CN113621475A

CN113621475A - Method and system for extracting, purifying and amplifying nucleic acid

Info

Publication number: CN113621475A
Application number: CN202010382233.8A
Authority: CN
Inventors: 吴保军; 王宏; 张方舟; 雷祝兵; 黄明垒; 樊丽
Original assignee: Suzhou Institute of Nano Tech and Nano Bionics of CAS
Current assignee: Suzhou Institute of Nano Tech and Nano Bionics of CAS
Priority date: 2020-05-08
Filing date: 2020-05-08
Publication date: 2021-11-09

Abstract

The invention discloses a method and a system for extracting, purifying and amplifying nucleic acid. The system comprises a nucleic acid extraction and purification device and a nucleic acid amplification detection device, wherein the nucleic acid extraction and purification device comprises: the extraction mechanism comprises a first component and a second component which are tightly matched, at least a partial area of the first component can reciprocate in a channel of the second component, and a liquid pipeline communicated with the channel is arranged at the bottom end of the second component; the intercepting structure is arranged at the bottom end of the second component; the liquid storage mechanism comprises a plurality of accommodating cavities, a protruding part is arranged on the bottom surface of each accommodating cavity, and the protruding parts can at least seal the liquid pipeline. The nucleic acid amplification detection device includes: the device comprises a micro-fluidic chip, a temperature control unit, an excitation unit and a detection unit. The invention can realize nucleic acid extraction and purification, and realize multicolor fluorescence detection by combining a plurality of physical cameras of the smart phone with the LED light source to perform nucleic acid amplification and real-time analysis.

Description

Method and system for extracting, purifying and amplifying nucleic acid

Technical Field

The invention relates to a method and a system for extracting, purifying and amplifying and detecting nucleic acid, in particular to a method and a device for extracting and purifying nucleic acid by using a smart phone with low cost, and a method and a device for amplifying nucleic acid and detecting fluorescence, belonging to the technical field of nucleic acid amplification and detection.

Background

Nucleic acid is a biological macromolecular compound polymerized by a plurality of nucleotide monomers, is one of the most basic substances of life, comprises deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), and is widely present in all animals, plants, microorganisms and organisms. Nucleic acids bind to proteins to form nucleoproteins. Different nucleic acids differ in their chemical composition and nucleotide arrangement. Nucleic acids are essential genetic material, and all organisms including animals and plants, bacterial viruses, etc. are inherited by nucleic acids, each species having its own specific nucleic acid sequence. By sequencing the nucleic acids, it is possible to determine which type of organism, for example an infection, is at all, what the small organism is responsible for by sequencing the nucleic acids and to use drugs to control the elimination of it. In the treatment of tumors, the genetic material of the tumor nucleic acid properties can also be used to determine what drug to use and what drug to use for sensitivity and thus to select a better drug.

The commonly used nucleic acid analysis methods include sequencing, amplification detection, hybridization detection, etc., and these analysis methods generally include steps of nucleic acid extraction, purification, amplification, detection, etc.

The technology for extracting and purifying nucleic acid is to separate the nucleic acid from biological macromolecular substances such as protein, polysaccharide, lipid and the like, ensure the integrity of nucleic acid molecules, is the basis for various researches in molecular biology and is a key technology for life science research and application. In the early 90 s of the 20 th century, the technology for extracting and purifying nucleic acid is a time-consuming and tedious technology, and meanwhile, toxic reagents such as phenol, chloroform and the like are required for extraction. With the advent of solid phase extraction technology and the availability and practicality of commercial kits, rapid and reliable assistance has been provided for nucleic acid extraction, thus promoting the development of techniques capable of extracting high quality nucleic acids from biological samples such as whole blood, serum, saliva, urine, stool and tissue. Commercial solid phase extraction techniques are mainly classified into two categories: centrifugal column purification and magnetic bead purification. The extraction and purification of nucleic acid by centrifugal column purification and magnetic bead purification usually include cell lysis, adsorption, washing and elution. The centrifugal column purification method is a method widely applied to the extraction of the current kit, and nucleic acid can be selectively adsorbed to the surface of a silicon hydroxyl membrane under a certain ionic environment to be separated from other biomolecules. The liquid added in each step in the purification process enters another centrifugal tube after being centrifuged, and is completely separated from the column containing the nucleic acid, so that the washing is thorough, and the purity stability is high. However, the purification process requires repeated centrifugation, and the operation is complicated and difficult to automate. The magnetic bead purification method utilizes the silicon hydroxyl surface of the superparamagnetic silicon oxide nanometer magnetic bead to combine with the nucleic acid with high salt and low pH value, and the low salt pH value is eluted to realize the purification of the nucleic acid; the superparamagnetism of the magnetic beads is utilized, the magnetic beads are separated from a liquid phase through a magnetic field without centrifugation, and the magnetic separation device has the advantage of easy automation.

The nucleic acid amplification detection is a method of amplifying a nucleic acid sequence to be detected by the action of an enzyme and then detecting the amplified nucleic acid sequence, and includes a thermal cycle amplification method typified by Polymerase Chain Reaction (PCR) and an isothermal amplification method typified by RPA and LAMP. PCR is carried out by combining a primer with a single strand at a low temperature (usually about 60 ℃) in a manner that DNA is denatured at a high temperature of 95 ℃ to become a single strand, adjusting the temperature to an optimum reaction temperature (about 72 ℃) for DNA polymerase, and synthesizing a complementary strand by DNA polymerase in a direction from phosphate to pentose (5 '-3'). In the fluorescent quantitative PCR, a fluorescent group is added into a reaction system, and the change of the quantity of each cycle of amplified products in the PCR amplification reaction is monitored in real time by using the change of a fluorescent signal.

Nucleic acid extraction, purification and amplification detection usually need to be performed in a professional molecular laboratory, and have severe requirements for both laboratory environment and professional operators. Thus, it is quite difficult for the PCR laboratory to penetrate into the interior of many biologically-relevant industries. Such as the recent outbreak of new coronavirus pneumonia and the recent outbreak of african swine fever, new demands are made for nucleic acid analysis in the field. In a nucleic acid detection system for rapid field inspection (POCT) developed in recent years, a nucleic acid analysis and detection mode of 'sample in and result out' is adopted to greatly simplify the operation flow, so that field deployment and operation of non-molecular inspection professionals are possible. The whole process of cell/bacterium/virus lysis, nucleic acid purification, reverse transcription, real-time quantitative PCR amplification, detection and other nucleic acid analysis is automatically completed in the cartridge by adopting a fully-sealed cartridge. Nucleic acid extraction, purification and amplification detection systems based on smart phones have become an economical and fast molecular diagnostic technique because they can provide a fast detection cycle, facilitate connection with remote experts and artificial intelligence systems through mobile communication, and provide accurate health information in resource-limited environments.

Therefore, how to combine nucleic acid extraction and purification, and amplification detection with a real-time nucleic acid detection device based on real-time quantitative PCR nucleic acid amplification and fluorescence detection technology, a method and a system for nucleic acid extraction and purification and nucleic acid amplification detection implemented by using a smart phone are sought, and have been the direction of efforts of researchers in the industry for a long time.

Disclosure of Invention

The main objective of the present invention is to provide a nucleic acid extraction and purification device and method to overcome the disadvantages of the prior art.

Another main object of the present invention is to provide a nucleic acid amplification detecting apparatus.

Another main objective of the present invention is to provide a method and system for nucleic acid purification and amplification detection.

In order to achieve the purpose, the technical scheme adopted by the invention comprises the following steps:

the embodiment of the invention provides a nucleic acid extraction and purification device, which comprises:

an extraction mechanism comprising a first component and a second component that are mated, the second component having a channel within which at least a partial region of the first component is capable of reciprocating and which allows a sample containing nucleic acids to be aspirated into the channel, a bottom end of the second component being provided with a liquid line, and the liquid line being in communication with the channel;

an intercepting structure disposed at a bottom end of the second module and intercepting the nucleic acid;

the liquid storage mechanism comprises a plurality of accommodating cavities, and a protruding part is arranged on the bottom surface of each accommodating cavity and at least can seal the liquid pipeline.

The embodiment of the invention also provides a nucleic acid extraction and purification method, which is implemented mainly based on the nucleic acid extraction and purification device, and the nucleic acid extraction and purification method comprises the following steps:

(1) placing the tail end of the liquid pipeline in the first reagent in the first accommodating cavity, applying pull force to the first assembly, and sucking a sample containing nucleic acid into a channel of the second assembly through the liquid pipeline and the intercepting structure, so that the nucleic acid is adsorbed on the inner wall of the second assembly;

(2) the outlet end of the liquid pipeline is in sealing contact with the surface of the protruding part, so that the liquid pipeline and the channel are sealed, and then thrust is applied to the first assembly, so that the liquid is injected into the first accommodating cavity through the intercepting structure;

(3) and (3) sequentially carrying out washing and elution treatment on the nucleic acid according to the steps (1) and (2) to obtain a pure nucleic acid solution.

The embodiment of the invention also provides a nucleic acid amplification detection device, which comprises:

the micro-fluidic chip is at least used for accommodating a primer, a probe and a PCR reaction system for detecting nucleic acid;

the temperature control unit is at least used for regulating and controlling the working temperature of the microfluidic chip;

the excitation unit is at least used for providing excitation light, and the excitation light irradiates on a PCR reaction product in the microfluidic chip to generate fluorescence; and the number of the first and second groups,

and the detection unit is at least used for collecting the generated fluorescence and analyzing and detecting the fluorescence.

Furthermore, the nucleic acid amplification detection device also comprises a microprocessor unit which is at least used for controlling the working states of the temperature control unit, the excitation unit and the detection unit.

The embodiment of the invention also provides an application of the nucleic acid amplification detection device in the field of nucleic acid amplification detection, and the method for carrying out nucleic acid amplification detection by using the nucleic acid amplification detection device comprises the following steps:

placing a sample to be detected in the sample adding hole, placing a PCR reaction system in a reaction chamber, and placing a primer and a probe for detecting nucleic acid in a distribution chamber;

the temperature control unit is adopted to heat or refrigerate the microfluidic chip to complete thermal cycle, so that the temperature condition in the reaction chamber can carry out PCR reaction, the temperature sensor is adopted to detect the temperature of the reaction chamber, and the automatic control algorithm of the microprocessor unit is adopted to carry out temperature control, so that the temperature is controlled in the PCR reaction range;

irradiating the PCR reaction product in the microfluidic chip by using excitation units to generate fluorescence;

and collecting the generated fluorescence by using a detection unit, and analyzing and detecting.

Correspondingly, the embodiment of the invention also provides a system for extracting, purifying and amplifying and detecting nucleic acid, which comprises:

the nucleic acid extraction and purification apparatus; and

the nucleic acid amplification detection apparatus.

Correspondingly, the embodiment of the invention also provides a method for extracting, purifying and amplifying and detecting nucleic acid, which comprises the following steps:

extracting and purifying nucleic acid according to the method; and

nucleic acid amplification and detection were performed as described previously.

The invention provides a method and a device for extracting, purifying and amplifying and detecting nucleic acid based on a smart phone, and compared with the prior art, the method and the device have the advantages that:

1) the present invention provides a low-cost nucleic acid extraction method and apparatus in which the extraction liquid can be passed through a liquid pipe having a large inner diameter and a silicone-based membrane, while the push-back liquid can be passed only through the silicone-based membrane. Compared with the method that both the liquid extraction and the liquid pushing-out can only pass through the silicon hydroxyl basement membrane, the vacuum suction force formed by pulling the piston open is far smaller than the pressure formed by pushing the piston, so that the method is favorable for the relatively viscous liquid, such as tissue cracking products, to complete the processes of adsorption and elution through the silicon hydroxyl membrane; compared with a magnetic bead method which needs to repeatedly move a magnet to realize solid-liquid separation, the device is simpler, the nucleic acid extraction method can be manually realized, and can also be completed by operating a plurality of stepping motors through the cooperation of a smart phone and a singlechip;

2) the invention utilizes the intelligent mobile phone to control thermal cycle and fluorescence collection, utilizes a plurality of physical cameras of the intelligent mobile phone to combine a plurality of LEDs with different central wavelengths to realize multicolor fluorescence detection, compared with the traditional mode of switching different fluorescence channels through mechanical motion, the excitation light source of the multicolor fluorescence detection in the invention controls the switch and the intensity in an electronic mode, the collection is independently carried out by matching the plurality of physical cameras of the intelligent mobile phone with the emission color filter, 4-6 channel fluorescence collection can be realized, the multi-channel emission color filter is utilized, the fluorescence channel can be expanded to 12-18 channels, no mechanical motion part is provided, the system failure rate is reduced, and the portability is improved.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments described in the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.

FIG. 1a and FIG. 1b are schematic views showing the structure of a nucleic acid purification apparatus according to an exemplary embodiment of the present invention.

FIGS. 2a and 2b are schematic views showing the structure of a nucleic acid isolation apparatus in which a first module is located at the bottom end of a second module according to an exemplary embodiment of the present invention.

FIGS. 2c and 2d are schematic views showing the structure of a nucleic acid extracting apparatus according to an exemplary embodiment of the present invention when the first member is drawn upward.

FIGS. 2e and 2f are schematic views showing the structure of a first module of a nucleic acid isolation apparatus during bolus-down injection according to an exemplary embodiment of the present invention.

FIG. 3 is a schematic diagram showing the structure of a nucleic acid amplification detecting apparatus according to an exemplary embodiment of the present invention.

FIGS. 4a and 4b are schematic diagrams illustrating a method for detecting nucleic acid amplification according to an exemplary embodiment of the present invention.

FIG. 5 is a block diagram showing the control of the light source drive and the thermostat control of the nucleic acid amplification detecting apparatus according to the exemplary embodiment of the present invention.

FIG. 6a is an assembly diagram of a micro-fluidic chip and a thermoelectric semiconductor refrigeration device in a nucleic acid amplification detection apparatus according to an exemplary embodiment of the present invention.

Fig. 6b is a schematic structural diagram of the microfluidic chip.

FIGS. 7a and 7b are graphs showing fluorescence changes with time after amplification of 100. mu.L of pseudovirus (about 10e4 pseudovirus) or negative control (water) as a sample in example 3 of the present invention, and analysis by software.

Description of reference numerals: 10-piston, 20-hollow cylinder, 21-channel, 30-liquid pipeline, 40-silicon hydroxyl basal membrane, 50-liquid storage mechanism, 51-first liquid storage tank, 51 a-heating sleeve, 52-second liquid storage tank, 53-third liquid storage tank, 54-fourth liquid storage tank, 55-bulge and 56-connection motor shaft; 100-microfluidic chip, 100 a-chip in-and-out bin assembly, 110-reaction chamber, 120-distribution chamber, 130-sample adding hole, 140-overflow hole, 200-excitation unit, 210-four-color LED light source, 211-constant current drive, 220-three-band-pass filter, 221-band-pass filter, 222-multi-pass filter, 300-temperature control unit, 310-Peltier, 311-high-power bidirectional H-bridge drive, 320-thermocouple sensor, 321-thermocouple temperature measurement converter, 330-heat conducting pad, 340-aluminum heat dissipation block, 350-fan, 400-detection unit, 410-smart phone, 410 a-smart phone support, 411-built-in camera, 412-switching power supply module, 413-Bluetooth module, 414-low dropout linear regulator, 420-band pass filter, 422-plano-convex lens, 500-microprocessor unit, 501-self-made PCB control board, A-optical component, 1-equipment shell.

Detailed Description

By means of rapid development of smart phones, more and more smart phones have a plurality of built-in cameras, each built-in physical camera can be independently controlled from the 9.0 version of an Android system, the resolution of the built-in camera is higher and higher, and meanwhile, a memory and a processor of the smart phone are powerful enough to be used for real-time image processing, so that the built-in cameras of the smart phones can be used for acquiring multi-channel fluorescence images, and the fluorescence intensity is calculated through an image processing algorithm.

In view of the shortcomings and drawbacks of the prior art, the present inventors have conducted extensive research and practice to provide a method and an apparatus for extracting nucleic acid at low cost, which comprises extracting liquid from a liquid reservoir (binding liquid, washing liquid and eluent), and then pushing the liquid back to the liquid reservoir through a silicon hydroxyl membrane to complete the processes of adsorption, washing and elution of nucleic acid. It features that the pumped liquid can pass through the pipeline with relatively large inner diameter and the silicon hydroxide film, while the pushed liquid can pass through the silicon hydroxide film only. Compared with the method that both the liquid extraction and the liquid pushing-out can only pass through the silicon hydroxyl basement membrane, the vacuum suction force formed by pulling the piston open is far smaller than the pressure formed by pushing the piston, so that the method is favorable for the relatively viscous liquid, such as tissue cracking products, to complete the processes of adsorption and elution through the silicon hydroxyl membrane; compared with a magnetic bead method which needs to move a magnet repeatedly to realize solid-liquid separation, the device is simpler. The nucleic acid extraction method can be realized manually, and can also be completed by operating a plurality of stepping motors through the cooperation of a smart phone and a singlechip.

The invention utilizes the smart phone to control thermal cycling and fluorescence collection. The multi-color fluorescence detection system has the advantages that the multi-color fluorescence detection is realized by combining the plurality of physical cameras of the smart phone with the plurality of LEDs with different central wavelengths, compared with the traditional mode of switching different fluorescence channels through mechanical motion, the excitation light source of the multi-color fluorescence detection controls the switch and the intensity in an electronic mode, the collection is independently carried out by matching the plurality of physical cameras of the smart phone with the emission color filter, the 4-6 channel fluorescence collection can be realized, the multi-channel emission color filter is utilized, the fluorescence channels can be expanded to 12-18 channels, mechanical motion parts are avoided, the system failure rate is reduced, and the portability is improved.

The technical solution, its implementation and principles, etc. will be further explained as follows.

The technical solution of the present invention will be explained in more detail below. It is to be understood, however, that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with one another to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.

One aspect of the embodiments of the present invention provides a nucleic acid extraction and purification apparatus, including:

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

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

In some embodiments, the nucleic acid extraction and purification device further comprises a fixing member, and the interception structure is arranged at the bottom end of the second component through the fixing member.

Further, the fixing member may be a fixing clip, but is not limited thereto.

In some embodiments, the intercepting structure comprises a porous membrane that can adsorb and elute nucleic acids under different conditions, for example, a silicone-based membrane (e.g., Whatman GF/F, etc.) that may preferably have a pore size of 0.1 μm to 10 μm, but is not limited thereto.

In some embodiments, the projections are disposed in one-to-one correspondence with the outlet ends of the liquid lines.

Further, the size of the projection is not smaller than the diameter of the liquid pipe.

In some embodiments, the reservoir mechanism comprises:

a first holding chamber in which a first reagent is stored, the first reagent including a sample containing a nucleic acid;

a second accommodating chamber in which a second reagent is stored, the second reagent including a cleaning solution;

a third holding chamber in which a third reagent is stored, the third reagent including an eluent.

Further, the number of the second accommodating cavities can be one or more than two, and the number is not limited.

In some embodiments, the nucleic acid extraction and purification device further comprises a heating mechanism, and the heating mechanism is annularly arranged outside the first accommodating cavity.

Further, the heating mechanism may be a heating collar, but is not limited thereto.

Further, the nucleic acid extraction and purification device also comprises a control mechanism, and the control mechanism is connected with an intelligent mobile terminal through a microprocessor unit.

In some more specific embodiments, the nucleic acid extracting and purifying apparatus may specifically include: a structure similar to a syringe and a liquid storage mechanism are provided, in which a sample containing nucleic acid (also referred to as a binding liquid), a first cleaning liquid, a second cleaning liquid and an eluent are sequentially stored. The liquid can be added before purification begins, or can be pre-stored in the liquid storage mechanism, the liquid storage mechanism with the pre-stored liquid is sealed by an aluminum foil, and the liquid is punctured before extraction begins or is punctured one by one according to requirements in the extraction process.

Furthermore, a structure in which a heating collar can be placed is provided around a holding chamber (also referred to as a liquid storage tank) for storing a sample containing nucleic acid, so that the liquid can be heated as required. The bottom of the containing cavity is also provided with a sealing bulge which is used for sealing the pipeline for sucking the liquid when the liquid is injected. The structure similar to the syringe comprises a piston, an empty cylinder, a liquid pipeline, a silicon hydroxyl-based membrane (such as Whatman GF/F and the like), a fixed compression ring and the like. The vacuum suction created by pulling the plunger open is much less than the pressure created by pushing the plunger open, and the fluid line facilitates the drawing of viscous fluids, such as tissue lysate, into the hollow cylinder.

Another aspect of the embodiments of the present invention provides a method for extracting and purifying nucleic acid, which is implemented mainly based on the foregoing apparatus for extracting and purifying nucleic acid, and the method for extracting and purifying nucleic acid includes:

Further, the nucleic acid extraction process comprises: the front end of the extraction mechanism, namely the tail end of the liquid pipeline is placed into the combined liquid storage tank, the piston is pulled out, and liquid can be sucked into the hollow cylinder through the liquid pipeline with larger inner diameter and the silicon hydroxyl film; rotating the hollow cylinder by 90 degrees and pressing down to the protruding structure of the liquid storage tank to seal the liquid channel; the piston is pushed in and the liquid can only be pushed back to the reservoir through the silicon hydroxyl membrane. Rotating the liquid storage tank, and finishing the same operation in the liquid storage tanks of the first cleaning solution, the second cleaning solution and the eluent in sequence. The eluent liquid storage pool can be used as an amplification reaction cavity. The eluent liquid storage tank can only store molecular biology grade water, a syringe is used for pumping the water, a pushing piston pushes the water through a silicon hydroxyl membrane, and the nucleic acid after elution and purification is transferred to another reaction cavity for amplification reaction.

Further, the nucleic acid extraction method can be realized manually, and can also be completed by controlling a plurality of stepping motors through the cooperation of a smart phone and a single chip microcomputer.

Another aspect of the embodiments of the present invention also provides a nucleic acid amplification detecting apparatus, including:

In some embodiments, the nucleic acid amplification detection apparatus further comprises a microprocessor unit for controlling at least the operation states of the temperature control unit, the excitation unit and the detection unit.

In some embodiments, the microfluidic chip includes a reaction chamber, a distribution chamber, a micro channel at least for communicating the chambers, a sample well, and an overflow hole, wherein the reaction chamber is at least used for storing a PCR reaction system, and the distribution chamber is at least used for storing a primer and a probe for detecting a nucleic acid.

In some embodiments, the excitation unit includes an LED light source disposed above a filter assembly, the filter assembly at least configured to generate excitation light of a desired wavelength.

Further, the LED light source includes a four-color LED light source, but is not limited thereto.

Further, the filtering component includes a single pass filter, a two pass filter or a multi pass filter, and particularly preferably a three pass filter, but is not limited thereto.

Further, the LED light source is connected to the microprocessor unit via an LED driving assembly (e.g., a constant current LED drive may be employed).

In some embodiments, the detection unit includes an intelligent mobile terminal having more than one fluorescent image acquisition assembly, and more than one filter assembly, the fluorescent image acquisition assemblies are disposed above the filter assembly in a one-to-one correspondence, and a plano-convex lens is disposed between the fluorescent image acquisition assemblies and the filter assembly, and is at least used for converging fluorescence onto the fluorescent image acquisition assemblies.

Furthermore, the intelligent mobile terminal is connected with the microprocessor unit through a Bluetooth module.

Furthermore, the detection system further comprises a switch power supply module or a high-capacity charging power supply, preferably a switch power supply module, which is at least used for providing power for the intelligent mobile terminal and the nucleic acid amplification detection device.

Further, the switching power supply module or the charging power supply is electrically connected with the microprocessor unit, preferably, a low dropout regulator is adopted for connection.

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

Further, the fluorescence image acquisition assembly comprises a camera assembly, and a built-in camera is preferably adopted.

In some embodiments, the temperature control unit comprises a heating and cooling assembly and a temperature sensing assembly, wherein the heating and cooling assembly is at least used for rapidly heating or rapidly cooling and enabling the temperature in the reaction chamber to rapidly change so as to achieve rapid thermal cycling required by PCR reaction.

Further, the heating and cooling assembly includes a thermoelectric semiconductor cooling device, but is not limited thereto.

Further, the temperature sensing assembly includes a thermocouple sensor, but is not limited thereto.

In some embodiments, the heating and cooling assembly is connected with the microprocessor unit through a high-power bidirectional H-bridge driving assembly, and the temperature sensing assembly is connected with the microprocessor unit through a thermocouple temperature measuring converter.

Further, the microprocessor unit includes a single chip microcomputer, but is not limited thereto.

In some more specific embodiments, the nucleic acid amplification detecting apparatus based on real-time quantitative PCR may include:

(1) the smart phone is provided with a plurality of built-in cameras, and each physical camera can independently control the acquisition of images;

(2) according to the original application (close-up shooting, telephoto shooting and the like) of the mobile phone camera, a lens is fixed in front of the camera according to the requirement, so that the micro-fluidic chip about 10cm in front of the camera can clearly image;

(3) fixing an emission color filter with a certain bandwidth, such as a full width at half maximum (FWHM) of 1nm-100nm, in front of the camera or the camera and the lens; the color filter can be single-pass, double-pass or multi-pass and is used for collecting specified fluorescent signals;

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

(5) fixing an excitation color filter with a certain bandwidth, such as a full width at half maximum (FWHM) of 1nm-100nm, in front of the LED light source, wherein the excitation color filter is matched with the central wavelength of the light source; or a plurality of LED light sources share a multi-pass color filter matched with the respective central wavelength;

(6) the peltier (thermoelectric semiconductor cooler) has very small thermal inertia and fast cooling and heating time. In combination with real-time temperature detection, the singlechip drives the Peltier through a high-power H-bridge driving assembly, and the rapid temperature thermal cycle required by PCR amplification reaction is realized through an automatic temperature control algorithm;

(7) the disposable microfluidic chip can perform PCR reaction in a reaction cavity of the chip;

(8) the temperature probe is a thermocouple or a thermistor temperature detection sensor and the like;

(9) auxiliary hardware, such as chip in-out motor, position sensor, etc.;

(10) the microprocessor unit is used for acquiring temperature and controlling auxiliary hardware and the like; the smart phone is communicated with the singlechip through Bluetooth;

(11) the smart phone application comprises a communication module with a single chip microcomputer, an image acquisition and analysis module, a user interface module and a central database/expert system communication module;

(12) the system can be powered by a high-capacity or rechargeable lithium battery of a switching power supply module;

(13) a portable case that securely holds all the components together to form a compact device; forming a closed fluorescent signal collecting space.

In another aspect, the present invention also provides the use of the aforementioned nucleic acid amplification detection device in the field of nucleic acid amplification detection.

In another aspect of the embodiments of the present invention, there is provided a method for detecting nucleic acid amplification using the nucleic acid amplification detection apparatus, including:

In some more specific embodiments, the detection method includes:

placing a sample to be detected in the sample adding hole, and placing a PCR reaction system in a reaction chamber;

the method is characterized in that a temperature control unit is adopted to carry out multi-period rapid thermal cycle on the micro-fluidic chip by taking three stages of high-temperature denaturation (usually about 95 ℃), low-temperature annealing (usually about 60 ℃), isothermal amplification (usually about 72 ℃) and the like as a period, so that the temperature in a reaction chamber is rapidly changed to carry out PCR reaction, a temperature sensor is adopted to detect the temperature of the reaction chamber in real time, and a singlechip is used for realizing automatic temperature control by driving Peltier through a high-power bidirectional H bridge through an automatic control algorithm.

Further, the method comprises: and (3) monitoring and collecting the fluorescence intensity of the PCR reaction product in real time, and analyzing the result through an amplification curve of which the fluorescence intensity changes along with time.

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

More specifically, the inventor designs 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 provided with a small thermal cycler, an LED four-color power light source, a fixed multi-channel filtering component and a multi-camera smart phone-based multi-channel fluorescence real-time detection system. Firstly, the device improves portability by eliminating mechanical moving parts, and adopts a fixed multi-channel filter to improve the speed of multi-color fluorescence detection; a multicolor fluorescence detection method is developed, a multicolor LED light source with adjustable brightness and switchable color and a plurality of individually controllable rear cameras in a smart phone synchronously switch fluorescence detection channels, and the multicolor fluorescence sequential detection method is realized. Secondly, in order to realize the rapid temperature thermal cycle of PCR, a single chip microcomputer is adopted to control a Peltier (thermoelectric semiconductor cooler), and a thermal cycle instrument capable of rapidly heating and cooling is realized through an automatic temperature control algorithm. And finally, Android APP software is developed on the smart phone, a plurality of built-in cameras of the smart phone are controlled to acquire fluorescence images and perform real-time image processing, and the self-made PCB control board is controlled through a Bluetooth communication technology to realize rapid thermal cycle, light source brightness adjustment, color switching and nucleic acid extraction. In view of the advantages of high integration and high mobility of functions, the device based on the smart phone is very suitable for rapid nucleic acid amplification and real-time quantitative analysis in a non-laboratory environment with limited resources.

The invention realizes multicolor fluorescence detection by combining a plurality of physical cameras of the smart phone with a plurality of LEDs with different central wavelengths, compared with the traditional mode of switching different fluorescence channels through mechanical motion, the excitation light source of the multicolor fluorescence detection controls the switch and the intensity in an electronic mode, the collection is independently carried out by matching the plurality of physical cameras of the smart phone with the emission color filter, the 4-6 channel fluorescence collection can be realized, the multi-pass emission color filter is utilized, the fluorescence channel can be expanded to 12-18 channels, no mechanical motion part is provided, the system failure rate is reduced, and the portability is improved.

The technical solutions in the embodiments of the present invention will be described in detail below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be obtained by a person skilled in the art without any inventive step based on the embodiments of the present invention, are within the scope of the present invention.

A low-cost nucleic acid extraction method and apparatus provided in an embodiment of the present invention is shown in FIGS. 1 a-1 b, and the nucleic acid extraction and purification apparatus includes an extraction mechanism, an interception mechanism, and a liquid storage mechanism.

The extraction mechanism comprises a first component and a second component which are tightly matched, wherein the first component can be a piston 10, the second component can be a cylindrical structure with a hollow structure, namely an empty cylinder 20, the empty cylinder 20 is provided with a channel 21, the piston 10 can reciprocate (i.e. suck and inject) in the channel 21, the channel 21 allows a sample containing nucleic acid to be sucked into the channel, the bottom end of the empty cylinder 20 is provided with a liquid pipeline 30, and the liquid pipeline 30 is communicated with the channel 21. The interception mechanism can be a silicon hydroxyl film 40, and the silicon hydroxyl film 40 is fixedly arranged at the bottom end of the hollow cylinder 20 through a fixed pressing ring 41. The protruding portion 55 is arranged corresponding to the outlet end of the liquid pipeline 30 one by one, and the size of the protruding portion 55 is not smaller than the diameter of the liquid pipeline 30, so that sealing and fitting are guaranteed.

The liquid storage mechanism 50 includes a first containing chamber (i.e., a first liquid reservoir 51) in which a sample containing nucleic acid is stored, a second containing chamber (i.e., a second liquid reservoir 52) in which a cleaning liquid is stored, a third containing chamber (i.e., a third liquid reservoir 53), and a third containing chamber (i.e., a fourth liquid reservoir 54) in which an eluent is stored.

The nucleic acid extracting and purifying device can further comprise a heating mechanism, the heating mechanism is annularly arranged outside the first liquid storage tank 51, and the heating mechanism can select a heating sleeve 51 a. The nucleic acid extraction and purification device further comprises a control mechanism, and the control mechanism is connected with an intelligent mobile terminal through a micro-processing unit.

The nucleic acid extraction device comprises a structure similar to a syringe and a plurality of liquid storage tanks, wherein a binding solution (a sample containing nucleic acid), a first cleaning solution, a second cleaning solution and an eluent are sequentially stored in a first liquid storage tank 51, a second liquid storage tank 52, a third liquid storage tank 53 and a fourth liquid storage tank 54. The liquid can be added before the purification is started, or can be pre-stored in a liquid storage tank, the liquid storage tank in which the liquid is pre-stored is sealed by an aluminum foil, and the liquid is punctured before the extraction is started or punctured one by one according to the requirement in the extraction process. The liquid storage tank for storing the combined liquid is also provided with a structure capable of placing a heating collar, so that the liquid can be heated as required. The bottom of the liquid storage tank is also provided with a sealing protrusion (bulge) for closing the pipeline for sucking the liquid when injecting the liquid. The structure similar to the syringe comprises a piston, an empty cylinder, a suction liquid pipeline, a silicon hydroxyl-based membrane (such as Whatman GF/F and the like), a fixed compression ring and the like. The vacuum suction created by pulling the plunger open is much less than the pressure created by pushing the plunger open and the aspiration fluid line facilitates the drawing of viscous fluids, such as tissue lysate, into the hollow barrel 20.

In a nucleic acid extraction and purification method provided in an embodiment of the present invention, as shown in fig. 2 a-2 f, the front end of the extraction mechanism, i.e., the end of the liquid pipeline 30, is placed in the first liquid reservoir 51 (fig. 2 a-2 b) containing the binding liquid, the piston 10 is pulled out, and the liquid can be sucked into the hollow cylinder 20 through the liquid pipeline 30 with a larger inner diameter and the silicon hydroxyl-based membrane 40 (fig. 2 c-2 d); rotating the empty cartridge 90 degrees and pressing down on the raised structure (i.e., the protrusion 55) of the first reservoir 51, sealing the fluid passage; the piston 10 is pushed in and the liquid can only be pushed back to the first reservoir 51 through the silicone membrane 40 (fig. 2 e-2 f). The reservoir mechanism 50 is rotated to perform the same operations in the second reservoir 52, the third reservoir 53 and the fourth reservoir 54 of the first cleaning solution, the second cleaning solution and the eluent in sequence. The fourth reservoir 54 containing the elution solution can be used as an amplification reaction chamber. The fourth reservoir 54 containing the eluent can also store only molecular biology grade water, the water is extracted by a syringe, the piston 10 is pushed to push the water through the silicon hydroxyl membrane 40, and the eluted and purified nucleic acid is transferred to another reaction cavity for amplification reaction.

The nucleic acid extraction method can be realized manually, or can be completed by operating a plurality of stepping motors through the cooperation of a smart phone and a singlechip, and the stepping electrodes are arranged on the connecting motor shaft 56.

Referring to fig. 5, a nucleic acid amplification detecting apparatus according to an embodiment of the present invention includes a microfluidic chip 100, an excitation unit 200, a temperature control unit 300, a detecting unit 400, and a microprocessor unit 500.

The microfluidic chip 100 includes a sample adding hole 130, an overflow hole 140, a distribution chamber 120, and a reaction chamber 110, as shown in fig. 6 b. The reaction chamber 110 is at least used for storing a PCR reaction system, and the distribution chamber 120 is at least used for storing primers and probes for detecting nucleic acids.

The excitation unit 200 includes four-color LED light sources 210 and a three-band-pass filter 220, and the four-color LED light sources 210 are disposed above the three-band-pass filter 220. The four color LED light sources 210 are connected to the microprocessor unit 500 through constant current LED drivers 211.

The detection unit 400 comprises a smart phone 410 and three band-pass filters 420, the smart phone 410 is provided with three built-in cameras 411, the built-in cameras 411 are arranged above the band-pass filters 420 in a one-to-one correspondence manner, and plano-convex lenses 422 are arranged between the built-in cameras 411 and the band-pass filters 420. The smart phone 410 is connected with the microprocessor unit 500 through a bluetooth module 413. The nucleic acid amplification detection apparatus further comprises a switching power supply module 412 or a charging power supply, which is at least used for providing power for the smart phone 410. The switching power supply module 412 is electrically connected to the microprocessor unit 500 through a low dropout regulator 414.

The temperature control unit 300 includes a peltier 310 and a thermocouple sensor 320, and the peltier 310 is used at least to heat and cool and thermally cycle the reaction chamber to a temperature required for a PCR reaction. The peltier 310 is connected with the microprocessor unit 500 through a high-power bidirectional H-bridge drive 311, and the thermocouple sensor 320 is connected with the microprocessor unit 500 through a thermocouple temperature measurement converter 321.

Wherein, the color filters in the dotted line area in fig. 5 can be collectively referred to as the optical assembly a area, and the constant current LED driver, the thermocouple temperature measurement converter, the switching power supply module, the bluetooth module, the low dropout regulator, and the like are all disposed on the self-made PCB control board 501 including the microprocessor unit, the temperature control unit, the bluetooth module, and the like.

Embodiment 1 Portable smart phone nucleic acid amplification detection device

The overall size of the nucleic acid amplification detecting apparatus is about 180mm × 90mm × 110mm, the weight is about 300g, the apparatus is designed by computer aided design (3D CAD) made by a 3D printer, the 3D printing material uses photosensitive resin, and the appearance of the apparatus after complete assembly is shown in FIG. 3. The smart phone adopts Hua is company Mate 20, and its back has 3 independent physical cameras, and operating system is Android 9.0, can independently control 3 physical cameras. The telephoto lens of Mate 20 has a long focal length and cannot clearly image within a distance of 10 cm. The plano-convex lens is selected for correcting focal length, such as Edmund Optics, #67-146, and a 60mm focal length plano-convex lens, so that 3 physical cameras can simultaneously image the microfluidic chip near 60 mm. Emission color filters, such as square filters of 8mm x 8mm selected from Newport, are respectively arranged in front of the 3 physical cameras, the central wavelengths are 525nm (#90037532),564nm (#90037535) and 685nm (#90063981), and the emission wavelengths correspond to FAM, HEX and Cy5 respectively (as shown in FIG. 4a and FIG. 4 b).

The fluorescence excitation light source selects a light macro 12W 4 color LED module which comprises 3 narrow-band LEDs, namely 460-465nm (50lm),520-525nm (160lm) and 620-625nm (90 lm); and 1 white LED with color temperature of 6000-7000K (220 lm). And the self-made PCB control panel is used for brightness adjustment and color switching. Excitation light of the desired wavelength is obtained with a multi-channel narrow-band filter, such as a three-channel filter, Edmund Optics, #87-237, 22@457nm, 20@530nm, 28@628nm (bandwidth @ center wavelength), corresponding to the excitation wavelengths of FAM, HEX and Cy5, respectively (see FIG. 4 a).

The fluorescence excitation light source can also be 3 LED light sources with center wavelengths of 465nm,520nm and 620nm respectively, and a square color filter with the center wavelength of newport of 8mm x 8mm is used, and the center wavelengths of 470nm (#90037531), 525nm (#90037532) and 637.5nm (#90063980) respectively correspond to the excitation wavelengths of FAM, HEX and Cy5 respectively (as shown in FIG. 4 b).

A control block diagram for implementing the functions of fast thermal cycling, LED light sources, etc., as shown in fig. 5. The Blue ball control panel based on the STM32F103 Microcontroller (MCU) is used as a control core to control functions of four-color LED light source adjustment, temperature rapid thermal cycle, stepping motor, temperature detection and the like, and the Blue ball control panel is communicated with the smart phone through a Bluetooth module. The self-made PCR control board has rich functions, and comprises an LED constant current driver (STCS05, ST Microelectronics), a thermocouple digital converter (MAX31856, Maxim Inc.) and a high-power bidirectional H-bridge driver (VNH5019, ST Microelectronics) for controlling Peltier. The microfluidic chip sample temperature was measured using a type K thermocouple (TT-K-20, OMEGA Engineering Inc.). The single chip microcomputer provides positive and negative power supplies for the Peltier by controlling the high-power bidirectional H-bridge driver to determine the heating and refrigerating states of the Peltier, and determines the heating quantity or refrigerating quantity output by the Peltier by controlling the duty ratio of Pulse Width Modulation (PWM). The microprocessor unit calculates the temperature state (heating and refrigerating) of the Peltier and the heating amount or the refrigerating amount required to be output through an automatic control PID (proportion integration differentiation) algorithm. Meanwhile, the microprocessor unit drives the LED constant-current driver through a Pulse Width Modulation (PWM) signal with fixed frequency so as to realize brightness control and color switching of the four-color LED light source. Through bluetooth module, the self-control PCB control panel can carry out wireless communication with the Android smart mobile phone. The stepping motor driving circuit is composed of a driver (L6470, ST Microelectronics).

The assembly of the disposable microfluidic chip and the peltier device is shown in fig. 6a, which is sequentially from top to bottom a disposable microfluidic chip 100, a thermal pad 330, a peltier 310, an aluminum heat sink (aluminum heat slug 340) and a turbo fan 350. Peltier (TEC1-12712, HB Inc.) is a thermoelectric semiconductor cooling device that achieves heating or cooling by the peltier effect. The peltier top is covered by a copper plate with a thermally conductive pad, which minimizes heat loss and eases heat transfer to the microfluidic chip to provide a uniform reaction temperature. The copper plate and peltier are all wrapped so that they fit closely together to promote heat conduction and form a compact heat capacity space. In addition, a 0.02 inch diameter K-type thermocouple (TT-K-20, OMEGA Engineering Inc.) was placed immediately below the microfluidic chip to measure temperature. To remove the excess heat and cool the bottom surface of the peltier, an aluminum heat sink and a turbofan are placed in close proximity to the peltier bottom surface.

The microfluidic chip comprises a sample adding hole 130, an overflow hole 140, 6 groups of distribution chambers 120 and 6 groups of reaction chambers 110, as shown in fig. 6 b. The cover sheet layer of the microfluidic chip is an optical film coated with pressure-sensitive adhesive, and before pressure is applied to seal a micro-channel and a reaction chamber, sequence-specific primers, fluorescent probes and magnesium acetate are added into a distribution chamber, and the mixture is heated and evaporated to dryness; and finally, sealing by using a pressure-sensitive optical film.

The mobile smart phone can provide a user interface with good operation experience, and can also be used for temperature and light source control, real-time quantitative fluorescence intensity detection and automatic storage control management of detection data. The inventor of the scheme uses Android Studio 3.2.1 to develop control software on a smart phone, can perform hardware control such as rapid thermal cycle and LED light source, and uses OPENCV 3.4.1 (open source computer vision library) on an Android system to perform image processing. The new camera functions recently released in Android Pie (9.0) and API version 28 allow control of a single physical camera in a multi-camera smart phone, so that independent control of multiple cameras built in the smart phone can be realized, different control parameters such as exposure time, white balance and focus position can be set for different cameras, independent control can be realized through self-development software, and finally real-time fluorescence collection of three channels can be realized by respectively setting three built-in cameras.

Example 2 nucleic acid extraction

Nucleic acid extraction reagent adopts

Reagents in Viral RNAmini kit. The sample is FNV-2019-NCoV-abeN pseudovirus provided by a compound Australian organism. Reservoir 61 stored 560 μ L100% ethanol, reservoir 62 stored 500 μ L AW1, reservoir 63 stored 500 μ L AW2, and reservoir 64 stored 100 μ L RNAse free water.

mu.L of pseudovirus (approx. 10e4 pseudovirus) or negative control (water) was added to 560. mu.LAVL and after incubation for 10 min at room temperature, added to reservoir 61 and mixed well. Using the nucleic acid extraction apparatus shown in fig. 1 a-1 b, a solution is sucked from the first reservoir 51 (as shown in fig. 2 c-2 d), the extraction apparatus is rotated 90 degrees, after the liquid pipeline 30 is compressed, the piston 10 is pushed to make the liquid pass through 1 to 5 layers of Whatman GF/F silicon hydroxyl basement membranes to carry out nucleic acid binding (as shown in fig. 2 e-2F); then the empty cylinder 20 of the extraction device is rotated 90 degrees in the opposite direction, the extraction device is moved to the second liquid storage tank 52, the solution is sucked (as shown in figures 2 c-2 d), the empty cylinder 20 of the extraction device is rotated 90 degrees, after the liquid pipeline 30 is pressed, the piston 10 is pushed to enable the liquid to pass through 1 to 5 layers of Whatman GF/F silicon hydroxyl basement membranes, and the first cleaning is carried out (as shown in figures 2 e-2F); then the empty cylinder 20 of the extraction device is rotated 90 degrees in the opposite direction, and is moved to the third liquid storage tank 53, the solution is sucked (as shown in fig. 2 c-2 d), the empty cylinder 20 of the extraction device is rotated 90 degrees, after the liquid pipeline 30 is pressed, the piston 10 is pushed to enable the liquid to pass through 1 to 5 layers of Whatman GF/F silicon hydroxyl basement membranes, and the second cleaning is carried out (as shown in fig. 2 e-2F); then the empty tube 20 of the extraction device is rotated 90 degrees in the opposite direction, and moved to the fourth reservoir 64, the solution is aspirated (as shown in fig. 2 c-2 d), the empty tube 20 of the extraction device is rotated 90 degrees, after the liquid line 30 is compressed, the piston 10 is pushed to make the liquid pass through 1 to 5 layers of Whatman GF/F silicon hydroxyl basement membrane, and the nucleic acid is eluted.

Example 3 RT-qPCR assay

RT-qPCR detection A one-step RT-qPCR kit RR064A from Takara was used. The reaction solution was prepared in the following ratio of table 1, with no water added. The primer and probe sequences are shown in Table 2 (the sequences are RT-qPCR primer and probe sequences for detecting the novel coronavirus pneumonia recommended by the U.S. CDC, 68% of the volume of a reaction cavity is added with a corresponding volume of reaction liquid, and the reaction liquid is sealed by a pressure-sensitive optical membrane after freeze-drying.

The nucleic acid extracted and purified as described in example 2 was transferred to a well, temperature cycling was done by a smartphone setting temperature program, (45 ℃/5min,95 ℃ 10sec,40x (95 ℃ 10sec,58 ℃ 30sec), by a lower computer control circuit and a Peltier heating/cooling device.three cameras of the cell phone sequentially collected one fluorescence image at the last 10 seconds of the extension temperature (58 ℃), image processing was performed using OPENCV 3.4.1 (open source computer vision library) on Android systems The position of each hole is calculated. After amplification of 100. mu.L of pseudovirus (approximately 10e4 pseudovirus) or negative control (water) as samples, the fluorescence was plotted as a function of time after analysis by software, as shown in FIGS. 7a and 7b, respectively.

TABLE 1 reaction solution Components and proportions

TABLE 2 primer and Probe sequences

In summary, according to the technical scheme, the invention can realize nucleic acid extraction and purification, the smart phone is used for controlling thermal cycle and fluorescence collection, the plurality of physical cameras of the smart phone are used for combining with the plurality of LEDs with different central wavelengths to realize multicolor fluorescence detection, compared with the traditional mode of switching different fluorescence channels through mechanical motion, an excitation light source of the multicolor fluorescence detection controls the switch and the intensity in an electronic mode, the collection is independently carried out through the cooperation of the plurality of physical cameras of the smart phone and an emission color filter, 4-6 channel fluorescence collection can be realized, the multi-pass emission color filter is used, the fluorescence channels can be expanded to 12-18 channels, mechanical motion parts are not used, the system failure rate is reduced, and the portability is improved.

The aspects, embodiments, features and examples of the present invention should be considered as illustrative in all respects and not intended to be limiting of the invention, the scope of which 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.

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

Throughout this specification, where a composition is described as having, containing, or comprising specific components or where a process is described as having, containing, or comprising specific process steps, it is contemplated that the composition of the present teachings also consist essentially of, or consist of, the recited components, and the process of the present teachings also consist essentially of, or consist of, the recited process steps.

It should be understood that the order of steps or the order in which particular actions are performed is not critical, so long as the teachings of the invention remain operable. Further, two or more steps or actions may be performed simultaneously.

In addition, the inventors of the present invention have also made experiments with other materials, process operations, and process conditions described in the present specification with reference to the above examples, and have obtained preferable results.

While the invention has been described with reference to illustrative embodiments, it will be understood by those skilled in the art that various other changes, omissions and/or additions may be made and substantial equivalents may be substituted for elements thereof without departing from the spirit and scope of the invention. In addition, many modifications may be made to adapt a particular situation or material to the teachings of the invention without departing from its scope. Therefore, it is intended that the invention not be limited to the particular embodiment disclosed for carrying out this invention, but that the invention will include all embodiments falling within the scope of the appended claims.

Claims

1. A nucleic acid extraction and purification device, characterized by comprising:

2. The nucleic acid extraction and purification device according to claim 1, wherein: the first assembly comprises a piston member; and/or the second component comprises a cylindrical structure with a hollow structure.

3. The nucleic acid extraction and purification device according to claim 1, further comprising a fixing member, wherein the intercepting structure is disposed at the bottom end of the second module through the fixing member; preferably, the fixing piece comprises a fixing compression ring;

and/or the intercepting structure comprises a porous membrane capable of adsorbing and eluting nucleic acids, preferably a silicone-based membrane having a pore size of 0.1 μm to 10 μm.

4. The nucleic acid extraction and purification device according to claim 1, wherein: the protruding parts are arranged in one-to-one correspondence with the outlet ends of the liquid pipelines; and/or the size of the bulge is not smaller than the diameter of the liquid pipeline.

5. The nucleic acid extraction and purification device according to claim 1, wherein the liquid storage mechanism comprises:

6. The nucleic acid extraction and purification device according to claim 5, further comprising a heating mechanism, wherein the heating mechanism is annularly arranged outside the first accommodating cavity; preferably, the heating mechanism comprises a heating collar; and/or, the nucleic acid extraction and purification device further comprises a control mechanism, and the control mechanism is connected with an intelligent mobile terminal through a microprocessor unit.

7. A method for extracting and purifying nucleic acid, which is mainly performed based on the nucleic acid extraction and purification apparatus according to any one of claims 1 to 6, and which comprises:

8. A nucleic acid amplification detecting apparatus characterized by comprising:

9. The apparatus for detecting nucleic acid amplification of claim 8, further comprising a microprocessor unit for controlling at least the operation states of the temperature control unit, the excitation unit, and the detection unit.

10. The nucleic acid amplification detection apparatus according to claim 8, wherein: the microfluidic chip comprises a reaction chamber, a distribution chamber, a micro-channel at least used for communicating the chambers, a sample adding hole and an overflow hole, wherein the reaction chamber and the distribution chamber are communicated with each other, the reaction chamber is at least used for storing a PCR reaction system, and the distribution chamber is at least used for storing primers and probes used for detecting nucleic acid.

11. The nucleic acid amplification detection apparatus according to claim 9, wherein: the excitation unit comprises an LED light source and a filter component, the LED light source is arranged above the filter component, and the filter component is at least used for generating excitation light with required wavelength; preferably, the LED light source comprises a four-color LED light source; preferably, the filtering component comprises a single-pass filter or a multi-pass filter, particularly preferably a three-pass filter; preferably, the LED light source is connected to the microprocessor unit through an LED driving assembly.

12. The nucleic acid amplification detection apparatus according to claim 8, wherein: the detecting unit comprises an intelligent mobile terminal with more than one fluorescent image collecting assembly and more than one filtering assembly, wherein the fluorescent image collecting assemblies are in one-to-one correspondence with the filtering assemblies, and a plano-convex lens is arranged between the fluorescent image collecting assemblies and the filtering assemblies.

13. The nucleic acid amplification detecting apparatus according to claim 12, wherein: the intelligent mobile terminal is connected with the microprocessor unit through a Bluetooth module; and/or, the detection system further comprises a switch power supply module or a charging power supply, preferably a switch power supply module, at least used for providing power supply for the intelligent mobile terminal and the nucleic acid amplification detection device, and the switch power supply module or the charging power supply is electrically connected with the microprocessor unit;

and/or the intelligent mobile terminal comprises an intelligent mobile phone, a tablet personal computer or an ARM-based microcomputer mainboard, and the fluorescent image acquisition assembly comprises a camera assembly, preferably a built-in camera.

14. The nucleic acid amplification detection apparatus according to claim 8, wherein: the temperature control unit comprises a heating and refrigerating assembly and a temperature sensing assembly, wherein the heating and refrigerating assembly is at least used for rapidly heating or rapidly refrigerating and enabling the temperature in the reaction chamber to rapidly change so as to achieve rapid thermal cycle required by PCR reaction; preferably, the heating and cooling assembly comprises a thermoelectric semiconductor cooling device; preferably, the temperature sensing assembly comprises a thermocouple sensor; preferably, the heating and refrigerating assembly is connected with the microprocessor unit through a bidirectional H-bridge driving assembly, and the temperature sensing assembly is connected with the microprocessor unit through a thermocouple temperature measuring converter; and/or the microprocessor unit comprises a singlechip.

15. Use of the nucleic acid amplification test device of any one of claims 8-14 in the field of nucleic acid amplification test, wherein the method for performing nucleic acid amplification test by using the nucleic acid amplification test device comprises:

16. Use according to claim 15, characterized in that the method comprises: and (3) monitoring and collecting the fluorescence intensity of the PCR reaction product in real time, and analyzing the result through an amplification curve of which the fluorescence intensity changes along with time.

17. A system for extracting, purifying and amplifying nucleic acid, which is characterized by comprising:

the nucleic acid extraction and purification apparatus according to any one of claims 1 to 6; and

the nucleic acid amplification detection apparatus according to any one of claims 8 to 14.

18. A method for extracting, purifying and amplifying nucleic acid, which is characterized by comprising the following steps:

performing nucleic acid extraction and purification according to the method of claim 7; and

nucleic acid amplification and detection according to the method of claim 15 or 16.