test, molecular biology test often take a long time, and do not have the effect quickly detected；Though and colloid gold test paper can be realized fastly
Speed detection, but cannot achieve the quantitative detection of virus；ELISA only can qualitative detection virus, and needed in operating process for a long time
It is incubated for and rinses, take a long time.
Utility model content
The technical problem to be solved by the utility model is to how quick and quantitative detection canid virus, and it is same
Shi Shixian DNA is extracted and quantitative fluorescent PCR is completed on a chip.
To solve the above problems, based on DNA extraction and quantitative fluorescent PCR principle, in conjunction with microflow control technique, the utility model
Provide a kind of canine virus fluorescence quantitative PCR detection micro-fluidic chip, which is characterized in that the micro-fluidic chip by substrate,
Upper cover plate is bonded with lower cover plate；
It is provided with sample holes A on substrate, fluid channel, DNA separation chamber, triangle vavle, waste liquid chamber, sample holes B, mixing chamber,
PCR reaction zone, fluorescence detection room；
Upper cover plate reserves the position of corresponding sample holes A, sample holes B and triangle vavle, corresponding waste liquid chamber position setting protrusion
Open chamber, upper cover plate are bonded to the upper surface of substrate；Lower cover plate is bonded to the lower surface of substrate, and base lower surface is all sealed
It closes；Substrate, upper cover plate and lower cover plate collectively form DNA separation chamber, waste liquid chamber, mixing chamber and the accommodation space of fluorescence detection room,
The fluid channel of fluid channel and PCR reaction zone；
DNA separation chamber is connected with sample holes A, triangle vavle respectively by being located at the fluid channel of base plate bottom；Waste liquid chamber is logical
Fluid channel is crossed to be connected with triangle vavle；Mixing chamber is connected by fluid channel with sample holes B, triangle vavle；PCR reaction zone passes through micro-
Runner is connected with triangle vavle and fluorescence detection room；
By the rotation of triangle vavle, the outlet of fluid channel of the DNA separation chamber in triangle vavle can be led to triangle vavle useless
Liquid chamber or the outlet of the fluid channel of mixing chamber are connected, to control the flow direction of waste liquid or DNA eluent；
By the rotation of triangle vavle, it is anti-PCR can be led to triangle vavle in the outlet of fluid channel of the mixing chamber in triangle vavle
It answers the fluid channel in area to export to connect, to control PCR reaction process.
The material of the substrate, upper cover plate and lower cover plate can be selected from glass, PMMA, PC, COC or COP.Upper cover plate or lower cover
Plate with a thickness of 0.1-0.5mm.Reaction chamber bottom surface, fluid channel inner surface can carry out hydrophilic or hydrophobic treatment.
Being connected to sample holes, the width of the fluid channel of chamber and triangle vavle and/or depth is 50-300 μm；PCR reaction zone
Fluid channel width and/or depth are 50-100 μm.
The volume of the DNA separation chamber is 50-500 μ l；The volume of the waste liquid chamber is 200-1000 μ l；The mixing chamber
Volume be 20-100 μ l；Can also in mixing chamber preset magnetic rotor, be easy to use magnetic agitation instrument to reaction reagent into
Row is sufficiently mixed.
The PCR reaction zone is made of three groups of fluid channels, and three groups of fluid channels respectively correspond heating module different in PCR instrument
Position；3-5 group is arranged in the S-shaped fluid channel of corresponding nucleic acid cleavage area heating module；The S-shaped of corresponding circular response area heating module
15-30 group is arranged in fluid channel；3-5 group is arranged in the S-shaped fluid channel of corresponding extension area heating module.
The canine virus rapid quantitative detection micro-fluidic chip further includes Rule plug or plug for closing sample holes.
The canine virus rapid quantitative detection micro-fluidic chip can separate magnetic by preset DNA in DNA separation chamber
Pearl；Can also in reaction chamber preset magnetic rotor, be easy to use magnetic agitation instrument to be sufficiently mixed to reaction reagent or DNA
Separation.
Canine virus described in the utility model can be selected from the coronal disease of canine distemper virus, canine parvovirus, rabies viruses, dog
Poison, dog encephalomyocarditis virus, canine parainfluenza virus or hepatitis infectiosa canis virus.
The utility model has an advantage that compared with prior art
1, the utility model can realize DNA separation and quantitative fluorescent PCR reaction on a chip, can be directed to micro sample
This is detected, convenient for users to from latter step operation is sampled you can learn that testing result, is realized to the highly sensitive of canine virus
Degree, quick, quantitative detection.
2, the utility model itself setting closing waste liquid pool, more convenient robotic operation, avoids to instrument
Pollution.
3, the utility model had both been applicable to large automatic detecting instrument, and also applicable small device is grasped manually
Make, operating process is simple and efficient, strong flexibility.
Detailed description of the invention
The positive structure schematic of Fig. 1 the utility model canine virus fluorescence quantitative PCR detection micro-fluidic chip, in figure
Label: 11- sample holes A, 12- fluid channel, the separation chamber 13-DNA, 14- triangle vavle, 15- waste liquid chamber, 16- sample holes B, 17- mixing
Room, 18-PCR reaction zone (in dotted line frame), 19- fluorescence detection room.
The upper cover plate structural schematic diagram of Fig. 2 the utility model canine virus fluorescence quantitative PCR detection micro-fluidic chip, figure
Middle label: 20- upper cover plate.
The vertical section structure of Fig. 3 the utility model canine virus fluorescence quantitative PCR detection micro-fluidic chip waste liquid chamber side
Schematic diagram, marked in the figure: upper figure: 1- canine virus fluorescence quantitative PCR detection micro-fluidic chip, along a line vertical profile；The following figure: 10- base
Plate, 20- upper cover plate, 30- lower cover plate, 11- sample holes A, 12- fluid channel, the separation chamber 13-DNA, 14- triangle vavle, 15- waste liquid
Room.
Fig. 4 the utility model canine virus fluorescence quantitative PCR detection micro-fluidic chip corresponds to the structure of instrument heating module
Schematic diagram, marked in the figure: 41- nucleic acid cleavage area heating module (dotted line frame), 42- circular response area heating module 1 (dotted line frame),
43- circular response area heating module 2 (dotted line frame), 44- circular response area heating module 3 (dotted line frame), 45- extension area add
Thermal modules (dotted line frame).
Specific embodiment
The utility model is further described with reference to the accompanying drawings and embodiments.It should be noted that in embodiment not in detail
The normal condition and method of explanation, the experiment condition and method routinely used according to fields personnel carry out.
Embodiment:
As shown in Figure 1-Figure 3, a kind of canine virus fluorescence quantitative PCR detection micro-fluidic chip, the micro-fluidic chip by
Substrate (10), upper cover plate (20) are bonded with lower cover plate (30)；Sample holes A (11), fluid channel are provided on substrate (10)
(12), DNA separation chamber (13), triangle vavle (14), waste liquid chamber (15), sample holes B (16), mixing chamber (17), PCR reaction zone
(18), fluorescence detection room (19)；Upper cover plate (20) reserves corresponding sample holes A (11), sample holes B (16) and triangle vavle (14)
Position, the opening chamber of corresponding waste liquid chamber (15) position setting protrusion, upper cover plate (20) are bonded to the upper surface of substrate (10)；Under
Cover board (30) is bonded to the lower surface of substrate (10), and substrate (10) lower surface is all closed；Substrate (10), upper cover plate (20) with
Lower cover plate (30) collectively forms the receiving sky of DNA separation chamber (13), waste liquid chamber (15), mixing chamber (17) and fluorescence detection room (19)
Between, the fluid channel of fluid channel (12) and PCR reaction zone (18).
DNA separation chamber (13) by be located at base plate bottom fluid channel (12) respectively with sample holes A (11), triangle vavle (14)
It is connected；Waste liquid chamber (15) is connected by fluid channel (12) with triangle vavle (14)；Mixing chamber (17) by fluid channel (12) with
Sample holes B (16), triangle vavle (14) are connected；PCR reaction zone (18) is examined by fluid channel (12) and triangle vavle (14) and fluorescence
Room (19) are surveyed to be connected；It, can be by miniflow of the DNA separation chamber (13) in triangle vavle (14) by the rotation of triangle vavle (14)
The fluid channel outlet that waste liquid chamber (15) or mixing chamber (17) are led to triangle vavle (14) in road outlet is connected, thus control waste liquid or
The flow direction of DNA eluent；It, can be by fluid channel of the mixing chamber (17) in triangle vavle (14) by the rotation of triangle vavle (14)
The fluid channel outlet that PCR reaction zone (18) is led to triangle vavle (14) in outlet is connected, to control PCR reaction process.
The material of the substrate (10), upper cover plate (20) and lower cover plate (30) can be selected from glass, PMMA, PC, COC or
COP；It is preferred that COC or COP；Upper cover plate (20) with a thickness of 0.2mm, lower cover plate (30) with a thickness of 0.1-0.5mm.
The width and/or depth for connecting the fluid channel (12) of each chamber and sample holes are 50-100 μm；PCR reaction zone
(18) fluid channel width and/or depth is 50-80 μm.
The volume of DNA separation chamber (13) is 100 μ l；The volume of waste liquid chamber (15) is 500 μ l；The volume of mixing chamber (17) is
100μl。
The PCR reaction zone (18) is made of three groups of fluid channels, and three groups of fluid channels respectively correspond heating different in PCR instrument
The position of module；3-5 group is arranged in the S-shaped fluid channel of corresponding nucleic acid cleavage area heating module (41)；Corresponding circular response area heating
15-30 group is arranged in the S-shaped fluid channel of module (42), (43) and (44)；The S-shaped miniflow of corresponding extension area heating module (45)
3-5 group is arranged in road.
The canine virus fluorescence quantitative PCR detection micro-fluidic chip is suitable for canine distemper virus, canine parvovirus, mad
Dog disease poison, canine coronavirus, dog encephalomyocarditis virus, canine parainfluenza virus and/or hepatitis infectiosa canis virus quantitative detection.
The application method of canine virus fluorescence quantitative PCR detection micro-fluidic chip described in the utility model:
1, the protective film at sample holes A is torn, DNA separation magnetic bead 2-5 μ l is injected into DNA separation chamber；
2, the samples to be tested such as animal blood serum, saliva, urine 50-100 μ l is injected into DNA separation chamber, reaction chamber from sample holes A
Interior air surveys through-hole discharge from triangle vavle, avoids blowing out bubble in reaction chamber as far as possible, closes sample introduction using Rule plug or plug
Hole, control triangle vavle close the through-hole for connecting DNA separation chamber；
3, micro-fluidic chip is placed on magnetic force blending instrument, opens switch, drive DNA to separate magnetic bead in sample by magnetic force
In flow repeatedly, DNA is adsorbed on magnetic bead, this step about 5-10 minutes；
4, DNA separation chamber is connected to by control triangle vavle with waste liquid chamber, buffer is injected from sample holes A, by remaining sample
It is discharged into waste liquid chamber, closing the interior through-hole for connecting DNA separation chamber of triangle vavle, (magnetic force blending instrument is always on during this, guarantees to inhale
The magnetic bead of attached DNA will not be flushed away)；
5, magnetic bead 30-60s is rinsed using buffer, DNA separation chamber is connected to waste liquid chamber, waste liquid is arranged by control triangle vavle
Enter waste liquid chamber；It repeats this flushing process 1-3 times, waste liquid is all discharged using air pump；
6, DNA eluent is injected from sample holes A, it is identical as above-mentioned rinsing step, DNA is eluted；
7, DNA separation chamber is connected to by control triangle vavle with mixing chamber, is injected the DNA of elution in mixing chamber using air pump,
PCR reaction reagent mixture is injected into mixing chamber from sample holes B, mixing can be shaken manually, can also preset magnetic rotor in chip,
It is mixed using magnetic force blending instrument；
8, mixing chamber is connected to by control triangle vavle with PCR reaction zone, micro- in reaction zone using air pump control PCR reaction system
Each heating module is flowed through in runner, completes DNA unwinding, is annealed, and is extended, it is final to import fluorescence detection room.Air pump can control liquid
The flowing velocity of body different zones in fluid channel guarantees the accurate completion of PCR program；Different heating modules is arranged different
Temperature, corresponding PCR reaction process demand.
9, by fluorescence detection room, instrument readings calculate viral load according to standard curve.
The above is only the preferred embodiment of the utility model only, is not intended to limit the utility model, all at this
Within the spirit and principle of utility model, any modification, equivalent replacement, improvement and so on should be included in the utility model
Protection scope within.
Claims (8)
1. a kind of canine virus fluorescence quantitative PCR detection micro-fluidic chip, which is characterized in that the micro-fluidic chip is by substrate
(10), upper cover plate (20) is bonded with lower cover plate (30)；
It is provided on substrate (10) sample holes A (11), fluid channel (12), DNA separation chamber (13), triangle vavle (14), waste liquid chamber
(15), (16) sample holes B, mixing chamber (17), PCR reaction zone (18), fluorescence detection room (19)；
Upper cover plate (20) reserves the position of corresponding sample holes A (11), sample holes B (16) and triangle vavle (14), corresponding waste liquid chamber
(15) the opening chamber of position setting protrusion, upper cover plate (20) are bonded to the upper surface of substrate (10)；
Lower cover plate (30) is bonded to the lower surface of substrate (10), and substrate (10) lower surface is all closed；
Substrate (10), upper cover plate (20) and lower cover plate (30) collectively form DNA separation chamber (13), waste liquid chamber (15), mixing chamber
(17) and the fluid channel of the accommodation space of fluorescence detection room (19), fluid channel (12) and PCR reaction zone (18)；
DNA separation chamber (13) is connected with sample holes A (11), triangle vavle (14) respectively by being located at the fluid channel (12) of base plate bottom
It is logical；Waste liquid chamber (15) is connected by fluid channel (12) with triangle vavle (14)；Mixing chamber (17) passes through fluid channel (12) and sample introduction
Hole B (16), triangle vavle (14) are connected；PCR reaction zone (18) passes through fluid channel (12) and triangle vavle (14) and fluorescence detection room
(19) it is connected；
It, can be by the outlet of fluid channel of the DNA separation chamber (13) in triangle vavle (14) and triangle by the rotation of triangle vavle (14)
Valve (14) leads to waste liquid chamber (15) or the fluid channel outlet of mixing chamber (17) is connected, to control the stream of waste liquid or DNA eluent
To；
It, can be by the outlet of fluid channel of the mixing chamber (17) in triangle vavle (14) and triangle vavle by the rotation of triangle vavle (14)
(14) the fluid channel outlet for leading to PCR reaction zone (18) is connected, to control PCR reaction process.
2. canine virus fluorescence quantitative PCR detection micro-fluidic chip according to claim 1, which is characterized in that the base
The material of plate (10), upper cover plate (20) and lower cover plate (30) can be selected from glass, PMMA, PC, COC or COP；Upper cover plate (20) or under
Cover board (30) with a thickness of 0.1-0.5mm.
3. canine virus fluorescence quantitative PCR detection micro-fluidic chip according to claim 1, which is characterized in that fluid channel
(12) width and/or depth is 50-300 μm；The fluid channel width and/or depth of PCR reaction zone (18) are 50-100 μm.
4. canine virus fluorescence quantitative PCR detection micro-fluidic chip according to claim 1, which is characterized in that DNA separation
The volume of room (13) is 50-500 μ l.
5. canine virus fluorescence quantitative PCR detection micro-fluidic chip according to claim 1, which is characterized in that waste liquid chamber
(15) volume is 200-1000 μ l.
6. canine virus fluorescence quantitative PCR detection micro-fluidic chip according to claim 1, which is characterized in that mixing chamber
(17) volume is 20-100 μ l.
7. canine virus fluorescence quantitative PCR detection micro-fluidic chip according to claim 1, which is characterized in that mixing
Preset magnetic rotor in room (17).
8. canine virus fluorescence quantitative PCR detection micro-fluidic chip according to claim 1, which is characterized in that the PCR
Reaction zone (18) is made of three groups of fluid channels, and three groups of fluid channels respectively correspond the position of heating module different in PCR instrument；It is corresponding
3-5 group is arranged in the S-shaped fluid channel of nucleic acid cleavage area heating module (41)；Corresponding circular response area heating module (42), (43) and
(44) 15-30 group is arranged in S-shaped fluid channel；3-5 group is arranged in the S-shaped fluid channel of corresponding extension area heating module (45).
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111929460A (en) * | 2020-08-19 | 2020-11-13 | 河南科技大学 | Sampling, chip and liquid inlet control device suitable for microfluidic automatic detection |
| CN114018787A (en) * | 2021-10-23 | 2022-02-08 | 广州市艾贝泰生物科技有限公司 | Particle detection unit, mixing system and mixing method |
-
2019
- 2019-01-25 CN CN201920129800.1U patent/CN209669229U/en not_active Expired - Fee Related
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111929460A (en) * | 2020-08-19 | 2020-11-13 | 河南科技大学 | Sampling, chip and liquid inlet control device suitable for microfluidic automatic detection |
| CN111929460B (en) * | 2020-08-19 | 2024-03-01 | 河南科技大学 | Sampling, chip and liquid inlet control device suitable for microfluidic automatic detection |
| CN114018787A (en) * | 2021-10-23 | 2022-02-08 | 广州市艾贝泰生物科技有限公司 | Particle detection unit, mixing system and mixing method |
| CN114018787B (en) * | 2021-10-23 | 2023-10-20 | 广州市艾贝泰生物科技有限公司 | Particle detection unit, mixing system and mixing method |
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| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20191122 Termination date: 20210125 |