CN112226350B - Centrifugal type nuclear extraction and amplification integrated system and detection method - Google Patents
Centrifugal type nuclear extraction and amplification integrated system and detection method Download PDFInfo
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- 238000001514 detection method Methods 0.000 title claims abstract description 148
- 230000003321 amplification Effects 0.000 title claims description 27
- 238000000605 extraction Methods 0.000 title claims description 27
- 238000003199 nucleic acid amplification method Methods 0.000 title claims description 27
- 108020004707 nucleic acids Proteins 0.000 claims abstract description 132
- 150000007523 nucleic acids Chemical class 0.000 claims abstract description 132
- 102000039446 nucleic acids Human genes 0.000 claims abstract description 132
- 238000006243 chemical reaction Methods 0.000 claims abstract description 65
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- 230000005284 excitation Effects 0.000 claims description 33
- 238000002844 melting Methods 0.000 claims description 28
- 230000008018 melting Effects 0.000 claims description 28
- 238000010306 acid treatment Methods 0.000 claims description 22
- 239000002699 waste material Substances 0.000 claims description 20
- 239000011324 bead Substances 0.000 claims description 14
- 238000011161 development Methods 0.000 claims description 14
- 238000001917 fluorescence detection Methods 0.000 claims description 14
- 238000002347 injection Methods 0.000 claims description 14
- 239000007924 injection Substances 0.000 claims description 14
- 239000003153 chemical reaction reagent Substances 0.000 claims description 12
- 239000013307 optical fiber Substances 0.000 claims description 10
- 230000005540 biological transmission Effects 0.000 claims description 7
- 239000000463 material Substances 0.000 claims description 6
- 239000000843 powder Substances 0.000 claims description 6
- 238000003860 storage Methods 0.000 claims description 6
- 238000000746 purification Methods 0.000 claims description 4
- 238000007789 sealing Methods 0.000 claims description 4
- 238000001746 injection moulding Methods 0.000 claims description 3
- 238000001179 sorption measurement Methods 0.000 claims description 3
- 238000003466 welding Methods 0.000 claims description 3
- 239000003292 glue Substances 0.000 claims description 2
- 239000000126 substance Substances 0.000 claims description 2
- 229920000297 Rayon Polymers 0.000 claims 1
- 238000012864 cross contamination Methods 0.000 abstract description 5
- 238000003752 polymerase chain reaction Methods 0.000 description 15
- 238000010586 diagram Methods 0.000 description 8
- 239000003480 eluent Substances 0.000 description 7
- 238000004140 cleaning Methods 0.000 description 6
- 239000000243 solution Substances 0.000 description 5
- 238000004458 analytical method Methods 0.000 description 4
- 238000003776 cleavage reaction Methods 0.000 description 4
- 238000005336 cracking Methods 0.000 description 4
- 230000007017 scission Effects 0.000 description 4
- 239000000853 adhesive Substances 0.000 description 3
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- 238000012408 PCR amplification Methods 0.000 description 2
- 239000012295 chemical reaction liquid Substances 0.000 description 2
- 238000003384 imaging method Methods 0.000 description 2
- 230000010354 integration Effects 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 238000011897 real-time detection Methods 0.000 description 2
- 230000002277 temperature effect Effects 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 1
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- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L7/00—Heating or cooling apparatus; Heat insulating devices
- B01L7/52—Heating or cooling apparatus; Heat insulating devices with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
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- 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
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- 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
- C12Q1/686—Polymerase chain reaction [PCR]
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- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
- B01L2200/10—Integrating sample preparation and analysis in single entity, e.g. lab-on-a-chip concept
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- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2300/00—Additional constructional details
- B01L2300/18—Means for temperature control
- B01L2300/1805—Conductive heating, heat from thermostatted solids is conducted to receptacles, e.g. heating plates, blocks
- B01L2300/1822—Conductive heating, heat from thermostatted solids is conducted to receptacles, e.g. heating plates, blocks using Peltier elements
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Abstract
The invention discloses a centrifugal type nuclear lifting and amplifying integrated system, which comprises a centrifugal type nuclear lifting and amplifying integrated instrument and a cartridge for carrying out nuclear lifting and amplifying integrated reaction, wherein the cartridge comprises a nucleic acid processing area and a nucleic acid detection area, the nucleic acid processing area is communicated with the nucleic acid detection area through a channel, and a plugged wax valve is arranged in the channel; the centrifugal type nuclear lifting and amplifying integrated instrument is provided with a cartridge supporting mechanism, a centrifugal motion driving device, a temperature control reaction mechanism, an optical detection mechanism and a circuit control system; the system has simple operation, small volume and high degree of automation; the system can be operated in a full-automatic process by only manually adding samples, and the whole process instrument is operated without liquid; the extract is not required to be manually transferred, so that cross contamination is avoided; the risk of false positives is reduced.
Description
Technical Field
The invention relates to the technical field of biomedical gene analysis instruments, in particular to a centrifugal type nuclear lifting and amplifying integrated system and a detection method.
Background
Nucleic acid extraction and Polymerase Chain Reaction (PCR) are common analytical means for molecular biology. In the past, traditional nucleic acid extraction and PCR are carried out in separate instruments, after sample pretreatment is carried out by experimenters, the sample loading of the nuclear lifting needs to be carried out manually, and after the nuclear lifting is finished, a PCR experiment system is prepared manually, and the whole process is carried out by on-machine detection, so that at least half a day is needed, the analysis and detection time is too long, and time and labor are wasted.
In addition, the separate process of the nuclear-mentioned PCR is easy to cause cross contamination and environmental pollution, and brings false positives of detection results and influences analysis of the results, so that development of a system integrating nucleic acid extraction and PCR amplification detection is urgently needed.
Disclosure of Invention
Aiming at the technical problems in the related art, the invention provides a centrifugal type nuclear extraction and amplification integrated system, which can overcome the defects in the prior art, shorten the analysis and detection time, reduce the possibility of cross contamination and have more accurate detection results.
In order to achieve the technical purpose, the technical scheme of the invention is realized as follows: the centrifugal type nuclear lifting and amplifying integrated system comprises a centrifugal type nuclear lifting and amplifying integrated instrument and a cartridge for carrying out nuclear lifting and amplifying integrated reaction, wherein the cartridge comprises a nucleic acid processing area and a nucleic acid detection area, the nucleic acid processing area is communicated with the nucleic acid detection area through a channel, and a plugged wax valve is arranged in the channel;
the centrifugal type nuclear lifting and amplifying integrated instrument is provided with a cartridge supporting mechanism, a centrifugal motion driving device, a temperature control reaction mechanism, an optical detection mechanism and a circuit control system.
Further, a liquid storage end formed by injection molding of a chemical inert material is arranged on the card box, a channel and an opening connected with the liquid storage end are sealed by a diaphragm or a slide of the inert material, and the sealing mode is adhesive, UV adhesive exposure or ultrasonic welding.
Further, the number of the nucleic acid detection areas is one or more, the nucleic acid detection areas are pre-embedded with a reaction reagent, and the reaction reagent is in a dry powder, freeze-dried ball or liquid reaction system.
Further, the cartridge further comprises a waste liquid area and a liquid injection area; the liquid injection area is communicated with the nucleic acid treatment area and the nucleic acid treatment area is communicated with the waste liquid area through channels, and a plugged wax valve is arranged in each channel; the number of the liquid injection areas is one or more, nuclear extraction reaction reagents are pre-buried in the liquid injection areas, and the nuclear extraction reaction reagents are in the form of dry powder, freeze-dried balls or liquid reaction systems.
Further, the cartridge can adopt a magnetic bead method or a centrifugal column method for nucleic acid extraction and purification; the centrifugal type nuclear extraction and amplification integrated instrument is provided with a magnetic bead adsorption mechanism aligned to the magnetic beads of the nucleic acid treatment area; and a centrifugal column is added in the middle of the nucleic acid treatment area in the way of extracting and purifying nucleic acid of the centrifugal column.
Further, the cartridges are fixed above the cartridge supporting mechanism, at least two cartridges are carried, and the two cartridges are arranged in an axisymmetric or centrosymmetric manner;
the temperature control reaction mechanism is positioned above the cartridge supporting mechanism, the nucleic acid detection area of the cartridge is in close contact with the temperature control reaction mechanism, and the optical detection mechanism is fixed on the outer side of the centrifugal motion driving device.
Further, the device also comprises a wax melting mechanism, wherein the wax melting mechanism adopts contact type wax melting or non-contact type wax melting; the wax melting mechanism adopts contact type wax melting, and the upper surface of the cartridge supporting mechanism and the position of the cartridge wax valve are provided with temperature control points which are in corresponding contact; the non-contact wax melting mode is characterized in that the wax melting mechanism is fixed on the outer side of the centrifugal mechanism and can be heated by infrared irradiation.
Further, the cartridge supporting mechanism performs centrifugal rotary motion at a rotation speed set by the circuit control system, and each nucleic acid detection area of the cartridge sequentially performs optical signal detection through the optical detection mechanism.
In another aspect of the invention, a centrifugal type nuclear extraction and amplification integrated detection method is provided, including a fluorescence detection method and a chromogenic detection method;
In the fluorescence detection method, the optical detection mechanism is connected with a detection light path through an optical fiber, the optical fiber is fixedly aligned with one circle of a nucleic acid detection area, when the nucleic acid detection area rotates through the path of the optical fiber, an excitation light source of the detection light path emits light to enter the nucleic acid detection area to be emitted into a detection device, and the detection device is converted into an electric signal to be transmitted to a circuit control system;
in the color development detection method, an excitation light source and a detection device are respectively arranged on the upper side and the lower side or the inner side and the outer side of the nucleic acid detection area, light emitted by the excitation light source is injected into the nucleic acid detection area, and light absorbed by a color development system is injected into the detection device on the other side of the excitation light source.
Further, in the fluorescence detection method, the excitation light source and the detection device are respectively arranged in different directions from the circumference of the nucleic acid detection area, the excitation light obliquely irradiates the nucleic acid detection area, and the emitted light irradiates the detection device arranged on the other side through the filter disc;
In the color development detection method, the excitation light source and the detection device are provided with angles, light emitted by the excitation light source enters the nucleic acid detection area, and light absorbed by the color development system enters the detection device;
In the fluorescence detection method, the optical detection mechanism performs detection of at least one optical channel.
The invention has the beneficial effects that: the system has simple operation, small volume and high degree of automation; the system can be operated in a full-automatic process by only manually adding samples, and the whole process instrument is operated without liquid; the extract is not required to be manually transferred, so that cross contamination is avoided; reducing the risk of false positives in the results; the extracted nucleic acid templates are directly subjected to amplification reaction on the same cartridge, so that reaction liquids in different reaction cartridges are centrifuged once and enter relevant areas, and the time of liquid transfer is reduced while automatic operation is realized; the rotary mode enables the cartridge to sequentially pass through the PCR optical detection mechanism, so that the scanning time or the imaging time of the optical mechanism in the traditional mode is reduced. In addition, the system is particularly suitable for non-specific PCR laboratory environments such as emergency treatment, medium and small hospitals and the like, and has no professional requirements on operators.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a schematic view of a cartridge according to an embodiment of the present invention
FIG. 2 is a schematic diagram of a cartridge employing a spin column according to an embodiment of the present invention;
FIG. 3 is a schematic diagram of a cartridge employing a magnetic bead method according to an embodiment of the present invention;
FIG. 4 is a schematic cross-sectional view of a centrifugal type nuclear lifting amplification integrated instrument according to an embodiment of the present invention;
FIG. 5 shows a schematic diagram of a fluorescence detection structure according to an embodiment of the present invention;
FIG. 6 is a schematic diagram of a fluorescence detection structure according to an embodiment of the present invention;
FIG. 7 is a schematic diagram of a transmission detection structure according to an embodiment of the present invention;
FIG. 8 is a schematic diagram II of a transmission detection structure according to an embodiment of the present invention;
FIG. 9 is a schematic diagram of a color development method reflection detection structure according to an embodiment of the present invention;
fig. 10 is a schematic diagram of a reflection detection structure according to a chromogenic method according to an embodiment of the present invention.
In the figure: 10. a cartridge; 10-1, a waste liquid zone; 10-2, a liquid injection area; 10-3, sample area; 10-4, cleavage binding region; 10-5, cleaning liquid area; 10-6, eluent zone; 20. centrifugal type nuclear lifting and amplifying integrated instrument; 1. a nucleic acid treatment region; 1-1, a centrifugal column; 1-2, a magnet; 1-3, a magnet movement track; 2. a nucleic acid detection zone; 3. a wax valve; 4. a cartridge support mechanism; 5. a centrifugal motion driving device; 6. a temperature control reaction mechanism; 7. an optical detection mechanism; 8. a temperature control reaction mechanism; 7-1, optical fiber.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which are derived by a person skilled in the art based on the embodiments of the invention, fall within the scope of protection of the invention.
As shown in fig. 1 to 4, a centrifugal type integrated system for nuclear lifting and amplifying according to an embodiment of the present invention comprises a centrifugal type integrated instrument for nuclear lifting and amplifying 20 and a cartridge 10 for carrying out integrated reaction for nuclear lifting and amplifying, wherein the cartridge 10 comprises a nucleic acid processing area 1 and a nucleic acid detecting area 2, the nucleic acid processing area 1 and the nucleic acid detecting area 2 are communicated through a channel, and a plugged wax valve 3 is arranged in the channel;
the centrifugal type nuclear lifting and amplifying integrated instrument 20 is provided with a cartridge supporting mechanism 4, a centrifugal motion driving device 5, a temperature control reaction mechanism 6, an optical detection mechanism 7 and a circuit control system.
In one embodiment of the present invention, the cartridge 10 is provided with a liquid storage end formed by injection molding of a chemically inert material, a channel and an opening connected with the liquid storage end are sealed by a membrane or a glass slide made of the inert material, and the sealing mode is adhesive, UV glue exposure or ultrasonic welding.
In one embodiment of the present invention, the number of the nucleic acid detecting areas 2 is one or more, and the nucleic acid detecting areas 2 are pre-embedded with a reaction reagent, where the reaction reagent is in a form of dry powder, freeze-dried balls or a liquid reaction system.
In one embodiment of the present invention, the cartridge 10 further comprises a waste liquid zone 10-1 and a liquid injection zone 10-2; the liquid injection region 10-2 is communicated with the nucleic acid treatment region 1 and the nucleic acid treatment region 1 is communicated with the waste liquid region 10-1 through a channel, and a plugged wax valve 3 is arranged in the channel; the number of the liquid injection areas 10-2 is one or more, the liquid injection areas 10-2 are pre-embedded with nuclear extraction reaction reagents, and the nuclear extraction reaction reagents are in the form of dry powder, freeze-dried balls or liquid reaction systems.
In one embodiment of the present invention, the cartridge 10 may employ a magnetic bead method or a spin column method for nucleic acid extraction and purification; the centrifugal type nucleic acid extraction and amplification integrated instrument 20 is provided with a magnetic bead adsorption mechanism aligned to the magnetic beads in the nucleic acid treatment area 1 in the magnetic bead method nucleic acid extraction and purification mode; and a centrifugal column is added in the middle of the nucleic acid treatment area 1 in the way of extracting and purifying nucleic acid of the centrifugal column.
In one embodiment of the present invention, the cartridge 10 is fixed above the cartridge supporting mechanism 4, the cartridge 4 carries at least two cartridges 10, and the two cartridges 10 are arranged in an axisymmetric or centrosymmetric manner;
The temperature control reaction mechanism 6 is located above the cartridge supporting mechanism 4, the nucleic acid detection area 2 of the cartridge 10 is in close contact with the temperature control reaction mechanism 6, and the optical detection mechanism 7 is fixed on the outer side of the centrifugal motion driving device 5.
In one embodiment of the invention, the device further comprises a wax melting mechanism, wherein the wax melting mechanism adopts contact type wax melting or non-contact type wax melting; the wax melting mechanism adopts contact type wax melting, and the upper surface of the cartridge supporting mechanism 4 and the position of the cartridge 10 wax valve are provided with temperature control points which are in corresponding contact; the non-contact wax melting mode is characterized in that the wax melting mechanism is fixed on the outer side of the centrifugal mechanism and can be heated by infrared irradiation.
In one embodiment of the present invention, the cartridge support mechanism 4 performs centrifugal rotation at a rotation speed set by the circuit control system, and each of the nucleic acid detecting areas 2 of the cartridge 10 sequentially performs optical signal detection by the optical detection mechanism 7.
As shown in fig. 5 to 10, in another aspect of the present invention, there is provided a centrifugal type nuclear lifting amplification integrated detection method including a fluorescence detection method and a chromogenic detection method;
in the fluorescence detection method, the optical detection mechanism 7 is connected with a detection light path through an optical fiber 7-1, the optical fiber 7-1 is fixedly aligned with one circle of the nucleic acid detection area 2, when the nucleic acid detection area 2 rotates through the path of the optical fiber 7-1, an excitation light source of the detection light path emits light to enter the nucleic acid detection area 2 to be emitted into a detection device, and the detection device is converted into an electric signal to be transmitted to a circuit control system;
in the color development detection method, an excitation light source and a detection device are respectively arranged on the upper side and the lower side or the inner side and the outer side of the nucleic acid detection area 2, light emitted by the excitation light source is injected into the nucleic acid detection area 2, and light absorbed by a color development system is injected into the detection device on the other side of the excitation light source.
In one embodiment of the present invention, in the fluorescence detection method, the excitation light source and the detection device are respectively disposed in different directions from the circumference of the nucleic acid detection area 2, the excitation light is obliquely incident into the nucleic acid detection area 2, and the emitted light is incident into the detection device disposed on the other side through the filter;
in the color development detection method, the excitation light source and the detection device are provided with angles, light emitted by the excitation light source enters the nucleic acid detection area 2, and light absorbed by a color development system enters the detection device;
in the fluorescence detection method, the optical detection means 7 performs detection of at least one optical channel.
In one embodiment of the present invention, the nucleic acid detecting area 2 is provided with 4 detecting pools, and the nucleic acid processed by the nucleic acid processing area 1 can enter a plurality of nucleic acid detecting pools in series or in parallel through the communicating channels, and react with a system pre-embedded in the detecting pools, so that single-sample multi-target detection can be performed.
In order to facilitate understanding of the above technical solutions of the present invention, the following describes the above technical solutions of the present invention in detail by a specific usage manner.
The cartridge 10 is a carrier for automatic nuclear-mention amplification reaction, and comprises a nucleic acid processing area 1, a nucleic acid detection area 2 and a waste liquid area 10-1, wherein all the areas are communicated through a channel, and a wax valve is arranged in the channel for sealing.
The liquid injection zone 10-2 comprises a sample zone 10-3, a cleavage and combination zone 10-4, a cleaning liquid zone 10-5 and an eluent zone 10-6;
the centrifugal type nuclear lifting and amplifying integrated instrument comprises a cartridge supporting mechanism 4, a centrifugal motion driving device 5, a temperature control reaction mechanism 6, an optical detection mechanism 7 and a circuit control system.
The nucleic acid detection reagent pre-buried in the nucleic acid detection area of the cartridge 10 adopts a fluorescent PCR amplification detection method, and the cartridge 10 is provided with 4 detection areas, namely, the nucleic acid extracted by the integrated system can be distributed to 4 detection areas for nucleic acid amplification and detection.
The cartridge 10 is loaded on the cartridge supporting mechanism 4, the cartridge 10 rotates along with the cartridge supporting mechanism 4 under the drive of the centrifugal motion driving device 5, a plurality of temperature control points are arranged at positions, which are closely contacted with the cartridge 10, on the cartridge supporting mechanism 4, the positions of the temperature control points correspond to the positions of the wax valves of the cartridge 10, the wax valves can be heated respectively under the control of the circuit control system, heat is transferred to the wax valves at the corresponding positions of the cartridge 10 through contact, and the wax valves are opened and communicated with channels between different closed areas of the wax valves.
The temperature control reaction mechanism 6 provides a temperature environment required by nucleic acid extraction and amplification reaction for the nucleic acid detection area of the contacted cartridge 10, and the optical detection mechanism 7 excites a reaction system of the nucleic acid detection area through an excitation light source and acquires fluorescent signals.
The cartridge supporting mechanism 4 is composed of a platform motor, a transmission device and a platform supporting component, wherein the transmission device in the embodiment is a belt wheel and a synchronous belt, and the platform motor drives the cartridge supporting mechanism 4 to perform circular motion through the transmission device.
The temperature control reaction mechanism 6 provides a temperature environment of 4-100 ℃ for the PCR reaction and comprises a PCR reaction tank adapter, a temperature sensor, a Peltier and a radiating fin. Wherein, the Peltier provides a lifting Wen Dongli source, the temperature sensor feeds back the PCR reaction tank adapter in real time, the circuit control system can control the temperature of the Peltier through PID control according to the feedback temperature of the temperature sensor, and then the PCR reaction tank adapter is controlled to rise and fall at high speed; the heat sink can conduct out the heat generated by the peltier.
The optical detection mechanism 7 can detect 4 groups of optical paths, each optical path is provided with an independent optical detection module, each optical detection module is provided with an excitation light source, excitation light emitted by the excitation light source irradiates the nucleic acid detection area 2 of the cartridge 10 through an excitation light source, fluorescent light is emitted by a reaction system in the excitation reaction tank, the excited fluorescent light enters the dichroic mirror through the collimation of the excitation light source through the convergence lens, and then enters the detector through the convergence lens and the excitation light filter at the transmitting end to be collected; the 4 optical detection channels are provided with excitation light sources with different spectrums, filter disc groups and detection devices, so that independent light paths are formed. The 4 groups of light paths are arranged in parallel.
The centrifugal column method is adopted for nuclear extraction:
Specifically, before the reaction, a sample is added into a sample area 10-3, the position of a wax valve of a nucleic acid processing area 1 is heated, a closed channel is communicated, the sample is transferred into the nucleic acid processing area 1 through centrifugal movement, a cracking binding area 10-4 with a cracking binding system is pre-packaged, and the sample enters the nucleic acid processing area 1 through centrifugal movement to realize the mixed reaction with the sample;
After the reaction, the reaction solution is rapidly centrifuged at a certain rotating speed, and the reaction solution passes through the centrifugal column 1-1, so that nucleic acid is positioned in the centrifugal column 1-1, and the waste liquid passing through the centrifugal column enters the waste liquid zone 10-1;
the wax valve 3 of the cleaning liquid area 10-5 is opened by hot melting, the cleaning liquid area 10-5 enters the nucleic acid treatment area 1 through a channel, the quick centrifugation is carried out, and the cleaned waste liquid enters the waste liquid area 10-1;
The wax valve 3 of the eluent area 10-6 is opened by hot melting, the eluent area 10-6 enters the nucleic acid treatment area 1 through a channel, and is rapidly centrifuged, the eluted nucleic acid is positioned at the bottom of the nucleic acid treatment area 1 after passing through the centrifugal column 1-1, and the channel communicated with the waste liquid area 10-1 is positioned at the high position of the nucleic acid treatment area 1 due to less eluent, so that the centrifugal rotating speed is controlled, and the eluted nucleic acid cannot enter the waste liquid area 10-1.
Opening a wax valve 3 between the nucleic acid treatment area 1 and the nucleic acid detection area 2, and reversely rotating, so that nucleic acid passes through the communication channel to sequentially pass through the nucleic acid detection area 2, and redundant nucleic acid enters a nucleic acid waste liquid area; the nucleic acid is respectively mixed with an amplification system preset in the nucleic acid detection area 23, the PCR reaction is carried out under the temperature effect of the temperature control reaction mechanism 6, and in the amplification reaction process, the real-time detection of fluorescent signals of amplification products in the nucleic acid detection area 2 can be realized through the cooperative operation of the cartridge supporting mechanism 4 and the optical detection mechanism 7, so that the whole process of nucleic acid extraction and amplification integration is completed.
The process of nuclear extraction by a magnetic bead method comprises the following steps:
Specifically, before the reaction, a sample is added into a sample area 10-3, the position of a wax valve of a nucleic acid processing area 1 is heated, a closed channel is communicated, the sample is transferred into the nucleic acid processing area 1 through centrifugal movement, a cracking binding area 10-4 with a cracking binding system is pre-packaged, and the sample enters the nucleic acid processing area 1 through centrifugal movement to realize the mixed reaction with the sample;
The cleavage and binding region 10-4, which is pre-packaged with the cleavage and binding system, enters the nucleic acid processing region 1 by centrifugal movement to effect a mixing reaction with the sample. The system can be uniformly mixed in the forward and reverse directions in the reaction process.
After the reaction, the nucleic acid adsorbed on the surface of the magnetic beads is quickly centrifuged at a certain rotating speed, the magnet 1-2 positioned in the magnet moving track 1-3 magnetically adsorbs the nucleic acid to one side of the nucleic acid treatment area 1, and the waste liquid enters the waste liquid area 10-1 through a channel;
The wax valve 3 of the cleaning liquid area 10-5 is opened by hot melting, the cleaning liquid area 10-5 enters the nucleic acid treatment area 1 through a channel, a uniform mixing system is rotated in the forward and reverse directions, the centrifugal is performed rapidly, and the cleaned waste liquid enters the waste liquid area 10-1;
The wax valve 3 of the eluent area 10-6 is opened by hot melting, the eluent area 10-6 enters the nucleic acid treatment area 1 through a channel, after a certain time, the eluted nucleic acid is positioned at the bottom of the nucleic acid treatment area 1, the wax valve 3 is opened, the magnetic beads in the system are reversely rotated and adsorbed by the magnet 1-2, the nucleic acid sequentially enters the nucleic acid detection area 2 through the channel, and the redundant nucleic acid enters the nucleic acid waste liquid area;
The nucleic acid is respectively mixed with an amplification system preset in the nucleic acid detection area 2, the PCR reaction is carried out under the temperature effect of the temperature control reaction mechanism 6, and in the amplification reaction process, the real-time detection of fluorescent signals of amplification products in the nucleic acid detection area 2 can be realized through the cooperative operation of the cartridge supporting mechanism 4 and the optical detection mechanism 7, so that the whole process of nucleic acid extraction and amplification integration is completed.
In summary, by means of the technical scheme, the system is simple to operate, small in size and high in automation degree; the system can be operated in a full-automatic process by only manually adding samples, and the whole process instrument is operated without liquid; the extract is not required to be manually transferred, so that cross contamination is avoided; reducing the risk of false positives in the results;
the extracted nucleic acid templates are directly subjected to amplification reaction on the same cartridge, so that reaction liquids in different reaction cartridges are centrifuged once and enter relevant areas, and the time for transferring the liquids is reduced while the automatic operation is realized; the rotary mode enables the cartridge to sequentially pass through the PCR optical detection mechanism, so that the scanning time or the imaging time of the optical mechanism in the traditional mode is reduced. In addition, the system is particularly suitable for non-specific PCR laboratory environments such as emergency treatment, medium and small hospitals and the like, and has no professional requirements on operators.
The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, alternatives, and improvements that fall within the spirit and scope of the invention.
Claims (9)
1. The centrifugal type nuclear lifting and amplifying integrated system is characterized by comprising a centrifugal type nuclear lifting and amplifying integrated instrument (20) and a cartridge (10) for carrying out nuclear lifting and amplifying integrated reaction, wherein the cartridge (10) comprises a nucleic acid processing area (1) and a nucleic acid detection area (2), the nucleic acid processing area (1) and the nucleic acid detection area (2) are communicated through a channel, and a plugged wax valve (3) is arranged in the channel;
the centrifugal type nuclear lifting and amplifying integrated instrument (20) is provided with a cartridge supporting mechanism (4), a centrifugal motion driving device (5), a temperature control reaction mechanism (6), an optical detection mechanism (7) and a circuit control system;
The device also comprises a wax melting mechanism, wherein the wax melting mechanism adopts contact type wax melting or non-contact type wax melting; the wax melting mechanism adopts contact type wax melting, and the upper surface of the cartridge supporting mechanism (4) and the position of the cartridge (10) wax valve are provided with temperature control points which are in corresponding contact; the non-contact wax melting mode is characterized in that the wax melting mechanism is fixed on the outer side of the centrifugal mechanism and is heated by infrared irradiation;
The card box supporting mechanism (4) consists of a platform motor, a transmission device and a platform supporting assembly, and the platform motor drives the card box supporting mechanism (4) to perform circular motion through the transmission device.
2. The integrated centrifugal nuclear lifting and amplifying system according to claim 1, wherein the cartridge (10) is provided with a liquid storage end formed by injection molding of a chemical inert material, a channel and an opening connected with the liquid storage end are sealed by a diaphragm or a slide of the inert material, and the sealing mode is viscose, UV glue exposure or ultrasonic welding.
3. The integrated system for centrifugal nuclear extraction and amplification according to claim 1, wherein the number of the nucleic acid detection areas (2) is one or more, and the nucleic acid detection areas (2) are pre-embedded with a reaction reagent in the form of a dry powder, a freeze-dried ball or a liquid reaction system.
4. The integrated centrifugal nuclear lifting and amplifying system according to claim 1, wherein the cartridge (10) further comprises a waste liquid zone (10-1) and a liquid injection zone (10-2); the liquid injection region (10-2) is communicated with the nucleic acid treatment region (1) and the nucleic acid treatment region (1) is communicated with the waste liquid region (10-1) through a channel, and a plugged wax valve (3) is arranged in the channel; the number of the liquid injection areas (10-2) is one or more, the liquid injection areas (10-2) are pre-embedded with nuclear extraction reaction reagents, and the nuclear extraction reaction reagents are in the form of dry powder, freeze-dried balls or liquid reaction systems.
5. The integrated system for centrifugal nuclear extraction and amplification according to claim 1, wherein said cartridge (10) employs a magnetic bead method or a centrifugal column method for nucleic acid extraction and purification; the centrifugal type nuclear extraction and amplification integrated instrument (20) is provided with a magnetic bead adsorption mechanism aligned to the magnetic beads of the nucleic acid treatment area (1); and a centrifugal column is added in the middle of the nucleic acid treatment area (1) in the way of extracting and purifying nucleic acid of the centrifugal column.
6. The integrated system for centrifugal nuclear lifting and amplification according to claim 1, wherein the cartridge (10) is fixed above the cartridge supporting mechanism (4), the cartridge (4) carries at least two cartridges (10), and the two cartridges (10) are arranged in an axisymmetric or centrosymmetric manner;
The temperature control reaction mechanism (6) is located above the cartridge supporting mechanism (4), the nucleic acid detection area (2) of the cartridge (10) is in close contact with the temperature control reaction mechanism (6), and the optical detection mechanism (7) is fixed on the outer side of the centrifugal motion driving device (5).
7. The integrated system for centrifugal nuclear lifting and amplification according to claim 1, wherein the cartridge supporting mechanism (4) performs centrifugal rotation movement at a rotation speed set by the circuit control system, and each nucleic acid detection area (2) of the cartridge (10) sequentially performs optical signal detection through the optical detection mechanism (7).
8. A detection method for a centrifugal type nuclear lifting amplification integrated system according to any one of claims 1 to 7, which is characterized by comprising a fluorescence detection method and a chromogenic detection method,
In the fluorescence detection method, the optical detection mechanism (7) is connected with a detection light path through an optical fiber (7-1), the optical fiber (7-1) is fixedly aligned with one circle of a nucleic acid detection area (2), when the nucleic acid detection area (2) rotates to pass through the path of the optical fiber (7-1), an excitation light source of the detection light path emits light to enter the nucleic acid detection area (2) to be injected into a detection device, and the detection device is converted into an electric signal to be transmitted to a circuit control system;
In the color development detection method, an excitation light source and a detection device are respectively arranged on the upper side and the lower side or the inner side and the outer side of the nucleic acid detection area (2), light emitted by the excitation light source is injected into the nucleic acid detection area (2), and light absorbed by a color development system is injected into the detection device on the other side of the excitation light source.
9. The detection method according to claim 8, wherein in the fluorescence detection method, the excitation light source and the detection device are respectively disposed in different directions from the circumference of the nucleic acid detection region (2), the excitation light is obliquely incident to the nucleic acid detection region (2), and the emitted light is incident to the detection device disposed on the other side through a filter;
In the color development detection method, the excitation light source and the detection device are provided with angles, light emitted by the excitation light source enters the nucleic acid detection area (2), and light absorbed by a color development system enters the detection device;
In the fluorescence detection method, the optical detection mechanism (7) performs detection of at least one optical channel.
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| CN113388513A (en) * | 2021-06-28 | 2021-09-14 | 清华大学深圳国际研究生院 | Micro-fluidic chip for nucleic acid extraction, amplification and detection |
| CN113604328B (en) * | 2021-08-09 | 2024-04-09 | 圣湘生物科技股份有限公司 | Amplification detection device and amplification detection method |
| CN116218631A (en) * | 2021-12-02 | 2023-06-06 | 上海微创惟微诊断技术有限公司 | Nucleic acid extraction device and method based on closed microfluidic chip |
| CN113913289B (en) * | 2021-12-07 | 2022-03-22 | 北京芯源视界生物科技有限公司 | Detection kit and detection method for pathogenic nucleic acid under airtight condition |
| CN114634869A (en) * | 2022-03-21 | 2022-06-17 | 诺美纳瑞(广州)医疗技术有限公司 | PCR detection system |
| CN115044461A (en) * | 2022-05-07 | 2022-09-13 | 贝迪嘉睿基因科技(苏州)有限公司 | A nucleic acid extraction and amplification cartridge based on magnetic beads and valve-controlled pipetting |
| CN116004357A (en) * | 2023-01-03 | 2023-04-25 | 杭州绿洁科技股份有限公司 | Microfluidic chip and centrifugal device for nucleic acid extraction and nucleic acid amplification detection |
| CN117264750B (en) * | 2023-11-23 | 2024-01-30 | 中国科学院空天信息创新研究院 | Detection cartridge and method of use thereof |
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