A device, system and method for real-time fluorescence quantitative nucleic acid amplification detection

Abstract

A device, system and method for real-time fluorescence quantitative nucleic acid amplification detection, the device includes a microfluidic unit, a sampling unit, a temperature control unit and a driving unit, the microfluidic unit is provided with one or more parallel annular flow channels, the annular flow channel has a liquid inlet and a liquid outlet, the sampling unit is arranged at the liquid inlet of the annular flow channel, and is used to inject samples into the annular flow channel, the temperature control unit is arranged in coordination with the microfluidic unit, the driving unit is arranged in coordination with the microfluidic unit, and is used to drive the movement of the microfluid in the annular flow channel, at least one section of the annular flow channel is made of transparent material for fluorescence detection, wherein the temperature control unit is configured so that different flow sections of the annular flow channel have different predetermined temperatures, and the microfluid realizes variable temperature operation by moving flow sections with different temperatures in the annular flow channel. The present invention simplifies nucleic acid amplification and detection operations, has flexible control, reduces detection time and material costs, and improves detection efficiency.

Description

Technical Field

[0001] The present invention relates to the technical field of nucleic acid amplification detection, and in particular to a device, system and method for real-time fluorescence quantitative nucleic acid amplification detection. Background Art

[0002] Polymerase chain reaction (PCR) is a widely used technique in molecular biology. It uses DNA polymerase reaction to specifically amplify a DNA fragment by simulating the DNA replication conditions in organisms. PCR technology is essential for gene sequencing, molecular cloning and gene mutation analysis in human diagnosis. The PCR reaction process usually involves three steps: denaturation, annealing and extension. These steps require the reaction system to be operated with variable temperature and temperature control. PCR instruments or thermal cyclers are used for amplification and detection of nucleic acid samples by adjusting the temperature in the cycle program. According to the scale, complexity and application requirements, the mechanical technology of PCR instruments has been continuously improved in the decades since the advent of PCR instruments; the safety of the equipment, the reliability of the heat block and the overall design specifications have been continuously updated and developed, and a variety of PCR equipment has emerged. For example, quantitative PCR (qPCR) provides quantitative data based on reference standards; digital PCR (dPCR) provides high-sensitivity quantitative analysis without standards; digital droplet PCR (ddPCR) uses a large number of sample partitions to generate high-throughput data. Among them, quantitative PCR equipment combined with microfluidic technology can achieve rapid nucleic acid amplification and high-throughput detection, and is currently at the forefront of PCR equipment development.

[0003] A microfluidic chip is a set of microchannels that are etched or molded onto a specific material (glass, silicon, or polymers such as polydimethylsiloxane). The microchannels that form a microfluidic chip are connected together to achieve the desired functions such as mixing, pumping, sorting, or controlling the biochemical environment. In the biomedical field, the vision of laboratories on a chip can be realized by integrating multiple medical tests on a single chip through microfluidic technology. In addition, the application of microfluidic technology can reduce the consumption of expensive reagents, simplify complex workflows, and improve its inherent clinical value.

[0004] Quantitative PCR (qPCR) has excellent detection threshold, signal analysis response and dynamic range. However, complex temperature control procedures and a single injection method are still the core issues that restrict the development and updating of qPCR devices. Most commercially available real-time qPCR platforms still require frequent temperature control operations, which increases the time consumed for nucleic acid amplification and detection and reduces reaction efficiency. During the operation of traditional qPCR equipment, multiple samples must be batch reacted, uniformly tested, and ended at the same time, which makes it difficult for traditional qPCR equipment to achieve flexible arrangements for single or different batches of samples, and thus cannot be a true high-throughput test. Summary of the invention

[0005] The main purpose of the present invention is to provide a device, system and method for real-time fluorescent quantitative nucleic acid amplification detection, to optimize nucleic acid amplification detection, especially its temperature control operation, to achieve rapid and flexible control of nucleic acid amplification and detection, to simplify the operation while reducing time and material costs, and to improve detection efficiency.

[0006] To achieve the above object, the present invention adopts the following technical solutions:

[0007] A device for real-time fluorescence quantitative nucleic acid amplification detection, comprising a microfluidic unit, an injection unit, a temperature control unit and a drive unit, wherein the microfluidic unit is provided with one or more parallel annular flow channels, the annular flow channel having a liquid inlet and a liquid outlet, the injection unit is arranged at the liquid inlet of the annular flow channel, and is used to inject samples into the annular flow channel, the temperature control unit is arranged in coordination with the microfluidic unit, and is used to perform segmented temperature control on the annular flow channel, the drive unit is arranged in coordination with the microfluidic unit, and is used to drive the movement of microfluids in the annular flow channel, at least one section of the annular flow channel is made of transparent material to facilitate fluorescence detection, wherein the temperature control unit is configured so that different flow sections of the annular flow channel have different predetermined temperatures, and the microfluid achieves temperature-changing operation by moving flow sections of different temperatures in the annular flow channel.

[0008] Further:

[0009] The temperature control unit includes three temperature control plates corresponding to the three different flow sections covering the annular flow channel. The segmented temperature control is implemented by controlling each temperature control plate, and different temperatures are set corresponding to different requirements of nucleic acid denaturation, annealing and extension in the microfluid. A fluorescence detection groove corresponding to the transparent section of the annular flow channel is arranged on the temperature control plate or between the temperature control plates. Preferably, the transparent section and the fluorescence detection groove are arranged at the starting point of the annular flow channel to detect the fluorescence value of the microfluid after each flow cycle.

[0010] Each temperature control plate is fan-shaped, and the three temperature control plates are formed into an annular structure corresponding to the annular flow channel.

[0011] The injection unit includes a microfluid generator and an injection pump. The microfluid generator is connected to the liquid inlet of the annular flow channel through the injection pump. The PCR reaction solution containing the sample and the magnetic fluid are input into the microfluid generator. In the microfluid generator, the magnetic fluid droplets are wrapped in the outer layer of the sample to form magnetic microfluid droplets, and the microfluid droplets are pumped into the annular flow channel by the injection pump. Preferably, it also includes a discharge pump arranged at the liquid outlet.

[0012] The driving unit is driven by air pressure, low-speed centrifugal force, mechanical force or magnetic force.

[0013] The driving unit comprises an air pump and an air hole arranged on the annular flow channel and connected to the air pump.

[0014] The driving unit includes a rotating shaft arranged at the circumferential center of the annular flow channel and a rotating arm connected to the rotating shaft and radially extending above the annular flow channel. The rotating arm is a magnetic rotating arm or a magnetic device is arranged on the rotating arm. The microfluid is magnetic after modification.

[0015] A microfluidic real-time fluorescent quantitative nucleic acid amplification detection system comprises the device for real-time fluorescent quantitative nucleic acid amplification detection and a detection unit for performing real-time fluorescent nucleic acid amplification detection on the sample in the annular flow channel, wherein the detection unit comprises a fluorescence detector. Preferably, the fluorescence detector comprises an excitation light source, an imaging system and a CCD camera.

[0016] A microfluidic real-time fluorescent quantitative nucleic acid amplification detection method, using the microfluidic real-time fluorescent quantitative nucleic acid amplification detection system to perform real-time fluorescent quantitative nucleic acid amplification detection, the method comprising the following steps:

[0017] heating different flow sections of the annular flow channel of the microfluidic unit to a desired temperature by the temperature control unit;

[0018] Inputting the microfluid to be tested into the annular flow channel through the sampling unit;

[0019] The driving unit drives the microfluid to flow along the annular flow channel in a unidirectional cycle according to a set number of cycles. Each cycle passes through flow sections with different temperatures, and the reaction liquid in the microfluid is programmed to change temperature to perform an amplification reaction, and real-time fluorescent quantitative nucleic acid amplification detection is performed through the detection unit.

[0020] Furthermore, the method further comprises: after the set cycle period ends, the microfluid is discharged from the annular flow channel, and a cleaning fluid is pumped into the annular flow channel through the injection unit to clean the flow channel.

[0021] The present invention has the following beneficial effects:

[0022] The present invention provides a device, system and method for real-time fluorescence quantitative nucleic acid amplification detection. The device of the present invention can optimize and simplify the temperature change program of the nucleic acid amplification detector, significantly reduce the time and energy loss caused by the temperature increase and decrease operation, and realize fast, high-throughput, and flexible control of nucleic acid amplification and detection operations. The operator only needs to add the sample to be tested into the annular flow channel of the microfluidic unit, and the device can automatically perform the amplification and detection steps. In the automated operation of the device, a temperature control unit, preferably a plurality of temperature control plates covering the annular flow channel, is used to change the temperature of different flow sections in the same annular flow channel, and the microfluid driven by the driving unit moves in the same annular flow channel, preferably at a uniform speed, and passes through flow channel sections with different temperatures to realize program temperature change operation. The microfluid can realize multiple program temperature change cycles by unidirectional circulation in the same annular flow channel, and the fluorescence value of the microfluid after each cycle can be detected.

[0023] By means of the above scheme, the present invention has at least the following advantages over the prior art:

[0024] 1. This device has a high degree of automation, which greatly simplifies the operation of personnel.

[0025] 2. When nucleic acid is amplified in this device, frequent temperature increases and decreases are not required.

[0026] 3. This device can achieve rapid and seamless temperature switching of nucleic acids in the reaction, thereby enhancing reaction efficiency.

[0027] 4. Microfluidics technology can be used to reduce the volume of the reaction system and to add multiple batches of samples to the same flow channel to achieve high-throughput detection.

[0028] 5. Samples can be injected at any time and tested in real time while the samples are reacting in the flow channel.

[0029] 6. The time required for amplification reaction per unit sample is at least 3 minutes shorter than that of traditional qPCR instruments.

[0030] In summary, the present invention can achieve short-time, high-throughput, and highly controllable nucleic acid amplification and detection. Compared with traditional qPCR instruments, the reaction time is greatly shortened, thereby quickly obtaining detection results of multiple targets. The microfluidic real-time fluorescent quantitative nucleic acid amplification detection system of the present invention can replace traditional non-microfluidic qPCR instruments, and is widely applicable to nucleic acid molecular diagnosis, and reduces time and material costs, simplifies manual operations, and thus improves detection efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 The figure is an overall schematic diagram of a real-time fluorescent quantitative nucleic acid amplification detection system according to an embodiment of the present invention.

[0032] Figure 2Schematic diagram of a temperature control plate in an embodiment of the present invention.

[0033] Figure 3 Schematic diagram of a microfluid generator and a sampling pump in one embodiment of the present invention.

[0034] Figure 4 Schematic diagram of the rotating shaft of a driving unit in an embodiment of the present invention. DETAILED DESCRIPTION

[0035] The following is a detailed description of the embodiments of the present invention. It should be emphasized that the following description is only exemplary and is not intended to limit the scope and application of the present invention.

[0036] It should be noted that when a component is referred to as being "fixed to" or "disposed on" another component, it can be directly on the other component or indirectly on the other component. When an element is referred to as being "connected to" another element, it can be directly connected to the other element or indirectly connected to the other element. In addition, connection can be used for both fixing and circuit / signal communication.

[0037] It should be understood that the terms "length", "width", "up", "down", "front", "back", "left", "right", "vertical", "horizontal", "top", "bottom", "inside", "outside", etc., indicating the orientation or position relationship, are based on the orientation or position relationship shown in the accompanying drawings, and are only for the convenience of describing the embodiments of the present invention and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore cannot be understood as a limitation on the present invention.

[0038] In addition, the terms "first" and "second" are used for descriptive purposes only and should not be understood 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 the features. In the description of the embodiments of the present invention, the meaning of "plurality" is two or more, unless otherwise clearly and specifically defined.

[0039] Figure 1The overall schematic diagram of a microfluidic real-time fluorescence quantitative nucleic acid amplification detection system according to an embodiment of the present invention. The embodiment of the present invention proposes a device for real-time fluorescence quantitative nucleic acid amplification detection, comprising a microfluidic unit, a sample injection unit, a temperature control unit 2 and a drive unit 5, wherein the microfluidic unit is provided with one or more parallel annular flow channels 1, the annular flow channel 1 having a liquid inlet and a liquid outlet, the sample injection unit is provided at the liquid inlet of the annular flow channel 1, and is used to inject samples into the annular flow channel 1, the temperature control unit 2 is provided in cooperation with the microfluidic unit, and is used to perform segmented temperature control on the annular flow channel 1, the drive unit 5 is provided in cooperation with the microfluidic unit, and is used to drive the movement of the microfluid in the annular flow channel 1, at least one section of the annular flow channel 1 is made of a transparent material, so as to perform fluorescence detection, wherein the temperature control unit 2 is configured so that different flow sections of the annular flow channel 1 have different predetermined temperatures, and the microfluid realizes temperature-changing operation by moving flow sections of different temperatures in the annular flow channel 1.

[0040] During the test, the driving unit 5 can drive the microfluid to move at a constant speed in the flow channel sections with different temperatures in the same annular flow channel 1 to achieve a programmed temperature change operation. The microfluid can achieve multiple programmed temperature change cycles by unidirectionally circulating in the same annular flow channel 1.

[0041] Figure 2 Schematic diagram of a temperature control plate in an embodiment of the present invention. In a preferred embodiment, the temperature control unit 2 includes three temperature control plates corresponding to the three different flow sections outside the annular flow channel 1. The segmented temperature control is implemented by controlling each temperature control plate, and different temperatures are set corresponding to different requirements of nucleic acid denaturation, annealing, and extension in the microfluid. A fluorescence detection slot 11 corresponding to the transparent section of the annular flow channel 1 is provided on the temperature control plate or between the temperature control plates. Preferably, the transparent section and the fluorescence detection slot 11 are provided at the starting point of the annular flow channel 1 to detect the fluorescence value of the microfluid after each flow cycle. Preferably, each temperature control plate is fan-shaped, and the three temperature control plates form an annular structure corresponding to the annular flow channel 1.

[0042] Figure 3 Schematic diagram of a microfluidic generator and an injection pump in an embodiment of the present invention. In a preferred embodiment, the injection unit includes a microfluidic generator 3 and an injection pump 4, the microfluidic generator 3 is connected to the liquid inlet of the annular flow channel 1 through the injection pump 4, the PCR reaction liquid containing the sample 7 and the magnetic fluid are input into the microfluidic generator 3, in the microfluidic generator 3, the magnetic fluid droplets 8 are wrapped in the outer layer of the sample 7 to form magnetic microfluidic droplets 6, and the microfluidic droplets 6 are pumped into the annular flow channel 1 through the injection pump 4, and preferably, a discharge pump 12 is also included arranged at the liquid outlet.

[0043] The PCR reaction solution may contain amplification template, enzyme, primer, magnesium ion and reaction buffer, such as Figure 3 As shown, the sample 7 to be amplified and tested can be added to the microfluid generator 3 through the discharge gun. The magnetic fluid droplets 8 are wrapped around the outer layer of the sample 7 through the microfluid generator 3 to form magnetic microfluid droplets 6. After the magnetic microfluid droplets 6 are pumped into the annular microfluid flow channel 1 by the injection pump 4, they start to move at a uniform speed in a cycle under the drive of the drive unit 5.

[0044] In different embodiments, the driving unit 5 may be driven by air pressure, low-speed centrifugal force, mechanical force, or magnetic force.

[0045] In one embodiment, the driving unit 5 includes an air pump and an air hole disposed on the annular flow channel 1 and connected to the air pump.

[0046] See also Figure 1 and Figure 4 In one embodiment, the driving unit 5 includes a rotating shaft disposed at the circumferential center of the annular flow channel 1 and a rotating arm connected to the rotating shaft and radially extending above the annular flow channel 1, the rotating arm is a magnetic rotating arm, or a magnetic device 9 is disposed on the rotating arm, and the microfluid is modified to be magnetic, so that it can be driven by the magnetic device 9 to move along the annular flow channel 1 as the rotating arm rotates. The rotating shaft is connected to a driving source, such as a motor.

[0047] See also Figure 1 The embodiment of the present invention also provides a microfluidic real-time fluorescent quantitative nucleic acid amplification detection system, comprising a device for real-time fluorescent quantitative nucleic acid amplification detection of any of the aforementioned embodiments and a detection unit for performing real-time fluorescent nucleic acid amplification detection on a sample in the annular flow channel 1 of the device, wherein the detection unit comprises a fluorescent detector 10, preferably, the fluorescent detector 10 comprises an excitation light source, an imaging system and a CCD camera. Nucleic acid detection is based on real-time fluorescent nucleic acid amplification detection technology (RT-qPCR).

[0048] The embodiment of the present invention further provides a microfluidic real-time fluorescent quantitative nucleic acid amplification detection method, using the microfluidic real-time fluorescent quantitative nucleic acid amplification detection system to perform real-time fluorescent quantitative nucleic acid amplification detection, the method comprising the following steps:

[0049] The temperature control unit 2 heats different flow sections of the annular flow channel 1 of the microfluidic unit to a desired temperature;

[0050] The microfluid to be tested is introduced into the annular flow channel 1 through the sample injection unit;

[0051] The driving unit 5 drives the microfluid to flow along the annular flow channel 1 in a unidirectional cycle according to the set number of cycles. Each cycle passes through flow sections with different temperatures, and the reaction liquid in the microfluid is programmed to change temperature to perform an amplification reaction, and real-time fluorescent quantitative nucleic acid amplification detection is performed through the detection unit.

[0052] Furthermore, the method further comprises: after the set cycle period ends, the microfluid is discharged from the annular flow channel 1, and a cleaning liquid is pumped into the annular flow channel 1 through the injection unit to clean the flow channel.

[0053] The specific embodiments of the present invention are further described below in conjunction with the accompanying drawings.

[0054] like Figure 1 The microfluidic real-time fluorescent quantitative nucleic acid amplification detection system of the illustrated embodiment includes a microfluidic unit, an injection unit (which also serves as a cleaning unit in this embodiment), a temperature control unit 2, a drive unit 5, and a detection unit. The microfluidic unit is provided with a closed-loop annular flow channel 1 for the microfluid to move in the flow channel and undergo an amplification reaction. The injection unit is provided at one end of the annular flow channel 1, and involves external units such as a microfluidic generator 3, an injection pump 4, and a liquid discharge pump 12. Three temperature-changing plates are provided in the temperature control unit 2, which cover the microfluidic annular flow channel 1 and are used for segmented temperature control of the flow channel; the drive unit 5 is matched with the microfluidic unit, and can utilize the microfluidic magnetic, weight or surface tension characteristics to drive the microfluid to move through the rotating shaft; the detection unit is a fluorescence detection system, which is placed outside the microfluidic unit and may include an excitation light source, a CCD camera, and the like.

[0055] Among them, the temperature control unit 2 changes the temperature of different flow sections in the same annular flow channel 1 by covering the microfluidic annular flow channel 1 with multiple temperature control plates. The microfluid moves at a uniform speed through the flow channel sections with different temperatures in the same annular flow channel 1 to achieve programmed temperature change operation. The microfluid moves in a unidirectional cycle in the same annular flow channel 1 to achieve multiple programmed temperature change cycles. A fluorescence detection groove 11 is set on the temperature control plate or between the temperature control plates corresponding to the starting point and transparent section of the annular flow channel 1 to detect the fluorescence value of the microfluid after each cycle. At least one section of the annular flow channel 1 is set to be a transparent material for fluorescence detection.

[0056] Among them, the PCR mixed reaction liquid with template, enzyme, primer and magnesium ion premixed externally is used as the main body, and is wrapped into a microfluid with special properties through the microfluid generator 3, and the special properties include magnetization, surface modification, special material wrapping, etc. After the microfluid is formed, it enters the annular microfluid flow channel through the injection pump 4, and performs unidirectional circulation in the annular flow channel 1 through the driving system. The driving force of the driving system on the microfluid is realized by magnetic force, air pressure, rotational force or mechanical force.

[0057] The flow channel is a closed annular closed flow channel, which can be set as a single or multiple flow channels in parallel. The annular flow channel 1 is provided with a single or parallel injection hole for the injection pump 4 to pump the microfluid into the flow channel.

[0058] Among them, according to the characteristics of the designed microfluid, it is not limited to a certain microfluid driving method, and various methods such as low-speed centrifugation, external pressure, magnetic drive, etc. can be used, and high-throughput fluid driving methods are preferred. For example, if air pressure is used to drive the microfluid, the driving unit 5 includes an air pump, and the annular flow channel 1 is provided with corresponding air holes.

[0059] Preferably, magnetic force is used to drive the microfluid, the driving unit 5 comprises a rotating shaft and a rotating arm with magnetism, and the microfluid droplet 6 is modified to have magnetism.

[0060] According to the reaction conditions required by the PCR reaction solution in the microfluid, the microfluid is driven to flow at a uniform speed in the annular flow channel 1. If the annular flow channel 1 is sampled in parallel or the same flow channel is sampled multiple times, the microfluid drive system will keep all microfluids in the annular flow channel 1 flowing at a uniform speed.

[0061] Among them, different temperatures can be set according to different requirements of nucleic acid denaturation, annealing and extension in the microfluidic system, and at least three temperature control plates with different temperatures are arranged to cover the annular microfluidic channel.

[0062] The fluorescence detection system may include but is not limited to an excitation light source, a filter, a CCD camera, and an imaging system. The fluorescence detection system may be placed on one side of the annular microfluidic flow channel to detect the microfluidic fluorescence signal in the flow channel in real time and transmit the data to generate an image.

[0063] The materials used for the microfluidic unit, the injection unit and the drive unit 5 are not limited to common medical plastics or glass products such as polyvinyl chloride (PVC), polyethylene (PE), polypropylene (PP), polystyrene (PS), polycarbonate (PC), and ABS.

[0064] Use Figure 1 The method for performing nucleic acid amplification detection by the microfluidic real-time fluorescent quantitative nucleic acid amplification detection device of the embodiment shown may include the following steps:

[0065] (1) using an injection gun to inject the premixed PCR reaction solution into the microfluidic generator from the outside; at the same time, the temperature control plate starts to heat to the required temperature;

[0066] (2) The microfluid generator generates a microfluid containing a PCR mixed reaction solution with special properties, which is pumped into the annular microfluid flow channel by an injection pump 4;

[0067] (3) Driven by the microfluidic drive system, the microfluid begins to circulate unidirectionally along the flow channel. As the microfluid passes through flow channels with different temperatures, the reaction liquid in the microfluid achieves programmed temperature change, and the amplification reaction begins, and at the same time, the fluorescence detection system starts to work;

[0068] (4) The microfluid circulates in the annular flow channel 1 according to the set number of cycles, and the software in the instrument starts to work, transmitting the detected signal to the processor and visualizing it.

[0069] (5) Through the timing or counting algorithm of the control software, the microfluid is discharged after the appropriate cycle period ends. After all the microfluid is discharged, the cleaning liquid can be pumped into the flow channel through the injection pump 4 to clean the flow channel.

[0070] Detection Examples

[0071] According to the sample 7 to be amplified and tested, a magnetic microfluid is made, and the embodiment uses a magnetic device 9 to drive the microfluid to operate. However, the present invention is not limited to this embodiment.

[0072] In this embodiment, a pre-mixed PCR reaction solution containing amplification template, enzyme, primer, magnesium ion and reaction buffer is prepared. Figure 3 As shown in the figure, the sample 7 to be amplified and tested is added to the microfluidic generator 3 through a discharge gun. The magnetic fluid droplets 8 are wrapped around the outer layer of the sample 7 by the microfluidic generator to form magnetic microfluidic droplets 6. The microfluidic droplets 6 are pumped into the microfluidic annular channel 1 by the injection pump 4 and begin to move in a circular motion at a uniform speed. According to the experimental requirements, the PCR reaction requires a programmed temperature change: 94°C denaturation---50°C annealing---72°C extension as an example. The three module sections A, B, and C in the temperature control plate are preheated to A: 94°C, B: 50°C, and C: 72°C. The microfluidic droplets 6 are pumped into section A of the temperature control plate to start, and at the same time the drive unit 5 starts working, driving the microfluidic droplets 6 in the channel to start moving at a uniform speed through the magnetic device 9, as shown in FIG. Figure 1As shown. Controlled by external control software, the drive unit 5 controls the microfluid droplet 6 to complete the three sections A, B, and C in sequence at a uniform speed at a set time, that is, to complete a cycle. After one cycle is completed, the fluorescence detector 10 is used to detect the fluorescence value of the microfluid 6 through the detection slot 11 and upload the data to the analysis software. Then the microfluid droplet 6 re-enters the A section and starts the second round of circulation. This cycle repeats, and after 40 cycles, the amplification and detection reactions are all completed, and the control software analyzes the fluorescence increment map. At this time, the discharge pump 12 will be started to discharge the microfluid that has finished the reaction. If all microfluids in the annular microchannel have been discharged, the injection pump 3 will be started to pump in the cleaning fluid to clean all the channels. This example can also be realized when there are multiple microfluids reacting in the channel, and new samples are added to the parallel channel for detection in real time, without affecting the microfluid currently being amplified and detected. If multiple microfluid drive shafts can be set, it is assumed that after the microfluid reaction on one of the microfluid drive shafts is completed and discharged, the magnetic device 9 is vacated. At this time, new microfluidic droplets 6 can be added to carry out new amplification reactions.

[0073] The background section of the present invention may contain background information about the problem or environment of the present invention, but does not necessarily describe the prior art. Therefore, the content included in the background section is not an admission by the applicant that the prior art is available.

[0074] The above content is a further detailed description of the present invention in combination with specific / preferred embodiments, and it cannot be determined that the specific implementation of the present invention is limited to these descriptions. For ordinary technicians in the technical field to which the present invention belongs, without departing from the concept of the present invention, it can also make several substitutions or modifications to these described embodiments, and these substitutions or modifications should be regarded as belonging to the protection scope of the present invention. In the description of this specification, the description of the reference terms "an embodiment", "some embodiments", "preferred embodiments", "examples", "specific examples", or "some examples" means that the specific features, structures, materials or characteristics described in combination with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representation of the above terms does not necessarily target the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner. In the absence of mutual contradiction, those skilled in the art can combine and combine the different embodiments or examples described in this specification and the features of different embodiments or examples. Although the embodiments of the present invention and their advantages have been described in detail, it should be understood that various changes, substitutions and modifications can be made herein without departing from the scope of protection of the patent application.

Claims

1. A device for real-time fluorescence quantitative nucleic acid amplification detection, characterized in that , including a microfluidic unit, a sampling unit, a temperature control unit and a driving unit, wherein the microfluidic unit is provided with one or more parallel annular flow channels, the annular flow channel is a closed annular closed flow channel, the annular flow channel has a liquid inlet and a liquid outlet, the sampling unit is arranged at the liquid inlet of the annular flow channel, and is used to inject samples into the annular flow channel, the temperature control unit is arranged in cooperation with the microfluidic unit, and is used to perform segmented temperature control on the annular flow channel, the driving unit is arranged in cooperation with the microfluidic unit, and is used to drive the movement of the microfluid in the annular flow channel, at least one section of the annular flow channel is made of transparent material for fluorescence detection, wherein the temperature control unit is configured so that different flow sections of the annular flow channel have different predetermined temperatures, and the microfluid moves through the flow sections with different temperatures in the annular flow channel The microfluid enters one of the annular flow channels and moves through a unidirectional circulation in the same annular flow channel. Each unidirectional circulation passes through flow segments with different temperatures to realize multiple programmed temperature change cycles. The temperature control unit includes three temperature control plates corresponding to the three different flow segments covered outside the annular flow channel. The segmented temperature control is implemented by controlling each temperature control plate. Different temperatures are set corresponding to different requirements of nucleic acid denaturation, annealing and extension in the microfluid. A fluorescence detection groove corresponding to the transparent segment of the annular flow channel is arranged on the temperature control plate or between the temperature control plates. The transparent segment and the fluorescence detection groove are arranged at the starting point of the annular flow channel to detect the fluorescence value of the microfluid after each flow cycle. The temperature control plate is fan-shaped, and the three temperature control plates form an annular structure corresponding to the annular flow channel.

2. The device for real-time fluorescence quantitative nucleic acid amplification detection according to claim 1, characterized in that The injection unit includes a microfluidic generator and an injection pump. The microfluidic generator is connected to the liquid inlet of the annular flow channel through the injection pump. The PCR reaction solution containing the sample and the magnetic fluid are input into the microfluidic generator. In the microfluidic generator, the magnetic fluid droplets are wrapped in the outer layer of the sample to form magnetic microfluid droplets, and the microfluid droplets are pumped into the annular flow channel through the injection pump.

3. The device for real-time fluorescence quantitative nucleic acid amplification detection according to claim 2, characterized in that , and also includes a liquid discharge pump arranged at the liquid outlet.

4. The device for real-time fluorescence quantitative nucleic acid amplification detection according to claim 1, characterized in that ,The driving unit is driven by air pressure, low-speed centrifugal force, mechanical force or magnetic force.

5. The device for real-time fluorescence quantitative nucleic acid amplification detection according to claim 4, characterized in that The driving unit includes an air pump and an air hole connected to the air pump and arranged on the annular flow channel.

6. The device for real-time fluorescence quantitative nucleic acid amplification detection according to claim 4, characterized in that The driving unit includes a rotating shaft arranged at the circumferential center of the annular flow channel and a rotating arm connected to the rotating shaft and radially extending above the annular flow channel. The rotating arm is a magnetic rotating arm or a magnetic device is arranged on the rotating arm. The microfluid is magnetic after modification.

7. A microfluidic real-time fluorescence quantitative nucleic acid amplification detection system, characterized in that , comprising a device for real-time fluorescent quantitative nucleic acid amplification detection as described in any one of claims 1 to 6 and a detection unit for performing real-time fluorescent nucleic acid amplification detection on the sample in the annular flow channel, wherein the detection unit comprises a fluorescence detector.

8. The microfluidic real-time fluorescent quantitative nucleic acid amplification detection system according to claim 7, characterized in that: The fluorescence detector includes an excitation light source, an imaging system and a CCD camera.

9. A microfluidic real-time fluorescence quantitative nucleic acid amplification detection method, characterized in that , using the microfluidic real-time fluorescent quantitative nucleic acid amplification detection system as claimed in claim 7 to perform real-time fluorescent quantitative nucleic acid amplification detection, the method comprising the following steps: heating different flow sections of the annular flow channel of the microfluidic unit to a desired temperature by the temperature control unit; The microfluid to be tested is introduced into the annular flow channel through the sample injection unit; The driving unit drives the microfluid to flow along the annular flow channel in a unidirectional cycle according to a set number of cycles. Each cycle passes through flow sections with different temperatures, and the reaction liquid in the microfluid is programmed to change temperature to perform an amplification reaction, and real-time fluorescent quantitative nucleic acid amplification detection is performed through the detection unit.

10. The microfluidic real-time fluorescent quantitative nucleic acid amplification detection method according to claim 9, characterized in that , also includes: after the set cycle period ends, the microfluid is discharged from the annular flow channel, and a cleaning liquid is pumped into the annular flow channel through the injection unit to clean the flow channel.

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