Method and system for nucleic acid extraction, purification and amplification detection

Abstract

The present invention discloses a method and system for nucleic acid extraction, purification and amplification detection. The system includes a nucleic acid extraction and purification device and a nucleic acid amplification detection device, and the nucleic acid extraction and purification device includes: an extraction mechanism, which includes a first component and a second component that fit closely together, at least a partial area of ​​the first component can reciprocate in the channel of the second component, and the bottom end of the second component is provided with a liquid pipeline connected to the channel; an interception structure, which is arranged at the bottom end of the second component; a liquid storage mechanism, including a plurality of accommodating chambers, each of which is provided with a protrusion on the bottom surface, and the protrusion can at least close the liquid pipeline. The nucleic acid amplification detection device includes: a microfluidic chip, a temperature control unit, an excitation unit and a detection unit. The present invention can realize nucleic acid extraction and purification, and use multiple physical cameras of a smart phone in combination with an LED light source to realize multi-color fluorescence detection, nucleic acid amplification and real-time analysis.

Description

Technical Field

[0001] The present invention relates to a method and system for nucleic acid extraction, purification and amplification detection, and in particular to a low-cost method and device for nucleic acid extraction and purification using a smart phone, as well as a method and device for nucleic acid amplification and fluorescence detection, belonging to the technical field of nucleic acid amplification detection. Background Art

[0002] Nucleic acid is a biological macromolecule compound formed by the polymerization of many nucleotide monomers. It is one of the most basic substances of life, including two major categories: deoxyribonucleic acid DNA and ribonucleic acid RNA. It is widely present in all animals, plants, microorganisms and organisms. Nucleic acid combines with protein to form nucleoprotein. Different nucleic acids have different chemical compositions and nucleotide arrangement sequences. Nucleic acid is the basic genetic material. All organisms, including animals, plants, bacteria and viruses, rely on nucleic acids for inheritance. Each species has its own specific nucleic acid sequence. By detecting the sequence of nucleic acids, it is possible to determine what type of organism it is. For example, if there is an infection, it can be determined by sequencing nucleic acids what kind of small organism is causing it, and drugs can be used to control and eliminate it. In the treatment of tumors, it is also possible to determine what drugs to use and what drugs are sensitive to based on the nucleic acid properties of the tumor so as to select better drugs.

[0003] Commonly used nucleic acid analysis methods include sequencing, amplification detection and hybridization detection. These analysis methods generally include steps such as nucleic acid extraction and purification, amplification and detection.

[0004] The extraction and purification technology of nucleic acids refers to separating them from biomacromolecules such as proteins, polysaccharides, and lipids, and ensuring the integrity of nucleic acid molecules. It is the basis for all aspects of molecular biology research and a key technology for life science research and application. In the early 1990s, the extraction and purification technology of nucleic acids was still a time-consuming and cumbersome technology. At the same time, toxic reagents such as phenol and chloroform were required for extraction. With the emergence of solid phase extraction technology and the effectiveness and practicality of commercial kits, the extraction of nucleic acids has been faster and more reliable, thus promoting the development of high-quality nucleic acid technology that can be extracted from biological samples such as whole blood, serum, saliva, urine, feces and tissues. Commercial solid phase extraction technology is mainly divided into two categories: centrifugal column purification and magnetic bead purification. The extraction and purification of nucleic acids using centrifugal column purification and magnetic bead purification usually include steps such as cell lysis, adsorption, washing and elution. The centrifugal column purification method is currently a widely used method for kit extraction. It uses the fact that nucleic acids can be selectively adsorbed to the surface of silanol membranes under certain ionic environments and separated from other biomolecules. During the purification process, the liquid added in each step will enter another centrifuge tube after centrifugation, which is completely separated from the column containing nucleic acid, so the washing is thorough and the purity stability is very high. However, the purification process requires repeated centrifugation, which is complicated to operate and difficult to automate. The magnetic bead purification method uses the high salt and low pH of the silanol surface of superparamagnetic silica nanoparticles to bind nucleic acids and elute at low salt pH to achieve nucleic acid purification; the superparamagnetism of magnetic beads is used to separate magnetic beads from the liquid phase through a magnetic field without centrifugation, which has the advantage of easy automation.

[0005] Nucleic acid amplification detection is a method of amplifying the nucleic acid sequence to be tested through the action of an enzyme and then detecting it, including thermal cycling amplification methods represented by polymerase chain reaction (PCR), and constant temperature amplification methods represented by RPA and LAMP. PCR uses the fact that DNA denatures into single strands at a high temperature of 95°C. At low temperatures (usually around 60°C), primers and single strands combine according to the principle of base complementary pairing. The temperature is then adjusted to the optimal reaction temperature of DNA polymerase (around 72°C), and DNA polymerase synthesizes complementary chains along the direction from phosphate to pentose (5'-3'). Fluorescence quantitative PCR is to add fluorescent groups to the reaction system and use the changes in fluorescent signals to monitor the changes in the amount of amplified products in each cycle of the PCR amplification reaction in real time.

[0006] The extraction, purification and amplification of nucleic acids usually need to be carried out in professional molecular laboratories, which have strict requirements on laboratory environment and professional operators. Therefore, it is quite difficult for PCR laboratories to penetrate into many biological-related industries. For example, the recent outbreak of novel coronavirus pneumonia and the recent outbreak of African swine fever have put forward new demands for nucleic acid analysis on site. The on-site rapid test (POCT) nucleic acid detection system developed in recent years has greatly simplified the operation process by adopting the "sample in, result out" nucleic acid analysis and detection mode, making on-site deployment and operation by non-molecular testing professionals possible. The fully sealed cartridge is used to automatically complete the entire process of nucleic acid analysis, such as cell / bacteria / virus lysis, nucleic acid purification, reverse transcription and real-time quantitative PCR amplification and detection, in the cartridge. Smartphone-based nucleic acid extraction, purification and amplification detection systems have become an affordable and rapid molecular diagnostic technology because they can provide a fast detection cycle, facilitate connection with remote experts and artificial intelligence systems through mobile communications, and provide accurate health information in resource-limited environments.

[0007] Therefore, how to combine nucleic acid extraction and purification, and amplification detection with nucleic acid real-time detection equipment based on real-time quantitative PCR nucleic acid amplification and fluorescence detection technology, and seek a low-cost method and system for nucleic acid extraction and purification and nucleic acid amplification detection using smartphones has become the direction that industry researchers have been working towards for a long time. Summary of the invention

[0008] The main purpose of the present invention is to provide a nucleic acid extraction and purification device and method in view of the above situation, so as to overcome the deficiencies in the prior art.

[0009] Another main object of the present invention is to provide a nucleic acid amplification detection device.

[0010] Another main purpose of the present invention is to provide a method and system for nucleic acid extraction, purification and amplification detection.

[0011] In order to achieve the above-mentioned invention object, the technical solution adopted by the present invention includes:

[0012] The embodiment of the present invention provides a nucleic acid extraction and purification device, which includes:

[0013] An extraction mechanism, comprising a first component and a second component that fit closely together, wherein the second component has a channel, wherein at least a partial area of ​​the first component can reciprocate in the channel, and wherein the channel allows a sample containing nucleic acid to be sucked into the channel, and wherein a liquid pipeline is disposed at the bottom end of the second component, and wherein the liquid pipeline is in communication with the channel;

[0014] an interception structure, which is disposed at the bottom end of the second component and intercepts the nucleic acid;

[0015] The liquid storage mechanism comprises a plurality of accommodating chambers, each of the accommodating chambers having a protruding portion on its bottom surface, and the protruding portion can at least close the liquid pipeline.

[0016] The embodiment of the present invention further provides a method for extracting and purifying nucleic acid, which is mainly implemented based on the aforementioned nucleic acid extraction and purification device, and the nucleic acid extraction and purification method includes:

[0017] (1) placing the end of the liquid pipeline in the first reagent of the first accommodating chamber, applying a pulling force to the first component, so that the sample containing the nucleic acid is sucked into the channel of the second component through the liquid pipeline and the intercepting structure, so that the nucleic acid is adsorbed on the inner wall of the second component;

[0018] (2) sealingly contacting the outlet end of the liquid pipeline with the surface of the protruding portion, thereby sealing the liquid pipeline and the channel, and then applying a thrust to the first component so that the liquid is pushed into the first accommodating chamber through the intercepting structure;

[0019] (3) Cleaning and eluting the nucleic acid in accordance with steps (1) and (2) to obtain a pure nucleic acid solution.

[0020] The embodiment of the present invention also provides a nucleic acid amplification detection device, which includes:

[0021] A microfluidic chip, at least for accommodating primers, probes and a PCR reaction system for detecting nucleic acids;

[0022] A temperature control unit, at least for regulating the operating temperature of the microfluidic chip;

[0023] an excitation unit, at least for providing excitation light, wherein the excitation light is irradiated on the PCR reaction product in the microfluidic chip to generate fluorescence; and

[0024] The detection unit is at least used to collect the generated fluorescence and perform analysis and detection.

[0025] Furthermore, the nucleic acid amplification detection device also includes a microprocessor unit, which is at least used to control the working states of the temperature control unit, the excitation unit, and the detection unit.

[0026] The embodiment of the present invention further provides a use of the aforementioned nucleic acid amplification detection device in the field of nucleic acid amplification detection. The method of using the nucleic acid amplification detection device to perform nucleic acid amplification detection includes:

[0027] The sample to be detected is placed in the sample loading hole, the PCR reaction system is placed in the reaction chamber, and the primers and probes for detecting nucleic acid are placed in the distribution chamber;

[0028] A temperature control unit is used to heat or cool the microfluidic chip to complete a thermal cycle, so that the temperature condition in the reaction chamber can perform a PCR reaction, and a temperature sensor is used to detect the temperature of the reaction chamber, and an automatic control algorithm of a microprocessor unit is used to perform temperature control so that the temperature is controlled within the PCR reaction range;

[0029] Using an excitation unit to irradiate the PCR reaction product in the microfluidic chip with excitation light to generate fluorescence;

[0030] The generated fluorescence is collected by a detection unit and analyzed and detected.

[0031] Accordingly, an embodiment of the present invention further provides a system for nucleic acid extraction, purification and amplification detection, which includes:

[0032] The aforementioned nucleic acid extraction and purification device; and

[0033] The aforementioned nucleic acid amplification detection device.

[0034] Accordingly, an embodiment of the present invention further provides a method for nucleic acid extraction, purification and amplification detection, which comprises:

[0035] Extract and purify nucleic acid according to the above method; and

[0036] Nucleic acid amplification and detection were performed according to the aforementioned methods.

[0037] The present invention proposes a method and device for nucleic acid extraction, purification and amplification detection based on a smartphone. Compared with the prior art, the present invention has the following advantages:

[0038] 1) The present invention provides a low-cost nucleic acid extraction method and device, in which the extracted liquid can pass through a liquid pipe with a larger inner diameter and a silanol membrane, while the pushed-back liquid can only pass through the silanol membrane. Compared with the situation where both the extracted liquid and the pushed-out liquid can only pass through the silanol membrane, the vacuum suction force formed by pulling the piston apart is much smaller than the pressure formed by pushing the piston forward, which is beneficial for relatively viscous liquids, such as tissue lysis products, to complete the adsorption and elution process through the silanol membrane; compared with the magnetic bead method that requires repeated movement of magnets to achieve solid-liquid separation, the device is relatively simple. This nucleic acid extraction method can be implemented manually or by using a smart phone and a single-chip microcomputer to control several stepper motors;

[0039] 2) The present invention uses a smartphone to control thermal cycling and fluorescence acquisition, and uses multiple physical cameras of the smartphone in combination with multiple LEDs with different central wavelengths to achieve multi-color fluorescence detection. Compared with the traditional method of switching different fluorescence channels through mechanical movement, the excitation light source of the multi-color fluorescence detection in the present invention controls the switch and intensity electronically, and the acquisition is independently performed by multiple physical cameras of the smartphone in combination with emission filters, which can achieve 4-6 channel fluorescence acquisition. By using a multi-pass emission filter, the fluorescence channel can be expanded to 12-18 channels. There are no mechanical moving parts, which reduces the system failure rate and improves portability. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0041] Figure 1a and Figure 1b It is a schematic structural diagram of a nucleic acid extraction and purification device in a typical embodiment of the present invention.

[0042] Figure 2a and Figure 2b It is a structural schematic diagram of a nucleic acid extraction device in a typical embodiment of the present invention when the first component is located at the bottom of the second component.

[0043] Figure 2c and Figure 2d It is a schematic structural diagram of a first component of a nucleic acid extraction device in a typical embodiment of the present invention when it is pulled upward.

[0044] Figure 2e and Figure 2f It is a schematic structural diagram of a first component of a nucleic acid extraction device in a typical embodiment of the present invention when being pushed downward.

[0045] Figure 3 It is a schematic structural diagram of a nucleic acid amplification detection device in a typical embodiment of the present invention.

[0046] Figure 4a and Figure 4b It is a schematic diagram of the principle of a nucleic acid amplification detection method in a typical embodiment of the present invention.

[0047] Figure 5 It is a control block diagram for constant temperature control and light source driving of a nucleic acid amplification detection device in a typical implementation scheme of the present invention.

[0048] Figure 6aIt is an assembly diagram of a microfluidic chip and a thermoelectric semiconductor refrigeration device in a nucleic acid amplification detection device in a typical implementation scheme of the present invention.

[0049] Figure 6b It is a schematic diagram of the structure of the microfluidic chip.

[0050] Figure 7a and Figure 7b It is a graph showing the change of fluorescence over time after software analysis using 100 μL of pseudovirus (about 10e4 pseudovirus) or negative control (water) as a sample for amplification in Example 3 of the present invention.

[0051] Description of reference numerals: 10-piston, 20-empty cylinder, 21-channel, 30-liquid pipeline, 40-silicon hydroxyl membrane, 50-liquid storage mechanism, 51-first liquid storage tank, 51a-heating sleeve, 52-second liquid storage tank, 53-third liquid storage tank, 54-fourth liquid storage tank, 55-protrusion, 56-connecting motor shaft; 100-microfluidic chip, 100a-chip inlet and outlet chamber assembly, 110-reaction chamber, 120-distribution chamber, 130-sample addition hole, 140-overflow hole, 200-excitation unit, 210-four-color LED light source, 211-constant current drive, 220-three-bandpass filter, 221-bandpass filter, 222-multi-pass color filter, 300-temperature control unit, 310-Peltier, 311-high-power bidirectional H-bridge driver, 320-thermocouple sensor, 321-thermocouple temperature converter, 330-thermal conductive pad, 340-aluminum heat sink, 350-fan, 400-detection unit, 410-smartphone, 410a-smartphone holder, 411-built-in camera, 412-switching power supply module, 413-Bluetooth module, 414-low voltage drop linear regulator, 420-bandpass color filter, 422-plano-convex lens, 500-microprocessor unit, 501-self-made PCB control board, A-optical component, 1-equipment housing. DETAILED DESCRIPTION

[0052] With the rapid development of smartphones, more and more smartphones have multiple built-in cameras. Starting from version 9.0 of the Android system, each built-in physical camera can be independently controlled, and the resolution of built-in cameras is getting higher and higher. At the same time, the memory and processor of smartphones are powerful enough to be used for real-time image processing. Therefore, the built-in camera of a smartphone can be used to collect multi-channel fluorescence images, and the fluorescence intensity can be calculated through image processing algorithms.

[0053] In view of the shortcomings and defects of the prior art, the inventors of this case have proposed the technical solution of the present invention after long-term research and extensive practice, providing a low-cost nucleic acid extraction method and device, the core of which is to extract liquid from the liquid storage pool (binding liquid, cleaning liquid and elution liquid), and then push the liquid back to the liquid pool through the silicon hydroxyl membrane, and complete the adsorption, cleaning and elution process of nucleic acid in sequence. Its characteristics are that the extracted liquid can pass through the pipe with a larger inner diameter and the silicon hydroxyl membrane, while the pushed back liquid can only pass through the silicon hydroxyl membrane. Compared with the extraction and push out of liquid can only pass through the silicon hydroxyl membrane, the vacuum suction formed by pulling the piston is much smaller than the pressure formed by pushing the piston, which is conducive to relatively viscous liquids, such as tissue lysis products, to complete the adsorption and elution process through the silicon hydroxyl membrane; compared with the magnetic bead method that requires repeated movement of magnets to achieve solid-liquid separation, the device is relatively simple. This nucleic acid extraction method can be implemented manually, or it can be completed by a smart phone and a single-chip microcomputer to control several stepper motors.

[0054] The present invention uses a smart phone to control thermal cycling and fluorescence collection. The multiple physical cameras of the smart phone are combined with multiple LEDs with different central wavelengths to achieve multi-color fluorescence detection. Compared with the traditional method of switching different fluorescence channels by mechanical movement, the excitation light source of the multi-color fluorescence detection in the present invention controls the switch and intensity by electronic means, and the collection is independently carried out by multiple physical cameras of the smart phone in conjunction with emission filters, which can achieve 4-6 channel fluorescence collection. By using a multi-pass emission filter, the fluorescence channel can also be expanded to 12-18 channels. There are no mechanical moving parts, which reduces the system failure rate and improves portability.

[0055] The technical solution, its implementation process and principle will be further explained as follows.

[0056] The technical solution of the present invention will be explained in more detail below. However, it should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here.

[0057] One aspect of an embodiment of the present invention provides a nucleic acid extraction and purification device, comprising:

[0058] An extraction mechanism, comprising a first component and a second component that fit closely together, wherein the second component has a channel, wherein at least a partial area of ​​the first component can reciprocate in the channel, and wherein the channel allows a sample containing nucleic acid to be sucked into the channel, and wherein a liquid pipeline is disposed at the bottom end of the second component, and wherein the liquid pipeline is in communication with the channel;

[0059] an interception structure, which is disposed at the bottom end of the second component and intercepts the nucleic acid;

[0060] The liquid storage mechanism comprises a plurality of accommodating chambers, each of the accommodating chambers having a protruding portion on its bottom surface, and the protruding portion can at least close the liquid pipeline.

[0061] In some embodiments, the first component may be a piston member, but is not limited thereto.

[0062] In some embodiments, the second component may be a cylindrical structure having a hollow structure, but is not limited thereto.

[0063] In some embodiments, the nucleic acid extraction and purification device further includes a fixing member, and the intercepting structure is disposed at the bottom end of the second component through the fixing member.

[0064] Furthermore, the fixing member may be a fixing pressure ring, but is not limited thereto.

[0065] In some embodiments, the interception structure includes a porous membrane that can adsorb and elute nucleic acids under different conditions, for example, a silanol membrane with a pore size of 0.1 μm-10 μm (such as Whatman GF / F, etc.), but is not limited thereto.

[0066] In some embodiments, the protrusions are arranged in a one-to-one correspondence with the outlet ends of the liquid pipelines.

[0067] Furthermore, the size of the protrusion is not less than the diameter of the liquid pipeline.

[0068] In some embodiments, the liquid storage mechanism comprises:

[0069] A first containing chamber storing a first reagent, wherein the first reagent includes a sample containing nucleic acid;

[0070] A second containing chamber storing a second reagent, wherein the second reagent includes a cleaning solution;

[0071] A third containing chamber is provided for storing a third reagent, wherein the third reagent includes an eluent.

[0072] Furthermore, the number of the second accommodating cavities may be one or more than two, and the number is not limited.

[0073] In some embodiments, the nucleic acid extraction and purification device further includes a heating mechanism, and the heating mechanism is disposed around the outside of the first accommodating cavity.

[0074] Further, the heating mechanism may be a heating ring, but is not limited thereto.

[0075] Furthermore, the nucleic acid extraction and purification device also includes a control mechanism, and the control mechanism is connected to an intelligent mobile terminal through a microprocessor unit.

[0076] Among them, in some more specific implementation cases, the nucleic acid extraction and purification device may specifically include: a syringe-like structure and a liquid storage mechanism, in which a sample containing nucleic acid (also referred to as a binding solution), a first cleaning solution, a second cleaning solution, and an elution solution are stored in sequence. The liquid can be added before the start of purification, or it can be pre-stored in the liquid storage mechanism. The liquid storage mechanism pre-stored with liquid is sealed with aluminum foil, which is punctured before the start of extraction or punctured one by one as needed during the extraction process.

[0077] Furthermore, the storage chamber (also called a liquid storage tank) for storing samples containing nucleic acids is surrounded by a structure in which a heating collar can be placed, so that the liquid can be heated as needed. The bottom of the storage chamber is also provided with a sealing protrusion, which is used to close the pipeline for sucking liquid when the liquid is injected. The syringe-like structure includes a piston, an empty barrel, a liquid pipeline, a silanol membrane (such as Whatman GF / F, etc.) and a fixed pressure ring. The vacuum suction force formed by pulling open the piston is much smaller than the pressure formed by pushing the piston, and the liquid pipeline is conducive to drawing viscous liquids, such as tissue lysis products, into the empty barrel.

[0078] Another aspect of the embodiments of the present invention provides a method for extracting and purifying nucleic acid, which is mainly implemented based on the aforementioned nucleic acid extraction and purification device, and the nucleic acid extraction and purification method includes:

[0079] (1) placing the end of the liquid pipeline in the first reagent of the first accommodating chamber, applying a pulling force to the first component, so that the sample containing the nucleic acid is sucked into the channel of the second component through the liquid pipeline and the intercepting structure, so that the nucleic acid is adsorbed on the inner wall of the second component;

[0080] (2) sealingly contacting the outlet end of the liquid pipeline with the surface of the protruding portion, thereby sealing the liquid pipeline and the channel, and then applying a thrust to the first component so that the liquid is pushed into the first accommodating chamber through the intercepting structure;

[0081] (3) Cleaning and eluting the nucleic acid in accordance with steps (1) and (2) to obtain a pure nucleic acid solution.

[0082] Furthermore, the nucleic acid extraction process includes: placing the front end of the extraction mechanism, that is, the end of the liquid pipeline, into the binding liquid reservoir, pulling out the piston, and the liquid can be sucked into the empty cylinder through the liquid pipeline with a larger inner diameter and the silicon hydroxyl membrane; rotating the empty cylinder 90 degrees and pressing it down to the protruding structure of the reservoir to seal the liquid channel; pushing the piston, the liquid can only be pushed back to the reservoir through the silicon hydroxyl membrane. Rotate the reservoir, and perform the same operation in the reservoirs of the first cleaning liquid, the second cleaning liquid, and the eluent in turn. The eluent reservoir can be used as an amplification reaction chamber. The eluent reservoir can also only store molecular biology grade water, use a syringe to extract water, push the piston to push the water through the silicon hydroxyl membrane, and transfer the eluted and purified nucleic acid to another reaction chamber for amplification reaction.

[0083] Furthermore, the nucleic acid extraction method can be implemented manually or by controlling several stepper motors through a smartphone and a single-chip microcomputer.

[0084] Another aspect of the embodiments of the present invention further provides a nucleic acid amplification detection device, comprising:

[0085] A microfluidic chip, at least for accommodating primers, probes and a PCR reaction system for detecting nucleic acids;

[0086] A temperature control unit, at least for regulating the operating temperature of the microfluidic chip;

[0087] an excitation unit, at least for providing excitation light, wherein the excitation light is irradiated on the PCR reaction product in the microfluidic chip to generate fluorescence; and

[0088] The detection unit is at least used to collect the generated fluorescence and perform analysis and detection.

[0089] In some embodiments, the nucleic acid amplification detection device further includes a microprocessor unit, which is at least used to control the working states of the temperature control unit, the excitation unit, and the detection unit.

[0090] In some embodiments, the microfluidic chip includes interconnected reaction chambers, distribution chambers, at least microchannels for connecting the chambers, sample loading holes, and overflow holes. The reaction chamber is at least used to store the PCR reaction system, and the distribution chamber is at least used to store primers and probes for detecting nucleic acids.

[0091] In some embodiments, the excitation unit includes an LED light source and a filter component. The LED light source is disposed above the filter component, and the filter component is at least used to generate excitation light of a desired wavelength.

[0092] Furthermore, the LED light source includes a four-color LED light source, but is not limited thereto.

[0093] Furthermore, the filter assembly includes a single-pass filter, a two-pass filter or a multi-pass filter, and is particularly preferably a three-band pass filter, but is not limited thereto.

[0094] Furthermore, the LED light source is connected to the microprocessor unit via an LED driving component (eg, a constant current LED driver may be used).

[0095] In some embodiments, the detection unit includes an intelligent mobile terminal having one or more fluorescent image acquisition components and one or more filter components. The fluorescent image acquisition components are arranged one by one above the filter components. A plano-convex lens is arranged between the fluorescent image acquisition component and the filter component, which is at least used to converge the fluorescence onto the fluorescent image acquisition component.

[0096] Furthermore, the intelligent mobile terminal is connected to the microprocessor unit via a Bluetooth module.

[0097] Furthermore, the detection system also includes a switching power supply module or a large-capacity charging power supply, preferably a switching power supply module, which is at least used to provide power for the smart mobile terminal and the nucleic acid amplification detection device.

[0098] Furthermore, the switching power supply module or the charging power supply is electrically connected to the microprocessor unit, preferably using a low voltage drop linear regulator.

[0099] In some embodiments, the intelligent mobile terminal includes a smart phone, a tablet computer, and a microcomputer motherboard module based on an ARM processor, but is not limited thereto.

[0100] Furthermore, the fluorescent image acquisition component includes a camera component, preferably a built-in camera.

[0101] In some embodiments, the temperature control unit includes a heating and cooling component and a temperature sensing component, and the heating and cooling component is used at least for rapid heating or rapid cooling, and makes the temperature in the reaction chamber change rapidly to achieve the rapid thermal cycle required for the PCR reaction.

[0102] Furthermore, the heating and cooling assembly includes a thermoelectric semiconductor cooling device, but is not limited thereto.

[0103] Furthermore, the temperature sensing component includes a thermocouple sensor, but is not limited thereto.

[0104] In some embodiments, the heating and cooling component is connected to the microprocessor unit via a high-power bidirectional H-bridge driving component, and the temperature sensing component is connected to the microprocessor unit via a thermocouple temperature measuring converter.

[0105] Furthermore, the microprocessor unit includes a single chip microcomputer, but is not limited thereto.

[0106] Among them, in some more specific implementation cases, the nucleic acid amplification detection device based on real-time quantitative PCR technology can specifically include:

[0107] (1) A smartphone with multiple built-in cameras, where each physical camera can independently control the image acquisition;

[0108] (2) According to the original purpose of the mobile phone camera (close-up, telephoto, etc.), the lens is fixed in front of the camera as required so that the microfluidic chip about 10 cm in front of the camera can be clearly imaged;

[0109] (3) Fixing a certain bandwidth, such as an emission filter with a full width at half maximum (FWHM) of 1 nm to 100 nm, in front of the camera or the camera and lens; the filter can be single-pass, double-pass or multi-pass, for the specified fluorescence signal collection;

[0110] (4) One or more groups of LED light sources with different central wavelengths, such as 300nm-800nm, and a circuit board for independently switching and controlling the brightness of each LED light source;

[0111] (5) An excitation filter with a certain bandwidth, such as a full width at half maximum (FWHM) of 1 nm to 100 nm, fixed in front of the LED light source to match the central wavelength of the light source; or multiple LED light sources share a multi-pass filter that matches their respective central wavelengths;

[0112] (6) Peltier (thermoelectric semiconductor cooler) has very small thermal inertia and can cool and heat very quickly. Combined with real-time temperature detection, the microcontroller drives the Peltier through a high-power H-bridge driver component, and the temperature automatic control algorithm realizes the rapid temperature thermal cycle required for PCR amplification reaction;

[0113] (7) A disposable microfluidic chip that can perform PCR reactions in the reaction chamber of the chip;

[0114] (8) Temperature probe, such as a thermocouple or thermistor temperature detection sensor;

[0115] (9) Auxiliary hardware, such as chip in and out motors, position sensors, etc.;

[0116] (10) Microprocessor unit, used to collect temperature and control auxiliary hardware, etc. The smartphone and the microcontroller communicate via Bluetooth;

[0117] (11) Smartphone applications, including communication with microcontrollers, image acquisition and analysis, user interface and central database / expert system communication modules;

[0118] (12) The system can be powered by a large-capacity switching power supply module or a rechargeable lithium battery;

[0119] (13) A portable chassis that firmly holds all components together to form a compact device; forming an enclosed space for collecting fluorescence signals.

[0120] Another aspect of the embodiments of the present invention further provides use of the aforementioned nucleic acid amplification detection device in the field of nucleic acid amplification detection.

[0121] Another aspect of the embodiments of the present invention further provides a method for performing nucleic acid amplification detection using the nucleic acid amplification detection device, comprising:

[0122] The sample to be detected is placed in the sample loading hole, the PCR reaction system is placed in the reaction chamber, and the primers and probes for detecting nucleic acid are placed in the distribution chamber;

[0123] A temperature control unit is used to heat or cool the microfluidic chip to complete a thermal cycle, so that the temperature condition in the reaction chamber can perform a PCR reaction, and a temperature sensor is used to detect the temperature of the reaction chamber, and an automatic control algorithm of a microprocessor unit is used to perform temperature control so that the temperature is controlled within the PCR reaction range;

[0124] Using an excitation unit to irradiate the PCR reaction product in the microfluidic chip with excitation light to generate fluorescence;

[0125] The generated fluorescence is collected by a detection unit and analyzed and detected.

[0126] In some more specific embodiments, the detection method comprises:

[0127] The sample to be tested is placed in the sample addition hole, and the PCR reaction system is placed in the reaction chamber;

[0128] A temperature control unit is used to perform multi-cycle rapid thermal cycling on the microfluidic chip with three stages as one cycle, namely high-temperature denaturation (usually around 95°C), low-temperature annealing (usually around 60°C), and suitable temperature amplification (usually around 72°C), so that the temperature in the reaction chamber changes rapidly to perform PCR reaction, and a temperature sensor is used to detect the temperature of the reaction chamber in real time. The single-chip microcomputer drives the Peltier through an automatic control algorithm using a high-power bidirectional H-bridge to achieve automatic temperature control.

[0129] Using an excitation unit to irradiate the PCR reaction product in the microfluidic chip with excitation light to generate fluorescence;

[0130] The generated fluorescence is collected by a detection unit and analyzed and detected.

[0131] Furthermore, the method includes: real-time monitoring of the fluorescence intensity of the collected PCR reaction products, and analyzing the results through an amplification curve showing the change of the fluorescence intensity over time.

[0132] In summary, the present invention provides an integrated nucleic acid detection method, primers and fluorescent probes, a microfluidic chip and a detection system for realizing real-time fluorescence detection of PCR amplification based on microfluidic technology.

[0133] More specifically, the inventor of this case designed a handheld nucleic acid real-time detection prototype device based on real-time quantitative PCR nucleic acid amplification and fluorescence detection technology. The device is equipped with a small thermal cycler, LED four-color power light source and fixed multi-channel filter components, as well as a multi-channel fluorescence real-time detection system based on a multi-camera smartphone. First, the device improves portability by eliminating mechanical moving parts, and uses a fixed multi-channel filter to increase the speed of multi-color fluorescence detection; a multi-color fluorescence detection method is developed, which synchronizes the fluorescence detection channels of a multi-color LED light source with adjustable brightness and switchable colors with multiple independently controllable rear cameras in a smartphone to achieve a multi-color fluorescence sequential detection method. Secondly, in order to achieve PCR rapid temperature thermal cycling, a single-chip microcomputer is used to control the Peltier (thermoelectric semiconductor cooler), and a thermal cycler with rapid heating and cooling is realized through an automatic temperature control algorithm. Finally, an Android APP software is developed on the smartphone to control multiple built-in cameras of the smartphone to obtain fluorescence images and perform real-time image processing, and also controls the self-made PCB control board through Bluetooth communication technology to achieve rapid thermal cycling, light source brightness adjustment, color switching and nucleic acid extraction. Given the advantages of highly integrated functions and high mobility, the smartphone-based device of the inventors of this case is very suitable for rapid nucleic acid amplification and real-time quantitative analysis in non-laboratory environments with limited resources.

[0134] The present invention utilizes multiple physical cameras of a smartphone in combination with multiple LEDs with different central wavelengths to achieve multi-color fluorescence detection. Compared with the traditional method of switching different fluorescence channels through mechanical movement, the excitation light source of the multi-color fluorescence detection in the present invention controls the switch and intensity electronically, and the collection is independently performed by multiple physical cameras of the smartphone in conjunction with emission filters, which can achieve 4-6 channel fluorescence collection. By using a multi-pass emission filter, the fluorescence channels can be expanded to 12-18 channels. There are no mechanical moving parts, which reduces the system failure rate and improves portability.

[0135] The following will describe the technical solutions in the embodiments of the present invention in detail in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0136] A low-cost nucleic acid extraction method and device provided in a specific embodiment of the present invention is as follows Figure 1a-1b As shown, the nucleic acid extraction and purification device includes an extraction mechanism, an interception mechanism and a liquid storage mechanism.

[0137] The extraction mechanism includes a first component and a second component that fit closely together, the first component can be a piston 10, the second component can be a cylindrical structure with a hollow structure, that is, an empty cylinder 20, the empty cylinder 20 has a channel 21, the piston 10 can reciprocate (i.e., suction and injection) in the channel 21, and the channel 21 allows a sample containing nucleic acid to be sucked into the channel, and a liquid pipeline 30 is provided at the bottom end of the empty cylinder 20, and the liquid pipeline 30 is connected to the channel 21. The interception mechanism can be a silicone hydroxyl membrane 40, and the fixed pressure ring 41 fixes the silicone hydroxyl membrane 40 to the bottom end of the empty cylinder 20. The protrusion 55 is arranged one-to-one with the outlet end of the liquid pipeline 30, and the size of the protrusion 55 is not less than the diameter of the liquid pipeline 30 to ensure a sealed fit.

[0138] The liquid storage mechanism 50 includes a first accommodating chamber (i.e., a first liquid storage tank 51) for storing a sample containing nucleic acid, a second accommodating chamber (i.e., a second liquid storage tank 52) for storing a cleaning liquid, a third accommodating chamber (i.e., a third liquid storage tank 53), and a third accommodating chamber (i.e., a fourth liquid storage tank 54) for storing an eluent.

[0139] The nucleic acid extraction and purification device may further include a heating mechanism, which is disposed around the outside of the first liquid storage tank 51. The heating mechanism may be a heating sleeve 51a. The nucleic acid extraction and purification device may also include a control mechanism, which is connected to a smart mobile terminal via a microprocessor unit.

[0140] The nucleic acid extraction device includes a structure similar to a syringe and several liquid reservoirs, wherein the first liquid reservoir 51, the second liquid reservoir 52, the third liquid reservoir 53 and the fourth liquid reservoir 54 store binding liquid (a sample containing nucleic acid), a first cleaning liquid, a second cleaning liquid and an eluting liquid in sequence. The liquid can be added before the purification starts, or it can be pre-stored in the liquid reservoir. The liquid reservoir pre-stored with liquid is sealed with aluminum foil, which is punctured before the extraction starts or punctured one by one as required during the extraction process. The liquid reservoir storing the binding liquid also has a structure in which a heating collar can be placed, and the liquid can be heated as required. The bottom of the liquid reservoir also has a sealing protrusion (protrusion) for closing the pipeline for sucking liquid when pushing the liquid. The structure similar to the syringe includes a piston, an empty cylinder, a liquid suction pipeline, a silanol membrane (such as Whatman GF / F, etc.) and a fixed pressure ring. The vacuum suction force formed by pulling the piston apart is much smaller than the pressure formed by pushing the piston, and the liquid suction pipeline is conducive to sucking viscous liquids, such as tissue lysis products, into the empty cylinder 20.

[0141] A method for extracting and purifying nucleic acid is provided in a specific embodiment of the present invention. The nucleic acid extraction process is as follows: Figure 2a-2f As shown, the front end of the extraction mechanism, i.e., the end of the liquid pipeline 30, is placed in a first liquid storage tank 51 ( Figure 2a-2b ), pull out the piston 10, and the liquid can be sucked into the empty cylinder 20 through the liquid pipeline 30 with a larger inner diameter and the silanol membrane 40 ( Figure 2c-2d ); rotate the empty cylinder 90 degrees and press down to the protruding structure (i.e., the protrusion 55) of the first liquid reservoir 51 to seal the liquid channel; push the piston 10, and the liquid can only be pushed back to the first liquid reservoir 51 through the silanol membrane 40 ( Figure 2e-2f ). The liquid storage mechanism 50 is rotated to perform the same operation in the second liquid storage tank 52, the third liquid storage tank 53 and the fourth liquid storage tank 54 of the first cleaning liquid, the second cleaning liquid and the eluent in turn. The fourth liquid storage tank 54 containing the eluent can be used as an amplification reaction chamber. The fourth liquid storage tank 54 containing the eluent can also store only molecular biology grade water, and the water is extracted by a syringe, and the piston 10 is pushed to push the water through the silanol membrane 40, and the eluted and purified nucleic acid is transferred to another reaction chamber for amplification reaction.

[0142] The nucleic acid extraction method can be implemented manually or by controlling several stepper motors in cooperation with a smart phone and a single-chip microcomputer, wherein the stepper electrodes are arranged on the motor shaft 56 .

[0143] See also Figure 5 As shown, a nucleic acid amplification detection device provided in a specific embodiment of the present invention includes a microfluidic chip 100, an excitation unit 200, a temperature control unit 300, a detection unit 400 and a microprocessor unit 500.

[0144] The microfluidic chip 100 includes a sample addition hole 130, an overflow hole 140, a distribution chamber 120, and a reaction chamber 110. Figure 6b The reaction chamber 110 is used to store at least the PCR reaction system, and the distribution chamber 120 is used to store at least the primers and probes used to detect nucleic acids.

[0145] The excitation unit 200 includes a four-color LED light source 210 and a three-bandpass filter 220, and the four-color LED light source 210 is disposed above the three-bandpass filter 220. The four-color LED light source 210 is connected to the microprocessor unit 500 through a constant current LED driver 211.

[0146] The detection unit 400 includes a smart phone 410 and three bandpass filters 420. The smart phone 410 has three built-in cameras 411. The built-in cameras 411 are arranged one by one above the bandpass filters 420. A plano-convex lens 422 is arranged between the built-in cameras 411 and the bandpass filters 420. The smart phone 410 is connected to the microprocessor unit 500 via a Bluetooth module 413. The nucleic acid amplification detection device also includes a switching power supply module 412 or a charging power supply, which is at least used to provide power for the smart phone 410. The switching power supply module 412 is electrically connected to the microprocessor unit 500 via a low voltage drop linear regulator 414.

[0147] The temperature control unit 300 includes a Peltier 310 and a thermocouple sensor 320. The Peltier 310 is used at least for heating and cooling, and makes the reaction chamber reach the temperature required for the PCR reaction. The Peltier 310 is connected to the microprocessor unit 500 through a high-power bidirectional H-bridge driver 311, and the thermocouple sensor 320 is connected to the microprocessor unit 500 through a thermocouple temperature measurement converter 321.

[0148] in, Figure 5 The color filters in the middle dotted area can be collectively referred to as the optical component A area. The constant current LED driver, thermocouple temperature measurement converter, switching power supply module, Bluetooth module, low voltage difference linear regulator, etc. are all arranged on a self-made PCB control board 501 including a microprocessor unit, a temperature control unit and a Bluetooth module.

[0149] Example 1 Portable Smartphone Nucleic Acid Amplification Detection Device

[0150] The overall size of the nucleic acid amplification detection device is about 180mm×90mm×110mm, and the weight is about 300g. It is designed by computer-aided design (3D CAD) produced by a 3D printer. The 3D printing material uses photosensitive resin. The appearance of the fully assembled device is as follows Figure 3As shown. The smartphone used is Huawei's Mate 20, which has three independent physical cameras on the back. The operating system is Android 9.0, and the three physical cameras can be independently controlled. The telephoto lens of Mate 20 has a long focal length and cannot clearly image within a distance of 10 cm. A plano-convex lens is used to correct the focal length, such as Edmund Optics, #67-146, 60mm focal length plano-convex lens, so that the three physical cameras can simultaneously image the microfluidic chip around 60mm. Emission filters are installed in front of the three physical cameras respectively, such as Newport's 8mm x 8mm square filters, with central wavelengths of 525nm (#90037532), 564nm (#90037535) and 685nm (#90063981), corresponding to the emission wavelengths of FAM, HEX and Cy5 respectively (such as Figure 4a and Figure 4b ).

[0151] The fluorescence excitation light source uses a Guanghong 12W 4-color LED module, which includes 3 narrow-band LEDs: 460-465nm (50lm), 520-525nm (160lm), 620-625nm (90lm); and 1 white light LED with a color temperature of 6000-7000K (220lm). The brightness is adjusted and the color is switched by a self-made PCB control board. Multi-channel narrow-band filters are used to obtain the required wavelength of excitation light, such as three-channel filters, Edmund Optics, #87-237, 22@457nm, 20@530nm, 28@628nm (bandwidth @ center wavelength), corresponding to the excitation wavelengths of FAM, HEX and Cy5 (such as Figure 4a ).

[0152] The fluorescence excitation light source can also use three LED light sources, whose central wavelengths are 465nm, 520nm and 620nm, respectively. They use Newport's 8mm x 8mm square filters, and the central wavelengths are 470nm (#90037531), 525nm (#90037532) and 637.5nm (#90063980), respectively, corresponding to the excitation wavelengths of FAM, HEX and Cy5 (such as Figure 4b ).

[0153] Control block diagram used to realize functions such as rapid thermal cycle, LED light source, etc. Figure 5As shown. The Blue Pill control board based on the STM32F103 microcontroller (MCU) is used as the control core to control the functions such as four-color LED light source adjustment, temperature rapid thermal cycle, stepper motor, temperature detection, and communicate with the smart phone through the Bluetooth module. The homemade PCR control board of the present invention is rich in functions, including LED constant current driver (STCS05, ST Microelectronics), thermocouple digital converter (MAX31856, Maxim Inc.), and high-power bidirectional H-bridge driver (VNH5019, STMicroelectronics) for controlling the Peltier. The sample temperature of the microfluidic chip is measured using a K-type thermocouple (TT-K-20, OMEGA Engineering Inc.). The single-chip microcomputer controls the high-power bidirectional H-bridge driver to provide positive and negative power to the Peltier to determine the heating and cooling state of the Peltier, and determines the heating amount or cooling amount of the Peltier output by controlling the duty cycle of the pulse width modulation (PWM). The microprocessor unit calculates the temperature state (heating, cooling) of the Peltier and the required output heating or cooling capacity through the automatic control PID (i.e. proportional integral differential closed-loop feedback method) algorithm. At the same time, the microprocessor unit drives the LED constant current driver through a fixed-frequency pulse width modulation (PWM) signal to achieve brightness control and color switching of the four-color LED light source. Through the Bluetooth module, the homemade PCB control board can communicate wirelessly with the Android smartphone. The stepper motor drive circuit consists of a driver (L6470, ST Microelectronics).

[0154] The assembly diagram of disposable microfluidic chip and Peltier device is shown in the figure Figure 6a , from top to bottom are disposable microfluidic chip 100, thermal pad 330, Peltier 310, aluminum heat sink (aluminum heat sink block 340) and turbo fan 350. Peltier (TEC1-12712, HB Inc.) is a thermoelectric semiconductor cooling device that achieves heating or cooling through the Peltier effect. The upper part of the Peltier is covered with a copper plate with a thermal pad, which can minimize heat loss and easily transfer heat to the microfluidic chip to provide a uniform reaction temperature. The copper plate and Peltier are wrapped so that they fit tightly together to promote heat conduction and form a compact heat capacity space. In addition, a K-type thermocouple (TT-K-20, OMEGA Engineering Inc.) with a diameter of 0.02 inches is placed closely under the microfluidic chip to measure the temperature. In order to discharge excess heat and cool the bottom surface of the Peltier, an aluminum heat sink and turbo fan are placed closely on the bottom surface of the Peltier.

[0155] The microfluidic chip includes a sample addition hole 130, an overflow hole 140, 6 groups of distribution chambers 120, and 6 groups of reaction chambers 110. Figure 6b The cover layer of the microfluidic chip is an optical film coated with a pressure-sensitive adhesive. Before applying pressure to seal the microchannel and reaction chamber, sequence-specific primers, fluorescent probes and magnesium acetate are first added to the distribution chamber, heated and evaporated; finally, the pressure-sensitive optical film is used to seal it.

[0156] Mobile smartphones can provide a user interface with a good operating experience, and can also be used for temperature, light source control and real-time quantitative fluorescence intensity detection, as well as automatic storage control management of detection data. The inventor of this case used Android Studio 3.2.1 to develop control software on a smartphone, which can perform hardware control such as rapid thermal cycling and LED light sources, and use OPENCV 3.4.1 (open source computer vision library) on the Android system for image processing. Due to the new camera function recently released in Android Pie (9.0) and API version 28, it is allowed to control a single physical camera in a multi-camera smartphone, so that the separate control of multiple cameras built into the mobile phone can be achieved, so that different control parameters such as exposure time, white balance and focus position can be set for different cameras. Independent control can be achieved through self-developed software, and finally three channels of fluorescence real-time acquisition can be achieved by setting three built-in cameras separately.

[0157] Example 2 Nucleic acid extraction

[0158] Nucleic acid extraction reagents Reagents in the Viral RNAMini kit. The sample is the FNV-2019-NCoV-abEN pseudovirus provided by Fubo Bio. Reservoir 61 stores 560 μL of 100% ethanol, reservoir 62 stores 500 μL of AW1, reservoir 63 stores 500 μL of AW2, and reservoir 64 stores 100 μL of RNAse free water.

[0159] 100 μL of pseudovirus (about 10e4 pseudovirus) or negative control (water) was added to 560 μL AVL, incubated at room temperature for 10 minutes, and then added to reservoir 61 and mixed. Figure 1a-1b The nucleic acid extraction device shown in FIG. 1 draws a solution (such as Figure 2c-2d As shown), the extraction device is rotated 90 degrees, and after the liquid pipeline 30 is compressed, the piston 10 is pushed to allow the liquid to pass through 1 to 5 layers of Whatman GF / F silanol membranes for nucleic acid binding (as shown in FIG. Figure 2e-2f Then the empty cylinder 20 of the extraction device is rotated 90 degrees in the opposite direction and moved to the second liquid storage tank 52 to draw the solution (as shown); Figure 2c-2dAs shown), the empty cylinder 20 of the extraction device is rotated 90 degrees, and after the liquid pipeline 30 is compressed, the piston 10 is pushed to allow the liquid to pass through 1 to 5 layers of Whatman GF / F silanol membrane for the first cleaning (as shown in FIG. Figure 2e-2f Then the empty cylinder 20 of the extraction device is rotated 90 degrees in the opposite direction and moved to the third liquid storage tank 53 to draw the solution (as shown); Figure 2c-2d As shown), the empty cylinder 20 of the extraction device is rotated 90 degrees, and after the liquid pipeline 30 is compressed, the piston 10 is pushed to allow the liquid to pass through 1 to 5 layers of Whatman GF / F silanol membrane for the second cleaning (as shown in FIG. Figure 2e-2f Then the empty cylinder 20 of the extraction device is rotated 90 degrees in the opposite direction and moved to the fourth liquid storage tank 64 to draw the solution (as shown); Figure 2c-2d As shown), the empty cylinder 20 of the extraction device is rotated 90 degrees, and after the liquid pipeline 30 is compressed, the piston 10 is pushed to allow the liquid to pass through 1 to 5 layers of Whatman GF / F silanol membrane to elute the nucleic acid.

[0160] Example 3 RT-qPCR detection

[0161] RT-qPCR detection uses Takara's one-step RT-qPCR kit RR064A. Prepare the reaction solution according to the proportions in Table 1 below, without water. The primer and probe sequences are shown in Table 2 (the sequences are the RT-qPCR primer and probe sequences recommended by the US CDC for detecting new coronavirus pneumonia. Add the corresponding volume of reaction solution to 68% of the volume of the reaction chamber, freeze-dry and seal with a pressure-sensitive optical film.

[0162] The nucleic acid extracted and purified according to the method described in Example 2 was transferred to the sample well, and the temperature program was set by the smartphone (45°C / 5min, 95°C 10sec, 40x (95°C 10sec, 58°C 30sec). The temperature cycle was completed by the lower computer control circuit and the Peltier heating / cooling device. In the last 10 seconds of the extension temperature (58°C), the three cameras of the mobile phone collected a fluorescent image in turn, and the OPENCV on the Android system was used. 3.4.1 (open source computer vision library) for image processing. Fluorescence images are stored in lossless digital negative (DNG) format. First, the inventors of this case converted the fluorescence image from DNG format to Mat format; then, the top-hat method was used for background filtering, which can minimize the background interference of the fluorescence image caused by uneven illumination. Then, morphological methods (such as Hough transform) are used to quickly locate the marking points and holes on the microfluidic chip. The position of each hole is calculated by the ratio of the hole to the marking point. After 100μL pseudovirus (approximately 10e4 pseudovirus) or negative control (water) was amplified as a sample, the fluorescence changes over time were plotted after software analysis, as shown in the following figure. Figure 7a and Figure 7bshown.

[0163] Table 1 Reaction solution composition and ratio

[0164]

[0165]

[0166] Table 2 Primer and probe sequences

[0167]

[0168] In summary, through the above technical scheme, the present invention can realize nucleic acid extraction and purification, use a smart phone to control thermal cycling and fluorescence acquisition, and use multiple physical cameras of the smart phone in combination with multiple LEDs with different central wavelengths to realize multi-color fluorescence detection. Compared with the traditional method of switching different fluorescence channels through mechanical movement, the excitation light source of the multi-color fluorescence detection in the present invention controls the switch and intensity electronically, and the acquisition is independently carried out through multiple physical cameras of the smart phone in combination with emission filters, which can realize 4-6 channel fluorescence acquisition. By using a multi-pass emission filter, the fluorescence channel can also be expanded to 12-18 channels. There are no mechanical moving parts, which reduces the system failure rate and improves portability.

[0169] The various aspects, embodiments, features and examples of the present invention should be considered as illustrative in all aspects and are not intended to limit the present invention, the scope of the present invention is defined only by the claims. Other embodiments, modifications and uses will be apparent to those skilled in the art without departing from the spirit and scope of the claimed invention.

[0170] The use of headings and sections in this disclosure is not meant to limit the disclosure; each section may apply to any aspect, embodiment, or feature of the disclosure.

[0171] Throughout this disclosure, where a composition is described as having, containing, or comprising particular components, or where a process is described as having, containing, or comprising particular process steps, it is contemplated that the compositions taught by the present invention also consist essentially of or consist of the recited components, and that the processes taught by the present invention also consist essentially of or consist of the recited process steps.

[0172] It should be understood that the order of the various steps or the order in which a particular action is performed is not very important, as long as the teachings of the present invention remain operable. In addition, two or more steps or actions may be performed simultaneously.

[0173] In addition, the inventors of this case also referred to the aforementioned embodiments and conducted experiments with other raw materials, process operations, and process conditions described in this specification, and obtained relatively ideal results.

[0174] Although the present invention has been described with reference to illustrative embodiments, it will be appreciated by those skilled in the art that various other changes, omissions and / or additions may be made and that elements of the described embodiments may be substituted with substantial equivalents without departing from the spirit and scope of the present invention. In addition, many modifications may be made to adapt a particular situation or material to the teachings of the present invention without departing from the scope of the present invention. Therefore, it is not intended herein to limit the present invention to the specific disclosed embodiments for carrying out the present invention, but rather it is intended that the present invention will include all embodiments within the scope of the appended claims.

Claims

1. A nucleic acid extraction and purification device, characterized in that include: An extraction mechanism, comprising a first component and a second component that fit closely together, wherein the first component is a piston member, the second component has a channel, the second component is a cylindrical structure with a hollow structure, a local area of ​​the first component can reciprocate in the channel, and the channel allows a sample containing nucleic acid to be sucked into the channel, and a liquid pipeline is provided at the bottom end of the second component, and the liquid pipeline is connected to the channel; An interception structure, which is a porous membrane capable of adsorbing and eluting nucleic acids, is disposed at the bottom end of the second component and intercepts the nucleic acids; The liquid storage mechanism comprises a plurality of accommodating chambers, each of the accommodating chambers having a protrusion on its bottom surface, the protrusions being arranged in one-to-one correspondence with the outlet ends of the liquid pipelines, the size of the protrusions being not less than the diameter of the liquid pipelines, and the protrusions being capable of closing the liquid pipelines.

2. The nucleic acid extraction and purification device according to claim 1, characterized in that It also includes a fixing member, and the intercepting structure is arranged at the bottom end of the second component through the fixing member.

3. The nucleic acid extraction and purification device according to claim 2, characterized in that: The fixing member is a fixing pressure ring.

4. The nucleic acid extraction and purification device according to claim 1, characterized in that: The interception structure is a silanol membrane with a pore size of 0.1 μm-10 μm.

5. The nucleic acid extraction and purification device according to claim 1, characterized in that: The liquid storage mechanism comprises: A first containing chamber storing a first reagent, wherein the first reagent includes a sample containing nucleic acid; A second containing chamber storing a second reagent, wherein the second reagent includes a cleaning solution; A third containing chamber is provided for storing a third reagent, wherein the third reagent includes an eluent.

6. The nucleic acid extraction and purification device according to claim 5, characterized in that It also includes a heating mechanism, which is arranged around the outside of the first accommodating cavity.

7. The nucleic acid extraction and purification device according to claim 6, characterized in that: The heating mechanism is a heating ring.

8. The nucleic acid extraction and purification device according to claim 5, characterized in that: The nucleic acid extraction and purification device also includes a control mechanism, which is connected to an intelligent mobile terminal via a microprocessor unit.

9. A method for extracting and purifying nucleic acid, characterized in that: The method is implemented based on the nucleic acid extraction and purification device according to any one of claims 1 to 8, and the nucleic acid extraction and purification method comprises: (1) placing the end of the liquid pipeline in the first reagent of the first accommodating chamber, applying a pulling force to the first component, so that the sample containing the nucleic acid is sucked into the channel of the second component through the liquid pipeline and the intercepting structure, so that the nucleic acid is adsorbed on the inner wall of the second component; (2) sealingly contacting the outlet end of the liquid pipeline with the surface of the protruding portion, thereby sealing the liquid pipeline and the channel, and then applying a thrust to the first component so that the liquid is pushed into the first accommodating chamber through the intercepting structure; (3) Clean and elute the nucleic acid in accordance with steps (1) and (2) to obtain a pure nucleic acid solution.

10. A system for nucleic acid extraction, purification and amplification detection, characterized in that include: The nucleic acid extraction and purification device according to any one of claims 1 to 8; as well as Nucleic acid amplification detection device.

11. The system for nucleic acid extraction, purification and amplification detection according to claim 10, characterized in that: The nucleic acid amplification detection device comprises: A microfluidic chip, comprising interconnected reaction chambers, distribution chambers, microchannels for connecting the chambers, sample loading holes, and overflow holes, wherein the reaction chambers are used to store PCR reaction systems, and the distribution chambers are used to store primers and probes for detecting nucleic acids; A temperature control unit, used to regulate the operating temperature of the microfluidic chip; an excitation unit, for providing excitation light, wherein the excitation light is irradiated on the PCR reaction product in the microfluidic chip to generate fluorescence; and The detection unit is used to collect the generated fluorescence and perform analysis and detection.

12. The system for nucleic acid extraction, purification and amplification detection according to claim 11, characterized in that: The nucleic acid amplification detection device also includes a microprocessor unit, which is used to control the working states of the temperature control unit, the excitation unit, and the detection unit.

13. The system for nucleic acid extraction, purification and amplification detection according to claim 12, characterized in that: The excitation unit comprises an LED light source and a filter assembly. The LED light source is arranged above the filter assembly. The filter assembly is used to generate excitation light of a required wavelength. The LED light source is connected to the microprocessor unit via an LED driving assembly.

14. The system for nucleic acid extraction, purification and amplification detection according to claim 13, characterized in that: The LED light source is a four-color LED light source.

15. The system for nucleic acid extraction, purification and amplification detection according to claim 13, characterized in that: The filter assembly is a single-pass color filter or a multi-pass color filter.

16. The system for nucleic acid extraction, purification and amplification detection according to claim 15, characterized in that: The filter component is a three-bandpass color filter.

17. The system for nucleic acid extraction, purification and amplification detection according to claim 12, characterized in that: The detection unit includes an intelligent mobile terminal having one or more fluorescent image acquisition components and one or more filter components. The fluorescent image acquisition components are arranged one by one above the filter components, and a plano-convex lens is arranged between the fluorescent image acquisition components and the filter components.

18. The system for nucleic acid extraction, purification and amplification detection according to claim 17, characterized in that: The intelligent mobile terminal is connected to the microprocessor unit via a Bluetooth module.

19. The system for nucleic acid extraction, purification and amplification detection according to claim 17, characterized in that: The nucleic acid extraction, purification and amplification detection system also includes a switching power supply module or a charging power supply for providing power to the smart mobile terminal and the nucleic acid amplification detection device. The switching power supply module or the charging power supply is electrically connected to the microprocessor unit.

20. The system for nucleic acid extraction, purification and amplification detection according to claim 19, characterized in that: The intelligent mobile terminal is selected from a smart phone, a tablet computer or an ARM-based microcomputer motherboard, and the fluorescent image acquisition component is a camera component.

21. The system for nucleic acid extraction, purification and amplification detection according to claim 20, characterized in that: The fluorescent image acquisition component is a built-in camera.

22. The system for nucleic acid extraction, purification and amplification detection according to claim 12, characterized in that: The temperature control unit includes a heating and cooling component and a temperature sensing component. The heating and cooling component is used for rapid heating or rapid cooling, and makes the temperature in the reaction chamber change rapidly to achieve the rapid thermal cycle required for the PCR reaction.

23. The system for nucleic acid extraction, purification and amplification detection according to claim 22, characterized in that: The heating and cooling component is a thermoelectric semiconductor cooling device.

24. The system for nucleic acid extraction, purification and amplification detection according to claim 22, characterized in that: The temperature sensing component is a thermocouple sensor.

25. The system for nucleic acid extraction, purification and amplification detection according to claim 22, characterized in that: The heating and cooling component is connected to the microprocessor unit via a bidirectional H-bridge driving component, and the temperature sensing component is connected to the microprocessor unit via a thermocouple temperature measuring converter.

26. The system for nucleic acid extraction, purification and amplification detection according to claim 12, characterized in that: The microprocessor unit is a single chip microcomputer.

27. A method for nucleic acid extraction, purification and amplification detection, characterized in that: The method is implemented based on the system for nucleic acid extraction, purification and amplification detection according to any one of claims 12 to 26, and the method comprises: Extracting and purifying nucleic acid according to the method for extracting and purifying nucleic acid according to claim 9; and A nucleic acid amplification and detection device is used to perform nucleic acid amplification and detection.

28. The method according to claim 27, characterized in that The method for performing nucleic acid amplification detection using the nucleic acid amplification detection device includes: The sample to be detected is placed in the sample loading hole, the PCR reaction system is placed in the reaction chamber, and the primers and probes for detecting nucleic acid are placed in the distribution chamber; A temperature control unit is used to heat or cool the microfluidic chip to complete a thermal cycle, so that the temperature condition in the reaction chamber can perform a PCR reaction, and a temperature sensor is used to detect the temperature of the reaction chamber, and an automatic control algorithm of a microprocessor unit is used to perform temperature control so that the temperature is controlled within the PCR reaction range; Using an excitation unit to irradiate the PCR reaction product in the microfluidic chip with excitation light to generate fluorescence; The generated fluorescence is collected by a detection unit and analyzed and detected.

29. The method according to claim 28, characterized in that The method comprises: real-time monitoring and collecting the fluorescence intensity of PCR reaction products, and analyzing the results through an amplification curve of the fluorescence intensity changing with time.

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