CN113322175A - Real-time fluorescence constant temperature nucleic acid amplification detection device - Google Patents
Real-time fluorescence constant temperature nucleic acid amplification detection device Download PDFInfo
- Publication number
- CN113322175A CN113322175A CN202110741819.3A CN202110741819A CN113322175A CN 113322175 A CN113322175 A CN 113322175A CN 202110741819 A CN202110741819 A CN 202110741819A CN 113322175 A CN113322175 A CN 113322175A
- Authority
- CN
- China
- Prior art keywords
- unit
- nucleic acid
- acid amplification
- excitation light
- real
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 238000001514 detection method Methods 0.000 title claims abstract description 67
- 150000007523 nucleic acids Chemical class 0.000 title claims abstract description 46
- 102000039446 nucleic acids Human genes 0.000 title claims abstract description 46
- 108020004707 nucleic acids Proteins 0.000 title claims abstract description 46
- 230000003321 amplification Effects 0.000 title claims abstract description 43
- 238000003199 nucleic acid amplification method Methods 0.000 title claims abstract description 43
- 230000005284 excitation Effects 0.000 claims abstract description 47
- 238000006243 chemical reaction Methods 0.000 claims abstract description 39
- 239000012295 chemical reaction liquid Substances 0.000 claims abstract description 22
- 239000007850 fluorescent dye Substances 0.000 claims abstract description 12
- 229910000838 Al alloy Inorganic materials 0.000 claims description 31
- 239000010410 layer Substances 0.000 claims description 28
- 239000011324 bead Substances 0.000 claims description 21
- 239000007788 liquid Substances 0.000 claims description 13
- 239000002344 surface layer Substances 0.000 claims description 11
- 239000004743 Polypropylene Substances 0.000 claims description 9
- 229920001155 polypropylene Polymers 0.000 claims description 9
- -1 Polyethylene Polymers 0.000 claims description 7
- 239000004698 Polyethylene Substances 0.000 claims description 6
- 229920000573 polyethylene Polymers 0.000 claims description 6
- 229920002379 silicone rubber Polymers 0.000 claims description 6
- 239000000758 substrate Substances 0.000 claims description 6
- 239000000853 adhesive Substances 0.000 claims description 4
- 230000001070 adhesive effect Effects 0.000 claims description 4
- 239000012530 fluid Substances 0.000 claims description 4
- 239000004417 polycarbonate Substances 0.000 claims description 4
- 229920000515 polycarbonate Polymers 0.000 claims description 4
- 239000004793 Polystyrene Substances 0.000 claims description 3
- 238000004026 adhesive bonding Methods 0.000 claims description 3
- 239000004800 polyvinyl chloride Substances 0.000 claims description 3
- 239000004945 silicone rubber Substances 0.000 claims 1
- 238000010586 diagram Methods 0.000 description 10
- 238000005516 engineering process Methods 0.000 description 9
- 239000000463 material Substances 0.000 description 6
- 238000000034 method Methods 0.000 description 6
- 229920003023 plastic Polymers 0.000 description 6
- 239000004033 plastic Substances 0.000 description 6
- 230000008569 process Effects 0.000 description 5
- 238000000926 separation method Methods 0.000 description 5
- 239000000243 solution Substances 0.000 description 5
- 239000002390 adhesive tape Substances 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 238000002073 fluorescence micrograph Methods 0.000 description 3
- 238000010438 heat treatment Methods 0.000 description 3
- 238000006467 substitution reaction Methods 0.000 description 3
- 238000005119 centrifugation Methods 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 238000001917 fluorescence detection Methods 0.000 description 2
- 239000003292 glue Substances 0.000 description 2
- 238000001746 injection moulding Methods 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000000465 moulding Methods 0.000 description 2
- 238000007789 sealing Methods 0.000 description 2
- 238000007397 LAMP assay Methods 0.000 description 1
- 102000018120 Recombinases Human genes 0.000 description 1
- 108010091086 Recombinases Proteins 0.000 description 1
- 229910000831 Steel Inorganic materials 0.000 description 1
- 230000003044 adaptive effect Effects 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000005540 biological transmission Effects 0.000 description 1
- 238000011109 contamination Methods 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 230000002950 deficient Effects 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000004880 explosion Methods 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 229920006015 heat resistant resin Polymers 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 238000009434 installation Methods 0.000 description 1
- 230000001678 irradiating effect Effects 0.000 description 1
- 238000011901 isothermal amplification Methods 0.000 description 1
- 238000002032 lab-on-a-chip Methods 0.000 description 1
- 238000004377 microelectronic Methods 0.000 description 1
- 238000010137 moulding (plastic) Methods 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 239000003973 paint Substances 0.000 description 1
- 238000003752 polymerase chain reaction Methods 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 239000010959 steel Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000009757 thermoplastic moulding Methods 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
- B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
- B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
- 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
Landscapes
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Wood Science & Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Zoology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Analytical Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Dispersion Chemistry (AREA)
- Biotechnology (AREA)
- Immunology (AREA)
- Hematology (AREA)
- Molecular Biology (AREA)
- Physics & Mathematics (AREA)
- Biophysics (AREA)
- Clinical Laboratory Science (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
- Apparatus Associated With Microorganisms And Enzymes (AREA)
Abstract
A real-time fluorescence constant-temperature nucleic acid amplification detection device comprises a microfluidic control unit, a temperature control unit, an excitation light unit, a detection unit and a mounting rod, wherein reaction liquid containing fluorescent dye is used for carrying out fluorescence constant-temperature nucleic acid amplification reaction in the microfluidic control unit, the temperature control unit provides constant temperature required by nucleic acid amplification for the microfluidic control unit, the excitation light unit generates excitation light to excite the fluorescent dye in the reaction liquid in the microfluidic control unit to generate fluorescence, and the detection unit collects fluorescence information generated by the reaction liquid in the microfluidic control unit to obtain a detection result; the micro-fluidic unit, the exciting light unit and the detection unit are mounted on the mounting rod together, the exciting light unit and the detection unit are located above the micro-fluidic unit, and at least the position and/or angle of the detection unit on the mounting rod relative to the micro-fluidic 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 fluorescence isothermal nucleic acid amplification detection device.
Background
The concept of miniature Total Analysis Systems was first proposed since the 90 s of the 20 th century, and then the microfluidic technology was rapidly developed on the basis of microelectronics, micromachines, bioengineering, and nanotechnology, becoming one of the leading-edge scientific and technological fields in the world. The core technology of the prior art is a microfluidic chip based on the microfluidic technology, which is also called a Lab-on-a-chip (Lab on chip). The microfluidic chip technology integrates basic operation units of sample preparation, reaction, separation, detection and the like in biological, chemical and medical analysis processes into a micron-scale chip, and automatically completes the whole analysis process. The microfluidic chip has the advantages of low consumption, low cost, high throughput, automatic operation and the like, and is widely applied to the field of biomedicine, wherein an important application is the nucleic acid detection technology based on the microfluidic chip.
The traditional nucleic acid detection technology mainly uses PCR technology (Polymerase Chain Reaction, English) and performs repeated conversion among three different temperatures to complete rapid amplification of nucleic acid, but has high requirements on equipment, needs at least one to two hours and has low efficiency. 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 has the disadvantages of complex structure, complex operation, high requirement on control, insufficient flexibility and convenience in use, high cost and still large optimization space.
It is to 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 constitute prior art known to a person of ordinary skill in the art.
Disclosure of Invention
The main purpose of the present invention is to overcome the above mentioned drawbacks of the background art and to provide a real-time fluorescence isothermal nucleic acid amplification detection device.
In order to achieve the purpose, the invention adopts the following technical scheme:
a real-time fluorescence constant-temperature nucleic acid amplification detection device comprises a microfluidic control unit, a temperature control unit, an excitation light unit, a detection unit and a mounting rod, wherein reaction liquid containing fluorescent dye carries out fluorescence constant-temperature nucleic acid amplification reaction in the microfluidic control unit, the temperature control unit provides constant temperature required by nucleic acid amplification for the microfluidic control unit, the excitation light unit generates excitation light to excite the fluorescent dye in the reaction liquid in the microfluidic control unit to generate fluorescence, and the detection unit collects fluorescence information generated by the reaction liquid in the microfluidic control unit to obtain a detection result; the micro-fluidic 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 located above the micro-fluidic unit, and at least the position and/or angle of the detection unit on the mounting rod relative to the micro-fluidic unit are adjustable.
Further:
the bottom of the mounting rod is provided with a bottom plate which plays a role in supporting the ground.
The temperature control unit is installed on the installation rod, and the micro-fluidic unit is arranged on the temperature control unit.
The temperature control unit comprises a supporting plate, a heat conduction shell, a heater and a temperature sensor, the supporting plate is connected with the mounting rod, the heat conduction shell is located on the upper side of the supporting plate, the heater is arranged in the heat conduction shell, the microfluidic control unit is arranged on the heat conduction shell, the temperature sensor is tightly attached to the surface of one side, close to the microfluidic control unit, of the heat conduction shell, and an external temperature controller is connected with the heater and the temperature sensor to control the temperature so as to keep the constant temperature required by the 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 to the inner wall of the lower aluminum alloy shell in an adhesive mode, and the temperature sensor is fixedly connected to 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 arranged in the light source shell, 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 exciting 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 circular ring, an outer circular ring with a stud at the upper end, a cylindrical part 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 part, a knob with a rod, a strip with teeth, an intermediate connecting piece and an L-shaped part, wherein the connecting structural part 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 part, the rod with the knob is provided with the teeth, is meshed with the strip with the teeth to drive the strip to move up and down, the intermediate connecting piece is positioned between the strip with the teeth and the L-shaped part, is connected with the strip with the teeth, is connected with one side of the L-shaped part, and the other side with holes of the L-shaped part is positioned between the cylindrical part and the outer circular ring, the stud penetrates through the hole to be in threaded connection with the inner ring of the cylindrical piece, the inner ring is located between the outer ring and the structural rod piece and is in interference fit with the outer ring, a gap exists between the structural rod piece and the inner ring, so that the structural rod piece can rotate in the inner ring, a round hole is formed in the corresponding position of the inner ring and the outer ring, the second screw is in threaded connection with the round hole and fixes the position of the structural rod piece, a raised cylinder is arranged at the front end of the structural rod piece, the lens support can rotate around the cylinder and is fixed on the raised cylinder through the third screw, and the camera module is installed on the lens support.
The camera module comprises a lens and an industrial camera, wherein the front end of the lens is provided with an emission light filter, the lens is installed at the front end of the industrial camera, and the lens and the industrial camera are clamped on corresponding positions of the lens support.
The microfluidic chip comprises a basal layer, a middle layer and a surface layer which are sequentially laminated together from bottom to top, wherein the middle layer comprises a plurality of sample adding holes, a plurality of air holes, a plurality of liquid separating cavities, a plurality of reaction cavities and flow channels, the sample adding holes are used for adding reaction liquid and flowing to the liquid separating cavities, the air holes enable fluid to flow in the chip, and the liquid separating cavities distribute the reaction liquid to the plurality of 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 base layer, the surface layer and the middle layer are bonded in an adhesive mode, 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 mu l.
The invention has the following beneficial effects:
the invention provides a real-time fluorescence constant-temperature nucleic acid amplification detection device, which realizes the constant-temperature amplification and real-time fluorescence detection of nucleic acid in a sample to be detected. The invention can well meet the requirements of clinical and epidemic disease detection and other rapid detection, and is particularly suitable for being applied to resource-deficient areas.
Drawings
FIG. 1 is a schematic structural diagram of a real-time fluorescence isothermal nucleic acid amplification detection apparatus 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 structural 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 diagram of a specific structure of the excitation light source device shown in fig. 4;
FIG. 6 is a schematic structural diagram of the detecting unit shown in FIG. 1;
FIG. 7 is an exploded view of the detection unit shown in FIG. 1;
FIG. 8 is a graph of real-time fluorescence intensity measured in accordance with an embodiment of the present invention.
Detailed Description
The embodiments of the present invention will be described in detail below. It should be emphasized that the following description is merely exemplary in nature and is not intended to limit the scope of the invention or its application.
It will be understood that when an element is referred to as being "secured to" 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 either a fixed or coupled or communicating function.
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 used in an orientation or positional relationship indicated in the drawings for convenience in describing the embodiments of the present invention and to simplify the description, and are not intended to indicate or imply that the referenced device or element must have a particular orientation, be constructed in a particular orientation, and be in any way limiting of the present invention.
Furthermore, the terms "first", "second" and "first" are used for descriptive purposes only and are not to be construed as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. In the description of the embodiments of the present invention, "a plurality" means two or more unless specifically limited otherwise.
Referring to fig. 1, an embodiment of the present invention provides a real-time fluorescence isothermal nucleic acid amplification detection apparatus, which includes a microfluidic unit 1, a temperature control unit 2, an excitation light unit 3, a detection unit 4, and a mounting rod 18, wherein a reaction solution containing a fluorescent dye performs a fluorescence isothermal nucleic acid amplification reaction in the microfluidic unit 1, the temperature control unit 2 provides the microfluidic unit 1 with a constant temperature required for nucleic acid amplification, 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, the detection unit 4 collects fluorescence information generated by the reaction solution in the microfluidic unit 1, and a detection result can be obtained by analyzing the collected fluorescence information; the micro-fluidic 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 micro-fluidic unit 1, and at least the position and/or angle of the detection unit 4 on the mounting rod 18 relative to the micro-fluidic unit 1 is adjustable.
In a preferred embodiment, the temperature control unit 2 is mounted on the mounting bar 18, and provides a support platform for the microfluidic unit 1 to indirectly mount the microfluidic unit 1 on the mounting bar 18 while providing a constant temperature required for nucleic acid amplification under the microfluidic unit 1. In other embodiments, the microfluidic cell 1 may also be directly mounted to the mounting bar 18 by mounting components, and the temperature control unit 2 may or may not be attached to the mounting bar 18, as long as isothermal reaction conditions are provided for the microfluidic cell 1.
The embodiment of the invention can conveniently realize the fluorescent constant-temperature nucleic acid amplification reaction and detection, has flexible and convenient operation and control, has simple structure and lower cost, can simultaneously detect a plurality of indexes, and ensures the reliable, stable and quick acquisition of the detection result.
FIG. 1 shows a real-time fluorescence isothermal nucleic acid amplification detecting device according to an exemplary embodiment, which includes a mounting rod 18, and a microfluidic unit 1, a temperature control unit 2, an excitation light unit 3 and a detecting unit 4 mounted on the mounting rod 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, and the substrate layer 5 and the intermediate layer 10 are bonded by gluing to ensure the sealing property and the thermal stability; the surface layer 11 and the intermediate layer 10 are glued to seal the air holes 9 and the sample application holes 6 and prevent contamination. 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 during centrifugation, the liquid separating cavities 7 equally divide the reaction liquid into the plurality of reaction cavities 8 through connected flow channels, and then the reaction cavities 8 contain reaction liquid containing fluorescent dye and can carry out nucleic acid amplification reaction.
The middle layer 10 is made of common transparent medical plastics, such as polyvinyl chloride (PVC), Polyethylene (PE), polypropylene (PP), Polystyrene (PS), and polycarbonate PC, and is molded by molding thermoplastic molding, injection molding, and other plastic molding methods; for example, an injection molding method is adopted to pre-process a mold, then a polypropylene (PP) material is melted in a constant temperature charging barrel at the temperature of 220 ℃ and 280 ℃, then the melted PP material is injected into the mold under the pressure of 800 ℃ and 140MPa, and then pressure maintaining and cooling molding are carried out. The substrate layer 5 and the surface layer 11 are both ultrathin adhesive tapes with good bonding effect, and the thickness of the ultrathin adhesive tapes is 0.02-0.1 mm. The volume of the reaction cavity 8 in the microfluidic chip is 5-10 mul. The fluid in the microfluidic chip is driven by the centrifuge and flows from the liquid separation cavity 7 to the reaction cavity 8 through the connected flow channel, so that liquid separation is completed. The rotating speed of the centrifugal machine is 1000-5000 rpm, the time is 10-60 s, and only one time of centrifugation is needed.
Fig. 3 is a schematic structural diagram of a temperature control unit 2, located below a microfluidic chip, for providing a temperature required for isothermal nucleic acid amplification, and including a bottom plate 12, a support plate 13, a lower aluminum alloy casing 14, a silicon rubber heater 15, a temperature sensor 16, an upper aluminum alloy casing 17, a mounting rod 18, and an external temperature controller. The bottom plate 12 is connected with the mounting rod 18 through threads, and plays a role of 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 support plate 13; the upper aluminum alloy shell 17 is positioned on the upper side of the lower aluminum alloy shell 14 and plays a role in heat conduction and temperature uniformity improvement. The silicon rubber heater 15 is a heating element, clings to the inner wall of the lower aluminum alloy shell 14 and is connected in an adhesive mode. The temperature sensor 16 is closely attached to the inner surface of the upper aluminum alloy case 17 and fixed by heat-resistant glue. The corresponding positions of the upper aluminum alloy shell 17, the silicon rubber heater 15, the lower aluminum alloy shell 14 and the support plate 13 are provided with holes and can be fixed together through four screws and nuts. The external temperature controller is used for temperature control to maintain a constant temperature required for the reaction.
The bottom plate 12 is made of steel and is located below 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 of the type K thermocouple, pt100 temperature sensor, or the like. The upper surface of the upper aluminum alloy shell 17 is sprayed with a layer of paint to increase diffuse reflection and reduce the influence on the fluorescent image.
The implementation process of the temperature control unit 2 is as follows: setting the constant temperature of the amplification reaction on an external temperature controller, then heating the air inside the upper aluminum alloy shell 17 and the lower aluminum alloy shell 14 by the silicon rubber heater 13 to increase the temperature of the upper surface of the upper aluminum alloy shell 17, then heating the reaction liquid in the reaction chamber 8 of the microfluidic chip to reach the constant temperature of the amplification reaction, and carrying out real-time temperature feedback by the temperature sensor 16 to carry out temperature regulation so as to keep the reaction liquid in the reaction chamber 8 at the constant temperature and continuously carry out the nucleic acid amplification reaction.
As shown in fig. 4, which is a schematic diagram of an overall structure of the excitation light unit 3, the excitation light unit is used for exciting a fluorescent dye in a reaction solution in a 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 a corresponding position on 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, which can be realized by gluing.
Fig. 5 is a schematic diagram of a specific structure of the excitation light source device 21 in fig. 4, which includes 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 bead 23 is welded on the lamp bead base 22 to generate exciting light. And the exciting light filter 24 is placed on the front side of the lamp bead 23 and plays a role in filtering light. The condenser lens 25 is placed on the front side of the excitation light filter 24 and performs a condensing function. The excitation light filter 24 and the condenser lens 25 are disposed at respective positions of the light source housing 26. The light source housing 26 includes five sides, is made of black aluminum alloy, and plays a role in fixing, supporting and dissipating 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 exciting light filter 24, the condensing lens 25 and the center of the microfluidic chip and the vertical line is 15-75 degrees, and the distance between the condensing lens 25 and the center of the microfluidic chip is 5-30 cm. The light source housing 26 is attached to each side by screws. The external light source power controller controls the power of the lamp bead 23.
The detection unit 4 is used for collecting fluorescence image information generated by 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, which is a schematic structural diagram of the detection unit 4, and fig. 7 is a schematic structural diagram of an explosion of the detection unit 4, the detection 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 threads at the inner ring, a structural rod 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 member 37, a knob 38 with a rod, a strip 39 with teeth, an intermediate connecting member 40, and an L-shaped member 41. The connection structural member 37 is fixed to the mounting rod 18 by the fourth screw 36 so that the detection unit 4 can move along the mounting rod 18. A knob 38 with a rod with teeth is placed in the connecting structural member 37, engaging with the strip 39 with teeth to move the strip up and down. The intermediate connecting member 40 is provided between the strip 39 with teeth and the L-shaped member 41, the intermediate connecting member 40 and the strip 39 with teeth are connected by screws and nuts, and the intermediate connecting member 40 and the L-shaped member 41 are connected by screws. The other side of the L-shaped part 41 is arranged between the cylindrical part 30 with the threads on the inner ring and the aluminum alloy outer ring 29 with the stud at the upper end, and the centers of the three parts are on the same axis, so that the aluminum alloy outer ring can rotate, and after the position is determined, the three parts 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 mode between the aluminum alloy outer ring 29 with the stud at the upper end and the structural rod piece 31, and a gap is reserved 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 stud at the upper end are provided with round holes at corresponding positions, the aluminum alloy outer ring 29 with the stud at the upper end is provided with threads at corresponding positions, the second screw 27 fixes the position of the structural rod piece 31 in a threaded connection mode, and the structural rod piece 31 can rotate in the plastic inner ring 28. The front end of the structural rod 31 is provided with a convex cylinder, and the lens holder 34 can rotate around the cylinder and is fixed on the convex cylinder through a third screw 32. The lens 33 with the emission light filter at the front end is installed at the front end of the industrial camera 35, and then the two are clamped on the corresponding position of the lens bracket 34 to play a role in collecting fluorescence information.
The industrial camera 35 is of a CCD or CMOS type, the lens is a zoom lens with a focal length of 2.8 mm-12 mm, and the wavelength of an emission light filter at the front end of the lens is selected to be adaptive to the emission wavelength of the fluorescent dye. In addition to the industrial camera and lens, the coordination of the remaining components provides a position adjustable gimbal for the industrial camera.
The detection unit 4 is used for acquiring a fluorescence image generated by irradiating the reaction liquid in the reaction cavity 8 in the microfluidic chip with the excitation light generated by the excitation light unit 3, and then performing fluorescence image transmission and image processing to obtain real-time fluorescence intensity information in the reaction cavities 8, namely a detection result, wherein the detection result can be used for judging whether the reaction liquid is positive or not.
The following briefly describes the process of nucleic acid detection using the real-time fluorescence isothermal nucleic acid amplification detection device of the embodiment of the present invention:
1) respectively pouring a proper amount of reaction liquid into a plurality of liquid separation cavities 7 from a plurality of sample adding holes 6 of a middle layer 10 at the speed of 200 mu L/min by using an injection gun, then putting the microfluidic chip on a centrifuge, centrifuging for a period of time at a certain rotating speed to ensure that all liquid in the liquid separation cavities 7 flows into the reaction cavities 8, and then sealing the sample adding holes 6 and the air holes 9 by using a surface layer 11;
2) placing the micro-fluidic chip on the surface of the upper aluminum alloy shell 17, turning on a main power supply, starting the exciting light unit 3, the temperature control unit 2 and the detection unit 4 to work, setting a constant temperature, starting real-time fluorescence detection, and obtaining a detection result after 20-30 min;
3) and after the detection is finished, the power supply is turned off, and the microfluidic chip can be taken out.
FIG. 8 shows a real-time fluorescence intensity profile, which is the result of nucleic acid detection, and it can be determined whether the reaction solution is positive. The nucleic acid amplification reaction suitable for the nucleic acid detection device comprises isothermal nucleic acid amplification technologies such as LAMP (loop-mediated isothermal amplification), RPA (recombinase polymerase amplification) and the like.
The invention has the advantages of rapidness, simplicity, stability, reliability, flexible and convenient control and the like when being used for carrying out the constant temperature amplification and detection of the fluorescent nucleic acid, and can well meet the requirements of clinical detection, epidemic disease detection and other rapid detections.
The background of the present invention may contain background information related to the problem or environment of the present invention and does not necessarily describe the prior art. Accordingly, the inclusion in the background section is not an admission of prior art by the applicant.
The foregoing is a more detailed description of the invention in connection with specific/preferred embodiments and is not intended to limit the practice of the invention to those descriptions. It will be apparent to those skilled in the art that various substitutions and modifications can be made to the described embodiments without departing from the spirit of the invention, and these substitutions and modifications should be considered to fall within the scope of the invention. In the description herein, references to the description of the term "one embodiment," "some embodiments," "preferred embodiments," "an example," "a specific example," or "some examples" or the like are intended to mean 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, the schematic representations of the terms used above are not necessarily intended to refer 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. Various embodiments or examples and features of various embodiments or examples described in this specification can be combined and combined by one skilled in the art 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 claims.
Claims (10)
1. A real-time fluorescence constant-temperature nucleic acid amplification detection device is characterized by comprising a microfluidic unit, a temperature control unit, an excitation light unit, a detection unit and a mounting rod, wherein reaction liquid containing fluorescent dye is used for carrying out fluorescence constant-temperature nucleic acid amplification reaction in the microfluidic unit, the temperature control unit provides constant temperature required by nucleic acid amplification for the microfluidic unit, 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 collects fluorescence information generated by the reaction liquid in the microfluidic unit to obtain a detection result; the micro-fluidic 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 located above the micro-fluidic unit, and at least the position and/or angle of the detection unit on the mounting rod relative to the micro-fluidic unit are adjustable.
2. The real-time fluorescent isothermal nucleic acid amplification detecting device according to claim 1, wherein a bottom plate for supporting the ground is provided at the bottom of the mounting rod.
3. The real-time fluorescent isothermal nucleic acid amplification detecting device according to claim 1 or 2, wherein the temperature control unit is mounted on the mounting rod, and the microfluidic unit is disposed on the temperature control unit.
4. The real-time fluorescence isothermal nucleic acid amplification detecting device according to 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 with the mounting rod, the heat conducting housing is located on the upper side of the support plate, the heater is arranged in the heat conducting housing, the microfluidic unit is arranged on the heat conducting housing, the temperature sensor is tightly attached to the surface of the heat conducting housing close to one side of the microfluidic unit, and an external temperature controller is connected with the heater and the temperature sensor for temperature control so as to maintain a constant temperature required by the reaction.
5. The real-time fluorescence isothermal nucleic acid amplification detecting device according to claim 4, wherein the heat conducting casing comprises a lower aluminum alloy casing and an upper aluminum alloy casing assembled together, the heater is a silicone rubber heater adhesively connected to the inner wall of the lower aluminum alloy casing, and the temperature sensor is fixedly connected to the inner surface of the upper aluminum alloy casing by a heat-resistant adhesive.
6. The real-time fluorescence isothermal nucleic acid amplification detecting 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 on the excitation light source device, the excitation light source device comprises a light source housing, a lamp bead base arranged in the light source housing, a lamp bead, an excitation light filter and a condenser 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 condenser 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 exciting 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.
7. The real-time fluorescence isothermal nucleic acid amplification detecting device according to any one of claims 1 to 6, wherein the detecting unit comprises a second screw, an inner ring, an outer ring having a stud at an upper end thereof, a cylindrical member having a thread at an inner ring thereof, a structural member, a third screw, a lens holder, a camera module, a fourth screw, a connecting structure, a knob having a rod, a bar having teeth, an intermediate connecting member and an L-shaped member, the connecting structure being fixed to the mounting rod by the fourth screw so that the detecting unit can move along the mounting rod, the knob being provided on the connecting structure, the rod having the knob being provided with teeth to engage with the bar having teeth to move the bar up and down, the intermediate connecting member being provided between the bar having teeth and the L-shaped member, the intermediate connecting member being connected to the bar having teeth, the middle connecting piece is connected with one side of the L-shaped piece, the other side of the L-shaped piece, which is provided with a hole, is positioned between the cylindrical piece and the outer ring, the stud penetrates through the hole to be connected with the inner ring of the cylindrical piece in a threaded manner, the inner ring is positioned between the outer ring and the structural rod piece and is in interference fit with the outer ring, a gap exists between the stud and the structural rod piece, so that the structural rod piece can rotate in the inner ring, round holes are formed in the corresponding positions of the inner ring and the outer ring, the second screw is in threaded connection with the round holes and fixes the position of the structural rod piece, a raised cylinder is arranged at the front end of the structural rod piece, the lens support can rotate around the cylinder and is fixed on the raised cylinder through the third screw, and the camera module is installed on the lens support.
8. The real-time fluorescence isothermal nucleic acid amplification detecting device according to claim 7, wherein the camera module comprises a lens with an emission light filter at the 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 on corresponding positions of the lens holder.
9. The real-time fluorescence isothermal nucleic acid amplification detecting device according to any one of claims 1 to 8, wherein the microfluidic chip comprises a substrate layer, an intermediate layer and a surface layer sequentially laminated from bottom to top, the intermediate layer comprises a plurality of sample adding holes, a plurality of air holes, a plurality of liquid separating cavities, a plurality of reaction cavities and flow channels, the sample adding holes are used for adding reaction liquid and flowing to the liquid separating cavities, the air holes enable the fluid to flow in the chip, and the liquid separating cavities distribute the reaction liquid to the plurality of reaction cavities through the flow channels.
10. The real-time fluorescence isothermal nucleic acid amplification detection device according to claim 9, wherein the intermediate layer is made of any one of polyvinyl chloride (PVC), Polyethylene (PE), polypropylene (PP), Polystyrene (PS) and Polycarbonate (PC), the substrate layer, the surface layer and the intermediate layer are bonded by gluing, the substrate layer and the surface layer are ultra-thin tapes with a thickness of 0.02 to 0.1mm, and the volume of the reaction chamber is 5 to 10 μ l.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110741819.3A CN113322175B (en) | 2021-07-01 | 2021-07-01 | Real-time fluorescence constant temperature nucleic acid amplification detection device |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110741819.3A CN113322175B (en) | 2021-07-01 | 2021-07-01 | Real-time fluorescence constant temperature nucleic acid amplification detection device |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN113322175A true CN113322175A (en) | 2021-08-31 |
| CN113322175B CN113322175B (en) | 2024-01-26 |
Family
ID=77425346
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202110741819.3A Active CN113322175B (en) | 2021-07-01 | 2021-07-01 | Real-time fluorescence constant temperature nucleic acid amplification detection device |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN113322175B (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114733587A (en) * | 2022-04-01 | 2022-07-12 | 合肥诺迈基生物科技有限公司 | Fluorescence detection chip, fluorescence detection system, fluorescence detection method and application thereof |
Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20030096877A (en) * | 2002-06-18 | 2003-12-31 | (주)바이오니아 | Apparatus for real time monitoring of products of nucleic acid amplification reaction |
| CN105823729A (en) * | 2016-03-21 | 2016-08-03 | 北京金诺美生物技术有限公司 | Automatic nucleic acid detecting device |
| CN106497779A (en) * | 2016-12-05 | 2017-03-15 | 天津伊科斯迪科技有限公司 | The colibacillary detection means of quick detection |
| CN106906137A (en) * | 2017-03-19 | 2017-06-30 | 北京化工大学 | A kind of high-throughput nucleic acid analysis device based on micro-fluidic chip |
| CN107456627A (en) * | 2017-08-03 | 2017-12-12 | 东莞市森斯电子机械科技有限公司 | A kind of full-automatic pricking device for realizing plastic blood bag and inactivation of virus bag safety |
| WO2018137513A1 (en) * | 2017-01-24 | 2018-08-02 | 北京万泰生物药业股份有限公司 | System for detecting convective pcr amplification and method for detecting convective pcr amplification |
| CN111925930A (en) * | 2020-08-07 | 2020-11-13 | 单洪瑞 | Virus detector |
| KR20210002317A (en) * | 2019-06-30 | 2021-01-07 | 주식회사 진시스템 | Multiplex pcr apparatus |
| CN212770649U (en) * | 2020-06-11 | 2021-03-23 | 天津诺迈科技有限公司 | Centrifugal micro-fluidic chip integrating nucleic acid extraction and fluorescence amplification |
-
2021
- 2021-07-01 CN CN202110741819.3A patent/CN113322175B/en active Active
Patent Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20030096877A (en) * | 2002-06-18 | 2003-12-31 | (주)바이오니아 | Apparatus for real time monitoring of products of nucleic acid amplification reaction |
| CN105823729A (en) * | 2016-03-21 | 2016-08-03 | 北京金诺美生物技术有限公司 | Automatic nucleic acid detecting device |
| CN106497779A (en) * | 2016-12-05 | 2017-03-15 | 天津伊科斯迪科技有限公司 | The colibacillary detection means of quick detection |
| WO2018137513A1 (en) * | 2017-01-24 | 2018-08-02 | 北京万泰生物药业股份有限公司 | System for detecting convective pcr amplification and method for detecting convective pcr amplification |
| CN106906137A (en) * | 2017-03-19 | 2017-06-30 | 北京化工大学 | A kind of high-throughput nucleic acid analysis device based on micro-fluidic chip |
| CN107456627A (en) * | 2017-08-03 | 2017-12-12 | 东莞市森斯电子机械科技有限公司 | A kind of full-automatic pricking device for realizing plastic blood bag and inactivation of virus bag safety |
| KR20210002317A (en) * | 2019-06-30 | 2021-01-07 | 주식회사 진시스템 | Multiplex pcr apparatus |
| CN212770649U (en) * | 2020-06-11 | 2021-03-23 | 天津诺迈科技有限公司 | Centrifugal micro-fluidic chip integrating nucleic acid extraction and fluorescence amplification |
| CN111925930A (en) * | 2020-08-07 | 2020-11-13 | 单洪瑞 | Virus detector |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114733587A (en) * | 2022-04-01 | 2022-07-12 | 合肥诺迈基生物科技有限公司 | Fluorescence detection chip, fluorescence detection system, fluorescence detection method and application thereof |
| CN114733587B (en) * | 2022-04-01 | 2024-02-20 | 合肥诺迈基生物科技有限公司 | Fluorescence detection chip, fluorescence detection system, fluorescence detection method and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN113322175B (en) | 2024-01-26 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| KR102426954B1 (en) | Convection PCR Amplification Detection System and Convection PCR Amplification Detection Method | |
| US8658099B2 (en) | Integrated apparatus for conducting and monitoring chemical reactions | |
| US10569273B2 (en) | Rapid thermal cycling for sample analyses and processing | |
| JP5650791B2 (en) | Chemical reaction assembly with heat exchange and optical detection | |
| CN110161003B (en) | Optical detection device and real-time fluorescence quantitative nucleic acid amplification detection system | |
| US7943088B2 (en) | Biochemical analyzer and method of controlling internal temperature of the biochemical analyzer | |
| CN107619781B (en) | Single-reaction temperature-control high-throughput micro-fluidic chip nucleic acid amplification device | |
| CN102768203A (en) | Space-oriented minitype cylindrical microfluidic PCR (polymerase chain reaction) real-time fluoroscopic detection system | |
| CN107051598B (en) | PCR microfluidic chip, preparation and use methods thereof and PCR equipment | |
| CN113322175A (en) | Real-time fluorescence constant temperature nucleic acid amplification detection device | |
| CN110804540B (en) | Microfluidic temperature control system and nucleic acid analysis system | |
| CN102604824A (en) | Space-oriented mini micro-fluidic real-time fluorescent PCR (Plymerase Chain Reaction) working system | |
| US20220340960A1 (en) | Nucleic acid amplification in-situ real-time detection system and method using microfluidic chip through optical fiber sensing | |
| CN109957506B (en) | Device for quantitative polymerase chain reaction by thermal convection through reagent container | |
| US9993819B2 (en) | Apparatus for actuating and reading a centrifugal microfluidic disk for biological and biochemical analyses, and use of the apparatus | |
| EP3463668A1 (en) | Rapid thermal cycling for sample analyses and processing | |
| US10245590B2 (en) | High-speed real-time PCR device based on lab-on-a-chip for detecting food-borne bacteria to agrifood, and methods for detecting food-borne bacteria to agrifood using the same | |
| CN116218653A (en) | Centrifugal fully-integrated high-flux nucleic acid detection micro-fluidic chip and detection method | |
| TWI725686B (en) | Tubular structure for producing droplets and method for producing droplets | |
| CN115340946B (en) | Amplification device for nucleic acid detection and detection method based on amplification device | |
| JP2007003409A (en) | Microchip inspection device | |
| CN212270116U (en) | Molecular detection system | |
| CN217351365U (en) | Portable ultra-fast real-time fluorescence quantitative PCR instrument | |
| KR102003784B1 (en) | micro chip for polymerase chain reaction and real-time PCR device comprising the same | |
| CN212246964U (en) | Centrifugal microfluidic chip and system for SAT |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |