CN209669229U - A kind of canine virus fluorescence quantitative PCR detection micro-fluidic chip - Google Patents

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

A microfluidic chip for detection of canine virus by fluorescent quantitative PCR

technical field

The utility model relates to the technical field of animal epidemic detection, in particular to a microfluidic chip for fluorescent quantitative PCR detection of canine viruses.

Background technique

Compared with other microorganisms, viruses are significantly different in structure, chemical composition, mode of reproduction and sensitivity to drugs. Animal viral diseases are highly contagious, widespread, serious harm and high incidence, especially pet viral diseases, which may also affect pet breeders. Therefore how rapid diagnosis, quantitative diagnosis become the key factor of controlling and treating these animal virus diseases.

Under current circumstances, veterinary laboratories and animal hospitals mainly use serological tests, molecular biology tests, and test strip cards or ELISA designed based on the principle of colloidal gold to detect animal viral diseases. However, these methods have disadvantages. Serological tests and molecular biology tests are often time-consuming and cannot be used for rapid detection; although colloidal gold test paper can achieve rapid detection, it cannot achieve quantitative detection of viruses; ELISA can only Qualitative detection of viruses requires long incubation and washing during operation, which takes a long time.

Utility model content

The technical problem to be solved by the utility model is: how to quickly and quantitatively detect canine virus, and simultaneously realize DNA extraction and fluorescent quantitative PCR to be completed on one chip.

In order to solve the above problems, based on the principles of DNA extraction and fluorescent quantitative PCR, combined with microfluidic technology, the utility model provides a microfluidic chip for canine virus fluorescent quantitative PCR detection, which is characterized in that the microfluidic chip consists of The substrate, the upper cover and the lower cover are bonded together;

The substrate is provided with injection hole A, micro-channel, DNA separation chamber, triangular valve, waste liquid chamber, injection hole B, mixing chamber, PCR reaction area, and fluorescence detection chamber;

The upper cover leaves the positions corresponding to the injection hole A, the injection hole B and the triangular valve, and a raised open chamber is set corresponding to the position of the waste liquid chamber. The upper cover is bonded to the upper surface of the substrate; the lower cover is bonded On the lower surface of the substrate, the lower surface of the substrate is completely closed; the substrate, the upper cover and the lower cover together constitute the accommodation space of the DNA separation chamber, the waste liquid chamber, the mixing chamber and the fluorescence detection chamber, the micro flow channel and the PCR reaction area. Microchannel;

The DNA separation chamber is connected to the injection hole A and the triangle valve through the microchannel at the bottom of the substrate; the waste liquid chamber is connected to the triangle valve through the microchannel; the mixing chamber is connected to the injection hole B and the triangle valve through the microchannel. The PCR reaction area is connected with the triangular valve and the fluorescence detection chamber through the micro flow channel;

Through the rotation of the triangular valve, the microchannel outlet of the DNA separation chamber in the triangular valve can be connected with the microchannel outlet of the triangular valve leading to the waste liquid chamber or the mixing chamber, thereby controlling the flow direction of the waste liquid or DNA eluent ;

Through the rotation of the triangular valve, the outlet of the microfluidic channel in the triangular valve of the mixing chamber can be connected with the microfluidic outlet of the triangular valve leading to the PCR reaction area, thereby controlling the PCR reaction process.

The materials of the substrate, the upper cover and the lower cover can be selected from glass, PMMA, PC, COC or COP. The thickness of the upper cover or the lower cover is 0.1-0.5mm. The bottom surface of the reaction chamber and the inner surface of the microchannel can be treated with hydrophilic or hydrophobic treatment.

The width and/or depth of the micro flow channel connecting the injection hole, the chamber and the triangular valve is 50-300 μm; the width and/or depth of the micro flow channel in the PCR reaction area is 50-100 μm.

The volume of the DNA separation chamber is 50-500 μl; the volume of the waste liquid chamber is 200-1000 μl; the volume of the mixing chamber is 20-100 μl; a magnetic rotor can also be preset in the mixing chamber to facilitate the use of a magnetic stirrer Mix the reagents thoroughly.

The PCR reaction area is composed of three groups of micro-channels, and the three groups of micro-channels correspond to the positions of different heating modules on the PCR instrument respectively; 3-5 groups of S-shaped micro-channels corresponding to the heating modules in the nucleic acid lysis zone are set; There are 15-30 groups of S-shaped micro-channels of the heating module in the reaction zone; 3-5 groups of S-shaped micro-channels corresponding to the heating module of the extended reaction zone.

The microfluidic chip for rapid quantitative detection of canine virus also includes a luer plug or plug for sealing the injection hole.

The microfluidic chip for rapid quantitative detection of canine virus can be preset with DNA separation magnetic beads in the DNA separation chamber; it can also be preset with a magnetic rotor in the reaction chamber to facilitate sufficient mixing of reaction reagents or DNA separation using a magnetic stirrer. .

The canine virus described in the utility model can be selected from canine distemper virus, canine parvovirus, rabies virus, canine coronavirus, canine encephalomyocarditis virus, canine parainfluenza virus or canine adenovirus.

Compared with the prior art, the utility model has the following advantages:

1. The utility model can realize DNA separation and fluorescent quantitative PCR reaction on one chip, and can detect micro samples, which is convenient for users to obtain the detection results in one step after sampling, and realizes high sensitivity to canine viruses, Rapid, quantitative detection.

2. The utility model is equipped with a closed waste liquid pool, which is more convenient for the automatic operation of the manipulator and avoids the pollution of the instrument.

3. The utility model is not only suitable for large-scale automatic detection instruments, but also suitable for manual operation of small-scale equipment. The operation process is simple and fast, and the flexibility is strong.

Description of drawings

Fig. 1. The schematic diagram of the front structure of the utility model canine virus fluorescent quantitative PCR detection microfluidic chip, the marks in the figure: 11-injection hole A, 12-micro flow channel, 13-DNA separation chamber, 14-triangular valve, 15-waste liquid chamber, 16-injection hole B, 17-mixing chamber, 18-PCR reaction area (in the dotted line frame), 19-fluorescence detection chamber.

Fig. 2. Schematic diagram of the structure of the upper cover plate of the microfluidic chip for detection of canine virus by fluorescence quantitative PCR of the present invention, marked in the figure: 20-upper cover plate.

Figure 3. Schematic diagram of the longitudinal section structure of the waste liquid chamber of the microfluidic chip for detection of canine virus by fluorescence quantitative PCR of the utility model, marked in the figure: upper figure: 1-microfluidic chip for detection of canine virus by fluorescence quantitative PCR, along the Longitudinal section of line a; the figure below: 10-substrate, 20-upper cover, 30-lower cover, 11-injection hole A, 12-microchannel, 13-DNA separation chamber, 14-triangular valve, 15- waste chamber.

Fig. 4. Schematic diagram of the structure of the heating module of the microfluidic chip corresponding to the utility model canine virus fluorescence quantitative PCR detection, marked in the figure: 41-nucleic acid lysis zone heating module (dotted line frame), 42-circulation reaction zone heating module 1 ( Dotted line box), 43-circulation reaction zone heating module 2 (dashed line box), 44-circulation reaction zone heating module 3 (dashed line box), 45-extended reaction zone heating module (dashed line box).

Detailed ways

Below in conjunction with accompanying drawing and embodiment the utility model is described further. It should be noted that the routine conditions and methods not described in detail in the examples are all carried out according to the experimental conditions and methods routinely adopted by those skilled in the art.

Example:

As shown in Figures 1-3, a microfluidic chip for canine virus fluorescence quantitative PCR detection, the microfluidic chip is bonded by a substrate (10), an upper cover (20) and a lower cover (30) formed; the substrate (10) is provided with an injection hole A (11), a micro-channel (12), a DNA separation chamber (13), a triangular valve (14), a waste liquid chamber (15), and an injection hole B ( 16), mixing chamber (17), PCR reaction area (18), fluorescence detection chamber (19); upper cover plate (20) leaves corresponding injection hole A (11), injection hole B (16) and triangular valve The position of (14), corresponding to the position of the waste liquid chamber (15), a raised open chamber is set, and the upper cover plate (20) is bonded to the upper surface of the substrate (10); the lower cover plate (30) is bonded to the substrate ( 10) the lower surface, completely seal the lower surface of the substrate (10); the substrate (10), the upper cover (20) and the lower cover (30) together constitute the DNA separation chamber (13), the waste liquid chamber (15), The accommodation space of the mixing chamber (17) and the fluorescence detection chamber (19), the microfluidic channel (12) and the microfluidic channel of the PCR reaction area (18).

The DNA separation chamber (13) communicates with the injection hole A (11) and the triangular valve (14) respectively through the micro-channel (12) located at the bottom of the substrate; the waste liquid chamber (15) communicates with the triangular valve through the micro-channel (12) The valve (14) is connected; the mixing chamber (17) is connected with the injection hole B (16) and the triangle valve (14) through the micro-channel (12); the PCR reaction area (18) is connected with the micro-channel (12) The triangular valve (14) is connected with the fluorescent detection chamber (19); by the rotation of the triangular valve (14), the microfluidic channel outlet of the DNA separation chamber (13) in the triangular valve (14) can be connected with the triangular valve (14) The outlet of the microfluidic channel leading to the waste liquid chamber (15) or the mixing chamber (17) is connected, thereby controlling the flow direction of the waste liquid or DNA eluent; by the rotation of the triangular valve (14), the mixing chamber (17) can be The micro-channel outlet in the triangular valve (14) is connected with the micro-channel outlet of the triangular valve (14) leading to the PCR reaction zone (18), thereby controlling the 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; COC or COP is preferred; the thickness of the upper cover plate (20) is 0.2mm , the thickness of the lower cover plate (30) is 0.1-0.5mm.

The width and/or depth of the micro-channel (12) connecting each chamber and the injection hole is 50-100 μm; the width and/or depth of the micro-channel in the PCR reaction area (18) is 50-80 μm.

The volume of the DNA separation chamber (13) is 100 μl; the volume of the waste liquid chamber (15) is 500 μl; the volume of the mixing chamber (17) is 100 μl.

Described PCR reaction area (18) is made up of three groups of micro-flow channels, and three groups of micro-flow channels correspond to the position of different heating modules on the PCR instrument respectively; -5 groups; 15-30 groups of S-shaped micro-channels corresponding to the heating modules (42), (43) and (44) of the circulating reaction zone; 3 groups of S-shaped micro-channels corresponding to the heating module (45) of the extended reaction zone -5 groups.

The canine virus fluorescent quantitative PCR detection microfluidic chip is suitable for the quantitative detection of canine distemper virus, canine parvovirus, rabies virus, canine coronavirus, canine encephalomyocarditis virus, canine parainfluenza virus and/or canine adenovirus .

The usage method of the canine virus fluorescent quantitative PCR detection microfluidic chip described in the utility model:

1. Tear off the protective film at the injection hole A, and inject 2-5 μl of DNA separation magnetic beads into the DNA separation chamber;

2. Inject 50-100 μl of animal serum, saliva, and urine into the DNA separation chamber from the injection hole A, and the air in the reaction chamber is discharged from the through hole of the triangular valve. Try to avoid blowing out air bubbles in the reaction chamber. Use a Luer plug or The plug closes the injection hole, and the control triangular valve closes the through hole connected to the DNA separation chamber;

3. Place the microfluidic chip on the magnetic mixer, turn on the switch, and drive the DNA separation magnetic beads to flow repeatedly in the sample through magnetic force, and adsorb the DNA on the magnetic beads. This step takes about 5-10 minutes;

4. Control the triangular valve to connect the DNA separation chamber with the waste liquid chamber, inject the buffer solution from the injection hole A, discharge the remaining samples into the waste liquid chamber, and close the through hole connecting the DNA separation chamber in the triangular valve (during this process, the magnetic force The mixer is always on to ensure that the magnetic beads adsorbing DNA will not be washed away);

5. Use the buffer solution to wash the magnetic beads for 30-60s, control the triangular valve to connect the DNA separation chamber with the waste liquid chamber, and discharge the waste liquid into the waste liquid chamber; repeat this washing process 1-3 times, and use the air pump to discharge all the waste liquid ;

6. Inject the DNA eluent from the injection hole A, and elute the DNA in the same way as the washing steps above;

7. Control the triangular valve to connect the DNA separation chamber with the mixing chamber, use the air pump to inject the eluted DNA into the mixing chamber, and inject the PCR reaction reagent mixture from the injection hole B into the mixing chamber, which can be shaken manually or preset in the chip. Magnetic rotor, use a magnetic mixer to mix;

8. Control the triangular valve to connect the mixing chamber with the PCR reaction area, and use the air pump to control the PCR reaction system to flow through each heating module in the microchannel of the reaction area to complete DNA melting, annealing, extension, and finally into the fluorescence detection room. The air pump can control the flow rate of the liquid in different areas of the microchannel to ensure the accurate completion of the PCR program; different heating modules set different temperatures to correspond to the needs of the PCR reaction process.

9. Through the fluorescence detection room, the instrument reads, and calculates the number of viruses according to the standard curve.

The above descriptions are only preferred embodiments of the present utility model, and are not intended to limit the present utility model. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present utility model shall be included in the Within the protection scope of the present utility model.

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).