WO2021134511A1 - Full-integrated small-scale chip type digital pcr detection system and detection method - Google Patents

Abstract

A full-integrated small-scale chip type digital PCR detection system and detection method. The PCR detection system comprises a pump valve machine module, a temperature control module, an optical detection module, a circuit control module, and a PCR detection chip; the pump valve machine module comprises a chip feeding part and an air pressure driving part; the temperature control module is provided on the chip feeding part, moves along with the PCR detection chip in parallel, and is used for controlling a reaction temperature of the PCR detection chip; the optical detection module comprises one or more optical detection means; a reaction reagent is injected in the PCR detection chip in advance, and a blood sample and the reaction reagent undergo a PCR reaction in the PCR detection chip; the circuit control module is used for transmitting an electric signal and completing the execution of a control command.

Description

A fully integrated and miniaturized chip-type digital PCR detection system and detection method Technical field

The application belongs to the technical field of nucleic acid detection, and relates to a PCR detection system and detection method, and in particular to a fully integrated and miniaturized chip-type digital PCR detection system and detection method.

Background technique

The application of in vitro polymerase chain reaction (PCR) makes in vitro DNA amplification possible. Qualitative analysis of DNA amplified products can be achieved by agarose gel electrophoresis after PCR amplification. As the demand for quantitative DNA detection continues to grow, traditional PCR based on electrophoretic analysis is gradually replaced by quantitative PCR (qPCR) analysis, that is, real-time quantitative PCR (RT-qPCR). However, fluorescent quantitative PCR generally uses PCR tubes to react and collect fluorescence. All nucleic acid templates exist in the same reaction system. Non-specific amplification will increase false positive results and background noise. Due to its sensitivity limitations, it is impossible to distinguish low-power nucleic acids. The copy number is variable, so it is still impossible to obtain an absolute quantitative test result. In order to achieve the purpose of absolute quantitative detection, digital PCR came into being. In digital PCR (dPCR), the DNA template is dispersed into thousands of microsystems for amplification in microdroplets or microcompartments separated from each other, which has low noise and the ability to capture low mutation signals.

The process of digital PCR for nucleic acid detection is generally divided into four steps: nucleic acid sample extraction, sample dispersion, sample amplification, and amplification signal detection. Up to now, commercial digital PCR instruments are mainly based on technologies such as water-in-oil (W/O) droplets, microplate chips, and microfluidic channels.

CN107904156A discloses an integrated fully automated digital PCR detection system. The system includes a left chamber and a right chamber. The left chamber and the right chamber are separated by an automatic isolation door device; the left chamber is provided with a droplet generating device The right chamber is provided with a PCR amplification device and a biochip reading device; the droplet generating device is used to extract the premix, and finally the premix is added to the liquid biological reaction system, thereby realizing the loading of nucleic acid samples into the biological In the chip; the PCR amplification device is used to amplify the nucleic acid of the biochip; the biochip reading device is used to automatically identify the nucleic acid information carried by the amplified biochip nucleic acid sample; also includes a negative pressure device, left Both the chamber and the right chamber are in communication with the inlet of the negative pressure device. The negative pressure device makes the left and right chambers form a negative pressure, and the air is discharged through the negative pressure device.

CN106834114A An automated detection system based on PCR applications, which consists of a punching system, an automatic reagent addition system, a mechanical arm system, a centrifuge, an intelligent control system, a transfer mechanism, a PCR detection system and a data analysis system; the punching system The system is responsible for making filter paper dried blood spots and extracting genetic material; the automatic reagent addition system is responsible for adding trace reagents to the reaction vessel; the robotic arm system is responsible for grabbing the reaction vessel; the centrifuge is responsible for fixing and carrying The reaction container performs centrifugal movement to complete the solid-liquid separation work; the conveying mechanism is responsible for conveying the reaction container to the robotic arm system, centrifuge, automatic reagent addition system and PCR detection system; the intelligent control system is connected to the printer via an interface. Hole system, robotic arm system, centrifuge, conveying mechanism, automatic reagent addition system and PCR detection system, and set its working parameters and control working status; the PCR detection system includes fluorescence detection components, heating devices and thermal sensors 10, Responsible for providing a different constant temperature reaction environment for the supernatant with genetic material and real-time detection of the amount of fluorescent signal in the sample; the data analysis system comprehensively processes and analyzes the data transmitted by the PCR detection system to obtain the immunodeficiency disease Test results.

CN208949317U discloses a digital PCR chip and a digital PCR detection system. The digital PCR chip includes a chip body with a droplet storage cavity and a liquid inlet provided on the chip body. The digital PCR chip also includes a chip body and A containing cavity connected to the liquid inlet, and a liquid discharge port provided on the chip body; the chip body also includes a first channel that respectively communicates the liquid inlet and the droplet storage cavity, the liquid discharge port, and the droplet storage cavity The connected second channel, the first channel has a first inner channel located inside the chip body, and the second channel has a second inner channel located inside the chip body. After the droplets are generated in the containing cavity, they enter the droplet storage cavity through the liquid inlet and the first channel. The droplets can maintain good stability and sealing during the transportation process and realize the droplets in the droplet storage cavity. Medium and uniform single-layer or multi-layer tiling is conducive to obtaining significantly more accurate detection results.

However, these digital PCR instruments currently have some shortcomings. For example, the operating procedures are complicated. Generally, three separate devices are required to complete droplet generation, nucleic acid amplification, and result detection. In addition, the split-type design requires professionals to operate and increase The labor cost is reduced; the multi-step sample transfer operation caused by the split design is prone to pollution, which makes the test results lose credibility; the fully integrated, user-friendly integrated design can meet the testing needs in a variety of scenarios to the greatest extent. The above reasons all limit the development of digital PCR instruments and the further widespread application of digital PCR technology to a certain extent. It is of great significance to design and develop an integrated "sample in-result out" digital PCR detection system that integrates droplet generation, nucleic acid amplification and amplification signal detection.

Application content

In view of the shortcomings of the prior art, the purpose of this application is to provide a fully integrated and miniaturized chip-based digital PCR detection system and detection method, which integrates the integration of droplet generation, nucleic acid amplification and amplification signal detection The "sample in-result out" digital PCR detection system has a compact structure, simple operation, can effectively avoid contamination, and completes digital PCR amplification and detection in a short time. .

To achieve this goal, this application adopts the following technical solutions:

In the first aspect, this application provides a fully integrated and miniaturized chip-based digital PCR detection system. The PCR detection system includes a pump valve mechanical module, a temperature control module, an optical detection module, a circuit control module, and a PCR detection chip.

The pump valve mechanical module includes a chip feeding part and an air pressure driving part. The chip feeding part is used to feed the PCR detection chip into the designated area. After the PCR detection chip is sent to the designated area, the air pressure driving part The sequence flow of reaction reagents in the PCR detection chip is controlled by air pressure to complete the PCR reaction.

The temperature control module is arranged on the chip feeding component, and the temperature control module moves in parallel with the PCR detection chip for controlling the reaction temperature of the PCR detection chip.

The optical detection module includes at least one optical detection device, and the optical detection device is used to take a picture of the PCR detection chip to obtain a fluorescence photo after the PCR reaction is completed.

Reaction reagents are pre-injected into the PCR detection chip, and the blood sample and the reaction reagents undergo PCR reaction in the PCR detection chip.

The circuit control module is used to transmit electrical signals and complete the execution of control commands.

The fully integrated and miniaturized chip-based digital PCR system provided by this application avoids the use of three instruments (droplet generator, thermal cycler, and The transfer steps included in the reader) may cause cross-contamination and human error of nucleic acid samples. In addition, the system integrates nucleic acid extraction and purification, microdroplet formation, digital nucleic acid amplification, and result detection. It realizes the "sample in-result out" detection mode, which greatly simplifies human operation steps, saves manpower, and improves Improve the detection efficiency and shorten the detection cycle. At the same time, the open structure design is compatible with the droplet-type digital PCR detection chip, which can meet the PCR temperature control and fluorescence imaging requirements including the droplet-type and chip-type digital PCR chip.

As a preferred technical solution of the present application, the chip feeding component includes a bottom plate on which a ball screw and a driving motor connected to one end of the ball screw are fixed.

Preferably, the driving motor drives the ball screw to rotate through a synchronous pulley.

Preferably, a chip tray is provided on the ball screw, and the chip tray moves along the ball screw.

Preferably, sensors are provided at both ends of the ball screw, and the sensors are used to detect the position of the chip tray on the ball screw.

Preferably, the pneumatic driving component includes a positive pressure air pump and a lower pressure shut-off valve array connected with the positive pressure air pump.

Preferably, the positive pressure air pump includes a cylinder, a solenoid valve and a separate air pressure interface connected in sequence.

Preferably, the lower pressure cut-off valve array includes 8 lower pressure cut-off valves independent of each other.

Preferably, the separate air pressure ports are independently connected to the down-pressure shut-off valve.

Preferably, the lower pressure cut-off valve includes a lower pressure head, and the lower pressure head is driven by the air pressure to move in a vertical direction so as to control the liquid flow in the PCR detection chip.

Preferably, the lower indenter is externally connected with a stepping motor, and the stepper motor is used to control the displacement distance of the lower indenter in the vertical direction.

Preferably, a contact switch is arranged above the stepping motor, and the contact switch is used for positioning the zero position of the lower indenter in the vertical direction.

Preferably, the pneumatic drive component further includes a limit plate, and the limit plate is provided with a same number of positioning holes as the lower indenter, and the lower indenter penetrates into the positioning hole to limit the lower position. The relative position between the indenters.

As a preferred technical solution of the present application, the temperature control module is fixed at the bottom of the chip tray, and the temperature control module moves in parallel along the ball screw with the chip tray.

In this application, the chip feeding component is integrated with the temperature control module, and the temperature control module can move inside the instrument along with the chip, which can avoid problems such as uneven heating caused by separate heating or affecting the focus of subsequent photographing.

Preferably, the temperature control module includes a temperature control component and a heat dissipation component, the temperature control component is fixed on the bottom of the chip tray, and the heat dissipation component is located on both sides of the temperature control component.

Preferably, the temperature control component includes a semiconductor heating chip and/or a semiconductor refrigeration chip.

Preferably, the heat dissipating component includes a heat dissipating fin and a heat dissipating fan located at one end of the heat dissipating fin.

Preferably, the temperature control module further includes a temperature sensor, and the temperature sensor is close to the bottom of the temperature control component.

As a preferred technical solution of the present application, the optical detection module includes an optical path support and at least two optical detection devices fixed side by side on the optical path support.

Preferably, the optical detection device includes a housing, and a detection optical path module is arranged inside the housing.

Preferably, the detection light path module includes a transmitting end module and a receiving end module, and the excitation light emitted by the transmitting end module is reflected by the PCR detection chip and then received and imaged by the receiving end module.

Preferably, the emitting end module includes an LED light source, a homogenizing mirror, a narrow band filter at the emitting end, and a dichroic mirror arranged at intervals along the excitation light path. The dichroic mirror is arranged obliquely, The excitation light filtered by the phase color mirror is irradiated to the PCR detection chip to be reflected.

Preferably, the receiving end module includes a receiving end narrow-band filter, lens and camera arranged in sequence along the reflected light optical path.

Preferably, the housing is made of aluminum alloy material.

As a preferred technical solution of the present application, the circuit control module includes a power supply circuit, a drive circuit, a control circuit, an information processing and transmission circuit, and embedded software.

Preferably, the power supply circuit is connected to direct current and is electrically connected to each power consumption module.

Preferably, the driving circuit is used to amplify the signal of the control circuit.

Preferably, the control circuit is used to control the movement of each module.

Preferably, the information processing and transmission circuit is connected to the client system, and the information processing and transmission circuit is used to transmit the detection results and the fluorescent photos obtained by shooting to the client.

Preferably, the embedded software includes instrument software and middleware, and the instrument software is written into the control circuit for behavior control and command execution of drive-type equipment; the middleware writes information processing and In the transmission circuit, it is used to complete the data communication and resource transmission between the digital PCR detection system and the client.

As a preferred technical solution of the present application, the PCR detection chip includes an upper layer board and a lower layer board arranged in a stack.

Preferably, the thickness of the upper layer board is 2-6mm, for example, it can be 2mm, 3mm, 4mm, 5mm or 6mm, but it is not limited to the listed values, and other unlisted values within this range of values are also applicable; further Preferably, the thickness of the upper layer board is 2 mm.

Preferably, the thickness of the lower layer board is 4-8mm, for example, it can be 4mm, 5mm, 6mm, 7mm or 8mm, but it is not limited to the listed values, and other unlisted values within this range of values are also applicable; further Preferably, the thickness of the lower plate is 5mm.

Preferably, the material of the upper layer board and the lower layer board are both polymethyl methacrylate.

As a preferred technical solution of the present application, the upper layer plate is provided with 8 baffle holes, and the position of the baffle holes corresponds to the position of the lower pressure head one-to-one.

Preferably, the diameter of the blocking hole is 2 to 3 mm, for example, it can be 2.0 mm, 2.1 mm, 2.2 mm, 2.3 mm, 2.4 mm, 2.5 mm, 2.6 mm, 2.7 mm, 2.8 mm, 2.9 mm or 3.0 mm. , But not limited to the listed values, and other unlisted values within this range of values are also applicable; further preferably, the diameter of the choke hole is 2.5 mm.

Preferably, a diaphragm is fixed in the choke hole, and the lower pressing head is located above the diaphragm, and as the lower pressing head reciprocates in the vertical direction, the diaphragm is pressed down to deform or return to its original shape.

Preferably, the material of the diaphragm is polydimethylsiloxane.

Preferably, the upper layer plate is also provided with sample loading holes, exhaust holes and air pressure holes, and the air pressure holes are combined with the separated air pressure interface.

As a preferred technical solution of the present application, the lower board is provided with four unit liquid storage tanks, a cleaning liquid tank and a waste liquid tank.

Preferably, the bottoms of the unit liquid storage tank, the cleaning liquid tank and the waste liquid tank are all provided with drain holes.

Preferably, the diameter of the drain hole is 1 to 2 mm, for example, it can be 1.0 mm, 1.1 mm, 1.2 mm, 1.3 mm, 1.4 mm, 1.5 mm, 1.6 mm, 1.7 mm, 1.8 mm, 1.9 mm or 2.0. mm, but not limited to the listed values, other unlisted values within this range of values are also applicable.

Preferably, the unit liquid storage tank, the cleaning liquid tank and the waste liquid tank are all S-shaped structural grooves.

Preferably, an air pressure channel is provided along the outer edge of the lower board, and the air pressure channel communicates with the air pressure hole opened on the upper board.

Preferably, the unit liquid storage tank and the cleaning liquid tank are respectively independently connected to the air pressure channel.

Preferably, the width of the air pressure channel is 1-2mm, for example, it can be 1.0mm, 1.1mm, 1.2mm, 1.3mm, 1.4mm, 1.5mm, 1.6mm, 1.7mm, 1.8mm, 1.9mm or 2.0mm. , But not limited to the listed values, and other unlisted values within this range of values are also applicable.

Preferably, the depth of the air pressure channel is 0.5 to 1 mm, for example, it can be 0.5 mm, 0.6 mm, 0.7 mm, 0.8 mm, 0.9 mm, or 1.0 mm, but it is not limited to the listed values, and other values within the range Values not listed also apply.

Preferably, the lower plate is also provided with 4 reaction chambers connected in sequence.

Preferably, the depth of the reaction chamber is 2 to 3 mm, for example, it can be 2.0 mm, 2.1 mm, 2.2 mm, 2.3 mm, 2.4 mm, 2.5 mm, 2.6 mm, 2.7 mm, 2.8 mm, 2.9 mm or 3.0. mm, but not limited to the listed values, other unlisted values within this range of values are also applicable.

Preferably, different reaction reagents are pre-stored in the unit liquid storage tank.

Preferably, according to different stored reaction reagents, the unit storage tank is divided into a cell lysis reaction reagent storage tank, a nucleic acid extraction reaction reagent storage tank, a nucleic acid purification reaction reagent storage tank, and a PCR reagent storage tank.

Preferably, the reaction chamber is divided into a cell lysis reaction chamber, a nucleic acid extraction reaction chamber, a nucleic acid purification reaction chamber, and a PCR reagent mixing chamber, which are sequentially connected according to the different PCR reaction stages.

Preferably, an FTA card is pre-placed in the nucleic acid extraction reaction chamber, and the FTA card is used to grab and release nucleic acid.

Preferably, Mix, primers and probes required for PCR reaction are pre-placed in the PCR reagent mixing chamber.

Preferably, the four reaction chambers are independently connected to the corresponding unit liquid storage tanks, and the reaction reagents stored in the unit liquid storage tanks are injected into the corresponding reaction chambers through the reaction reagent channel to react with the blood sample.

Preferably, the reaction reagent channel is provided with a choke groove corresponding to the position of the diaphragm, and the shape of the choke groove matches the shape of the corresponding diaphragm after being pressed and deformed. The reset realizes the blocking or dredging of the choke groove so as to control the liquid flow in the reaction reagent channel.

Preferably, the cell lysis reaction chamber and the nucleic acid extraction reaction chamber are independently connected to the cleaning solution pool. After the reaction, the cleaning solution stored in the cleaning solution pool is injected into the cell lysis reaction chamber and the nucleic acid extraction reaction chamber through the cleaning solution channel. Clean.

Preferably, a choke groove corresponding to the position of the diaphragm is provided on the cleaning fluid channel, and the shape of the choke groove matches the shape of the corresponding diaphragm after being pressed and deformed. The reset realizes the blocking or dredging of the choke groove so as to control the liquid flow in the cleaning liquid channel.

Preferably, the cell lysis reaction chamber, the nucleic acid extraction reaction chamber and the nucleic acid purification reaction chamber are respectively independently connected to the waste liquid pool, and the waste liquid generated after the reaction flows into the waste liquid pool through the waste liquid recovery channel for centralized discharge.

Preferably, the waste liquid recovery channel is provided with a choke groove corresponding to the position of the diaphragm, and the shape of the choke groove matches the shape of the corresponding diaphragm after being pressed and deformed. Or reset to block or unclog the choke groove so as to control the liquid flow in the waste liquid recovery channel.

Preferably, the connecting channel between the nucleic acid purification reaction chamber and the PCR reagent mixing chamber is provided with a baffle groove corresponding to the position of the diaphragm, and the shape of the baffle groove is corresponding to the shape of the diaphragm after the corresponding diaphragm is pressed and deformed. The shape of the filter is matched, and the blocking or dredging of the choke groove is realized by pressing down or resetting the diaphragm, so as to control the liquid in the nucleic acid purification reaction chamber to flow into the PCR reagent mixing chamber.

Preferably, the lower plate is also provided with a chip slot communicating with the PCR reagent mixing chamber.

Preferably, a microporous digital PCR chip and PCR thermally conductive components are placed in the chip slot.

Preferably, the chip groove is a stepped groove, the microporous digital PCR chip is located in the upper groove, and the PCR thermally conductive component is located in the lower groove.

Preferably, the PCR thermally conductive component is a thermally conductive aluminum block.

Preferably, a strip-shaped pretreatment heat-conducting component is embedded at the bottom of the lower plate, and the pre-treatment heat-conducting component is located below the cell lysis reaction chamber, the nucleic acid extraction reaction chamber and the nucleic acid purification reaction chamber.

Preferably, the pretreatment heat-conducting component is a heat-conducting aluminum block.

In the second aspect, this application provides a fully integrated and miniaturized chip-based digital PCR detection method, which uses the chip-based digital PCR detection system described in the first aspect to perform digital PCR detection on blood samples.

The detection method includes:

(I) The blood sample is pretreated into a suspension sample, the suspension sample is injected into the PCR detection chip, and the chip feeding component sends the PCR detection chip into the biological reaction area;

(Ⅱ) The air pressure driving component controls the sequential flow of reaction reagents in the PCR detection chip by air pressure driving. The suspension sample and different reaction reagents sequentially undergo cell lysis, nucleic acid extraction, nucleic acid purification and PCR reactions. During this process, the temperature control module Heating the different reaction areas of the PCR detection chip;

(Ⅲ) After the reaction is over, the optical detection module emits a light source to irradiate the reaction sample to excite the fluorescent signal points, and the fluorescent signal is photographed and collected and transmitted to the circuit control module;

(IV) The circuit control module performs photoelectric signal conversion on the collected optical signals and transmits them to the client for data analysis.

As a preferred technical solution of the present application, in step (I), the suspension sample is injected into the PCR detection chip through the sample hole.

Preferably, the PCR detection chip injected with the suspension sample is fixed on the chip tray, and the chip feeding component sends the PCR detection chip under the pressure cut-off valve array.

Preferably, in step (II), the pneumatic drive control mode is:

The cylinder is continuously filled with gas to generate positive pressure, and the air pressure is connected to different pressure stop valves through the separate air pressure interface. The pressure is driven by the opening and closing of the solenoid valve to control the pressure to move the lower pressure head back and forth in the vertical direction, and the lower pressure head moves down. When the diaphragm is compressed, the diaphragm deforms to block the flow blocking groove and cut off the liquid flow channel where the flow block is located; the lower pressure head rises away from the diaphragm, and the diaphragm resets and separates from the flow block to clear the liquid flow channel where the flow block is located.

Preferably, step (II) specifically includes the following steps:

(1) The suspension sample flows into the cell lysis reaction chamber, and the reaction reagent channel between the cell lysis reaction reagent reservoir and the cell lysis reaction chamber is cleared through the described air pressure driving control method, and the reaction stored in the cell lysis reaction reagent reservoir is cleared. Reagents are injected into the cell lysis reaction chamber and the suspension sample undergoes a cell lysis reaction; after the reaction is completed, the cleaning solution channel between the cleaning solution pool and the cell lysis reaction chamber is cleared through the air pressure driving control method, and the cleaning solution stored in the cleaning solution pool Inject into the cell lysis reaction chamber for cleaning; after the cleaning, the waste liquid recovery channel between the waste liquid pool and the cell lysis reaction chamber is cleared through the air pressure drive control method, and the waste liquid in the cell lysis reaction chamber flows into the waste liquid pool;

(2) After the cell lysis reaction is completed, the suspension sample flows from the cell lysis reaction chamber into the nucleic acid extraction reaction chamber, and the reaction reagent channel between the nucleic acid extraction reaction reagent reservoir and the nucleic acid extraction reaction chamber is cleared through the described air pressure driving control method , The reaction reagents stored in the nucleic acid extraction reaction reagent reservoir are injected into the nucleic acid extraction reaction chamber and the suspension sample undergoes nucleic acid extraction reaction; after the reaction is completed, the air pressure drive control method is used to clear the gap between the cleaning solution pool and the nucleic acid extraction reaction chamber Cleaning fluid channel, the cleaning fluid stored in the cleaning fluid pool is injected into the nucleic acid extraction reaction chamber for cleaning; after cleaning, the waste fluid recovery channel between the waste fluid pool and the nucleic acid extraction reaction chamber is dredged through the air pressure driving control method, and nucleic acid extraction The waste liquid in the reaction chamber flows into the waste liquid pool;

(3) After the nucleic acid extraction reaction is completed, the suspension sample flows from the nucleic acid extraction reaction chamber into the nucleic acid purification reaction chamber, and the reaction reagent channel between the nucleic acid purification reaction reagent reservoir and the nucleic acid purification reaction chamber is cleared through the described air pressure driving control method , The reaction reagents stored in the nucleic acid purification reaction reagent storage tank are injected into the nucleic acid purification reaction chamber and the suspension sample undergoes nucleic acid purification reaction; after the reaction is completed, the air pressure drive control method is used to clear the gap between the waste liquid pool and the nucleic acid purification reaction chamber Waste liquid recovery channel, the waste liquid in the nucleic acid purification reaction chamber flows into the waste liquid pool;

(4) After the nucleic acid purification reaction is completed, the suspension sample flows from the nucleic acid purification reaction chamber into the PCR reagent mixing chamber, and the reaction reagent channel between the PCR reagent storage pool and the PCR reagent mixing chamber is cleared through the air pressure driving control method. The PCR reagent stored in the reagent storage pool is injected into the PCR reagent mixing chamber and mixed with the suspension sample to generate droplets;

(5) The droplet enters the chip slot and completes digital PCR amplification with the microporous digital PCR chip.

The numerical range described in this application not only includes the above-mentioned exemplified point values, but also includes any point value between the above-mentioned numerical ranges that are not exemplified. Due to the limitation of space and for the sake of brevity, this application will not exhaust all the points The specific point value included in the stated range.

The system refers to an equipment system, a device system, or a production device.

This application exemplarily provides a manual one-step implementation step of the PCR detection system, which specifically includes:

S1, connect the 28V power supply and data line, start the system power switch;

S2. Click the "IN/OUT" button on the front panel, and the chip tray will be out of the warehouse;

S3. Aspirate the sample to be tested and inject it into the injection hole on the PCR detection chip;

S4. Put the PCR detection chip into the chip tray, click the "IN/OUT" button on the front panel, and the chip tray will be put into the warehouse;

S5. Click the "MODE" button on the front panel and select the detection program ("MRN" microRNA or "MLT" methylation).

S6. Click the "START" button on the front panel to start the test (the "RUNNING" light is always on, which means the test is in progress).

S7. After the detection is over ("all indicator lights are always on"), click the "CCD-X" button on the front panel, select the photo path through the "CCD-1" and "CCD-2" indicator lights, and then click the "START" button , Control the light path to take pictures of the chip, read the pictures on the client and analyze them.

S8. Click the "IN/OUT" button on the front panel to release the chip tray;

S9. Take out the tested chip, click the "IN/OUT" button on the front panel, and the chip tray will enter the warehouse;

S10. Turn off the system power switch and power off.

Compared with the prior art, the beneficial effects of this application are:

(1) The fully integrated and miniaturized chip-based digital PCR system provided by this application avoids the use of three instruments (droplet generator, droplet generator, The transfer steps included in the thermal cycler and reader) may cause cross-contamination and human error of the nucleic acid sample.

(2) The fully integrated and miniaturized chip-based digital PCR system provided by this application integrates nucleic acid extraction and purification, microdroplet formation, digital nucleic acid amplification and result detection, and realizes the "sample in-result out" detection mode. It greatly simplifies the manual operation steps, saves manpower, improves the detection efficiency, and shortens the detection cycle.

(3) The fully integrated and miniaturized chip-based digital PCR system provided by this application greatly reduces the volume and quality of the digital PCR system, saves space and transportation costs, can be transferred easily, and has a wider range of application scenarios.

(4) The open structure design of the fully integrated and miniaturized chip-based digital PCR system provided by this application is compatible with the droplet-based digital PCR detection chip, and can meet the requirements of PCR temperature control, including droplet-based and chip-based digital PCR chips. Fluorescence imaging needs.

Description of the drawings

FIG. 1 is a schematic structural diagram of a PCR detection system provided by a specific embodiment of this application;

FIG. 2 is a schematic structural diagram of a pump valve mechanical module provided by a specific embodiment of this application;

FIG. 3 is a schematic diagram of the structure of a chip feeding component and a temperature control module provided by a specific embodiment of this application;

FIG. 4 is an internal light path diagram of a blue light optical detection device provided by a specific embodiment of this application;

FIG. 5 is an internal light path diagram of a green light optical detection device provided by a specific embodiment of this application;

6 is a schematic structural diagram of a blue light optical detection device provided by a specific embodiment of this application;

FIG. 7 is a schematic structural diagram of a green light optical detection device provided by a specific embodiment of this application;

FIG. 8 is a schematic diagram of the appearance of a PCR detection chip provided by a specific embodiment of this application;

FIG. 9 is a schematic structural diagram of an upper board provided by a specific embodiment of this application;

FIG. 10 is a schematic structural diagram of a lower board provided by a specific embodiment of this application;

FIG. 11 is a schematic diagram of the interface assembly of the separated air pressure interface and the PCR detection chip provided by a specific embodiment of this application;

Among them, 1-circuit control module; 2-PCR detection chip; 3-down pressure cut-off valve array; 4-light path bracket; 5-cylinder; 6-solenoid valve; 7-limit plate; 8-chip tray; 9-step Incoming motor; 10-sensor; 11-ball screw; 12- timing belt wheel; 13- heat sink; 14- cooling fan; 15- blue optical detection device; 16- green optical detection device; 17- bottom plate; 18- Blue LED light source array; 19-Green LED light source array; 20- Homogenizing mirror; 21-Narrowband filter at the emission end of the blue light path; 22-Narrowband filter at the emission end of the green light path; 23-Dichroic mirror for the blue light path ; 24-green light path dichroic mirror; 25-blue light path receiving end narrow-band filter; 26-green light path receiving end narrow-band filter; 27-lens; 28-camera; 29-upper plate; 30-resistance Orifice; 31- Membrane; 32- Lower pressure head; 33- Sample hole; 34- Vent hole; 35- Separate air pressure interface; 36- Air pressure hole; 37- Lower plate; 38- Unit reservoir; 39- waste liquid pool; 40- drainage hole; 41- air pressure channel; 42- reaction chamber; 43- reaction reagent channel; 44- choke tank; 45- chip tank; 46- microporous digital PCR chip; 47- PCR thermally conductive aluminum block; 48-pretreatment thermally conductive aluminum block; 49-shell.

Detailed ways

It should be understood that in the description of this application, the terms "center", "vertical", "horizontal", "upper", "lower", "front", "rear", "left", "right", " The orientation or positional relationship indicated by "vertical", "horizontal", "top", "bottom", "inner", "outer", etc. are based on the orientation or positional relationship shown in the drawings, and are only for the convenience of describing the application and The description is simplified, rather than indicating or implying that the pointed device or element must have a specific orientation, be constructed and operated in a specific orientation, and therefore cannot be understood as a limitation of the present application.

It should be noted that in the description of this application, unless otherwise clearly specified and limited, the terms "set", "connected", and "connected" should be understood in a broad sense, for example, it can be a fixed connection or a detachable connection. Connection or integral connection; it can be mechanical connection or electrical connection; it can be direct connection or indirect connection through an intermediate medium, and it can be the internal communication between two components. For those of ordinary skill in the art, the specific meaning of the above-mentioned terms in this application can be understood through specific circumstances.

The technical solutions of the present application will be further described below in conjunction with the drawings and specific implementations.

In a specific embodiment, the present application provides a fully integrated and miniaturized chip-based digital PCR detection system. As shown in FIG. 1, the PCR detection system includes a pump valve mechanical module, a temperature control module, and an optical detection module. , Circuit control module 1 and PCR detection chip 2.

The pump valve mechanical module is shown in Figure 2, including a chip feeding component and a pneumatic drive component. The chip feeding component is used to feed the PCR detection chip 2 into the designated area. After the PCR detection chip 2 is sent to the designated area, the The air pressure driving component controls the sequential flow of the reaction reagents in the PCR detection chip 2 by air pressure driving to complete the PCR reaction.

The chip feeding components are shown in Figure 3, including a bottom plate 17, on which a ball screw 11 and a stepping motor 9 connected to one end of the ball screw 11 are fixed. The stepping motor 9 is connected to the ball wire through a synchronous pulley 12 The bar 11 is connected for transmission. The ball screw 11 is provided with a chip tray 8, and the chip tray 8 moves along the ball screw 11. The two ends of the ball screw 11 are provided with sensors 10, and the sensors 10 are used to detect the position of the chip tray 8 on the ball screw 11.

The pneumatic driving component specifically includes a positive pressure air pump and a downward pressure shut-off valve array 3 that is pneumatically connected to the positive pressure air pump. The positive pressure air pump includes a cylinder 5, an electromagnetic valve 6 and a separate air pressure interface 35 connected in sequence. The down-pressure shut-off valve array 3 includes 8 down-pressure shut-off valves independent of each other. The separate air pressure ports 35 are independently connected to the lower pressure shut-off valve. The downward pressure stop valve includes a downward pressure head 32, which is driven by air pressure to move in a vertical direction so as to control the liquid flow in the PCR detection chip 2. The lower indenter 32 is externally connected to the stepper motor 9. The stepper motor 9 is used to control the displacement distance of the lower indenter 32 in the vertical direction. A contact switch is arranged above the stepper motor 9, and the contact switch is used to position the lower indenter 32 in the vertical direction. The zero position. The pneumatic drive component also includes a limit plate 7, and the limit plate 7 is provided with the same number of positioning holes as the lower indenter 32, and the lower indenter 32 penetrates into the positioning hole to limit the relative position between the lower indenter 32 .

The temperature control module is fixed at the bottom of the chip tray 8, and the temperature control module moves in parallel along the ball screw 11 with the chip tray 8 to control the temperature of the PCR detection chip 2. The temperature control module is shown in Fig. 3 and includes a temperature control component and a heat dissipation component. The temperature control component is fixed on the bottom of the chip tray 8 and the heat dissipation component is located on both sides of the temperature control component. The temperature control component includes a semiconductor heating fin and/or a semiconductor refrigeration fin, and the heat dissipation component includes a heat sink 13 and a heat dissipation fan 14 located at one end of the heat sink 13. The temperature control module also includes a temperature sensor, which is closely attached to the bottom of the temperature control component.

The optical detection module is shown in Figures 6 and 7, including the optical path bracket 4 and the blue optical detection device 15 and the green light optical detection device 16 fixed side by side on the optical path bracket 4. The optical detection module is used for PCR after the PCR reaction is completed. The detection chip 2 takes a photo to obtain a fluorescence photo. Both the blue light optical detection device 15 and the green light optical detection device 16 include a housing 49, the housing 49 is made of aluminum alloy material whose outer surface is painted black, and a detection optical path module is arranged inside the housing 49. As shown in Figures 4 and 5, the detection light path module includes a transmitting end module and a receiving end module. The excitation light emitted by the transmitting end module is reflected by the PCR detection chip 2 and then received and imaged by the receiving end module. As shown in FIG. 4, the emitting end module of the blue optical detection device 15 includes a blue LED light source array 18 (465 ~ 485nm, 3W) and a homogenizing mirror 20 (25×25mm, surface roughness) arranged at intervals along the excitation light path. 1.6), the narrowband filter 21 (470nm±15nm) at the emission end of the blue light path and the blue light path dichroic mirror 23 (passing below 500nm, reflecting above 500nm), the blue light path dichroic mirror 23 is set obliquely, passing through the blue light path two The excitation light filtered by the phase color mirror 23 is irradiated on the PCR detection chip 2 to be reflected. The receiving end module of the blue light optical detection device 15 includes a narrowband filter 25 (520nm±15nm), a lens 27 (F52D09, 51.9mm fixed focus) and a camera 28 (Germany) arranged in sequence along the reflected light path of the blue light optical path.

DMK 72BUC02, 5MP monochrome CMOS). As shown in Fig. 5, the emission end module of the green optical detection device 16 includes green LED light source array 19 (520～535nm, 3W) and homogenizing mirror 20 (25×25mm, surface The roughness is 1.6), the narrow band filter 22 (528nm±15nm) at the emission end of the green light path and the dichroic mirror 24 for the green light path (passing below 550nm and reflecting above 550nm), the dichroic mirror 24 for the green light path is set obliquely , The excitation light filtered by the dichroic mirror 24 of the green light path is irradiated to the PCR detection chip 2 to be reflected. The receiving end module of the green light optical detection device 16 includes a green light optical path receiving end narrowband filter 26 (560nm±15nm), lens 27 (F52D09, 51.9mm fixed focus) and camera 28 (Germany) arranged in sequence along the reflected light optical path.

DMK 72BUC02, 5MP monochrome CMOS).

The circuit control module 1 is used to transmit electrical signals and complete the execution of control commands. Specifically, the circuit control module 1 includes a power supply circuit, a drive circuit, a control circuit, an information processing and transmission circuit, and embedded software. Wherein, the power supply circuit is connected to direct current and is electrically connected with each power consumption module. The drive circuit is used to amplify the signal of the control circuit. The control circuit is used to control the movement of each module. The information processing and transmission circuit is connected to the client system, and the information processing and transmission circuit is used to transmit the detection results and the fluorescent photos obtained by shooting to the client. Embedded software includes instrument software and middleware. The instrument software is written into the control circuit for behavior control and command execution of drive equipment; the middleware is written into the information processing and transmission circuit to complete the digital PCR detection system and Data communication and resource transmission between clients.

The PCR detection chip 2 is arranged on the chip feeding component, and reaction reagents are pre-injected into the PCR detection chip 2. Specifically, the PCR detection chip 2 as shown in FIG. 8 includes an upper layer board 29 and a lower layer board 37 stacked in layers, the upper layer board 29 has a thickness of 2 mm, the lower layer board 37 has a thickness of 5 mm, and the materials of the upper layer board 29 and the lower layer board 37 All are polymethyl methacrylate.

As shown in Figure 9, the upper plate 29 is provided with 8 choke holes 30. The position of the choke holes 30 corresponds to the position of the lower pressure head 32. The diameter of the choke holes 30 is 2.5mm. A diaphragm 31 is fixed. As shown in FIG. 11, the lower pressing head 32 is located above the diaphragm 31. As the lower pressing head 32 reciprocates in the vertical direction, the diaphragm 31 is pushed down to deform or restore to its original shape. The material of the diaphragm 31 is polydimethyl silicon. Oxane. The upper plate 29 is also provided with a sample loading hole 33, an exhaust hole 34 and an air pressure hole 36, and the air pressure hole 36 is combined with a separate air pressure interface 35.

As shown in FIG. 10, four unit liquid storage tanks 38, a cleaning liquid tank and a waste liquid tank 39 are provided on the lower board 37. The bottoms of the unit liquid storage tank 38, the cleaning liquid tank and the waste liquid tank 39 are all provided with a drain hole 40 (as shown in FIG. 8), and the diameter of the drain hole 40 is 1 mm. The unit liquid storage tank 38, the cleaning liquid tank and the waste liquid tank 39 are all S-shaped structural grooves. An air pressure channel 41 is provided along the outer edge of the lower plate 37, and the air pressure channel 41 communicates with an air pressure hole 36 opened on the upper plate 29. The unit liquid storage tank 38 and the cleaning liquid tank are respectively independently connected to the air pressure channel 41, the width of the air pressure channel 41 is 1 mm, and the depth is 0.5 mm. The lower plate 37 is also provided with four reaction chambers 42 connected in sequence, and the depth of the reaction chamber 42 is 2 mm.

Different reaction reagents are pre-stored in the unit reservoir 38. According to the different reaction reagents stored, the unit reservoirs 38 are respectively a cell lysis reaction reagent reservoir, a nucleic acid extraction reaction reagent reservoir, and a nucleic acid purification reaction reagent reservoir. Pool and PCR reagent reservoir. The reaction chamber 42 is divided into a cell lysis reaction chamber, a nucleic acid extraction reaction chamber, a nucleic acid purification reaction chamber, and a PCR reagent mixing chamber, which are sequentially connected according to different PCR reaction stages. FTA cards are pre-placed in the nucleic acid extraction reaction chamber, and the FTA cards are used to grab and release nucleic acids. The Mix, primers and probes required for PCR reaction are placed in the PCR reagent mixing chamber in advance.

The four reaction chambers 42 are respectively independently connected to the corresponding unit storage tank 38. Specifically, the cell lysis reaction chamber is connected to the cell lysis reaction reagent storage tank, the nucleic acid extraction reaction chamber is connected to the nucleic acid extraction reaction reagent storage tank, and the nucleic acid purification reaction chamber Connect the nucleic acid purification reaction reagent storage pool, and the PCR reagent mixing chamber connects the PCR reagent storage pool. The reaction reagent stored in the unit reservoir 38 is injected into the corresponding reaction chamber 42 through the reaction reagent channel 43 to react with the blood sample. The reaction reagent channel 43 is provided with a baffle groove 44 corresponding to the position of the diaphragm 31. The shape of the baffle groove 44 matches the shape of the corresponding diaphragm 31 after being pressed and deformed. The diaphragm 31 is pressed down or reset to realize the resistance. The blocking or unblocking of the flow groove 44 controls the flow of the liquid in the reaction reagent channel 43.

The cell lysis reaction chamber and the nucleic acid extraction reaction chamber are independently connected to the cleaning solution pool. After the reaction, the cleaning solution stored in the cleaning solution pool is injected into the cell lysis reaction chamber and the nucleic acid extraction reaction chamber through the cleaning solution channel for cleaning. The cleaning fluid channel is provided with a baffle groove 44 corresponding to the position of the diaphragm 31. The shape of the baffle groove 44 matches the shape of the corresponding diaphragm 31 after being pressed and deformed, and the diaphragm 31 is pressed down or reset to achieve flow resistance. The blocking or unblocking of the groove 44 controls the flow of liquid in the cleaning liquid channel.

The cell lysis reaction chamber, the nucleic acid extraction reaction chamber, and the nucleic acid purification reaction chamber are respectively independently connected to the waste liquid pool 39, and the waste liquid generated after the reaction flows into the waste liquid pool 39 through the waste liquid recovery channel for centralized discharge. The waste liquid recovery channel is provided with a baffle groove 44 corresponding to the position of the diaphragm 31. The shape of the baffle groove 44 matches the shape of the corresponding diaphragm 31 after being pressed and deformed. The diaphragm 31 is pressed down or reset to achieve resistance. The flow groove 44 is blocked or unblocked to control the liquid flow in the waste liquid recovery channel.

The connecting channel between the nucleic acid purification reaction chamber and the PCR reagent mixing chamber is provided with a baffle groove 44 corresponding to the position of the diaphragm 31, and the shape of the baffle groove 44 matches the shape of the corresponding diaphragm 31 after being pressed and deformed. The blocking or dredging of the blocking groove 44 is achieved by pressing down or resetting the diaphragm 31 to control the flow of the liquid in the nucleic acid purification reaction chamber into the PCR reagent mixing chamber.

The lower plate 37 is also provided with a chip slot 45 communicating with the PCR reagent mixing chamber, and a microporous digital PCR chip 46 and a PCR thermally conductive aluminum block 47 are placed in the chip slot 45. Specifically, the chip groove 45 is a stepped groove, the microporous digital PCR chip 46 is located in the upper groove, and the PCR thermally conductive aluminum block 47 is located in the lower groove. As shown in FIG. 10, a strip-shaped pretreatment thermally conductive aluminum block 48 is embedded at the bottom of the lower plate 37, and the pretreated thermally conductive aluminum block 48 is located below the cell lysis reaction chamber, the nucleic acid extraction reaction chamber, and the nucleic acid purification reaction chamber.

In another specific embodiment, the present application provides a fully integrated and miniaturized chip-based digital PCR detection method, which uses the chip-based digital PCR detection system provided in the foregoing specific embodiments to perform digital PCR detection on blood samples;

The detection method includes:

(Ⅰ) The blood sample is preprocessed into a suspension sample, the suspension sample is injected into the PCR detection chip 2 through the sample hole 33, the PCR detection chip 2 injected with the suspension sample is fixed on the chip tray 8, and the chip is fed The component sends the PCR detection chip 2 into the biological reaction area;

(Ⅱ) The pneumatic drive component controls the sequential flow of the reaction reagents in the PCR detection chip 2 through the pneumatic drive. The suspension sample and the different reaction reagents sequentially undergo cell lysis, nucleic acid extraction, nucleic acid purification and PCR reactions. During this process, the temperature is controlled. The module heats the different reaction areas of the PCR detection chip;

The pneumatic drive control method is:

Cylinder 5 is continuously filled with gas to generate positive pressure. The air pressure is connected to different pressure cut-off valves through the separate air pressure interface 35. The pressure head 32 is driven to reciprocate in the vertical direction through the opening and closing and closing of the solenoid valve 6 to control the air pressure. The indenter 32 moves down to compress the diaphragm 31, the diaphragm 31 deforms to block the flow blocking groove 44, and cuts off the liquid flow channel where the flow blocking groove 44 is located; the lower pressure head 32 rises and leaves the diaphragm 31, and the diaphragm 31 resets and leaves the flow blocking groove. 44. Unblock the liquid flow channel where the choke groove 44 is located;

Further, step (II) specifically includes the following steps:

(1) The suspension sample flows into the cell lysis reaction chamber, and the reaction reagent channel 43 between the cell lysis reaction reagent reservoir and the cell lysis reaction chamber is cleared through the described air pressure driving control method, and the cell lysis reaction reagent reservoir is stored The reaction reagent is injected into the cell lysis reaction chamber and the suspension sample undergoes a cell lysis reaction; after the reaction is completed, the cleaning solution channel between the cleaning solution pool and the cell lysis reaction chamber is cleared through the air pressure drive control method, and the cleaning solution stored in the cleaning solution pool is cleaned The liquid is injected into the cell lysis reaction chamber for cleaning; after the cleaning is completed, the waste liquid recovery channel between the waste liquid pool 39 and the cell lysis reaction chamber is cleared through the air pressure drive control method, and the waste liquid in the cell lysis reaction chamber flows into the waste liquid pool. Within 39;

(2) After the cell lysis reaction is completed, the suspension sample flows from the cell lysis reaction chamber into the nucleic acid extraction reaction chamber, and the reaction reagent channel between the nucleic acid extraction reaction reagent reservoir and the nucleic acid extraction reaction chamber is cleared through the described air pressure driving control method 43. The reaction reagents stored in the nucleic acid extraction reaction reagent reservoir are injected into the nucleic acid extraction reaction chamber to undergo nucleic acid extraction reaction with the suspension sample; after the reaction is completed, the cleaning solution pool and the nucleic acid extraction reaction chamber are cleared through the air pressure driving control method. In the cleaning solution channel, the cleaning solution stored in the cleaning solution pool is injected into the nucleic acid extraction reaction chamber for cleaning; after cleaning, the waste solution recovery channel between the waste solution pool 39 and the nucleic acid extraction reaction chamber is dredged through the air pressure drive control method, The waste liquid in the nucleic acid extraction reaction chamber flows into the waste liquid pool 39;

(3) After the nucleic acid extraction reaction is completed, the suspension sample flows from the nucleic acid extraction reaction chamber into the nucleic acid purification reaction chamber, and the reaction reagent channel between the nucleic acid purification reaction reagent reservoir and the nucleic acid purification reaction chamber is cleared through the described air pressure driving control method 43. The reaction reagents stored in the nucleic acid purification reaction reagent storage tank are injected into the nucleic acid purification reaction chamber to undergo nucleic acid purification reaction with the suspension sample; after the reaction is completed, the waste liquid pool 39 and the nucleic acid purification reaction chamber are dredged through the air pressure drive control method. The waste liquid recovery channel in the middle, the waste liquid in the nucleic acid purification reaction chamber flows into the waste liquid pool 39;

(4) After the nucleic acid purification reaction is completed, the suspension sample flows from the nucleic acid purification reaction chamber into the PCR reagent mixing chamber, and the reaction reagent channel 43 between the PCR reagent storage pool and the PCR reagent mixing chamber is cleared through the air pressure driving control method. The PCR reagent stored in the PCR reagent reservoir is injected into the PCR reagent mixing chamber and mixed with the suspension sample to generate droplets;

(5) The droplet enters the chip slot 45, and completes the digital PCR amplification with the microporous digital PCR chip 46.

In this process, the temperature control module performs constant temperature or variable temperature heating on different reaction areas of the PCR detection chip 2;

(Ⅲ) After the reaction is completed, the optical detection module emits a light source to irradiate the reaction sample, and the reaction area of the PCR detection chip 2 will emit two kinds of weak fluorescent signal points with different spectra, and the number of fluorescent points of each color represents the nucleic acid labeled by it. The initial content of the sequence, the fluorescent spots are photographed and collected and transmitted to the circuit control module 1;

(IV) The circuit control module 1 converts the collected optical signals into electrical signals and sends them to the client for data analysis. At the same time, the drive control of each mechanical structure system is completed, and the position of each mechanical component is returned to zero. The client uses fully intelligent automatic algorithms to complete the gate and data analysis and processing, and output the test results. At the same time, it cooperates with the circuit control system to complete data communication with the host, and the detection ends.

The applicant declares that the above are only specific implementations of this application, but the scope of protection of this application is not limited to this, and those skilled in the art should understand that any person skilled in the art disclosed in this application Any changes or replacements that can be easily conceived within the technical scope fall within the scope of protection and disclosure of this application.

Claims (15)

1. A fully integrated and miniaturized chip-type digital PCR detection system, which includes a pump valve mechanical module, a temperature control module, an optical detection module, a circuit control module, and a PCR detection chip;

   The pump valve mechanical module includes a chip feeding part and an air pressure driving part. The chip feeding part is used to send the PCR detection chip into the designated area. After the PCR detection chip is sent to the designated area, the air pressure drive The components are driven by air pressure to control the sequential flow of reaction reagents in the PCR detection chip to complete the PCR reaction;

   The temperature control module is arranged on the chip feeding component, and the temperature control module moves in parallel with the PCR detection chip for controlling the reaction temperature of the PCR detection chip;

   The optical detection module includes at least one optical detection device, and the optical detection device is used to take a photo of the PCR detection chip to obtain a fluorescent photo after the PCR reaction is completed;

   Reaction reagents are pre-injected into the PCR detection chip, and the blood sample and the reaction reagents undergo a PCR reaction in the PCR detection chip;

   The circuit control module is used to transmit electrical signals and complete the execution of control commands.
2. The PCR detection system according to claim 1, wherein the chip feeding component comprises a bottom plate, and a ball screw and a driving motor connected to one end of the ball screw are fixed on the bottom plate.
3. 3. The PCR detection system according to claim 2, wherein the driving motor drives the ball screw to rotate through a synchronous pulley.
4. The PCR detection system according to claim 2 or 3, wherein a chip tray is provided on the ball screw, and the chip tray moves along the ball screw.
5. The PCR detection system according to any one of claims 2-3, wherein sensors are provided at both ends of the ball screw, and the sensors are used to detect the position of the chip tray on the ball screw.
6. 5. The PCR detection system according to any one of claims 1 to 5, wherein the air pressure driving component comprises a positive pressure air pump and a down-pressure shut-off valve array connected to the positive pressure air pump.
7. The PCR detection system according to claim 6, wherein the positive pressure air pump comprises a cylinder, a solenoid valve, and a separate air pressure interface connected in sequence;

   Preferably, the lower pressure cut-off valve array includes 8 lower pressure cut-off valves independent of each other;

   Preferably, the separate air pressure ports are independently connected to the down pressure stop valve;

   Preferably, the lower pressure cut-off valve includes a lower pressure head, and the lower pressure head is driven by air pressure to move in a vertical direction so as to control the liquid flow in the PCR detection chip;

   Preferably, the lower indenter is externally connected to a stepper motor, and the stepper motor is used to control the displacement distance of the lower indenter in the vertical direction;

   Preferably, a contact switch is arranged above the stepping motor, and the contact switch is used to position the zero position of the lower indenter in the vertical direction;

   Preferably, the pneumatic drive component further includes a limit plate, and the limit plate is provided with a same number of positioning holes as the lower indenter, and the lower indenter penetrates into the positioning hole to limit the lower position. The relative position between the indenters.
8. The PCR detection system according to any one of claims 1-7, wherein the temperature control module is fixed at the bottom of the chip tray, and the temperature control module moves in parallel along the ball screw with the chip tray;

   Preferably, the temperature control module includes a temperature control component and a heat dissipation component, the temperature control component is fixed on the bottom of the chip tray, and the heat dissipation component is located on both sides of the temperature control component;

   Preferably, the temperature control component includes a semiconductor heating chip and/or a semiconductor refrigeration chip;

   Preferably, the heat dissipation component includes a heat sink and a heat dissipation fan located at one end of the heat sink;

   Preferably, the temperature control module further includes a temperature sensor, and the temperature sensor is close to the bottom of the temperature control component.
9. 8. The PCR detection system according to any one of claims 1-8, wherein the optical detection module comprises a light path support and at least two optical detection devices fixed side by side on the light path support;

   Preferably, the optical detection device includes a housing, and a detection optical path module is arranged inside the housing;

   Preferably, the detection light path module includes a transmitting end module and a receiving end module, and the excitation light emitted by the transmitting end module is reflected by the PCR detection chip and then received and imaged by the receiving end module;

   Preferably, the emitting end module includes an LED light source, a homogenizing mirror, a narrow band filter at the emitting end, and a dichroic mirror arranged at intervals along the excitation light path. The dichroic mirror is arranged obliquely, and the dichroic mirror is arranged obliquely. The excitation light filtered by the phase color mirror is irradiated to the PCR detection chip to be reflected;

   Preferably, the receiving end module includes a receiving end narrow-band filter, lens and camera arranged in sequence along the reflected light optical path;

   Preferably, the housing is made of aluminum alloy material.
10. The PCR detection system according to any one of claims 1-9, wherein the circuit control module includes a power supply circuit, a drive circuit, a control circuit, an information processing and transmission circuit, and embedded software;

    Preferably, the power supply circuit is connected to direct current and is electrically connected to each power consumption module;

    Preferably, the driving circuit is used to amplify the signal of the control circuit;

    Preferably, the control circuit is used to control the movement of each module;

    Preferably, the information processing and transmission circuit is connected to the client system, and the information processing and transmission circuit is used to transmit the detection results and the fluorescent photos obtained by shooting to the client;

    Preferably, the embedded software includes instrument software and middleware, and the instrument software is written into the control circuit for behavior control and command execution of drive-type equipment; the middleware writes information processing and In the transmission circuit, it is used to complete the data communication and resource transmission between the digital PCR detection system and the client.
11. The PCR detection system according to any one of claims 1-10, wherein the PCR detection chip comprises an upper layer board and a lower layer board arranged in a stack;

    Preferably, the thickness of the upper layer board is 2-6 mm, and further preferably, the thickness of the upper layer board is 2 mm;

    Preferably, the thickness of the lower layer board is 4-8mm; further preferably, the thickness of the lower layer board is 5mm;

    Preferably, the material of the upper layer board and the lower layer board are both polymethyl methacrylate.
12. The chip-type digital PCR detection system according to any one of claims 1-11, wherein the upper plate is provided with 8 baffle holes, and the position of the baffle hole is the same as the position of the lower pressure head. One correspondence

    Preferably, the diameter of the choke hole is 2 to 3 mm, and more preferably, the diameter of the choke hole is 2.5 mm;

    Preferably, a diaphragm is fixed in the choke hole, and the lower pressure head is located above the diaphragm, and as the lower pressure head reciprocates in the vertical direction, the diaphragm is pressed down to deform or return to its original shape;

    Preferably, the material of the diaphragm is polydimethylsiloxane;

    Preferably, the upper layer plate is also provided with sample loading holes, exhaust holes and air pressure holes, and the air pressure holes are combined with the separated air pressure interface.
13. The PCR detection system according to any one of claims 1-12, wherein the lower plate is provided with four unit liquid storage tanks, a cleaning liquid tank and a waste liquid tank;

    Preferably, the bottoms of the unit liquid storage tank, the cleaning liquid tank and the waste liquid tank are all provided with drain holes;

    Preferably, the diameter of the drain hole is 1 to 2 mm;

    Preferably, the unit liquid storage tank, cleaning liquid tank and waste liquid tank are all S-shaped structural grooves;

    Preferably, an air pressure channel is provided along the outer edge of the lower board, and the air pressure channel communicates with an air pressure hole opened on the upper board;

    Preferably, the unit liquid storage tank and the cleaning liquid tank are separately connected to the air pressure channel;

    Preferably, the width of the air pressure channel is 1 to 2 mm;

    Preferably, the depth of the air pressure channel is 0.5-1 mm;

    Preferably, the lower board is provided with 4 reaction chambers connected in sequence;

    Preferably, the depth of the reaction chamber is 2 to 3 mm;

    Preferably, different reaction reagents are pre-stored in the unit reservoir;

    Preferably, according to different stored reaction reagents, the unit storage tank is divided into a cell lysis reaction reagent storage tank, a nucleic acid extraction reaction reagent storage tank, a nucleic acid purification reaction reagent storage tank, and a PCR reagent storage tank;

    Preferably, the reaction chamber is divided into a cell lysis reaction chamber, a nucleic acid extraction reaction chamber, a nucleic acid purification reaction chamber, and a PCR reagent mixing chamber, which are connected in sequence according to different PCR reaction stages;

    Preferably, an FTA card is placed in the nucleic acid extraction reaction chamber in advance, and the FTA card is used to grab and release nucleic acid;

    Preferably, Mix, primers and probes required for PCR reaction are placed in the PCR reagent mixing chamber in advance;

    Preferably, the four reaction chambers are respectively independently connected to the corresponding unit reservoir, and the reaction reagent stored in the unit reservoir is injected into the corresponding reaction chamber through the reaction reagent channel to react with the blood sample;

    Preferably, the reaction reagent channel is provided with a choke groove corresponding to the position of the diaphragm, and the shape of the choke groove matches the shape of the corresponding diaphragm after being pressed and deformed. Reset to block or unclog the choke groove so as to control the liquid flow in the reaction reagent channel;

    Preferably, the cell lysis reaction chamber and the nucleic acid extraction reaction chamber are independently connected to the cleaning solution pool. After the reaction, the cleaning solution stored in the cleaning solution pool is injected into the cell lysis reaction chamber and the nucleic acid extraction reaction chamber through the cleaning solution channel. Cleaning

    Preferably, a choke groove corresponding to the position of the diaphragm is provided on the cleaning fluid channel, and the shape of the choke groove matches the shape of the corresponding diaphragm after being pressed and deformed. Reset to block or unclog the choke groove so as to control the liquid flow in the cleaning fluid channel;

    Preferably, the cell lysis reaction chamber, the nucleic acid extraction reaction chamber, and the nucleic acid purification reaction chamber are respectively independently connected to a waste liquid pool, and the waste liquid generated after the reaction flows into the waste liquid pool through the waste liquid recovery channel for centralized discharge;

    Preferably, the waste liquid recovery channel is provided with a choke groove corresponding to the position of the diaphragm, and the shape of the choke groove matches the shape of the corresponding diaphragm after being pressed and deformed. Or reset to block or unclog the choke groove so as to control the liquid flow in the waste liquid recovery channel;

    Preferably, the connecting channel between the nucleic acid purification reaction chamber and the PCR reagent mixing chamber is provided with a baffle groove corresponding to the position of the diaphragm, and the shape of the baffle groove is corresponding to the shape of the diaphragm after the corresponding diaphragm is pressed and deformed. The shape of the filter is matched, and the blocking or dredging of the choke groove is realized by pressing down or resetting the diaphragm to control the liquid in the nucleic acid purification reaction chamber to flow into the PCR reagent mixing chamber;

    Preferably, the lower board is also provided with a chip slot communicating with the PCR reagent mixing chamber;

    Preferably, a microporous digital PCR chip and PCR thermally conductive components are placed in the chip slot;

    Preferably, the chip groove is a stepped groove, the microporous digital PCR chip is located in the upper groove, and the PCR thermally conductive component is located in the lower groove;

    Preferably, the PCR thermally conductive component is a thermally conductive aluminum block;

    Preferably, a strip-shaped pretreatment heat-conducting component is embedded at the bottom of the lower plate, and the pre-treatment heat-conducting component is located below the cell lysis reaction chamber, the nucleic acid extraction reaction chamber and the nucleic acid purification reaction chamber;

    Preferably, the pretreatment heat-conducting component is a heat-conducting aluminum block.
14. A fully integrated and miniaturized chip-based digital PCR detection method, which adopts the chip-based digital PCR detection system according to any one of claims 1-13 to perform digital PCR detection on blood samples;

    The detection method includes:

    (I) The blood sample is pretreated into a suspension sample, the suspension sample is injected into the PCR detection chip, and the chip feeding component sends the PCR detection chip into the biological reaction area;

    (Ⅱ) The air pressure driving component controls the sequential flow of reaction reagents in the PCR detection chip by air pressure driving. The suspension sample and different reaction reagents sequentially undergo cell lysis, nucleic acid extraction, nucleic acid purification and PCR reactions. During this process, the temperature control module Heating the different reaction areas of the PCR detection chip;

    (Ⅲ) After the reaction is over, the optical detection module emits a light source to irradiate the reaction sample to excite the fluorescent signal points, and the fluorescent signal is photographed and collected and transmitted to the circuit control module;

    (IV) The circuit control module performs photoelectric signal conversion on the collected optical signals and transmits them to the client for data analysis.
15. The detection method according to claim 14, wherein in step (I), the suspension sample is injected into the PCR detection chip through the sample hole;

    Preferably, the PCR detection chip injected with the suspension sample is fixed on the chip tray, and the chip feeding component sends the PCR detection chip under the down-pressure shut-off valve array;

    Preferably, in step (II), the pneumatic drive control mode is:

    The cylinder is continuously filled with gas to generate positive pressure, and the air pressure is connected to different pressure stop valves through the separate air pressure interface. The pressure is driven by the opening and closing of the solenoid valve to control the pressure to move the lower pressure head back and forth in the vertical direction, and the lower pressure head moves down. Compress the diaphragm, the diaphragm is deformed to block the flow blocking groove, and cut off the liquid flow path where the flow blocking groove is located; the lower pressure head rises away from the diaphragm, the diaphragm is reset and separated from the flow blocking groove to clear the liquid flow path where the flow blocking groove is located;

    Preferably, step (II) specifically includes the following steps:

    (1) The suspension sample flows into the cell lysis reaction chamber, and the reaction reagent channel between the cell lysis reaction reagent reservoir and the cell lysis reaction chamber is cleared through the described air pressure driving control method, and the reaction stored in the cell lysis reaction reagent reservoir is cleared. Reagents are injected into the cell lysis reaction chamber and the suspension sample undergoes a cell lysis reaction; after the reaction is completed, the cleaning solution channel between the cleaning solution pool and the cell lysis reaction chamber is cleared through the air pressure driving control method, and the cleaning solution stored in the cleaning solution pool Inject into the cell lysis reaction chamber for cleaning; after the cleaning, the waste liquid recovery channel between the waste liquid pool and the cell lysis reaction chamber is cleared through the air pressure drive control method, and the waste liquid in the cell lysis reaction chamber flows into the waste liquid pool;

    (2) After the cell lysis reaction is completed, the suspension sample flows from the cell lysis reaction chamber into the nucleic acid extraction reaction chamber, and the reaction reagent channel between the nucleic acid extraction reaction reagent reservoir and the nucleic acid extraction reaction chamber is cleared through the described air pressure driving control method , The reaction reagents stored in the nucleic acid extraction reaction reagent reservoir are injected into the nucleic acid extraction reaction chamber and the suspension sample undergoes nucleic acid extraction reaction; after the reaction is completed, the air pressure drive control method is used to clear the gap between the cleaning solution pool and the nucleic acid extraction reaction chamber Cleaning fluid channel, the cleaning fluid stored in the cleaning fluid pool is injected into the nucleic acid extraction reaction chamber for cleaning; after cleaning, the waste fluid recovery channel between the waste fluid pool and the nucleic acid extraction reaction chamber is dredged through the air pressure driving control method, and nucleic acid extraction The waste liquid in the reaction chamber flows into the waste liquid pool;

    (3) After the nucleic acid extraction reaction is completed, the suspension sample flows from the nucleic acid extraction reaction chamber into the nucleic acid purification reaction chamber, and the reaction reagent channel between the nucleic acid purification reaction reagent reservoir and the nucleic acid purification reaction chamber is cleared through the described air pressure driving control method , The reaction reagents stored in the nucleic acid purification reaction reagent storage tank are injected into the nucleic acid purification reaction chamber and the suspension sample undergoes nucleic acid purification reaction; after the reaction is completed, the air pressure drive control method is used to clear the gap between the waste liquid pool and the nucleic acid purification reaction chamber Waste liquid recovery channel, the waste liquid in the nucleic acid purification reaction chamber flows into the waste liquid pool;

    (4) After the nucleic acid purification reaction is completed, the suspension sample flows from the nucleic acid purification reaction chamber into the PCR reagent mixing chamber, and the reaction reagent channel between the PCR reagent storage pool and the PCR reagent mixing chamber is cleared through the air pressure driving control method. The PCR reagent stored in the reagent storage pool is injected into the PCR reagent mixing chamber and mixed with the suspension sample to generate droplets;

    (5) The droplet enters the chip slot and completes digital PCR amplification with the microporous digital PCR chip.

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