CN111909842A - Integrated digital PCR system and use method thereof - Google Patents

Integrated digital PCR system and use method thereof Download PDF

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Publication number
CN111909842A
CN111909842A CN202010863328.1A CN202010863328A CN111909842A CN 111909842 A CN111909842 A CN 111909842A CN 202010863328 A CN202010863328 A CN 202010863328A CN 111909842 A CN111909842 A CN 111909842A
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digital pcr
sample
micro
valve
cavity
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李刚
谢腾宝
武银
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Chongqing University
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Chongqing University
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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B01L3/502769—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by multiphase flow arrangements
    • B01L3/502784—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by multiphase flow arrangements specially adapted for droplet or plug flow, e.g. digital microfluidics
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L7/00—Heating or cooling apparatus; Heat insulating devices
    • B01L7/52—Heating or cooling apparatus; Heat insulating devices with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6851—Quantitative amplification
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0861—Configuration of multiple channels and/or chambers in a single devices
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/18—Means for temperature control
    • B01L2300/1838—Means for temperature control using fluid heat transfer medium
    • B01L2300/185—Means for temperature control using fluid heat transfer medium using a liquid as fluid
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00—Moving or stopping fluids
    • B01L2400/04—Moving fluids with specific forces or mechanical means
    • B01L2400/0475—Moving fluids with specific forces or mechanical means specific mechanical means and fluid pressure
    • B01L2400/0487—Moving fluids with specific forces or mechanical means specific mechanical means and fluid pressure fluid pressure, pneumatics

Abstract

The invention discloses an integrated digital PCR system and a using method thereof, wherein the system comprises a high-density microcavity array type digital PCR chip with an integrated cavity, an optical detection module, a hot water circulation temperature control module, a fluid control module and a central control module; when the system is applied to nucleic acid sample detection, firstly, a digital PCR chip is fixed on an optical detection module objective table, and a through pipe communicated with a sample tube and a hot water tank is assembled at each interface of the chip; then, start central control module, set up the relevant parameter of control program, realize in proper order through control program control fluid control module, hot water circulation temperature control module and optical detection module: sample filling, sample discretization, hot water circulation amplification, chip scanning record, data analysis and other operations. Based on the combination of the microcavity array and the integrated cavity structure of the digital PCR chip, the system can complete all the digital PCR detection processes on a single instrument, thereby improving the detection efficiency and the reliability of results.

Description

Integrated digital PCR system and use method thereof
Technical Field
The invention belongs to the technical field of microfluidic chip analysis, and particularly relates to an integrated digital PCR system and a using method thereof.
Background
The digital PCR (digital PCR, dPCR) technology is a third generation Quantitative nucleic acid detection technology following the Real-time fluorescent Quantitative PCR (Quantitative Real-time PCR) technology, is an absolute nucleic acid Quantitative technology, and has the following outstanding advantages compared with the traditional PCR: can realize absolute nucleic acid quantification without depending on a standard curve, has higher tolerance to inhibitors, can analyze complex mixtures, and can realize detection of trace nucleic acid samples, rare mutation detection under complex backgrounds, identification of small difference of expression levels and the like. The working principle is that a sample is decomposed into thousands of even millions of droplets (each droplet is used as an independent PCR reaction unit) until each droplet contains one or zero target analysis, after all the droplets are subjected to PCR amplification simultaneously, the droplets containing the target analysis generate positive signals due to the target analysis amplification, and the droplets without target molecules do not have signals, and finally, the original concentration or the content of the target molecules of the sample can be calculated by counting the positive signals and calculating according to a Poisson distribution formula.
Due to its great technical advantages, the digital PCR technology has received wide attention from domestic and foreign research institutions and enterprises in recent years, and a plurality of digital PCR instrument systems have been developed in succession. Typical commercial digital PCR systems currently include BioMark, marketed by FluidigmTMSystem, Quantstudio by Life TechnologiesTMSystem, QX100 by Bio-RadTMSystem and RainDrop from RainDance Technologies, IncTMProvided is a system. These systems all need multiple instruments to cooperate to complete the whole digital PCR detection process, for example, the core component of the BioMark system is PDMS chip integrated with a series of complex micro-channels and micro-valves, the system needs two instruments to realize the complete digital PCR detection process, firstly, an integrated fluid channel Controller (IFC Controller) is combined with a large number of micro-valves integrated on the digital PCR chip to decompose the sample full of the micro-pipeline network into a large number of independent anti-reverse-flowApplying a unit, then transferring the digital PCR chip after the sample discretization is finished to a chip Reader (Biomark Reader), and performing thermal cycle amplification and PCR reaction result reading analysis; QuantStaudioTMThe core component of the system is a silicon-based Chip integrated with a large number of through holes, the system needs three instruments for realizing the complete Digital PCR detection process, firstly, a Chip sample adding instrument (Digital PCR Chip Loader) is utilized, based on the wettability difference of hydrophilic through holes and hydrophobic surfaces, the liquid sample distribution in all the through holes is realized through the action of surface tension, and then, the Chip completing the sample distribution is transferred to a flat-plate thermal cycler (ProFlex)TM2x Flat PCR System), realizing thermal cycle amplification, and finally transferring the amplified chip to a chip analyzer (Quantstrudio)TM3D Digital PCR Instrument) was performed. Unlike BioMark and QuantStaudioTMSystem, QX100TM System and RaindropTMIn the system, a droplet type microfluidic chip is used for decomposing a sample into thousands of nano-liter-level droplets, and after PCR amplification, a detector similar to a flow cytometer is used for detecting each droplet one by one to obtain a detection result. The whole system at least comprises a combination of three instruments: a micro-droplet generator, a thermal cycler, and a micro-droplet analyzer. In recent years, many researchers have conducted extensive studies on individual administration of tumors, gene copy number variation, and the like using the above system. However, there are some technical and cost limitations to the large-scale deployment of the above system. For example, the systems all need additional instruments to realize sample introduction or sample discretization, and the digital PCR chip after sample discretization needs manual operation to realize chip transfer between the instruments, resulting in relatively complex flow, long time-consuming detection process, and increased risk of human error and sample contamination. In addition, BioMarkTM and QuantStaudioTMThe digital PCR chips adopted by the system have low decomposition density, so that the detection precision is relatively insufficient, the measurable dynamic range is limited, and the manufacturing cost of the chips is high; and QX100TM System and RaindropTMThe system uses a droplet-type microfluidic chip to decompose the sample, in order to maintain the decomposition of the dropletsDispersibility, the need to add surfactants, and such additives may have an effect or interfere with certain experimental systems, thereby affecting the confidence of the results.
Therefore, there is an urgent need to develop a novel and integrated digital PCR system, which can realize full automation and one-stop operation of the whole digital PCR process of sample introduction, sample discretization, thermal cycle amplification and result reading analysis, simplify the digital PCR detection experiment process, reduce the operation cost, improve the reliability of the detection result and further improve the detection precision, so that the digital PCR technology is changed from a "expensive" and "professional" laboratory technology to a conventional tool for common laboratories and clinical examinations.
Disclosure of Invention
Aiming at the defects in the prior art, the integrated digital PCR system and the using method thereof provided by the invention solve the problems of complex using process, high operating cost, low detection efficiency and insufficient reliability of the existing digital PCR system.
In order to achieve the purpose of the invention, the invention adopts the technical scheme that: an integrated digital PCR system comprises a digital PCR chip, an optical detection module, a fluid control module, a hot water circulation temperature control module and a central control module;
the digital PCR chip is placed on a two-dimensional step scanning platform on the optical detection module and is connected with the hot water circulation temperature control module and the fluid control module, the fluid control module is also respectively connected with the hot water circulation temperature control module and the central control module, and the hot water circulation temperature control module and the optical detection module are both connected with the central control module.
Furthermore, the digital PCR chip is a high-density microcavity array structure integrating a cavity;
the digital PCR chip comprises a top layer glass cover plate, a thin film layer, a cavity layer and a bottom layer glass substrate;
the top layer glass cover plate is provided with a sample inlet through hole, a sample outlet through hole, a first water inlet through hole and a first water outlet through hole;
the film layer is provided with a polydimethylsiloxane micro-cavity array structure, a sample injection port, a waste liquid sample outlet, a second water inlet through hole and a second water outlet through hole, the polydimethylsiloxane micro-cavity array structure is formed by connecting a plurality of micro-pipelines in parallel in series with a plurality of micro-cavities, and two ends of each micro-pipeline are respectively connected with the sample injection port and the waste liquid sample outlet; the second water inlet through hole and the second water outlet through hole are respectively arranged on two sides of the polydimethylsiloxane micro-cavity array structure; one side of the film layer, which is provided with the polydimethylsiloxane micro-cavity array structure, is a structural surface, and the other side of the film layer is a non-structural surface;
the cavity layer is provided with a third water inlet, a third water outlet and a cavity area, and the projection area of the cavity area on the thin film layer covers the polydimethylsiloxane micro-cavity array structure area on the cavity area;
the first water inlet through hole, the second water inlet through hole and the third water inlet are in one-to-one correspondence and are mutually communicated, the first water outlet through hole, the second water outlet through hole and the third water outlet are in one-to-one correspondence and are mutually communicated, the sample inlet through hole corresponds to the sample inlet and is mutually communicated, and the sample outlet through hole corresponds to the waste liquid sample outlet and is mutually communicated;
the top layer glass cover plate and the structural surface of the film layer are bonded to form a closed micro-pipeline system, and the non-structural surface of the film layer, the cavity layer and the bottom layer glass substrate are bonded to form a closed cavity;
the sample inlet through hole of the digital PCR chip is connected with the outlet of the sample tube through a through tube, and a first valve is arranged on the through tube.
Further, at least 1000 micro-cavities are included in the polydimethylsiloxane micro-cavity array structure, and the geometric shape and the size of each micro-cavity are consistent;
the polydimethylsiloxane micro-cavity array structure is a non-transparent structure, a polydimethylsiloxane film at the bottom of each micro-cavity forms an isolation film between the micro-cavity and the cavity layer, and the thickness of the isolation film is 20-200 microns.
Further, the cavity layer is a transparent structure, the cavity layer is made of polydimethylsiloxane, double-sided tape or film through carving, and the thickness of the cavity layer is 30-1000 microns.
Further, the optical detection module comprises a two-dimensional step scanning platform, a bright field optical assembly and a fluorescence microscopy optical assembly;
the bright field optical assembly is arranged above the two-dimensional stepping scanning platform, the fluorescence microscopic optical assembly is arranged below the two-dimensional stepping scanning platform, and the two-dimensional stepping scanning platform is connected with the central control module;
the two-dimensional step scanning platform is used for placing a digital PCR chip;
the bright field optical element is used for observing the integrity of the sample introduction of the digital PCR chip;
the fluorescence microscopic optical assembly is used for detecting the PCR result.
Further, the fluid control module comprises a fluid control circuit, a vacuum pump, a second valve and a waste liquid bottle;
the fluid control circuit is respectively connected with the control end of the first valve and the central control module;
the fluid control circuit still respectively with three way valve among vacuum pump, second valve and the hot water circulation temperature control module is connected, the vacuum pump air inlet pass through the siphunculus with the gas outlet of waste liquid bottle is connected, the output port of second valve pass through the siphunculus with the feed inlet of waste liquid bottle is connected, two input ports of second valve respectively with the appearance mouth through-hole and the first delivery port of digital PCR chip are connected.
Furthermore, the hot water circulation temperature control module comprises a first constant-temperature water tank, a second constant-temperature water tank, a third constant-temperature water tank, a three-way valve and a temperature control circuit;
the input of temperature control circuit with central control module connects, temperature control circuit's output is connected with the temperature control end of first constant temperature water pitcher, second constant temperature water pitcher and third constant temperature water pitcher respectively, three input of three-way valve respectively through a siphunculus with first constant temperature water pitcher, second constant temperature water pitcher and third constant temperature water pitcher are connected, three-way valve's output through a siphunculus with water inlet on the digital PCR chip is connected, three-way valve's control end with fluid control circuit connects.
A method of using an integrated digital PCR system, comprising the steps of:
s1, preparation: fixing a digital PCR chip on a two-dimensional step scanning platform, assembling and connecting all through pipes according to the requirements of all interfaces, simultaneously starting a central control module, setting parameters for regulating and controlling fluid control and temperature control, starting a temperature control circuit, and controlling the temperature of a constant-temperature water tank in a hot water circulation temperature control module according to the detection requirements of the digital PCR;
s2, filling: adjusting the opening state of a valve based on the digital PCR detection requirement, and pumping air in each micro-pipeline and micro-cavity in the digital PCR chip by using a vacuum pump, so that the sample liquid in the sample tube is sucked into the digital PCR chip until the sample liquid is filled in all the micro-pipelines and micro-cavities in the digital PCR chip;
s3, sample discretization: adjusting the opening state of a valve based on the digital PCR detection requirement, introducing the oil phase in the sample tube into the digital PCR chip by using a vacuum pump, removing the sample liquid in each micro-pipeline, and isolating the oil phase from the sample liquid in each micro-cavity;
s4, thermal cycle amplification: adjusting the opening state of a valve based on the digital PCR detection requirement, and driving water in a constant-temperature water tank at a preset temperature to continuously pass through the digital PCR chip integrated cavity through a vacuum pump so as to realize a thermal cycle nucleic acid amplification reaction of sample liquid in each microcavity in the digital PCR chip;
s5, image processing and data analysis: and controlling the valves and the vacuum pump to be closed, starting the optical detection module, scanning the optical detection module to obtain all micrographs of the digital PCR chip, splicing the micrographs by using the central control module to obtain a fluorescence picture of the whole microcavity reaction region of the digital PCR chip, and performing image processing and data analysis on the fluorescence picture to obtain a final detection result.
Further, the PCR detection requirements of step S1 include conventional digital PCR detection and isothermal digital PCR detection;
when the detection requirement is conventional digital PCR detection, three constant-temperature water tanks in the hot water circulation temperature control module are respectively heated to a preset temperature and are kept stable;
when the detection requirement is isothermal digital PCR detection, controlling the temperature of two constant temperature water tanks in the hot water circulation temperature control module to a preset temperature respectively and maintaining the temperature to be stable, and leaving the other constant temperature water tank empty; wherein, one of the two thermostatic water tanks which are controlled by temperature is a thermostatic hot water tank, and the other thermostatic cold water tank.
Further, in step S2:
when the detection requirement is conventional digital PCR detection, the sample filling process specifically comprises the following steps:
controlling the first valve to be opened and the second valve to be opened towards the first water outlet, and pumping air in each micro-pipeline and micro-cavity in the digital PCR chip by using a vacuum pump, so that the sample liquid in the sample tube is sucked into the digital PCR chip until the sample liquid is filled in all the micro-pipelines and micro-cavities in the digital PCR chip;
when the detection requirement is isothermal digital PCR detection, the sample filling process specifically comprises the following steps:
controlling the second valve to open towards the first water outlet, pumping air in each micro-pipeline and micro-cavity in the digital PCR chip by using a vacuum pump, then controlling the first valve to open, the second valve to open towards the first water outlet and the three-way valve to open towards the direction of the constant-temperature cold water tank, and sucking the sample liquid in the sample tube into the digital PCR chip until the sample liquid is filled in all the micro-pipelines and micro-cavities in the digital PCR chip;
in the step S3:
when the detection requirement is conventional digital PCR detection, the mode of adjusting the opening state of the valve is as follows: controlling the first valve to open and the second valve to open towards the direction of the through hole of the sample outlet;
when the detection requirement is isothermal digital PCR detection, the mode of adjusting the opening state of the valve is as follows: controlling the first valve to open, the second valve to open towards the two inlet directions simultaneously and the three-way valve to open towards the direction of the constant-temperature cold water tank;
in the step S4:
when the detection requirement is conventional digital PCR detection, the thermal cycle amplification method specifically comprises the following steps:
the three-way valve is controlled to be opened towards the three constant-temperature water tanks and the second valve is controlled to be opened towards the first water outlet, and the three kinds of hot water with different temperatures in the three constant-temperature water tanks are driven by the vacuum pump to sequentially circulate through the digital PCR chip integrated cavity according to a set sequence and period, so that the sample liquid in each microcavity in the digital PCR chip realizes the thermal cycle nucleic acid amplification reaction;
when the detection requirement is isothermal digital PCR detection, the thermal cycle amplification method specifically comprises the following steps:
and controlling the second valve to open towards the first water outlet and the three-way valve to open towards the constant-temperature hot water tank, and continuously passing constant-temperature water in the constant-temperature hot water tank through the cavity area by using the vacuum pump so as to realize thermal cycle nucleic acid amplification reaction of sample liquid in each microcavity in the digital PCR chip.
The invention has the beneficial effects that:
the integrated digital PCR system and the using method thereof provided by the invention have the advantages of simple and convenient operation, low cost, high detection efficiency and good result reliability, and are embodied in the following points:
(1) the integrated digital PCR system can realize full automation and 'fool' operation of sample introduction, sample discretization, thermal cycle amplification and result reading analysis of the whole digital PCR detection process on a single instrument, greatly reduces the complexity of the digital PCR detection operation, reduces the probability of human error and sample pollution, improves the reliability of the detection result, and avoids the dependence of the digital PCR detection operation on professional technicians;
(2) based on the negative pressure sample introduction mode of the Polydimethylsiloxane (PDMS) film gas permeability, the sample in the sample filling micro-pipeline is only wasted by filling the micro-pipeline network and the microcavity array of the digital PCR chip and removing redundant samples in the sample filling micro-pipeline, so that the utilization rate of the samples is greatly improved compared with the conventional digital PCR system;
(3) compared with the existing thermal cycle amplification mode based on hot water circulation of an integrated microcavity, the thermal cycle amplification mode based on hot water circulation of the integrated microcavity does not need a complex and precise rapid temperature rise and fall control design, avoids time-consuming temperature rise and fall processes, avoids the problem of uneven temperature caused by incomplete contact and lamination of a chip and a hot plate due to an integrated structure, can greatly reduce the complexity of a temperature control module, shorten the thermal cycle amplification time and improve the thermal cycle reaction uniformity of the chip;
(4) the integrated cavity structure of the chip can also utilize the water molecule permeability of the PDMS film to play a role in water compensation in the thermal cycle amplification process, so that the problem of water loss caused by high-temperature volatilization of reaction liquid in the microcavity due to thermal cycle is avoided, and the reliability of PCR reaction is further ensured.
Drawings
FIG. 1 is a block diagram of an integrated digital PCR system according to the present invention.
FIG. 2 is an exploded view of the digital PCR chip structure provided by the present invention.
FIG. 3 is a schematic diagram of the assembly of the digital PCR chip according to the present invention.
FIG. 4 is a schematic top view of the digital PCR chip according to the present invention.
FIG. 5 is a flow chart of a method for using the integrated digital PCR system of the present invention.
FIG. 6 is a schematic diagram of a conventional digital PCR application process of the integrated digital PCR system provided by the present invention.
FIG. 7 is a diagram of the detection result obtained by applying the integrated digital PCR system provided by the present invention to conventional digital PCR.
FIG. 8 is a schematic diagram of the usage flow of the integrated digital PCR system applied to isothermal digital PCR.
Wherein: 1. a digital PCR chip; 2. an optical detection module; 3. a fluid control module; 4. a hot water circulation temperature control module; 5. a central control module; 6. a sample tube; 7. a first valve; 1-1, a top glass cover sheet; 1-2, a thin film layer; 1-3, a cavity layer; 1-4, a bottom glass substrate; 1-11, a sample inlet through hole; 1-12, sample outlet through holes; 1-13, a first water inlet through hole; 1-14, a first water outlet through hole; 1-21, polydimethylsiloxane micro-cavity array structure; 1-211, micro-tubes; 1-212, microcavity; 1-22, sample injection port; 1-23, a waste liquid sample outlet; 1-24 and a second water inlet through hole; 1-25, a second water outlet through hole; 1-31, a third water inlet; 1-32 and a third water outlet; 1-33, a cavity area; 2-1, a light source; 2-2, a first convex lens; 2-3, a first reflector; 2-4, a second convex lens; 2-5, a first electronic shutter; 2-6, a two-dimensional step scanning platform; 2-7, a microscope objective; 2-8, dichroic mirror; 2-9, a third convex lens; 2-10, a second electronic shutter; 2-11, mercury lamps; 2-12, a second reflector; 2-13, an image sensor; 2-14, exciting an optical filter; 2-15, an emission filter; 3-1, a fluid control circuit; 3-2, a vacuum pump; 3-3, a second valve; 3-4, a waste liquid bottle; 4-1, a first constant-temperature water tank; 4-2, a second constant-temperature water tank; 4-3, a third constant-temperature water tank; 4-4, a three-way valve; 4-5, a temperature control circuit.
Detailed Description
The following description of the embodiments of the present invention is provided to facilitate the understanding of the present invention by those skilled in the art, but it should be understood that the present invention is not limited to the scope of the embodiments, and it will be apparent to those skilled in the art that various changes may be made without departing from the spirit and scope of the invention as defined and defined in the appended claims, and all matters produced by the invention using the inventive concept are protected.
Example 1:
as shown in fig. 1, an integrated digital PCR system includes a digital PCR chip 1, an optical detection module 2, a fluid control module 3, a hot water circulation temperature control module 4 and a central control module 5;
the digital PCR chip 1 is placed on a two-dimensional step scanning platform 2-6 on the optical detection module 2 and is connected with a hot water circulation temperature control module 4 and a fluid control module 3, the fluid control module 3 is also connected with the hot water circulation temperature control module 4 and a central control module 5 respectively, and the hot water circulation temperature control module 4 and the optical detection module 2 are both connected with the central control module 5.
The digital PCR chip 1 is used for realizing automatic decomposition and quantitative uniform distribution of an input sample, a complex macro-micro interface is not required to be configured, and the complexity and the running cost of a digital PCR system are reduced; the optical detection module 2 can realize program-controlled switching between bright field and fluorescence microscopic observation modes, can realize program-controlled automatic focusing, and is convenient for accurately acquiring reaction images in the digital PCR chip 1; the hot water circulation temperature control module 4 comprises three constant temperature water tanks which are controlled by a program to realize heating, cooling or constant temperature control through a temperature control circuit 4-5 so as to provide proper temperature conditions for sample amplification reaction in the digital PCR chip 1; the fluid control module 3 is used for realizing the filling and discretization of the samples in the digital PCR chip 1 and controlling the sequence and time of liquid in different constant-temperature water tanks flowing through the digital PCR chip 1, and provides environmental support for the reaction of the samples in the digital PCR chip 1.
As shown in fig. 2-4, the digital PCR chip 1 in this embodiment is a high-density microcavity array structure with integrated cavities;
the digital PCR chip 1 comprises a top layer glass cover plate 1-1, a thin film layer 1-2, a cavity layer 1-3 and a bottom layer glass substrate 1-4;
the top layer glass cover plate 1-1 is provided with a sample inlet through hole 1-11, a sample outlet through hole 1-12, a first water inlet through hole 1-13 and a first water outlet through hole 1-14;
the film layer 1-2 is provided with a polydimethylsiloxane micro-cavity array structure 1-21, a sample inlet 1-22, a waste liquid outlet 1-23, a second water inlet through hole 1-24 and a second water outlet through hole 1-25, the polydimethylsiloxane micro-cavity array structure 2-1 is formed by connecting a plurality of parallel micro-pipelines 2-211 in series with a plurality of micro-cavities 2-212, and two ends of each micro-pipeline are respectively connected with the sample inlet 1-22 and the waste liquid outlet 1-23; the second water inlet through holes 1-24 and the second water outlet through holes 1-25 are respectively arranged at two sides of the polydimethylsiloxane micro-cavity array structure 1-21; one side of the film layer 1-2, which is provided with the polydimethylsiloxane micro-cavity array structure 1-21, is a structural surface, and the other side is a non-structural surface;
the cavity layer 1-3 is provided with a third water inlet 1-31, a third water outlet 1-32 and a cavity area 1-33, the projection area of the cavity area 1-33 on the film layer 1-2 covers the polydimethylsiloxane micro-cavity array structure 1-21 area on the film layer, and the projection area is slightly larger than the polydimethylsiloxane micro-cavity array structure 1-21 area;
the first water inlet through holes 1-13, the second water inlet through holes 1-24 and the third water inlets 1-31 are in one-to-one correspondence and are mutually communicated, the first water outlet through holes 1-14, the second water outlet through holes 1-25 and the third water outlets 1-32 are in one-to-one correspondence and are mutually communicated, the sample inlet through holes 1-11 are in correspondence and are mutually communicated with the sample inlets 1-22, and the sample outlet through holes 1-12 are in correspondence and are mutually communicated with the waste liquid sample outlets 1-23;
the top glass cover plate 1-1 and the structural surface of the film layer 1-2 are bonded to form a closed micro-pipeline system, the non-structural surface of the film layer 1-2, the cavity layer 1-3 and the bottom glass substrate 1-4 are bonded to form a closed cavity, wherein in the figure 3, after the digital PCR chip 1 is assembled, the first water inlet through hole 1-13, the second water inlet through hole 1-24 and the third water inlet 1-31 form a water inlet, the first water outlet through hole 1-14, the second water outlet through hole 1-25 and the third water outlet 1-32 form a water outlet, the sample inlet through hole 1-11 and the sample inlet 1-22 form a sample inlet, and the sample outlet through hole 1-12 and the waste liquid sample outlet 1-23 form a sample outlet;
in addition, the sample inlet through holes 1-11 of the digital PCR chip 1 are connected with the outlet of the sample tube 6 through a through tube, and the through tube is provided with a first valve 7.
Specifically, the polydimethylsiloxane micro-cavity array structure 1-21 comprises at least 1000 micro-cavities 2-212, and the geometric shapes and sizes of the micro-cavities 2-212 are consistent, so that the digital PCR chip 1 can realize basic digital PCR analysis; (ii) a
The polydimethylsiloxane micro-cavity array structure 1-21 is a non-transparent structure, the polydimethylsiloxane thin film at the bottom of each micro-cavity 2-212 forms an isolation film between the micro-cavity 2-212 and the cavity layer 1-3, the thickness of the isolation film is 20-200 micrometers, when the thickness of the isolation film is set, when the vacuum negative pressure is applied to the cavity layer 1-3 due to the fact that the isolation film is too thin, deformation of the film is too large, the sample filling effect is affected, and if the isolation film is too thick, the air permeation rate is slow, and further the negative pressure sampling rate is slow.
The cavity layer 1-3 is made by engraving polydimethylsiloxane, a double-sided adhesive tape or a film, the thickness of the cavity layer 1-3 is 30-1000 microns, and the cavity layer 1-3 is used for connecting a vacuum source and realizing bubble-free sample injection through negative pressure; as a hot water circulation tank, realizing thermal circulation amplification reaction; the water replenishing tank is used for preventing the problem of water volatilization of the microcavity array reaction system in the thermal cycle amplification process.
As shown in fig. 1, the optical detection module 2 in the present embodiment includes a two-dimensional step-and-scan stage 2-6, a bright-field optical assembly, and a fluorescence microscopy optical assembly; the bright field optical assembly is arranged above the two-dimensional stepping scanning platform 2-6, the fluorescence microscopic optical assembly is arranged below the two-dimensional stepping scanning platform 2-6, and the two-dimensional stepping scanning platform 2-6 is connected with the central control module 5;
wherein, the two-dimensional step scanning platform 2-6 is used for placing the digital PCR chip 1; the bright field optical element is used for observing the integrity of the sample introduction of the digital PCR chip 1; and the fluorescence microscopic optical assembly is used for detecting the PCR result.
Specifically, the bright field optical assembly comprises a light source 2-1, a first convex lens 2-2, a first reflector 2-3, a second convex lens 2-4 and a first electronic shutter 2-5; the light source 2-1, the first convex lens 2-2, the first reflector 2-3, the second convex lens 2-4 and the first electronic shutter 2-5 are all arranged above the two-dimensional stepping scanning platform 2-6, and light emitted by the light source 2-1 sequentially passes through the first convex lens 2-2, the first reflector 2-3, the second convex lens 2-4 and the first electronic shutter 2-5 to irradiate the digital PCR chip 1;
the fluorescence micro-optical component comprises a micro-objective lens 2-7, a dichroic mirror 2-8, an excitation filter 2-14, an emission filter 2-15, a third convex lens 2-9, a second electronic shutter 2-10, a mercury lamp 2-11, a second reflecting mirror 2-12 and an image sensor 2-13; light emitted by the mercury lamp 2-11 sequentially passes through a second electronic shutter 2-10, a third convex lens 2-9, an excitation filter 2-14 and a dichroic mirror 2-8 to irradiate the digital PCR chip 1, and an imaging result of the digital PCR chip 1 by the microscope objective lens 2-7 is transmitted to the central control module 5 sequentially passes through the dichroic mirror 2-8, an emission filter 2-15, a second reflecting mirror 2-12 and an image sensor 2-13; the first electronic shutter 2-5 and the second electronic shutter 2-10 are both connected with the central control module 5; among them, the image sensors 2 to 13 are CCD elements or CMOS elements.
It should be noted that the above-mentioned optical detection module structure is only one structure capable of implementing the above-mentioned functions, and any bright field optical component and fluorescence microscopy optical component capable of implementing the above-mentioned functions are all within the scope of protection claimed in the present application.
As shown in fig. 1, the fluid control module 3 in the present embodiment includes a fluid control circuit 3-1, a vacuum pump 3-2, a second valve 3-3, and a waste liquid bottle 3-4;
the fluid control circuit 3-1 is respectively connected with the control end of the first valve 7 and the central control module 5;
the fluid control circuit 3-1 is also respectively connected with a vacuum pump 3-2, a second valve 3-3 and a three-way valve 4-4 in the hot water circulation temperature control module 4, an air inlet of the vacuum pump 3-2 is connected with a discharge hole of the waste liquid bottle 3-4 through a through pipe, an output port of the second valve 3-3 is connected with a feed inlet of the waste liquid bottle 3-4 through a through pipe, and two input ports of the second valve 3-3 are respectively connected with a sample outlet through hole 1-12 and a first water outlet of the PCR chip.
The vacuum pump is used for extracting air and liquid to be removed from the digital PCR chip, the waste liquid bottle is used for containing waste liquid discharged from the digital PCR chip and waste water solution in a thermal cycle process, the fluid control circuit is used for controlling the vacuum pump, a second valve and the work of a three-way valve in the hot water cycle temperature control module, the second valve is connected with a sample outlet of the digital PCR chip except for one branch shown in figure 1, and the two branch valves, one branch of which is connected with a water outlet of the digital PCR chip, can also be formed by combining two independent valves, so long as the independent control of two fluid passages can be realized, and the three-way valve can also be replaced by 3 independent valves respectively connected with three constant temperature water tanks in a similar way, so long as the independent control of the three fluid passages can be ensured; it should be noted that the function that needs to be implemented in this embodiment is limited only by the term "fluid control circuit", and any circuit that can implement the above function in the prior art is within the scope of the present application, so that the specific circuit structure of this embodiment need not be described.
As shown in fig. 1, the hot water circulation temperature control module 4 in this embodiment includes a first constant temperature water tank 4-1, a second constant temperature water tank 4-2, a third constant temperature water tank 4-3, a three-way valve 4-4, and a temperature control circuit 4-5;
the input end of a temperature control circuit 4-5 is connected with a central control module 5, the output end of the temperature control circuit 4-5 is respectively connected with the temperature control ends of a first constant temperature water tank 4-1, a second constant temperature water tank 4-2 and a third constant temperature water tank 4-3, the three input ends of a three-way valve 4-4 are respectively connected with the first constant temperature water tank 4-1, the second constant temperature water tank 4-2 and the third constant temperature water tank 4-3 through a through pipe, the output end of the three-way valve 4-4 is connected with a water inlet on a digital PCR chip 1 through a through pipe, and the control end of the three-way valve 4-4 is connected with a fluid control circuit 3-1.
Wherein, the three-way valve is used for controlling conducting of three constant temperature water pitchers under fluid control circuit's control, and temperature control circuit heats three constant temperature water pitchers according to central control module's control, and is similar to fluid control circuit, and the temperature control circuit in this embodiment sets for based on its realization function, as long as the circuit that can realize above-mentioned function all is in the scope that this application requested protection among the prior art, need not in this embodiment to describe its concrete circuit structure.
Example 2:
as shown in fig. 5, the present embodiment provides a method for using the digital PCR system of embodiment 1, which includes the following steps:
s1, preparation: fixing a digital PCR chip on a two-dimensional step scanning platform, assembling and connecting all through pipes according to the requirements of all interfaces, simultaneously starting a central control module, setting parameters for regulating and controlling fluid control and temperature control, starting a temperature control circuit, and controlling the temperature of a constant-temperature water tank in a hot water circulation temperature control module according to the detection requirements of the digital PCR;
s2, filling: adjusting the opening state of a valve based on the digital PCR detection requirement, and pumping air in each micro-pipeline and micro-cavity in the digital PCR chip by using a vacuum pump, so that the sample liquid in the sample tube is sucked into the digital PCR chip until the sample liquid is filled in all the micro-pipelines and micro-cavities in the digital PCR chip;
after the vacuum pump is started, air in each micro-tube and micro-cavity in the digital PCR chip is pumped by the vacuum pump by utilizing the air permeability of the PDMS film to form negative pressure, and then the sample liquid in the sample tube is sucked into the digital PCR chip;
s3, sample discretization: adjusting the opening state of a valve based on the digital PCR detection requirement, introducing the oil phase in the sample tube into the digital PCR chip by using a vacuum pump, removing the sample liquid in each micro-pipeline, and isolating the oil phase from the sample liquid in each micro-cavity;
s4, thermal cycle amplification: adjusting the opening state of a valve based on the digital PCR detection requirement, and driving water in a constant-temperature water tank at a preset temperature to continuously pass through the digital PCR chip integrated cavity through a vacuum pump so as to realize a thermal cycle nucleic acid amplification reaction of sample liquid in each microcavity in the digital PCR chip;
the integrated cavity in the digital PCR chip is a cavity area in the cavity layer, and the cavity area is integrated in the digital PCR chip and is a component of the digital PCR chip, so the integrated cavity is also called as the integrated cavity of the digital PCR chip;
s5, image processing and data analysis: and controlling the valves and the vacuum pump to be closed, starting the optical detection module, scanning the optical detection module to obtain all micrographs of the digital PCR chip, splicing the micrographs by using the central control module to obtain a fluorescence picture of the whole microcavity reaction region of the digital PCR chip, and performing image processing and data analysis on the fluorescence picture to obtain a final detection result.
Specifically, the PCR detection requirements of step S1 above include conventional digital PCR detection and isothermal digital PCR detection;
when the detection requirement is conventional digital PCR detection, three constant-temperature water tanks in the hot water circulation temperature control module are respectively heated to a preset temperature and are kept stable;
when the detection requirement is isothermal digital PCR detection, controlling the temperature of two constant temperature water tanks in the hot water circulation temperature control module to a preset temperature respectively and maintaining the temperature to be stable, and leaving the other constant temperature water tank empty; wherein, one of the two thermostatic water tanks which are controlled by temperature is a thermostatic hot water tank, and the other thermostatic cold water tank.
In the above step S2:
when the detection requirement is conventional digital PCR detection, the sample filling process specifically comprises the following steps:
controlling the first valve to be opened and the second valve to be opened towards the first water outlet, and pumping air in each micro-pipeline and micro-cavity in the digital PCR chip by using a vacuum pump, so that the sample liquid in the sample tube is sucked into the digital PCR chip until the sample liquid is filled in all the micro-pipelines and micro-cavities in the digital PCR chip;
when the detection requirement is isothermal digital PCR detection, the sample filling process specifically comprises the following steps:
controlling the second valve to open towards the first water outlet, pumping air in each micro-pipeline and micro-cavity in the digital PCR chip by using a vacuum pump, then controlling the first valve to open, the second valve to open towards the first water outlet and the three-way valve to open towards the direction of the constant-temperature cold water tank, and sucking the sample liquid in the sample tube into the digital PCR chip until the sample liquid is filled in all the micro-pipelines and micro-cavities in the digital PCR chip;
in the above step S3:
when the detection requirement is conventional digital PCR detection, the mode of adjusting the opening state of the valve is as follows: controlling the first valve to open and the second valve to open towards the direction of the through hole of the sample outlet;
when the detection requirement is isothermal digital PCR detection, the mode of adjusting the opening state of the valve is as follows: controlling the first valve to open, the second valve to open towards the two inlet directions simultaneously and the three-way valve to open towards the direction of the constant-temperature cold water tank;
in the above step S4:
when the detection requirement is conventional digital PCR detection, the thermal cycle amplification method specifically comprises the following steps:
the three-way valve is controlled to be opened towards the three constant-temperature water tanks and the second valve is controlled to be opened towards the first water outlet, and the three hot water with different temperatures in the three constant-temperature water tanks are driven by the vacuum pump to sequentially circulate through the digital PCR chip integrated cavity according to a set sequence and period, so that the sample liquid in each microcavity in the digital PCR chip realizes the thermal cycle nucleic acid amplification reaction;
when the detection requirement is isothermal digital PCR detection, the thermal cycle amplification method specifically comprises the following steps:
and controlling the second valve to open towards the first water outlet and the three-way valve to open towards the constant-temperature hot water tank, and continuously passing constant-temperature water in the constant-temperature hot water tank through the cavity area by using the vacuum pump so as to realize thermal cycle nucleic acid amplification reaction of sample liquid in each microcavity in the digital PCR chip.
It should be noted that, in the above process, when the control of the valve and the vacuum pump is mentioned in each step, except for the valve or the vacuum pump for controlling the opening, other valves or vacuum pumps are all in the closed state, for example, in step S2, when the first valve and the second valve are controlled to open toward the first water outlet and the vacuum pump is started, the three-way valve and the second valve are in the closed state toward the sample outlet through hole.
Example 3:
as shown in FIG. 6, the conventional digital PCR detection is performed by using the system of the present invention, taking the detection of EGFR L858R mutation in peripheral blood of a certain lung cancer patient as an example, the specific steps are as follows:
(1) chip, reagent and sample preparation
Manufacturing an integrated cavity digital PCR chip containing 20000 micro-cavities by combining a photoetching mold manufacturing process with PDMS (polydimethylsiloxane) pouring reverse mold and an oxygen plasma surface treatment bonding process; preparing a pair of specific primers aiming at EGFR L858R mutation synthesis; circulating DNA is extracted from peripheral blood serum of a patient with lung cancer, polymerase, a primer, a probe, a buffer solution and the like are added into a sample tube for storing the circulating DNA, a sample solution is prepared by mixing, and paraffin oil is added into the sample solution.
(2) Chip assembly
And (2) placing and fixing the digital PCR chip prepared in the step (1) on a two-dimensional step scanning platform, communicating a chip sample inlet to the sample tube prepared in the step (1) through a micro tube and a valve, communicating a chip water inlet to three constant-temperature water tanks through the micro tube and a three-branch switching valve, and communicating a chip sample outlet and a water outlet to a vacuum pump through the micro tube and a two-branch switching valve.
(3) System control parameter setting
Starting a central control module, setting the temperatures of three constant-temperature water tanks to be T1 ═ 95 ℃, T2 ═ 60 ℃ and T3 ═ 50 ℃ respectively by using a software interface, and setting relevant parameters of a fluid control process as follows: opening a valve 1 and a valve 2-II, closing a valve 2-I and a valve 3, and keeping the time for 3 min; opening the valve 1 and the valve 2-I, closing the valve 2-II and the valve 3, and keeping the time for 5 min; thirdly, opening the valves 3-III and 2-II, and closing the valve 1, the valve 2-I, the valve 3-I and the valve 3-II for 10 min; opening valves 3-I and 2-II, and closing valves 1, 2-I, 3-II and 3-III for 10 min; 45 circulation: opening the valve 3-I and the valve 2-II, and closing the valve 1, the valve 2-I, the valve 3-II and the valve 3-III for 40 s; the valves 3-II and 2-II are opened, and the valves 1, 2-I, 3-I and 3-III are closed for 30 s. (wherein, the valve 1 corresponds to a first valve, the valve 2 corresponds to a second valve, the valves 2-I and 2-II respectively correspond to the directions of a sample outlet and a first water outlet which are connected with the digital PCR chip by the second valve, the valve 3 corresponds to a three-way valve, and the valves 3-I, 3-II and 3-III respectively correspond to the directions of the three-way valve which is connected with three constant temperature water tanks);
(4) chip loading and sample discretization
Starting a temperature control circuit until each constant-temperature water tank is heated to a preset constant temperature; after each constant temperature water tank is heated to a preset temperature, starting a fluid control circuit, opening a micro valve for connecting a sample tube and a sample inlet of a digital PCR chip and a micro valve for connecting a water outlet of the digital PCR chip and a waste liquid bottle by using a preset program of control software, keeping other micro valves closed, starting a vacuum pump, sucking sample liquid in the sample tube into the digital PCR chip, and filling the whole micro pipeline network and the micro cavity array of the digital PCR chip with the sample liquid within 3 min; after the sample filling is finished, opening a micro valve for connecting the sample tube and the sample inlet of the digital PCR chip and a micro valve for connecting the sample outlet of the digital PCR chip and the waste liquid bottle by using a control software preset program, keeping other micro valves closed, introducing the upper oil phase in the sample tube into the digital PCR chip by using a vacuum pump to remove redundant sample liquid in the sample filling pipeline, and completely replacing the sample liquid in the sample filling pipeline by using the oil phase filled in 5min, thereby isolating the sample liquid in each microcavity and realizing the discretization of the sample liquid.
(5) Thermal cycling amplification reactions
After sample filling and sample liquid discretization are completed, a fluid control circuit is used, a control software is used for presetting a program to control hot water with three different temperatures to circulate through the chip integrated microcavity, so that the sample liquid in the microcavity array positioned above the integrated microcavity is subjected to thermal cycle amplification reaction, and specific fluid control is performed as the fluid control flow (c) - (v) in the step (3), namely, the thermal cycle curve for realizing PCR reaction is as follows: 50 ℃ for 10 minutes, 95 ℃ for 10 minutes, and 45 cycles of 95 ℃ for 45 seconds and 60 ℃ for 40 seconds.
(6) Signal reading and analysis
After the thermal cycle amplification reaction is completed, a central control module software interface is used for starting a fluorescence light path of the optical module and automatically focusing, a scanning starting point and a scanning range are set, the optical module is controlled by control software to scan and record a digital PCR chip microcavity array area fluorescence photo through a two-dimensional scanning mobile platform, the obtained scanning fluorescence photo is automatically spliced by the software, an integral fluorescence picture of the digital PCR chip microcavity array area is obtained, finally a positive signal of the fluorescence picture is extracted through the software, and the EGFR L858R mutation condition of a lung cancer patient is analyzed.
As shown in FIG. 7, the detection result obtained by the above method is shown, and it can be seen from the figure that the system can achieve high sensitivity, accurate absolute quantification of rare mutation of cancer patients, and the chip design based on 20000 micro-cavities can achieve a higher dynamic measurement range (10-10)4copies/μL)。
Example 4:
as shown in fig. 8, the isothermal digital PCR detection is performed by using the system of the present invention, taking listeria detection as an example, the specific steps are as follows:
(1) chip, reagent and sample preparation
Manufacturing an integrated cavity digital PCR chip containing 20000 micro-cavities by combining a photoetching mold manufacturing process with PDMS (polydimethylsiloxane) pouring reverse mold and an oxygen plasma surface treatment bonding process; preparing a pair of specific primers aiming at the synthesis of Listeria monocytogenes; placing a sample tube storing a sample to be detected in an ice water box, adding a primer, magnesium acetate, an exo reagent of TwistDx company, a buffer solution and the like into the sample tube, mixing to prepare a sample solution, and adding paraffin oil into the sample solution.
(2) Chip assembly
And (2) placing and fixing the digital PCR chip prepared in the step (1) on a two-dimensional step scanning platform, communicating a chip sample inlet to the sample tube prepared in the step (1) through a micro tube and a one-way valve, communicating a chip water inlet to three constant-temperature water tanks through the micro tube and a three-branch switching valve, and communicating a chip sample outlet and a water outlet to a vacuum pump through the micro tube and a two-branch switching valve.
(3) System control parameter setting
Starting a central control module, setting the temperatures of three constant-temperature water tanks to be T1 ═ 0 ℃, T2 ═ 39 ℃ and T3 ═ room temperature (without water and in a vacant mode) by using a software interface, and setting relevant parameters of a fluid control flow as follows: opening a valve 2-II, closing a valve 1, a valve 2-I and a valve 3 for 5 min; opening the valve 1, the valve 2-II and the valve 3-I, and closing the valve 2-I, the valve 3-II and the valve 3-III for 3 min; thirdly, opening the valve 1, the valve 2-I, the valve 2-II and the valve 3-I, and closing the valve 3-II and the valve 3-III for 5 min; opening valves 2-II and 3-II, and closing valves 1, 2-I, 3-I and 3-III for 25 min; (wherein, the valve 1 corresponds to a first valve, the valve 2 corresponds to a second valve, the valves 2-I and 2-II respectively correspond to the direction of a sample outlet and a first water outlet through hole which are connected with the digital PCR chip of the second valve, the valve 3 corresponds to a three-way valve, and the valves 3-I, 3-II and 3-III respectively correspond to a constant-temperature cold water tank, a constant-temperature hot water tank and a vacant constant-temperature water tank which are connected with the three-way valve);
(4) chip loading and sample discretization
Starting the temperature control circuit until each constant-temperature water tank is heated or cooled to a preset constant temperature; after the water temperature of each constant temperature water tank reaches a preset temperature, starting a fluid control circuit, opening a micro valve for connecting a water outlet of the digital PCR chip and the waste liquid bottle by using a preset program of control software, keeping other micro valves closed, starting a vacuum pump, and establishing negative pressure in a micro pipeline network and a microcavity array of the digital PCR chip by using the air permeability of a PDMS film; after 5min, opening a micro valve for connecting a sample inlet of the digital PCR chip and the sample tube, and sucking the sample liquid in the sample tube into the digital PCR chip through the established negative pressure; meanwhile, a micro valve for connecting a water inlet of the digital PCR chip and a water tank at 0 ℃ is opened, cold water is filled into the chip integrated cavity, and the sample liquid filled in the chip is temporarily inhibited from generating PCR amplification reaction; after the sample filling is finished, opening a micro valve for connecting a sample outlet of the digital PCR chip and a waste liquid bottle by using a control software preset program, introducing an upper oil phase in the sample tube into the digital PCR chip by using a vacuum pump to remove redundant sample liquid in the sample filling pipeline, and completely replacing the sample liquid in the sample filling pipeline by using the filled oil phase within 5min, so that the sample liquid in each microcavity is isolated, and the discretization of the sample liquid is realized.
(5) Thermal cycling amplification reactions
After the sample filling and the sample liquid discretization are completed, the micro valve connecting the water inlet of the digital PCR chip and the water tank at 39 ℃ and the micro valve connecting the water outlet of the digital PCR chip and the waste liquid bottle are opened by utilizing a preset program of control software through the fluid control circuit, other micro valves are kept closed, and 39 ℃ hot water continuously flows through the chip integration cavity, so that the sample liquid in the microcavity array positioned on the integration microcavity is subjected to isothermal PCR reaction.
(6) Signal reading and analysis
After 25min, completing PCR reaction, starting a fluorescence light path of the optical module by using a software interface of the central control module, automatically focusing, setting a scanning starting point and a scanning range, controlling the two-dimensional scanning moving platform of the optical module to scan and record a fluorescence photo of the microcavity array region of the digital PCR chip by using control software, automatically splicing the obtained scanning fluorescence photo by using the software to obtain an integral fluorescence picture of the microcavity array region of the digital PCR chip, finally extracting a positive signal of the fluorescence picture by using the software, analyzing the concentration of listeria monocytogenes in a sample, and providing help for food safety and environmental monitoring.
In the description of the present invention, it is to be understood that the terms "center", "thickness", "upper", "lower", "horizontal", "top", "bottom", "inner", "outer", "radial", and the like, indicate orientations and positional relationships based on the orientations and positional relationships shown in the drawings, and are used merely for convenience in describing the present invention and for simplicity in description, and do not indicate or imply that the referenced devices or elements must have a particular orientation, be constructed and operated in a particular orientation, and thus, are not to be construed as limiting the present invention. Furthermore, the terms "first," "second," and "third" are used for descriptive purposes only and are not to be construed as indicating or implying relative importance or an implicit indication of the number of technical features. Thus, features defined as "first", "second", "third" may explicitly or implicitly include one or more of the features.

Claims (10)

1. An integrated digital PCR system is characterized by comprising a digital PCR chip (1), an optical detection module (2), a fluid control module (3), a hot water circulation temperature control module (4) and a central control module (5);
the digital PCR chip (1) is placed on a two-dimensional stepping scanning platform (2-6) on the optical detection module (2) and is connected with the hot water circulation temperature control module (4) and the fluid control module (3), the fluid control module (3) is also connected with the hot water circulation temperature control module (4) and the central control module (5) respectively, and the hot water circulation temperature control module (4) and the optical detection module (2) are both connected with the central control module (5).
2. The integrated digital PCR system according to claim 1, wherein the digital PCR chip (1) is a high-density microcavity array structure of integrated cavities;
the digital PCR chip (1) comprises a top layer glass cover plate (1-1), a thin film layer (1-2), a cavity layer (1-3) and a bottom layer glass substrate (1-4);
the top layer glass cover plate (1-1) is provided with a sample inlet through hole (1-11), a sample outlet through hole (1-12), a first water inlet through hole (1-13) and a first water outlet through hole (1-14);
the film layer (1-2) is provided with a polydimethylsiloxane micro-cavity array structure (1-21), a sample injection port (1-22), a waste liquid sample outlet port (1-23), a second water inlet through hole (1-24) and a second water outlet through hole (1-25), the polydimethylsiloxane micro-cavity array structure (1-21) is formed by connecting a plurality of parallel micro-pipelines (1-211) in series with a plurality of micro-cavities (1-212), and two ends of each micro-pipeline (1-211) are respectively connected with the sample injection port (1-22) and the waste liquid sample outlet port (1-23); the second water inlet through holes (1-24) and the second water outlet through holes (1-25) are respectively arranged at two sides of the polydimethylsiloxane micro-cavity array structure (1-21); one side of the film layer (1-2) provided with the polydimethylsiloxane micro-cavity array structure (1-21) is a structural surface, and the other side is a non-structural surface;
the cavity layer (1-3) is provided with a third water inlet (1-31), a third water outlet (1-32) and a cavity area (1-33), and the projection area of the cavity area (1-33) on the thin film layer (1-2) covers the polydimethylsiloxane micro-cavity array structure (1-21) area;
the first water inlet through holes (1-13), the second water inlet through holes (1-24) and the third water inlets (1-31) are in one-to-one correspondence and are communicated with one another, the first water outlet through holes (1-14), the second water outlet through holes (1-25) and the third water outlets (1-32) are in one-to-one correspondence and are communicated with one another, the sample inlet through holes (1-11) are in correspondence and are communicated with the sample inlets (1-22), and the sample outlet through holes (1-12) are in correspondence and are communicated with the waste liquid sample outlets (1-23);
the top layer glass cover plate (1-1) and the structural surface of the film layer (1-2) are bonded to form a closed micro-pipeline system, and the non-structural surface of the film layer (1-2), the cavity layer (1-3) and the bottom layer glass substrate (1-4) are bonded to form a closed cavity;
the sample inlet through holes (1-11) of the digital PCR chip (1) are connected with the outlet of the sample tube (6) through a through tube, and a first valve (7) is arranged on the through tube.
3. The integrated digital PCR system according to claim 2, wherein at least 1000 micro-cavities are included in the polydimethylsiloxane micro-cavity array structure (1-21), and the geometrical shape and size of each micro-cavity are consistent;
the polydimethylsiloxane micro-cavity array structure (1-21) is a non-transparent structure, a polydimethylsiloxane film at the bottom of each micro-cavity (1-212) forms an isolation film between the micro-cavity (1-212) and the cavity layer (1-3), and the thickness of the isolation film is 20-200 micrometers.
4. The integrated digital PCR system according to claim 2, wherein the cavity layers (1-3) are through-penetrating structures, the cavity layers (1-3) are made of polydimethylsiloxane, double-sided tape or film, and the thickness of the cavity layers (1-3) is 30-1000 microns.
5. The integrated digital PCR system according to claim 2, wherein the optical detection module (2) comprises a two-dimensional step-and-scan platform (2-6), a bright-field optical assembly and a fluorescence microscopy optical assembly;
the bright field optical assembly is arranged above the two-dimensional stepping scanning platform (2-6), the fluorescence microscopic optical assembly is arranged below the two-dimensional stepping scanning platform (2-6), and the two-dimensional stepping scanning platform (2-6) is connected with the central control module (5);
the two-dimensional step scanning platform (2-6) is used for placing the digital PCR chip (1);
the bright field optical element is used for observing the integrity of the sample introduction of the digital PCR chip (1);
the fluorescence microscopic optical assembly is used for detecting the PCR result.
6. The integrated digital PCR system according to claim 2, wherein the fluid control module (3) comprises a fluid control circuit (3-1), a vacuum pump (3-2), a second valve (3-3) and a waste bottle (3-4);
the fluid control circuit (3-1) is respectively connected with the control end of the first valve (7) and the central control module (5);
the fluid control circuit (3-1) is further connected with a three-way valve (4-4) in the vacuum pump (3-2), the second valve (3-3) and the hot water circulation temperature control module (4) respectively, an air inlet of the vacuum pump (3-2) is connected with an air outlet of the waste liquid bottle (3-4) through a through pipe, an output port of the second valve (3-3) is connected with a feed inlet of the waste liquid bottle (3-4) through a through pipe, and two input ports of the second valve (3-3) are connected with a sample outlet through hole (1-12) and a first water outlet of the digital PCR chip (1) respectively.
7. The integrated digital PCR system according to claim 6, wherein the hot water circulation temperature control module (4) comprises a first constant temperature water tank (4-1), a second constant temperature water tank (4-2), a third constant temperature water tank (4-3), a three-way valve (4-4) and a temperature control circuit (4-5);
the input end of the temperature control circuit (4-5) is connected with the central control module (5), the output end of the temperature control circuit (4-5) is respectively connected with the temperature control ends of the first constant temperature water tank (4-1), the second constant temperature water tank (4-2) and the third constant temperature water tank (4-3), three input ends of the three-way valve (4-4) are respectively connected with the first constant temperature water tank (4-1), the second constant temperature water tank (4-2) and the third constant temperature water tank (4-3) through a through pipe, the output end of the three-way valve (4-4) is connected with the water inlet on the digital PCR chip (1) through a through pipe, the control end of the three-way valve (4-4) is connected with the fluid control circuit (3-1).
8. A method for using an integrated digital PCR system is characterized by comprising the following steps:
s1, preparation: fixing a digital PCR chip on a two-dimensional step scanning platform, assembling and connecting all through pipes according to the requirements of all interfaces, simultaneously starting a central control module, setting parameters for regulating and controlling fluid control and temperature control, starting a temperature control circuit, and controlling the temperature of a constant-temperature water tank in a hot water circulation temperature control module according to the detection requirements of the digital PCR;
s2, filling: adjusting the opening state of a valve based on the digital PCR detection requirement, and pumping air in each micro-pipeline and micro-cavity in the digital PCR chip by using a vacuum pump, so that the sample liquid in the sample tube is sucked into the digital PCR chip until the sample liquid is filled in all the micro-pipelines and micro-cavities in the digital PCR chip;
s3, sample discretization: adjusting the opening state of a valve based on the digital PCR detection requirement, introducing the oil phase in the sample tube into the digital PCR chip by using a vacuum pump, removing the sample liquid in each micro-pipeline, and isolating the oil phase from the sample liquid in each micro-cavity;
s4, thermal cycle amplification: adjusting the opening state of a valve based on the digital PCR detection requirement, and driving water in a constant-temperature water tank at a preset temperature to continuously pass through the digital PCR chip integrated cavity through a vacuum pump so as to realize a thermal cycle nucleic acid amplification reaction of sample liquid in each microcavity in the digital PCR chip;
s5, image processing and data analysis: and controlling the valves and the vacuum pump to be closed, starting the optical detection module, scanning the optical detection module to obtain all micrographs of the digital PCR chip, splicing the micrographs by using the central control module to obtain a fluorescence picture of the whole microcavity reaction region of the digital PCR chip, and performing image processing and data analysis on the fluorescence picture to obtain a final detection result.
9. The method of using the integrated digital PCR system of claim 8, wherein the PCR detection requirements of step S1 include conventional digital PCR detection and isothermal digital PCR detection;
when the detection requirement is conventional digital PCR detection, three constant-temperature water tanks in the hot water circulation temperature control module are respectively heated to a preset temperature and are kept stable;
when the detection requirement is isothermal digital PCR detection, controlling the temperature of two constant temperature water tanks in the hot water circulation temperature control module to a preset temperature respectively and maintaining the temperature to be stable, and leaving the other constant temperature water tank empty; wherein, one of the two thermostatic water tanks which are controlled by temperature is a thermostatic hot water tank, and the other thermostatic cold water tank.
10. The method of using the integrated digital PCR system according to claim 9,
in the step S2:
when the detection requirement is conventional digital PCR detection, the sample filling process specifically comprises the following steps:
controlling the first valve to be opened and the second valve to be opened towards the first water outlet, and pumping air in each micro-pipeline and micro-cavity in the digital PCR chip by using a vacuum pump, so that the sample liquid in the sample tube is sucked into the digital PCR chip until the sample liquid is filled in all the micro-pipelines and micro-cavities in the digital PCR chip;
when the detection requirement is isothermal digital PCR detection, the sample filling process specifically comprises the following steps:
controlling the second valve to open towards the first water outlet, pumping air in each micro-pipeline and micro-cavity in the digital PCR chip by using a vacuum pump, then controlling the first valve to open, the second valve to open towards the first water outlet and the three-way valve to open towards the direction of the constant-temperature cold water tank, and sucking the sample liquid in the sample tube into the digital PCR chip until the sample liquid is filled in all the micro-pipelines and micro-cavities in the digital PCR chip;
in the step S3:
when the detection requirement is conventional digital PCR detection, the mode of adjusting the opening state of the valve is as follows: controlling the first valve to open and the second valve to open towards the direction of the through hole of the sample outlet;
when the detection requirement is isothermal digital PCR detection, the mode of adjusting the opening state of the valve is as follows: controlling the first valve to open, the second valve to open towards the two inlet directions simultaneously and the three-way valve to open towards the direction of the constant-temperature cold water tank;
in the step S4:
when the detection requirement is conventional digital PCR detection, the thermal cycle amplification method specifically comprises the following steps:
the three-way valve is controlled to be opened towards the three constant-temperature water tanks and the second valve is controlled to be opened towards the first water outlet, the three hot water with different temperatures in the three constant-temperature water tanks are driven by the vacuum pump to sequentially circulate through the integrated cavity of the digital PCR chip according to a set sequence and period, so that the sample liquid in each microcavity in the digital PCR chip realizes the thermal cycle nucleic acid amplification reaction;
when the detection requirement is isothermal digital PCR detection, the thermal cycle amplification method specifically comprises the following steps:
and controlling the second valve to open towards the first water outlet and the three-way valve to open towards the constant-temperature hot water tank, and continuously passing constant-temperature water in the constant-temperature hot water tank through the cavity area by using the vacuum pump so as to realize thermal cycle nucleic acid amplification reaction of sample liquid in each microcavity in the digital PCR chip.
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