CN113322175B - Real-time fluorescence constant temperature nucleic acid amplification detection device - Google Patents
Real-time fluorescence constant temperature nucleic acid amplification detection device Download PDFInfo
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- CN113322175B CN113322175B CN202110741819.3A CN202110741819A CN113322175B CN 113322175 B CN113322175 B CN 113322175B CN 202110741819 A CN202110741819 A CN 202110741819A CN 113322175 B CN113322175 B CN 113322175B
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- B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
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Abstract
The real-time fluorescence isothermal nucleic acid amplification detection device comprises a microfluidic unit, a temperature control unit, an excitation light unit, a detection unit and an installation rod, wherein a reaction liquid containing fluorescent dye performs fluorescence isothermal nucleic acid amplification reaction in the microfluidic unit, the temperature control unit provides the microfluidic unit with constant temperature required by nucleic acid amplification, the excitation light unit generates excitation light to excite the fluorescent dye in the reaction liquid in the microfluidic unit to generate fluorescence, and the detection unit acquires fluorescence information generated by the reaction liquid in the microfluidic unit to obtain a detection result; the microfluidic unit, the excitation light unit and the detection unit are installed on the installation rod together, the excitation light unit and the detection unit are located above the microfluidic unit, and at least the position and/or angle of the detection unit on the installation rod relative to the microfluidic unit are adjustable. The device has the advantages of simple structure, low cost, flexible and convenient operation and adjustment, and reliable, stable and quick acquisition of detection results.
Description
Technical Field
The invention relates to the field of nucleic acid detection, in particular to a real-time fluorescent isothermal nucleic acid amplification detection device.
Background
The concept of a micro total analysis system (Miniaturized Total Analysis Systems) was first proposed in the 90 s of the 20 th century, and after that, on the basis of microelectronics, micromechanics, bioengineering and nanotechnology, microfluidic technology has rapidly developed, becoming one of the forefront technological fields of the world at present. At present, the core technology is a microfluidic chip based on the microfluidic technology, which is also called a Lab on chip (Lab on chip). The microfluidic chip technology (Microfluidics) integrates basic operation units of sample preparation, reaction, separation, detection and the like in biological, chemical and medical analysis processes on a micron-scale chip, and automatically completes the whole analysis process. The microfluidic chip is widely used in the biomedical field because of the advantages of low consumption, low cost, high throughput, automatic operation and the like, and one important application is a nucleic acid detection technology based on the microfluidic chip.
The traditional nucleic acid detection technology takes PCR technology (English: polymerase Chain Reaction Chinese: polymerase chain reaction) as the main technology, and performs repeated conversion between three different temperatures for multiple times to complete rapid amplification of nucleic acid, but has high equipment requirement, needs at least one to two hours, and has low efficiency. And the isothermal amplification technology only needs one temperature, so that the equipment requirement is reduced, and the efficiency is improved. However, the existing isothermal nucleic acid amplification equipment is complex in structure, complex in control, high in control requirement, inflexible and convenient to use, high in cost and still has a large optimization space.
It should be noted that the information disclosed in the above background section is only for understanding the background of the present application and thus may include information that does not form the prior art that is already known to those of ordinary skill in the art.
Disclosure of Invention
The invention aims to overcome the defects of the background technology and provide a real-time fluorescent isothermal nucleic acid amplification detection device.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
the real-time fluorescence isothermal nucleic acid amplification detection device comprises a microfluidic unit, a temperature control unit, an excitation light unit, a detection unit and an installation rod, wherein a reaction liquid containing fluorescent dye performs fluorescence isothermal nucleic acid amplification reaction in the microfluidic unit, the temperature control unit provides the microfluidic unit with constant temperature required by nucleic acid amplification, the excitation light unit generates excitation light to excite the fluorescent dye in the reaction liquid in the microfluidic unit to generate fluorescence, and the detection unit acquires fluorescence information generated by the reaction liquid in the microfluidic unit to obtain a detection result; the microfluidic unit, the excitation light unit and the detection unit are installed on the installation rod together, the excitation light unit and the detection unit are located above the microfluidic unit, and at least the position and/or angle of the detection unit on the installation rod relative to the microfluidic unit are adjustable.
Further:
the bottom of the mounting rod is provided with a bottom plate which plays a role in ground support.
The temperature control unit is installed on the installation rod, and the microfluidic unit is arranged on the temperature control unit.
The temperature control unit comprises a supporting plate, a heat conducting shell, a heater and a temperature sensor, wherein the supporting plate is connected with the mounting rod, the heat conducting shell is positioned on the upper side of the supporting plate, the heater is arranged in the heat conducting shell, the microfluidic unit is arranged on the heat conducting shell, the temperature sensor is tightly attached to the surface, close to one side of the microfluidic unit, of the heat conducting shell, and an external temperature controller is connected with the heater and the temperature sensor for temperature control so as to keep the constant temperature required by reaction.
The heat conduction shell comprises a lower aluminum alloy shell and an upper aluminum alloy shell which are assembled together, the heater is a silicon rubber heater which is connected with the inner wall of the lower aluminum alloy shell in an adhesive mode, and the temperature sensor is fixedly connected with the inner surface of the upper aluminum alloy shell through heat-resistant glue.
The excitation light unit comprises a bottom plate, a first screw, a light source support and an excitation light source device, one side of the light source support is fixed on the mounting rod through the first screw, the other side of the light source support is fixed with the excitation light source device, the excitation light source device comprises a light source shell, a lamp bead base, a lamp bead, an excitation light filter and a condensing lens, the lamp bead is arranged on the lamp bead base, the excitation light filter is arranged on the front side of the lamp bead, and the condensing lens is arranged on the front side of the excitation light filter; preferably, the included angle between the connecting line of the lamp bead, the excitation light filter, the condensing lens and the center of the microfluidic chip and the vertical line is 15-75 degrees, and the distance between the condensing lens and the center of the microfluidic chip is 5-30 cm.
The detection unit comprises a second screw, an inner ring, an outer ring with a stud at the upper end, a cylindrical member with threads at the inner ring, a structural rod piece, a third screw, a lens bracket, a camera module, a fourth screw, a connecting structural member, a knob with a rod, a strip with teeth, a middle connecting member and an L-shaped member, wherein the connecting structural member is fixed on the mounting rod through the fourth screw, so that the detection unit can move along the mounting rod, the knob is arranged on the connecting structural member, the rod carried by the knob is provided with teeth which are meshed with the strip with teeth to drive the strip to move up and down, the middle connecting member is positioned between the strip with teeth and the L-shaped member, the middle connecting member is connected with the strip with teeth, the middle connecting member is connected with one side of the L-shaped member, the other side of the L-shaped piece with the hole is positioned between the cylindrical piece and the outer ring, the stud penetrates through the hole to be in threaded connection with the inner ring of the cylindrical piece, the inner ring is positioned between the outer ring and the structural rod piece, is in interference fit with the outer ring, and has a gap with the structural rod piece so that the structural rod piece can rotate in the inner ring, the camera module comprises an inner ring, a second screw, a structural rod piece, a third screw, a camera module and a lens support, wherein the inner ring is provided with a round hole at the corresponding position of the outer ring, the second screw is in threaded connection with the round hole and fixes the position of the structural rod piece, the front end of the structural rod piece is provided with a convex cylinder, the lens support can rotate around the cylinder and is fixed on the convex cylinder through the third screw, and the camera module is mounted on the lens support.
The camera module comprises a lens with an emission light filter at the front end and an industrial camera, wherein the lens is arranged at the front end of the industrial camera, and the lens and the industrial camera are clamped at the corresponding positions of the lens bracket.
The microfluidic chip comprises a substrate layer, an intermediate layer and a surface layer which are sequentially laminated from bottom to top, wherein the intermediate layer comprises a plurality of sample adding holes, a plurality of air holes, a plurality of liquid distributing cavities, a plurality of reaction cavities and a plurality of flow channels, the sample adding holes are used for adding reaction liquid and flow to the liquid distributing cavities, the air holes enable the liquid to flow in the chip, and the liquid distributing cavities distribute the reaction liquid to the reaction cavities through the flow channels.
The material of the middle layer is selected from any one of polyvinyl chloride (PVC), polyethylene (PE), polypropylene (PP), polystyrene (PS) and Polycarbonate (PC), the basal layer, the surface layer and the middle layer are bonded in an adhesive mode, the basal layer and the surface layer are ultrathin adhesive tapes with the thickness of 0.02-0.1 mm, and the volume of the reaction cavity is 5-10 mu l.
The invention has the following beneficial effects:
the invention provides a real-time fluorescence isothermal nucleic acid amplification detection device for realizing isothermal amplification and real-time fluorescence detection of nucleic acid in a sample to be detected, wherein a microfluidic unit, an excitation light unit and a detection unit are arranged on a mounting rod together, the excitation light unit and the detection unit are positioned above the microfluidic unit, at least the position and/or angle of the detection unit relative to the microfluidic unit on the mounting rod are adjustable, and the device can realize convenient and rapid high-flux nucleic acid detection by matching the units arranged on the same mounting rod. The invention can well meet the requirements of rapid detection such as clinical and epidemic disease detection, and is particularly suitable for application in areas with lack of resources.
Drawings
FIG. 1 is a schematic diagram of a real-time fluorescence isothermal nucleic acid amplification detection device according to an embodiment of the present invention;
fig. 2 is a schematic structural view of the microfluidic cell shown in fig. 1;
FIG. 3 is a schematic view of the temperature control unit shown in FIG. 1;
FIG. 4 is a schematic diagram showing the overall structure of the excitation light unit shown in FIG. 1;
FIG. 5 is a schematic view showing a specific structure of the excitation light source device shown in FIG. 4;
FIG. 6 is a schematic structural view of the detecting unit shown in FIG. 1;
FIG. 7 is a schematic view of an exploded structure of the detection unit shown in FIG. 1;
FIG. 8 is a graph of real-time fluorescence intensity detected in accordance with an embodiment of the present invention.
Detailed Description
The following describes embodiments of the present invention in detail. It should be emphasized that the following description is merely exemplary in nature and is in no way intended to limit the scope of the invention or its applications.
It will be understood that when an element is referred to as being "mounted" or "disposed" on another element, it can be directly on the other element or be indirectly on the other element. When an element is referred to as being "connected to" another element, it can be directly connected to the other element or be indirectly connected to the other element. In addition, the connection may be for both a fixing action and a coupling or communication action.
It is to be understood that the terms "length," "width," "upper," "lower," "front," "rear," "left," "right," "vertical," "horizontal," "top," "bottom," "inner," "outer," and the like are merely for convenience in describing embodiments of the invention and to simplify the description by referring to the figures, rather than to indicate or imply that the devices or elements referred to must have a particular orientation, be constructed and operated in a particular orientation, and thus are not to be construed as limiting the invention.
Furthermore, the terms "first," "second," and the like, are used for descriptive purposes only and are not to be construed as indicating or implying a relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defining "a first" or "a second" may explicitly or implicitly include one or more such feature. In the description of the embodiments of the present invention, the meaning of "plurality" is two or more, unless explicitly defined otherwise.
Referring to fig. 1, an embodiment of the present invention provides a real-time fluorescent isothermal nucleic acid amplification detection device, where the nucleic acid amplification detection device includes a microfluidic unit 1, a temperature control unit 2, an excitation light unit 3, a detection unit 4, and a mounting rod 18, where a reaction solution containing a fluorescent dye performs a fluorescent isothermal nucleic acid amplification reaction in the microfluidic unit 1, the temperature control unit 2 provides a required constant temperature for nucleic acid amplification to the microfluidic unit 1, the excitation light unit 3 generates excitation light to excite the fluorescent dye in the reaction solution in the microfluidic unit 1 to generate fluorescence, and the detection unit 4 collects fluorescent information generated by the reaction solution in the microfluidic unit 1, and analyzes the collected fluorescent information to obtain a detection result; wherein the microfluidic unit 1, the excitation light unit 3 and the detection unit 4 are mounted on the mounting rod 18 together, the excitation light unit 3 and the detection unit 4 are located above the microfluidic unit 1, and at least the position and/or angle of the detection unit 4 on the mounting rod 18 relative to the microfluidic unit 1 are adjustable.
In a preferred embodiment, the temperature control unit 2 is mounted on a mounting bar 18, while providing the necessary constant temperature for nucleic acid amplification below the microfluidic unit 1, also provides a support platform for the microfluidic unit 1 to indirectly mount the microfluidic unit 1 to the mounting bar 18. In other embodiments, the microfluidic unit 1 may also be mounted directly to the mounting bar 18 by a mounting member, while the temperature control unit 2 may or may not be attached to the mounting bar 18, as long as isothermal reaction conditions can be provided to the microfluidic unit 1.
The embodiment of the invention can conveniently realize fluorescent isothermal nucleic acid amplification reaction and detection, has flexible and convenient operation control, has simple structure and lower cost, can simultaneously detect multiple indexes, and ensures reliable, stable and quick acquisition of detection results.
Referring to FIG. 1, a real-time fluorescent isothermal nucleic acid amplification detection device according to an exemplary embodiment includes a mounting bar 18, and a microfluidic unit 1, a temperature control unit 2, an excitation light unit 3, and a detection unit 4 mounted on the mounting bar 18.
As shown in fig. 2, which is a schematic structural diagram of the microfluidic unit 1, the microfluidic chip is composed of a substrate layer 5, an intermediate layer 10 and a surface layer 11, wherein the substrate layer 5 and the intermediate layer 10 are bonded by gluing to ensure tightness and thermal stability; the surface layer 11 and the middle layer 10 seal the air hole 9 and the sample application hole 6 by means of gluing, and prevent pollution. The base layer 5 is located on the lower side, the intermediate layer 10 is located on the upper side of the base layer 5, and the surface layer 11 is located on the upper side of the intermediate layer 10. The middle layer 10 comprises a plurality of sample adding holes 6, a plurality of air holes 9, a plurality of liquid separating cavities 7 and a plurality of reaction cavities 8, wherein the sample adding holes 6 are used for adding reaction liquid, the air holes 9 are used for enabling fluid to flow in a chip when centrifuging, the liquid separating cavities 7 are used for equally dividing the reaction liquid to the plurality of reaction cavities 8 through connected runners, and then the reaction cavities 8 contain the reaction liquid containing fluorescent dye, so that nucleic acid amplification reaction can be carried out.
The material of the middle layer 10 adopts common transparent medical plastics, such as polyvinyl chloride (PVC), polyethylene (PE), polypropylene (PP), polystyrene (PS) and polycarbonate PC, and adopts various plastic forming modes such as compression molding thermoplastic forming, injection molding forming and the like; for example, a method of injection molding is adopted, a mold is processed in advance, then polypropylene (PP) material is melted in a constant temperature charging barrel 220-280 ℃, then 800-140MPa is pressurized, the melted PP material is injected into the mold, and then pressure maintaining and cooling molding are carried out. The base layer 5 and the surface layer 11 are ultrathin adhesive tapes with good bonding effect, and the thickness is 0.02-0.1 mm. The volume of the reaction cavity 8 in the microfluidic chip is 5-10 μl. The fluid in the microfluidic chip is driven by a centrifugal machine to flow from the liquid separation cavity 7 to the reaction cavity 8 through a connected runner, so that liquid separation is completed. The rotating speed of the centrifugal machine is 1000-5000 rpm, the time is 10-60 s, and the centrifugal machine is only needed to be used once.
As shown in fig. 3, the temperature control unit 2 is schematically configured below the microfluidic chip and is used for providing a temperature required for isothermal nucleic acid amplification, and comprises a bottom plate 12, a support plate 13, a lower aluminum alloy housing 14, a silicon rubber heater 15, a temperature sensor 16, an upper aluminum alloy housing 17, a mounting rod 18 and an external temperature controller. The base plate 12 is threadably connected to the mounting rod 18 for ground support. The supporting plate 13 is connected with the mounting rod 18 through bolts and nuts, and plays a role in fixedly supporting the temperature control unit 2 and determining the position of the temperature control unit 2. The lower aluminum alloy shell 14 is positioned on the upper side of the supporting plate 13; the upper aluminum alloy housing 17 is located above the lower aluminum alloy housing 14, and serves to conduct heat and improve temperature uniformity. The silicon rubber heater 15 is a heating element, is tightly attached to the inner wall of the lower aluminum alloy shell 14, and is connected by gluing. The temperature sensor 16 is fixed by heat-resistant glue to the inner surface of the upper aluminum alloy housing 17. The corresponding positions of the upper aluminum alloy shell 17, the silicon rubber heater 15, the lower aluminum alloy shell 14 and the supporting plate 13 are all provided with holes, and the holes can be fixed together through four screw nuts. The external temperature controller is used for temperature control to maintain the desired constant temperature for the reaction.
The bottom plate 12 is made of steel and is positioned on the lower side of the supporting plate 13. The mounting rod 18 is made of aluminum alloy, the mounting rod 18 can be divided into two sections, and the two sections of rods are connected through threads. The material of the support plate 13 is black heat-resistant resin. The temperature sensor 16 may be a type K thermocouple, pt100 temperature sensor, or the like. The upper surface of the upper aluminum alloy shell 17 is coated with a layer of paint to increase diffuse reflection and reduce the influence on fluorescent images.
The implementation flow of the temperature control unit 2 is as follows: the constant temperature of the amplification reaction is set on an external temperature controller, then the silicon rubber heater 13 heats the air in the upper aluminum alloy shell 17 and the lower aluminum alloy shell 14, so that the upper surface temperature of the upper aluminum alloy shell 17 is increased, then the reaction liquid in the reaction cavity 8 of the microfluidic chip is heated again to reach the constant temperature of the amplification reaction, the temperature sensor 16 carries out real-time temperature feedback, temperature regulation is carried out, the reaction liquid in the reaction cavity 8 is kept constant, and the nucleic acid amplification reaction is continuously carried out.
As shown in fig. 4, the overall structure of the excitation light unit 3 is schematically shown, and the excitation light unit is used for exciting fluorescent dye in the reaction solution in the reaction chamber of the microfluidic chip to generate fluorescence, and includes a bottom plate 12, a mounting rod 18, an excitation light source device 21, a light source bracket 20, a first screw 19 and an external light source power controller. The light source bracket 20 is fixed at the corresponding position of the mounting rod 18 by a first screw 19 on one side, and the excitation light source device 19 is fixedly clamped on the other side, and can be realized by an adhesive mode.
Fig. 5 is a schematic diagram of a specific structure of the excitation light source device 21 in fig. 4, including a lamp bead base 22, a lamp bead 23, an excitation light filter 24, a condensing lens 25, and a light source housing 26. The lamp beads 23 are welded to the lamp bead base 22 to generate excitation light. The excitation light filter 24 is placed on the front side of the lamp bead 23 to perform a filtering function. The condensing lens 25 is disposed at the front side of the excitation filter 24 to perform a condensing function. The excitation light filter 24 and the condenser lens 25 are placed at the corresponding positions of the light source housing 26. The light source housing 26 comprises five sides, is made of black aluminum alloy, and has the functions of fixing, supporting and radiating heat.
The number of the lamp beads 23 is 1-20, the total power is 0.3-5W, and the wavelength selection of the excitation light filter 24 and the lamp beads 23 is adapted to the excitation light wavelength of the fluorescent dye. The included angle between the connecting line of the lamp bead 23, the excitation light filter 24, the condensing lens 25 and the center of the micro-fluidic chip and the vertical line is 15-75 degrees, and the distance between the condensing lens 25 and the center of the micro-fluidic chip is 5-30 cm. The sides of the light source housing 26 are connected by screws. The external light source power controller controls the power of the lamp beads 23.
The detection unit 4 is used for collecting fluorescent image information generated by the reaction liquid in the reaction cavity of the microfluidic chip through the industrial camera, and performing software analysis to obtain a detection result. Referring to fig. 6, a schematic structural diagram of the detecting unit 4 is shown, and fig. 7 is a schematic exploded structural diagram of the detecting unit 4. The detecting unit 4 includes a second screw 27, a plastic inner ring 28, an aluminum alloy outer ring 29 with a stud at the upper end, a cylindrical member 30 with a threaded inner ring, a structural rod member 31, a third screw 32, a lens 33 with an emission light filter at the front end, a lens holder 34, an industrial camera 35, a fourth screw 36, a connecting structural member 37, a knob 38 with a rod, a long strip 39 with teeth, a middle connecting member 40 and an L-shaped member 41. The connecting structure 37 is fixed to the mounting bar 18 by means of a fourth screw 36, so that the detection unit 4 can be moved along the mounting bar 18. A knob 38 with a rod is placed in the connecting structure 37, the rod being toothed, and engaging with a toothed strip 39 moves the strip up and down. The intermediate connecting member 40 is between the toothed bar 39 and the L-shaped member 41, the intermediate connecting member 40 is connected to the toothed bar 39 by a screw and a nut, and the intermediate connecting member 40 is connected to one side of the L-shaped member 41 by a screw. The other side of the L-shaped member 41 is arranged between the cylindrical member 30 with threads on the inner ring and the aluminum alloy outer ring 29 with studs on the upper end, and the centers of the cylindrical member 30 with threads on the upper end and the aluminum alloy outer ring are on the same axis, so that the aluminum alloy outer ring can rotate, and after the position is determined, the cylindrical member, the aluminum alloy outer ring and the aluminum alloy outer ring can be fixed through threaded connection. The plastic inner ring 28 is connected with the aluminum alloy outer ring 29 with the stud at the upper end in an interference fit manner between the aluminum alloy outer ring 29 with the stud at the upper end and the structural rod piece 31, and a gap exists between the plastic inner ring and the structural rod piece 31. The plastic inner ring 28 and the aluminum alloy outer ring 29 with the studs at the upper end are provided with round holes, threads are arranged in the corresponding positions of the aluminum alloy outer ring 29 with the studs at the upper end, the second screw 27 fixes the position of the structural rod member 31 in a threaded connection mode, and the structural rod member 31 can rotate in the plastic inner ring 28. The front end of the structural rod 31 has a convex cylinder, and the lens holder 34 is rotatable around the cylinder and fixed to the convex cylinder by a third screw 32. The lens 33 with the light-emitting filter at the front end is arranged at the front end of the industrial camera 35, and then the lens 33 and the industrial camera are clamped at the corresponding positions of the lens bracket 34, so that the fluorescent information collection function is realized.
The industrial camera 35 is of CCD or CMOS type, the lens is a zoom lens with a focal length of 2.8-12 mm, and the wavelength selection of the emission light filter at the front end of the lens is adapted to the emission wavelength of the fluorescent dye. The coordination of the remaining parts, except for the industrial camera and the lens, provides a position-adjustable gimbal for the industrial camera.
The detection unit 4 is configured to collect fluorescent images generated by the reaction solution in the reaction chambers 8 in the microfluidic chip by the excitation light generated by the excitation light unit 3, and then perform fluorescent image transmission and image processing to obtain real-time fluorescent intensity information in the reaction chambers 8, that is, detection results, where the detection results can be used to determine whether the reaction solution is positive.
The following briefly describes the procedure for nucleic acid detection using the real-time fluorescence isothermal nucleic acid amplification detection device according to an embodiment of the present invention:
1) Respectively pouring a proper amount of reaction liquid into a plurality of liquid separating cavities 7 from a plurality of sample adding holes 6 of a middle layer 10 at a speed of 200 mu L/min by using an injection gun, then placing a microfluidic chip on a centrifugal machine, centrifuging for a period of time at a certain rotating speed, so that all the liquid in the liquid separating cavities 7 flows into a reaction cavity 8, and then sealing the sample adding holes 6 and the air holes 9 by using a surface layer 11;
2) Placing a microfluidic chip on the surface of an aluminum alloy shell 17 at the upper side, turning on a main power supply, starting the excitation light unit 3, the temperature control unit 2 and the detection unit 4 to work, setting constant temperature, starting real-time fluorescence detection, and obtaining a detection result after 20-30 min;
3) After the detection is completed, the power supply is turned off, and the microfluidic chip can be taken out.
As shown in FIG. 8, the real-time fluorescence intensity graph, i.e., the result of nucleic acid detection, can determine whether the reaction solution is positive. The nucleic acid amplification reaction to which the nucleic acid detection device is applied includes isothermal nucleic acid amplification technologies such as LAMP (loop-mediated isothermal amplification technology) and RPA (recombinase polymerase amplification technology).
The invention is used for amplifying and detecting fluorescent nucleic acid at constant temperature, has the advantages of rapidness, simplicity, stability, reliability, flexible and convenient control and the like, and can well meet the requirements of rapid detection such as clinical detection, epidemic disease detection and the like.
The background section of the present invention may contain background information about the problems or environments of the present invention and is not necessarily descriptive of the prior art. Accordingly, inclusion in the background section is not an admission of prior art by the applicant.
The foregoing is a further detailed description of the invention in connection with specific/preferred embodiments, and it is not intended that the invention be limited to such description. It will be apparent to those skilled in the art that several alternatives or modifications can be made to the described embodiments without departing from the spirit of the invention, and these alternatives or modifications should be considered to be within the scope of the invention. In the description of the present specification, reference to the terms "one embodiment," "some embodiments," "preferred embodiments," "examples," "specific examples," or "some examples," etc., means that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, schematic representations of the above terms are not necessarily directed to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Those skilled in the art may combine and combine the features of the different embodiments or examples described in this specification and of the different embodiments or examples without contradiction. Although embodiments of the present invention and their advantages have been described in detail, it should be understood that various changes, substitutions and alterations can be made herein without departing from the scope of the invention as defined by the appended claims.
Claims (9)
1. The real-time fluorescence isothermal nucleic acid amplification detection device is characterized by comprising a microfluidic unit, a temperature control unit, an excitation light unit, a detection unit and an installation rod, wherein a reaction liquid containing fluorescent dye performs fluorescence isothermal nucleic acid amplification reaction in the microfluidic unit, the temperature control unit provides the microfluidic unit with constant temperature required by nucleic acid amplification, the excitation light unit generates excitation light to excite the fluorescent dye in the reaction liquid in the microfluidic unit so as to generate fluorescence, and the detection unit acquires fluorescence information generated by the reaction liquid in the microfluidic unit so as to obtain a detection result; the microfluidic unit, the excitation light unit and the detection unit are mounted on the mounting rod together, the excitation light unit and the detection unit are positioned above the microfluidic unit, and at least the position and/or angle of the detection unit on the mounting rod relative to the microfluidic unit are adjustable; the detecting unit comprises a second screw, an inner ring, an outer ring with a stud at the upper end, a cylindrical member with threads at the inner ring, a structural rod piece, a third screw, a lens support, a camera module, a fourth screw, a connecting structural member, a knob with a rod, a strip with teeth, a middle connecting member and an L-shaped member, wherein the connecting structural member is fixed on the mounting rod through the fourth screw, so that the detecting unit can move along the mounting rod, the knob is arranged on the connecting structural member, the rod carried by the knob is provided with teeth, the knob is meshed with the strip with teeth to drive the strip to move up and down, the middle connecting member is positioned between the strip with teeth and the L-shaped member, the middle connecting member is connected with one side of the L-shaped member, the other side of the L-shaped member is positioned between the cylindrical member and the outer ring, the stud passes through the hole and the cylindrical member, the cylindrical rod piece is meshed with the strip with teeth to drive the strip with the cylindrical rod piece, the cylindrical rod piece is in the clearance between the cylindrical ring and the cylindrical ring, the cylindrical ring is in the clearance fit with the cylindrical ring, and the cylindrical ring is in the clearance structure, and the cylindrical ring is fixed between the cylindrical ring and the cylindrical ring.
2. The apparatus of claim 1, wherein the bottom of the mounting bar is provided with a bottom plate for ground support.
3. The real-time fluorescent isothermal nucleic acid amplification detection device according to claim 1, wherein the temperature control unit is mounted on the mounting rod, and the microfluidic unit is disposed on the temperature control unit.
4. The apparatus of claim 3, wherein the temperature control unit comprises a support plate, a heat conducting housing, a heater, and a temperature sensor, the support plate is connected to the mounting rod, the heat conducting housing is located on the upper side of the support plate, the heater is disposed in the heat conducting housing, the microfluidic unit is disposed on the heat conducting housing, the temperature sensor is closely attached to a surface of the heat conducting housing near one side of the microfluidic unit, and an external temperature controller is connected to the heater and the temperature sensor for temperature control to maintain a constant temperature required for the reaction.
5. The apparatus of claim 4, wherein the heat conductive housing comprises a lower aluminum alloy housing and an upper aluminum alloy housing assembled together, the heater is a silicone rubber heater adhesively attached to an inner wall of the lower aluminum alloy housing, and the temperature sensor is fixedly attached to an inner surface of the upper aluminum alloy housing with a heat resistant adhesive.
6. The real-time fluorescent isothermal nucleic acid amplification detection device according to any one of claims 1 to 5, wherein the excitation light unit comprises a bottom plate, a first screw, a light source support and an excitation light source device, one side of the light source support is fixed on the mounting rod through the first screw, the other side of the light source support is fixed with the excitation light source device, the excitation light source device comprises a light source shell, a lamp bead base, a lamp bead, an excitation light filter and a condensing lens, the lamp bead is arranged in the light source shell, the lamp bead is arranged on the lamp bead base, the excitation light filter is arranged on the front side of the lamp bead, and the condensing lens is arranged on the front side of the excitation light filter; the included angle between the connecting line of the lamp bead, the excitation light filter and the condensing lens and the center of the microfluidic chip and the vertical line is 15-75 degrees, and the distance between the condensing lens and the center of the microfluidic chip is 5-30 cm.
7. The apparatus according to any one of claims 1 to 5, wherein the camera module comprises a lens with an emission filter at a front end and an industrial camera, the lens is mounted at the front end of the industrial camera, and the lens and the industrial camera are clamped at corresponding positions of the lens holder.
8. The real-time fluorescent isothermal nucleic acid amplification detection device according to any one of claims 1 to 5, wherein the microfluidic chip comprises a substrate layer, an intermediate layer and a surface layer which are sequentially stacked together from bottom to top, the intermediate layer comprises a plurality of sample addition holes for adding reaction liquid and flowing to the sample addition holes, a plurality of gas holes for allowing the liquid to flow in the chip, a plurality of liquid separation cavities for distributing the reaction liquid to the plurality of reaction cavities through the gas holes, and a plurality of reaction cavities and flow channels.
9. The real-time fluorescence isothermal nucleic acid amplification detecting device according to claim 8, wherein the material of the middle layer is selected from any one of polyvinyl chloride (PVC), polyethylene (PE), polypropylene (PP), polystyrene (PS) and Polycarbonate (PC), the base layer, the surface layer and the middle layer are bonded in an adhesive manner, the base layer and the surface layer are ultrathin adhesive tapes with the thickness of 0.02-0.1 mm, and the volume of the reaction cavity is 5-10 μl.
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