CN109536384B

CN109536384B - Digital PCR system for rapid absolute quantification of nucleic acid and application thereof

Info

Publication number: CN109536384B
Application number: CN201811582784.8A
Authority: CN
Inventors: 毛红菊; 程祖乐
Original assignee: Shanghai Institute of Microsystem and Information Technology of CAS
Current assignee: Shanghai Institute of Microsystem and Information Technology of CAS
Priority date: 2018-12-24
Filing date: 2018-12-24
Publication date: 2022-02-18
Anticipated expiration: 2038-12-24
Also published as: CN109536384A

Abstract

The invention provides a digital PCR system for rapid absolute quantification of nucleic acid and application thereof, wherein the system comprises: the sample reaction chip comprises a plurality of mutually independent sample storage cavities, wherein a plurality of micron-sized sample reaction units can be accommodated in the sample storage cavities; the micro heating disc array is used for heating the sample reaction chip, the micro heating disc array comprises a plurality of mutually independent micro heating discs, and one micro heating disc only corresponds to and heats one sample storage cavity; and a temperature control system connected with the micro heating disc for realizing temperature control; wherein, a single sample storage cavity and a corresponding single micro heating plate jointly form a rapid digital PCR detection unit. The digital PCR system provided by the invention has various advantages over the existing digital PCR system, can realize the heating rate of more than 100 ℃/s and the cooling rate of more than 50 ℃/s, has single cycle time of less than 10s, and thus realizes the rapid absolute quantitative detection of the digital PCR nucleic acid.

Description

Digital PCR system for rapid absolute quantification of nucleic acid and application thereof

Technical Field

The invention relates to a technology for rapid amplification and absolute quantitative detection of nucleic acid, in particular to a digital PCR system for rapid and absolute quantification of nucleic acid and application thereof.

Background

Polymerase Chain Reaction (PCR) is a gene amplification technique that can exponentially amplify a target DNA sequence over a period of time to obtain a large number of copies of the target DNA sequence.

The 40 amplification cycles of a nucleic acid sample on a conventional PCR instrument usually take more than one hour, and a large amount of time is consumed in the process of raising and lowering the temperature of the system and stabilizing the temperature, which not only consumes a large amount of time, but also needs a large volume of sample. In some specific PCR applications, the evaporation of the reaction solution and other serious problems can be caused.

The rapid PCR technique is expected to solve these problems. The research of rapid PCR dates back to 90 s in the last century, Carl W. et al found that the real time for the completion of denaturation and annealing reactions in the PCR reaction process is less than 1s, which is much shorter than the currently designed 15s, and that the extension time of a 200bp long sequence is only about 10s, which is much shorter than the designed 60s [ Biotechniques.10(1991) ]. Meanwhile, the research result also shows that for nucleic acid amplification, the long-time temperature transition process is easy to cause non-specific amplification and the activity of the nucleic acid amplification enzyme is reduced. Therefore, they have proposed a rapid PCR method for realizing highly specific amplification by shortening the temperature switching and stabilization time of a PCR system through a rapid temperature increase and decrease speed and a stabilization speed. Based on the principle, Neuzil et al utilizes a micro-processing method to prepare a temperature circulating device based on joule heat, utilizes the characteristic of low heat capacity of millimeter-scale micro-heating electrodes to realize the temperature rise of 175 ℃/s and the temperature reduction rate of 125 ℃/s, and successfully applies the temperature circulating device to a reaction system containing 100nl of reaction liquid and 1 ul of oil phase, thereby realizing the application of completing the real-time fluorescence quantitative experiment of Nucleic acid within 5 minutes at the fastest speed [ Nucleic Acids Res.34(2006) ]. However, when quantifying a target molecule, real-time fluorescence quantitative PCR requires drawing a standard curve according to the Ct value, and the detection accuracy is reduced due to slight variations in the Ct value. In the rapid qPCR, the amplification efficiency is easily affected due to the short amplification period, the reaction system is small, and the detection of a fluorescent signal is difficult, so that the obtained amplification curve does not have the traditional qPCR rule. For semi-quantitative application of the Ct value of an amplification curve to be analyzed, the Ct value is difficult to determine and changes greatly. Therefore, the rapid PCR still has the problem of data accuracy in the real-time fluorescence semi-quantitative detection of nucleic acid.

The digital PCR is an absolute quantitative nucleic acid detection technology developed by combining the traditional PCR technology with the microfluidic technology, and has great application prospect in the aspects of early disease detection and individualized diagnosis and treatment. The digital PCR is an end-point method detection technology, has small dependence on amplification efficiency, represents the copy number of target molecules by directly counting the number of positive and negative signals of each reaction unit, can accurately detect the target molecules without a standard curve, and realizes absolute quantitative analysis of the target molecules. Furthermore, the method has great advantages in the detection of low-abundance samples. At present, the research on digital PCR is mainly focused on droplet generation/cavity manufacturing, and the research on the thermal cycle process of the digital PCR is less, so that the reaction of the digital PCR still adopts a traditional PCR instrument, and the whole experiment time can be as long as one hour or more. In the case of digital PCR, long-term thermal cycling reaction is likely to cause the breakage of liquid droplets and evaporation of the reaction solution.

Therefore, considering the problems of the reaction time and the detection precision of PCR, developing a rapid digital PCR technology, shortening the reaction time of digital PCR, improving the detection efficiency of digital PCR, and realizing rapid absolute quantitative detection of nucleic acid is very important.

Disclosure of Invention

The invention aims to provide a digital PCR system for rapid absolute quantification of nucleic acid and application thereof, thereby solving the problems of overlong reaction time, lower detection efficiency and low detection precision of the digital PCR system in the prior art.

In order to solve the technical problems, the invention adopts the following technical scheme:

according to a first aspect of the present invention there is provided a digital PCR system for rapid absolute quantification of nucleic acids comprising: the sample reaction chip comprises a plurality of independent millimeter-scale sample storage cavities, and a plurality of micron-scale sample reaction units can be accommodated in a single sample storage cavity; the micro heating disc array is used for heating the sample reaction chip, the micro heating disc array comprises a plurality of mutually independent millimeter-scale micro heating discs, and one micro heating disc only corresponds to and heats one sample storage cavity; and a temperature control system connected with the micro heating disc for realizing temperature control; the single sample storage cavity and the corresponding single micro heating plate jointly form a rapid digital PCR detection unit, and the digital PCR system formed by a plurality of rapid digital PCR detection units can realize the temperature switching rate of more than 50 ℃/s and the single thermal cycle time of less than 20 s.

The sample reaction chip also has the function of dividing the sample into micron-sized sample reaction units.

Preferably, 2000-10000 micron-sized sample reaction units can be accommodated in a single sample storage cavity in the sample reaction chip.

Further preferably, the sample reaction unit in the sample storage cavity is in the form of water-in-oil droplets, and the diameter of the water-in-oil droplets is 20-100 μm.

Preferably, the sample reaction chip further has a droplet splitting function, and the droplet splitting function is realized by a microfluidic pipeline with gradually narrowed pipe diameter, so that the number of droplets entering each sample storage cavity is basically equal.

Specifically, after the sample inlet pipe and the oil phase inlet pipe are converged into one main microfluidic pipeline, the main microfluidic pipeline is divided into two secondary microfluidic pipelines, each secondary microfluidic pipeline is divided into two tertiary microfluidic pipelines, and the rest is done until the sample inlet pipe and the oil phase inlet pipe are connected to each sample storage cavity. Preferably, the diameter of each next-stage pipeline is 60-70% of the diameter of the adjacent previous-stage pipeline, and more preferably, 65-70% of the diameter of each next-stage pipeline, so that the liquid drops are uniformly divided into two smaller liquid drops when entering the next-stage pipeline from the previous-stage pipeline until the liquid drops enter the sample storage cavity.

According to the invention, the advantages of the design of the micro-fluidic pipeline with gradually narrowed pipe diameter comprise: 1) the generation frequency of liquid drops can be improved, the micro-fluidic operation time is shortened, and the whole micro-fluidic operation can be completed within 5 minutes; 2) but also ensures that the number and size of the droplets in each sample storage chamber are equal; 3) the design that the pipeline gradually narrows also increases the flow resistance of the pipeline, and the flow resistance is increased along with the gradual narrowing of the pipe diameter, so that the phenomena that liquid drops entering the sample storage cavity flow back to the pipeline in the subsequent heating process and liquid drops among the sample storage cavities flow are prevented.

Also preferably, the sample reaction unit in the sample storage cavity can be in a micro-cavity form, the diameter of the micro-cavity is 20-100 μm, and the height of the micro-cavity is 30-100 μm.

Each micro heating plate is integrated with a temperature measuring electrode and a heating electrode, and the temperature control system is respectively connected with the temperature measuring electrode and the heating electrode to realize the temperature control of the micro heating plate.

Preferably, the micro heating plate array comprises a plurality of micro heating plates prepared by etching, corroding and the like, each micro heating plate is connected with the main body framework through different narrow beams on two sides, the short and narrow connecting beams ensure the strength of the heating plates, and meanwhile, independent and uniform sample heating areas on the micro heating plates are also realized. Alternatively, the narrow beams have dimensions of 300 μm × 350 μm and 300 μm × 600 μm, respectively.

Still preferably, every little heating plate is integrated a temperature measurement electrode and a heating electrode, temperature measurement electrode and heating electrode communicate with the little heating plate through the narrow roof beam of little heating plate both sides respectively for separate each other between heating electrode circuit and the temperature measurement electrode circuit, reduce the possibility of crosstalking, this design has simplified the line layout of heating array simultaneously, have great wiring advantage when possessing more than or equal to eight heating plates quantity, and is further, pad and heating electrode have been shortened in this design, distance between the temperature measurement electrode, the extra heat consumption of system has been reduced.

Preferably, the sample reaction chip is made of any one or more materials selected from polydimethylsiloxane, epoxy resin, glass, quartz glass, polymethyl methacrylate, polycarbonate and the like.

Preferably, the sample reaction chip is formed by connecting and compounding at least two materials in a plasma bonding mode, an ultrasonic welding mode, a chemical bonding mode and the like.

Preferably, the substrate of the micro-heating disk array is made of any one or more materials selected from silicon, glass, quartz glass and the like, and the temperature measuring electrode and the heating electrode are respectively made of any one or more materials selected from platinum, gold, silver, copper, chromium, titanium and the like.

According to a preferred embodiment of the present invention, a temperature control system comprises: the temperature measuring device comprises a temperature measuring module based on a constant current source, a data acquisition card for data acquisition and communication, an upper computer for data processing, a PWM electronic switch and an actuator for providing heating power, such as a 12V power supply.

Preferably, the sample reaction chip further comprises a dielectric layer, the micro heating plate heats the sample in the sample storage chamber through the dielectric layer, and the micro heating plate is not in direct contact with the sample. Preferably, the thickness of the dielectric layer is 100-300 μm.

The dielectric layer can be made of any one or a plurality of materials selected from polydimethylsiloxane, epoxy resin, glass, quartz glass, polymethyl methacrylate, polycarbonate and the like.

It should be understood that the number of sample storage chambers on the sample reaction chip may be any suitable number, and may be any suitable arrangement.

It should also be understood that the number of the micro heating plates on the micro heating plate array is equal to the number of the sample storage chambers on the sample reaction chip, and the arrangement is basically the same, in other words, the number and arrangement of the micro heating plates on the micro heating plate array are determined according to the sample storage chambers on the sample reaction chip.

According to the second aspect of the present invention, there is also provided an application of the rapid digital PCR system as described above as a universal platform in the field of absolute quantification of nucleic acids, including but not limited to absolute quantitative detection of target molecules of nucleic acids (DNA and RNA, etc.), absolute quantitative detection of nucleic acid mutations, absolute quantitative detection of nucleic acid methylation, absolute quantitative detection of copy number variation, and absolute quantitative detection of nucleic acids such as accurate library construction of sequencing, in fields of scientific research, medical inspection, food inspection, environmental monitoring, national defense safety, etc.

According to a preferred embodiment of the invention, the application comprises the following steps: 1) providing a digital PCR system as described above; 2) dispersing the nucleic acid sample into uniform micron-sized sample reaction units by a microfluidic technology, and distributing the uniform micron-sized sample reaction units into each millimeter-sized sample storage cavity; 3) a sample storage cavity and a corresponding micro heating disc form a rapid digital PCR detection unit, and under the action of a temperature control system, each sample storage cavity is heated by the corresponding micro heating disc to realize rapid digital PCR; and 4) counting the number of sample units with fluorescence signals in each storage cavity to determine the accurate value of the initial concentration of the target nucleic acid, thereby realizing absolute quantification of the nucleic acid sample.

According to the digital PCR system provided by the invention, the PCR temperature rise and drop rate of more than 20 ℃/s can be realized, wherein the denaturation time is 1-5 s, the denaturation temperature is 90-98 ℃, the extension time is 5-15 s, the extension temperature is 48-72 ℃, the single cycle time is less than 20s, and the total time of 40 cycles is less than 15 minutes.

Preferably, a PCR heating rate of 50 ℃/s or greater and a cooling rate of 30 ℃/s or greater are provided.

Particularly preferably, the detection unit can realize a temperature rise speed of more than 100 ℃/s and a temperature fall speed of more than 50 ℃/s, the PCR reaction denaturation time is less than 2s, the extension time is less than 8s, and the single cycle time is less than 10 s.

According to the invention, when the sample reaction chip is subjected to thermal circulation, the oil phase injection port always maintains positive pressure, so that the pressure in the cavity is ensured to be balanced.

According to the digital PCR system provided by the invention, the sample reaction chip comprises a plurality of millimeter-scale sample storage cavities, each sample storage cavity can contain 2000-10000 micron-scale sample reaction units, the micro heating disc array is composed of a base material with good heat conductivity and a metal electrode, and the size of a single micro heating disc is in millimeter level. The single sample storage cavity and the corresponding single micro heating plate jointly form a rapid digital PCR detection unit, and under the action of the temperature control system, the single sample storage cavity is heated through the corresponding micro heating plate to realize rapid digital PCR. Because each detection unit of the digital PCR system has the characteristic of small heat capacity of the system and each micro heating disc is independent, the digital PCR system provided by the invention has various advantages over the existing digital PCR system, including rapid heating and cooling rate and accurate thermal control during PCR, even the heating rate of more than 100 ℃/s and the cooling rate of more than 50 ℃/s can be realized, the PCR reaction denaturation time is less than 2s, the extension time is less than 8s, and the single cycle time is less than 10s, thereby realizing the high-specificity rapid absolute quantitative detection of the digital PCR nucleic acid.

In conclusion, the invention provides a digital PCR system for rapid absolute quantification of nucleic acid with greatly shortened reaction time and improved detection efficiency and application thereof.

Drawings

FIG. 1 is a top view of a sample reaction chip in a digital PCR system according to a preferred embodiment of the present invention;

FIG. 2 is a cross-sectional view of the sample reaction chip shown in FIG. 1;

FIG. 3 is a top view of a micro-heated disk array of the digital PCR system according to the preferred embodiment;

FIG. 4 is a schematic diagram of the overall configuration of the digital PCR system according to the preferred embodiment;

FIG. 5 is a schematic diagram of the temperature cycling of the digital PCR system in the application of the amplification and detection of the GAPDH gene in a human umbilical vein endothelial cell cDNA sample, provided in accordance with the present invention;

FIG. 6 is a graph showing the results observed under natural light in the application of amplification and detection of the GAPDH gene in a human umbilical vein endothelial cell cDNA sample according to the preferred embodiment;

FIG. 7 is a graph of the local results observed under fluorescence for the application of amplification and detection of the GAPDH gene in a human umbilical vein endothelial cell cDNA sample according to the preferred embodiment;

FIG. 8(a), FIG. 8(b), FIG. 8(c), FIG. 8(d) are graphs showing the results of the copy number detection of GAPDH gene in samples of different concentrations according to the preferred embodiment.

Detailed Description

The present invention will be further described with reference to the following specific examples. It should be understood that the following examples are illustrative only and are not intended to limit the scope of the present invention.

According to a preferred embodiment of the present invention, there is provided a digital PCR system, the system mainly comprising: a sample reaction chip 1, a micro heating plate array 2 and a temperature control system 3.

As shown in FIG. 1, the sample reaction chip 1 comprises eight independent sample storage chambers 11, one sample injection port 12, one oil phase injection port 13 and a microfluidic pipeline 15 connecting the above structures, according to the preferred embodiment, the eight independent sample storage chambers 11 are divided into two rows and arranged on the sample reaction chip 1, the size of each sample storage chamber 11 in this embodiment is 2mm × 2mm, and actually, 2 to 5mm × 2 to 5mm may be preferred. The preferable arrangement route of the microfluidic pipeline 15 is shown in the figure, after a sample inlet pipe where a sample inlet 12 is located and an oil phase inlet pipe where an oil phase inlet 13 is located are converged into a main microfluidic pipeline, the main microfluidic pipeline is divided into two parts to form two secondary microfluidic pipelines, each secondary microfluidic pipeline is divided into two parts to form two three-stage microfluidic pipelines, and the like until the two secondary microfluidic pipelines are connected to each sample storage cavity, wherein the diameter of each next-stage pipeline is 70% of that of the adjacent previous-stage pipeline.

It should be understood that the number and arrangement of the sample storage chambers 11 and the arrangement of the microfluidic channels 15 are not limited to those shown in fig. 1, and are given by way of example only and are not limited thereto, and may be any other suitable number and any other suitable arrangement. According to the present invention, the size of the individual sample storage chambers 11 is preferably in the millimeter scale.

According to the preferred embodiment, when a nucleic acid sample is injected through the sample injection port 12, the nucleic acid sample is sheared by the oil phase from the oil phase injection port 13 to generate large-sized droplets, each time the nucleic acid sample passes through the classification of the narrower microfluidic channel, the large-sized droplets are uniformly divided into two smaller droplets, and so on, the nucleic acid sample is finally and uniformly distributed into each sample storage cavity 11 to form a plurality of micron-sized sample reaction units. Preferably, a single sample storage cavity contains 2000-10000 micron-sized sample reaction units.

It should also be understood that the dispersed form of the sample is not limited to the water-in-oil droplet form shown in this embodiment, but may be in the form of a microcavity.

As shown in fig. 2, the sample reaction chip 1 is formed by bonding a Polydimethylsiloxane (PDMS) chip 16 and a glass substrate 17 by means of plasma bonding.

It should be understood that the sample reaction chip 1 may be made of any one material selected from polydimethylsiloxane, epoxy resin, glass, quartz glass, polymethylmethacrylate, and polycarbonate, or a composite of a plurality of materials. It is also preferable that the sample reaction chip 1 is composed of at least two materials by one of plasma bonding, ultrasonic welding, chemical bonding, and the like.

As shown in fig. 3, the micro-heating plate array 2 is formed by etching through a silicon wafer by an etching process to form eight mutually independent micro-heating plates 21, the eight micro-heating plates 21 are divided into two rows and arranged neatly, and one micro-heating plate 21 only corresponds to and heats one sample storage cavity 11, so that the number of the micro-heating plates 21 is equal to the number of the sample storage cavities 11 on the sample reaction chip 1, and the arrangement mode is basically the same. It should be understood, however, that the number and arrangement of the micro-heating disks 21 are not limited to those shown in fig. 3, and are only given by way of example and not limitation, and in fact, the number and arrangement of the micro-heating disks 21 depend on the sample storage chamber 11 of the sample reaction chip 1.

Wherein, micro-heating plate 21 is still connected with the main body frame through the narrow beam 24, 24' of both sides, and this kind of short and narrow tie-beam has guaranteed the intensity of heating plate, also realizes independent and even sample heating region on the micro-heating plate simultaneously. Preferably, the narrow beams 24, 24' on both sides are 300 μm 350 μm and 300 μm 600 μm in size, respectively. Each micro-heating disk 21 is also connected to a temperature measuring electrode 22 and a heating electrode 23 through narrow beams 24, 24', respectively.

According to the preferred embodiment, the micro heating plates 21 are hollowed out, and as shown in the figure, a hollowed-out area 25 is formed between the micro heating plate 21 and the micro heating plate 21'. In the preferred embodiment, the heating electrodes 23 are also arranged in a ring shape. The mutual independence of temperature measurement and heating process between each micro heating plate 21 is fully guaranteed through the annular heating electrode 23 and the hollow-out treatment, the crosstalk is less, and therefore the single micro heating plate 21 can independently and accurately heat the single sample storage cavity 11.

According to a preferred embodiment of the present invention, the substrate of the micro-heating disk array 2 is made of any one or more materials selected from silicon, glass, and quartz glass, and the temperature measuring electrode 22 and the heating electrode 23 are made of any one or more materials selected from platinum, gold, silver, copper, chromium, and titanium.

According to a preferred embodiment of the present invention, the sample reaction chip 1 further comprises a dielectric layer through which the micro-heating plate 21 heats the sample in the sample storage chamber 11, and the micro-heating plate 21 is not in direct contact with the sample.

As shown in fig. 4, in the digital PCR system provided in the present embodiment, the temperature control system 3 mainly comprises a constant current source 31, a data acquisition card 32, an upper computer 33, a PWM electronic switch 34, and an actuator 35, where the actuator is optionally a 12V power supply. The working principle of the temperature control system 3 is as follows: the constant current source 31 is used as a temperature measuring circuit, the data acquisition card 32 is used for reading the voltage at the temperature measuring electrode 22 end and transmitting the voltage signal to the upper computer 33 end, the upper computer 33 processes the voltage signal and converts the voltage signal into real-time temperature, meanwhile, the PID control algorithm is used for obtaining adjusting parameters and generating PWM signals, and the PWM signals adjust the power output of the actuator 35 to the heating electrode through the PWM electronic switch 34, so that the heating of the sample reaction chip 1 is realized.

According to the digital PCR system provided by the preferred embodiment, the single sample storage cavity 11 and the corresponding single micro heating plate 21 jointly form a fast digital PCR detection unit, and under the action of the temperature control system 3, the single sample storage cavity 11 is heated by the corresponding single micro heating plate 21 to realize fast temperature change. Since each detection unit of the digital PCR system has the characteristic of small heat capacity of the system and each micro-heating disk 21 is independent of each other, the digital PCR system provided according to the present invention has various advantages over the current digital PCR system, such as a fast ramp-up rate and accurate thermal control during PCR, a temperature switching and settling time of less than 2s in this embodiment, a PCR heating rate of at least about 50 ℃/s or more and a ramp-down rate of 30 ℃/s or more, a single digital PCR cycle time of less than 20s, and a total digital PCR cycle time of less than 15 minutes.

According to a preferred embodiment of the present invention, the above digital PCR system is applied to the amplification and detection of the GAPDH gene in human umbilical vein endothelial cell cDNA. Wherein, the digital PCR amplification system is as follows:

reagent	Volume of
		Takara Premix Ex Taq	12.5μL
Forward Primer(10μM)	0.5μL
		Reverse Primer(10μM)	0.5μL
FAM-fluorescent probes	0.5μL
		cDNA sample	2.0μL
Water (W)	9.0μL

The rapid digital PCR program is shown in FIG. 5, and the hot start at 95 ℃ is carried out for 30s, 40 cycles are carried out, each cycle comprises 3s of denaturation reaction at 95 ℃, 15s of annealing extension reaction at 60 ℃, and the total amplification time is 12min and 30 s.

The primer sequences used were as follows:

F：5’GGGAAACTGTGGCGTGAT3’(SEQ ID NO.1)

R:5’GAGTGGGTGTCGCTGTTGA3’(SEQ ID NO.2)

the sequence of the amplification template is shown as SEQ ID NO. 3.

And (4) counting results: observing the amplified sample reaction chip under a microscope, counting the total number of droplets N and the number of droplets N with fluorescence signals under natural light (figure 6) and fluorescence (figure 7) respectively by adopting a FAM channel of an inverted microscope, and obtaining the copy number k of the GAPDH gene in the sample through Poisson distribution:

furthermore, the copy numbers of the GAPDH genes in different cDNA samples with known concentration gradients are detected by using the system, and the detection results are shown in fig. 8(a), fig. 8(b), fig. 8(c) and fig. 8(d), and the calculated copy numbers of the detected positive droplets have a good correspondence with the known sample concentrations, which indicates that the digital PCR method provided by the present invention is reliable in practical application.

The above-mentioned embodiments of the present invention are only preferred embodiments, and are not intended to limit the scope of the present invention, and since the solution adopted by the embodiments of the present invention is technically compatible with other applications in the claims, the above-mentioned embodiments of the present invention may be varied in various applications as described in the claims. All simple and equivalent changes and modifications made according to the claims and the content of the specification of the present application fall within the scope of the claims of the present patent application. The invention has not been described in detail in order to avoid obscuring the invention.

Sequence listing

<110> Shanghai microsystem and information technology institute of Chinese academy of sciences

<120> a digital PCR system for rapid absolute quantification of nucleic acids and applications thereof

<160> 3

<210> 1

<211> 18

<212> DNA

<213> Artificial sequence

<400> 1

gggaaactgt ggcgtgat 18

<210> 2

<211> 18

<212> DNA

<213> Artificial sequence

<400> 2

gagtgggtgt cgctgttga 19

<210> 3

<211> 299

<212> DNA

<213> GAPDH Gene

<400> 3

gggaaactgt ggcgtgatgg ccgcggggct ctccagaaca tcatccctgc ctctactggc 60

gctgccaagg ctgtgggcaa ggtcatccct gagctgaacg ggaagctcac tggcatggcc 120

ttccgtgtcc ccactgccaa cgtgtcagtg gtggacctga cctgccgtct agaaaaacct 180

gccaaatatg atgacatcaa gaaggtggtg aagcaggcgt cggagggccc cctcaagggc 240

atcctgggct acactgagca ccaggtggtc tcctctgact tcaacagcga cacccactc 299

Claims

1. A digital PCR system for rapid absolute quantification of nucleic acids, comprising:

the sample reaction chip comprises a plurality of independent millimeter-scale sample storage cavities, a plurality of micron-scale sample reaction units can be accommodated in a single sample storage cavity, the sample reaction chip has a liquid drop splitting function, the liquid drop splitting function is realized through a microfluidic pipeline with gradually narrowed pipe diameter, the number of liquid drops entering each sample storage cavity is basically equal, the diameter of each next-stage pipeline is 65% -70% of that of the adjacent previous-stage pipeline, and the size of the single sample storage cavity is 2-5 mm multiplied by 2-5 mm;

the micro heating disc array is used for heating the sample reaction chip, the micro heating disc array is formed by etching through a silicon wafer by adopting an etching process, the micro heating disc array comprises a plurality of mutually independent micro heating discs with millimeter scale, one micro heating disc only corresponds to and heats one sample storage cavity, each micro heating disc is integrated with one temperature measuring electrode and one heating electrode, the temperature measuring electrodes and the heating electrodes are respectively communicated with the micro heating discs through different narrow beams at two sides of the micro heating disc, and the micro heating discs are hollowed out to reduce the temperature transfer between every two adjacent micro heating discs; and

the temperature control system is connected with the micro heating disc to realize temperature control;

the single sample storage cavity on the sample reaction chip can contain 2000-10000 micron-sized sample reaction units, the micron-sized sample reaction units in the sample reaction chip comprise a water-in-oil droplet form or a micro-cavity form, the single sample storage cavity and the corresponding single micro-heating plate jointly form a rapid digital PCR detection unit, and the digital PCR system formed by the rapid digital PCR detection units can realize the temperature switching rate of more than 50 ℃/s and the single thermal cycle time of less than 20 s.

2. The digital PCR system of claim 1, wherein each micro heating plate is integrated with a temperature measuring electrode and a heating electrode, and the temperature measuring electrode and the heating electrode are respectively communicated with the micro heating plate through different narrow beams at two sides of the micro heating plate.

3. The digital PCR system of claim 1, wherein the sample reaction chip is made of any one or more materials selected from polydimethylsiloxane, epoxy resin, glass, quartz glass, polymethyl methacrylate and polycarbonate.

4. The digital PCR system of claim 1, wherein the substrate of the micro heating disk array is made of at least one material selected from the group consisting of silicon, glass, and quartz glass, and the temperature measuring electrode and the heating electrode are made of any one or more materials selected from the group consisting of platinum, gold, silver, copper, chromium, and titanium.

5. Use of the digital PCR system according to any one of claims 1-4, comprising absolute quantitative detection of nucleic acid target molecules, absolute quantitative detection of nucleic acid mutations, absolute quantitative detection of nucleic acid methylation, absolute quantitative determination of copy number variation, and accurate pooling for sequencing.

6. Use according to claim 5, characterized in that it comprises the following steps:

1) providing a digital PCR system according to any one of claims 1-4;

2) dispersing the nucleic acid sample into uniform micron-sized sample reaction units by a microfluidic technology, and uniformly distributing the micron-sized sample reaction units into sample storage cavities with various millimeter scales;

3) a sample storage cavity and a corresponding micro heating disc form a rapid digital PCR detection unit, and under the action of a temperature control system, each sample storage cavity is heated by the corresponding micro heating disc to realize rapid digital PCR; and

4) the accurate value of the initial concentration of the target nucleic acid can be determined by counting the number of sample units with the fluorescence signals in each storage cavity, so that the absolute quantification of the nucleic acid sample is realized.