CN110616144A - Liquid drop digital PCR chip and use method thereof - Google Patents
Liquid drop digital PCR chip and use method thereof Download PDFInfo
- Publication number
- CN110616144A CN110616144A CN201810970247.4A CN201810970247A CN110616144A CN 110616144 A CN110616144 A CN 110616144A CN 201810970247 A CN201810970247 A CN 201810970247A CN 110616144 A CN110616144 A CN 110616144A
- Authority
- CN
- China
- Prior art keywords
- chip
- pcr
- amplification
- pipeline
- detection position
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 239000007788 liquid Substances 0.000 title claims abstract description 52
- 238000007847 digital PCR Methods 0.000 title claims abstract description 22
- 238000000034 method Methods 0.000 title claims description 30
- 238000001514 detection method Methods 0.000 claims abstract description 71
- 230000003321 amplification Effects 0.000 claims abstract description 59
- 238000003199 nucleic acid amplification method Methods 0.000 claims abstract description 59
- 239000012807 PCR reagent Substances 0.000 claims abstract description 34
- 230000003068 static effect Effects 0.000 claims abstract description 22
- 230000015572 biosynthetic process Effects 0.000 claims abstract description 9
- 238000005755 formation reaction Methods 0.000 claims abstract description 9
- 230000003287 optical effect Effects 0.000 claims abstract description 5
- 238000006243 chemical reaction Methods 0.000 claims description 12
- 239000000758 substrate Substances 0.000 claims description 10
- WYTGDNHDOZPMIW-RCBQFDQVSA-N alstonine Natural products C1=CC2=C3C=CC=CC3=NC2=C2N1C[C@H]1[C@H](C)OC=C(C(=O)OC)[C@H]1C2 WYTGDNHDOZPMIW-RCBQFDQVSA-N 0.000 claims description 9
- 238000011304 droplet digital PCR Methods 0.000 claims description 9
- 239000012295 chemical reaction liquid Substances 0.000 claims description 8
- 239000003153 chemical reaction reagent Substances 0.000 claims description 7
- 238000007789 sealing Methods 0.000 claims description 4
- 238000011901 isothermal amplification Methods 0.000 claims description 3
- 229920003229 poly(methyl methacrylate) Polymers 0.000 claims description 3
- 239000004926 polymethyl methacrylate Substances 0.000 claims description 3
- 239000004205 dimethyl polysiloxane Substances 0.000 claims description 2
- 235000013870 dimethyl polysiloxane Nutrition 0.000 claims description 2
- CXQXSVUQTKDNFP-UHFFFAOYSA-N octamethyltrisiloxane Chemical compound C[Si](C)(C)O[Si](C)(C)O[Si](C)(C)C CXQXSVUQTKDNFP-UHFFFAOYSA-N 0.000 claims description 2
- 238000004987 plasma desorption mass spectroscopy Methods 0.000 claims description 2
- 229920000435 poly(dimethylsiloxane) Polymers 0.000 claims description 2
- 239000004810 polytetrafluoroethylene Substances 0.000 claims description 2
- 229920001343 polytetrafluoroethylene Polymers 0.000 claims description 2
- 238000013461 design Methods 0.000 abstract description 5
- 238000005516 engineering process Methods 0.000 description 9
- 238000000137 annealing Methods 0.000 description 5
- 238000004925 denaturation Methods 0.000 description 5
- 230000036425 denaturation Effects 0.000 description 5
- 238000011002 quantification Methods 0.000 description 5
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
- 239000000463 material Substances 0.000 description 4
- 238000004519 manufacturing process Methods 0.000 description 3
- 230000035772 mutation Effects 0.000 description 3
- 108020004707 nucleic acids Proteins 0.000 description 3
- 150000007523 nucleic acids Chemical class 0.000 description 3
- 102000039446 nucleic acids Human genes 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 239000000523 sample Substances 0.000 description 3
- 238000000926 separation method Methods 0.000 description 3
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 2
- 108010006785 Taq Polymerase Proteins 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 230000014509 gene expression Effects 0.000 description 2
- 238000007731 hot pressing Methods 0.000 description 2
- 230000002209 hydrophobic effect Effects 0.000 description 2
- 238000010191 image analysis Methods 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 238000004806 packaging method and process Methods 0.000 description 2
- 238000012545 processing Methods 0.000 description 2
- 230000035945 sensitivity Effects 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- GPRLSGONYQIRFK-MNYXATJNSA-N triton Chemical compound [3H+] GPRLSGONYQIRFK-MNYXATJNSA-N 0.000 description 2
- 238000009423 ventilation Methods 0.000 description 2
- 108700011259 MicroRNAs Proteins 0.000 description 1
- 108091034117 Oligonucleotide Proteins 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 238000011529 RT qPCR Methods 0.000 description 1
- 238000010923 batch production Methods 0.000 description 1
- 238000005842 biochemical reaction Methods 0.000 description 1
- 238000003759 clinical diagnosis Methods 0.000 description 1
- 230000002860 competitive effect Effects 0.000 description 1
- 239000013068 control sample Substances 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000001746 injection moulding Methods 0.000 description 1
- 239000002679 microRNA Substances 0.000 description 1
- 244000000010 microbial pathogen Species 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000007481 next generation sequencing Methods 0.000 description 1
- 239000011148 porous material Substances 0.000 description 1
- 239000002683 reaction inhibitor Substances 0.000 description 1
- 239000013074 reference sample Substances 0.000 description 1
- 239000004094 surface-active agent Substances 0.000 description 1
- 230000009261 transgenic effect Effects 0.000 description 1
- 239000000439 tumor marker Substances 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
Classifications
-
- 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
-
- 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
- B01L3/502707—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 the manufacture of the container or its components
-
- 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
-
- 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
Abstract
The invention designs a liquid drop digital PCR chip which integrates three functional units of liquid drop formation, amplification and detection. The chip droplet forming unit comprises an oil hole, a PCR reagent hole and corresponding pipelines, and the oil hole pipeline and the PCR reagent hole pipeline form a cross; the chip amplification unit is a snake-shaped pipeline; the chip detection unit comprises a dynamic detection position and a static detection position, wherein the dynamic detection position is a pipeline and is matched with the optical detection module to detect the amplified liquid drops in real time; and the static detection position collects all the amplified liquid drops, and a CCD camera is used for simultaneously detecting positive amplification.
Description
Technical Field
The invention relates to the technical field of digital PCR, in particular to a microfluidic chip integrating droplet formation, amplification and detection.
Background
As a nucleic acid in vitro amplification technology, the PCR technology has the advantages of strong specificity, high sensitivity, rapidness, simplicity, good repeatability, less template quantity and the like, and is widely applied to the fields of forensic detection, clinical diagnosis, molecular biology research and the like. The basic principle of PCR is to simulate the natural DNA copying process, rely on the specific complementation of oligonucleotide primer and template, and realize the exponential amplification of target segment through multiple denaturation-annealing-extension temperature cycle processes.
The digital PCR technology is a new nucleic acid detection and quantification technology, and is different from the traditional quantification PCR technology, the digital PCR adopts an absolute quantification mode, does not depend on a standard curve and a reference sample, and directly detects the copy number of a target sequence. This detection format is widely used because it has more excellent sensitivity, specificity and accuracy than conventional qPCR. The technology has been generally accepted for the advantages of trace nucleic acid sample detection, rare mutation detection and expression amount micro-difference identification under complex background, and has been paid more and more attention to the wide application prospect in gene expression research, microRNA research, genome copy number identification, cancer marker rare mutation detection, pathogenic microorganism identification, transgenic component identification, NGS sequencing library accurate quantification, result verification and other aspects.
The strategy adopted by digital PCR is very simple, namely, divide and conquer, a standard PCR reaction system is equally distributed into a large number of tiny reactors, and target molecules (DNA templates) with or without one or more copies are contained in each reactor, so that 'single-molecule template PCR amplification' is realized. Common methods of liquid separation include liquid drop methods and grid methods. The liquid drop method is to use oil and surfactant to wrap PCR reagent to form liquid drops with uniform size, and the grid method is to manufacture tiny pore channels on the chip to realize liquid separation. After the amplification is finished, the copy number of the target sequence is calculated through the number of 'luminous' reactors, so that accurate absolute quantification can be carried out without depending on a control sample and a standard curve; in addition, because the digital PCR only judges whether two amplification states exist or not when the result is judged, the intersection point of a fluorescence signal and a set threshold line does not need to be detected, and the identification of a Ct value is not relied on, the influence of the amplification efficiency on the reaction of the digital PCR is greatly reduced, and the tolerance capability on a PCR reaction inhibitor is greatly improved; the process of standard reaction system allocation in digital PCR experiments can greatly reduce the background sequence concentration which has a competitive effect with a target sequence, so that the digital PCR technology is particularly suitable for detecting rare mutation in a complex background.
The micro-fluidic chip technology realizes a multi-step biochemical reaction process by utilizing highly integrated functional units in a micro-scale space, reduces manual operation, realizes Sample in-result out (Sample in-Answer out), and provides an automatic solution for the fields of biomedical diagnosis, analytical chemistry, life science and the like. The microfluidic chip has the advantages of reducing the consumption of a reaction system and required reagents, reducing manual operation processes through automatic control and improving the operation consistency. Currently, the mainstream digital PCR products on the market include QX100 and QX200 from Bio-Rad; RainDrop, RainDance corporation; the Bio-Mark HD system from Fluidigm and the QuantStaudio system from Thermo Fisher Scientific. Such products typically contain more than 2 devices-a liquid separation (and amplification) device and a result reading device. Increasing the number of manual handling steps and risking exposure of the amplification product. In order to solve the problem, the invention designs an integrated chip which integrates the functions of droplet formation, amplification and detection.
Disclosure of Invention
The invention designs a liquid drop digital PCR chip which integrates three functional units of liquid drop formation, amplification and detection. The chip design is simplified, a complex structure and a micro valve are not needed, the processing difficulty is reduced, and the production is easy. Meanwhile, a using method of the liquid drop digital PCR chip is also provided.
The first aspect of the invention provides a droplet digital PCR chip.
The digital PCR chip for liquid drops integrates three functional units of liquid drop formation, amplification and detection; the chip droplet forming unit comprises an oil hole (1), a PCR reagent hole (2) and corresponding pipelines (3 and 4), wherein the oil hole pipeline and the PCR reagent hole pipeline form a cross (5); the chip amplification unit is a snake-shaped pipeline (6); the chip detection unit comprises a dynamic detection position (7) and a static detection position (8), wherein the dynamic detection position (7) is a pipeline, and the static detection position is a square bin (8); in addition, the chip comprises a vent hole (9).
Preferably, the chip comprises a cover plate and a substrate, wherein the cover plate at least comprises 1 PCR reagent hole (2) and 1 oil hole (1), and the substrate comprises a chamber and a pipeline.
Preferably, the amplification unit of the chip is coupled to an external temperature control system to form a temperature gradient zone.
Preferably, the chip adopts a flow type amplification method, PCR liquid drops are formed and then sequentially flow through a serpentine pipe (6) of an amplification area to realize the amplification process, and the diameter of the pipe only allows one liquid drop to pass through each time. The amplification mode comprises isothermal amplification and temperature-variable amplification. In isothermal amplification, an external temperature control system keeps constant temperature, and the generated PCR liquid drops can pass through a constant temperature area where the serpentine pipeline is located at a constant speed. Two symmetrical ends of the serpentine pipeline (6) in the temperature-variable amplification are respectively a denaturation temperature zone and an annealing and extension temperature zone, and the time of liquid drops passing through the corresponding temperature zones can be controlled by adjusting the flow rate.
Preferably, the surface of the chip is transparent, and the manufacturing material is one or more of PS, PC, COC, COP, PMMA, PTFE or PDMS.
Preferably, the chip can form 2 ten thousand to 2 million PCR droplets with the droplet size of 2 picoliters to 0.2 milliliters, and more preferably, can form 2 ten thousand to 1 million PCR droplets with the droplet size of 5 picoliters to 0.1 milliliters.
Preferably, the amplification results can be detected in two ways, including dynamic detection and static detection. The dynamic detection means that the amplified liquid drops are detected in real time by an optical detection module (10) matched with the dynamic detection position (7), and the static detection means that all the amplified liquid drops are collected at a static detection position (8) and are simultaneously detected by a CCD camera (11).
In another aspect, the invention provides a method for using a droplet digital PCR chip, comprising the following steps:
(a) injecting corresponding reagents into the PCR reagent hole (2) and the oil hole (1);
(b) sealing gaskets are covered on the PCR reagent hole (2) and the oil hole (1);
(c) driving the liquid in the reagent hole and the oil hole to flow to the cross (5) to form PCR reaction liquid drops with uniform size;
(d) the PCR reaction liquid drops sequentially flow through the amplified serpentine pipeline (6) to complete the amplification reaction;
(e) the positive liquid drops are counted in real time at the dynamic detection position (7), or the positive liquid drops are simultaneously detected at the static detection position (8) after the amplification reaction is completely finished.
The invention has the advantages that the microfluidic chip is utilized to integrate three functions of droplet formation, amplification and detection counting in the droplet digital PCR technology, a closed system is formed, and the product pollution after amplification is avoided. The design of the micro-fluidic chip is simplified, a complex structure and a micro valve are not needed, and the cost and the difficulty of batch production are reduced. Meanwhile, the complexity of an external control system is reduced, and the method can be applied to small-sized automatic equipment.
Drawings
FIG. 1 is a schematic diagram of a droplet digital PCR chip according to the present invention
FIG. 2 is a schematic diagram of the method for detecting the amplification result of a droplet digital PCR chip according to the present invention
Wherein the reference numbers: 1. oil hole, 2, PCR reagent hole, 3, pipeline one, 4, pipeline two, 5, cross, 6, snakelike pipeline, 7, dynamic detection position, 8, static detection position, 9, air vent, 10, optical detection module, 11, CCD camera.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are exemplary and in no way limit the present invention and its applications or uses. Meanwhile, the sizes of the respective portions shown in the drawings are not drawn in an actual proportional relationship for the convenience of description.
Example 1.
A droplet digital PCR chip integrates three functional units of droplet formation, amplification and detection, as shown in FIG. 1 and FIG. 2. Wherein, chip droplet forms the unit and contains 1 oilhole (1), 1 PCR reagent hole (2) and oilhole pipeline one (3) and pipeline two (4). The oil hole pipeline I (3), the pipeline II (4) and the PCR reagent hole pipeline form a cross (5); the chip amplification unit is a snake-shaped pipeline (6); the chip detection adopts a dynamic detection position (7), and a static detection position (8) can be used for storing liquid drops after detection. The dynamic detection position (7) is a pipeline, and the caliber of the dynamic detection position is consistent with that of the snake-shaped pipeline (6). In addition, the chip comprises a vent hole (9), and a hydrophobic film is adhered at the vent hole to prevent liquid leakage and realize a ventilation function.
The chip comprises cover plate and substrate, contains 1 PCR reagent hole (2), 1 oilhole (1) and 1 air vent (9) on the cover plate, contains pipeline one (3), pipeline two (4), PCR reagent hole pipeline, amplification unit's snakelike pipeline (6), dynamic detection position (7) and static detection position (8) of oilhole on the substrate.
The amplification units of the chip are coupled to an external temperature control system to form temperature bands, including 95 and 60 degrees.
The chip adopts a flow type amplification method, PCR liquid drops sequentially flow through a snake-shaped pipeline (6) of an amplification area after being formed, the amplification process is realized, and the diameter of the pipeline only allows one liquid drop to pass through each time. The temperature-variable amplification mode is adopted, 95 ℃ denaturation is carried out for 5 seconds, 60 ℃ annealing and extension are carried out for 15 seconds, and the cycle is carried out for 40 times. Two symmetrical ends of the serpentine pipeline (6) in the amplification process are respectively a denaturation temperature region and an annealing and extension temperature region.
The chip material is PMMA. And respectively processing the cover plate and the substrate, and then carrying out hot-pressing packaging.
The chip can form about 10 ten thousand PCR droplets with a volume of 100 pl. A single reaction requires 10ul PCR reagents and 180ul oil. The PCR system was Taq polymerase 0.25U/ul, 10 mM Tris-HCl (pH 8.9), 1.5 mM MgC120.5 mg/ml BSA, 0.1% NaCl, 0.1% Triton X-lOO, 0.8 mM dNTP mix, 14pmol primer 1,14pmol primer 2, 1xSYBR Green I and diluted template DNA.
The dynamic detection position (7) is used for detecting and counting the amplified liquid drops one by one. The dynamic detection means that an optical detection module (10) is matched outside the dynamic detection position (7) to detect the amplified liquid drops in real time.
The use method of the liquid drop digital PCR chip comprises the following steps:
(a) 10ul of PCR reagent and 180ul of oil are respectively injected into the PCR reagent hole (2) and the oil hole (1);
(b) sealing gaskets are covered on the PCR reagent hole (2) and the oil hole (1);
(c) driving the liquid in the PCR reagent hole (2) and the oil hole (1) to flow, and enabling the reagent in the oil hole to simultaneously flow out of the pipeline I (3) and the pipeline II (4) and to be converged with the PCR reagent at the cross (5) to form PCR reaction liquid drops with uniform size;
(d) the PCR reaction liquid drops sequentially flow through the amplified serpentine pipeline (6), and the amplification reaction is completed after 40 cycles;
(e) and counting the positive liquid drops in real time at the dynamic detection position (7) and calculating the absolute content of the template DNA.
Example 2.
A droplet digital PCR chip integrates three functional units of droplet formation, amplification and detection. Wherein, chip droplet forms the unit and contains 1 oilhole (1), 1 PCR reagent hole (2) and oilhole pipeline one (3) and pipeline two (4). The first pipeline (3) and the second pipeline (4) of the oil hole and the PCR reagent hole pipeline form a cross (5); the chip amplification unit is a snake-shaped pipeline (6); the chip detection adopts a static detection position (8), and after all liquid drops are amplified, the liquid drops enter the static detection position (8) and are tiled, a CCD camera is used for photographing and acquiring positive signals. In addition, the chip comprises a vent hole (9), and a hydrophobic film is adhered at the vent hole to prevent liquid leakage and realize a ventilation function.
The chip comprises a cover plate and a substrate, wherein the cover plate comprises 1 PCR reagent hole (2), 1 oil hole (1) and 1 vent hole (9), and the substrate comprises a first pipeline (3) of the oil hole, a second pipeline (4), a PCR reagent hole pipeline, a snake-shaped pipeline (6) of an amplification unit and a static detection position (8).
The amplification units of the chip are coupled to an external temperature control system to form temperature bands, including 95 and 60 degrees.
The chip adopts a flow type amplification method, PCR liquid drops sequentially flow through a snake-shaped pipeline (6) of an amplification area after being formed, the amplification process is realized, and the diameter of the pipeline only allows one liquid drop to pass through each time. The temperature-variable amplification mode is adopted, 95 ℃ denaturation is carried out for 5 seconds, 60 ℃ annealing and extension are carried out for 15 seconds, and the cycle is carried out for 40 times. Two symmetrical ends of the serpentine pipeline (6) in the amplification process are respectively a denaturation temperature region and an annealing and extension temperature region.
The chip material is COC. And respectively carrying out injection molding on the cover plate and the substrate, and then carrying out hot-pressing packaging.
The chip can form about 2 ten thousand PCR droplets with a volume of 1 nl. A single reaction required 20ul PCR reagents and 80ul oil volume. The PCR system was Taq polymerase 0.25U/ul, 10 mM Tris-HCl (pH 8.9), 1.5 mM MgC120.5 mg/ml BSA, 0.1% NaCl, 0.1% Triton X-lOO, 0.8 mM dNTP mix, 14pmol primer 1,14pmol primer 2, 1xSYBR Green I and diluted template DNA.
And (3) simultaneously detecting the amplified liquid drops by using a static detection position (8) and a matched CCD camera, and counting positive results by using image analysis.
The use method of the liquid drop digital PCR chip comprises the following steps:
(a) 20ul of PCR reagent and 80ul of oil are respectively injected into the PCR reagent hole (2) and the oil hole (1);
(b) sealing gaskets are covered on the PCR reagent hole (2) and the oil hole (1);
(c) driving the liquid in the PCR reagent hole (2) and the oil hole (1) to flow, and enabling the reagent in the oil hole to simultaneously flow out of the pipeline I (3) and the pipeline II (4) and to be converged with the PCR reagent at the cross (5) to form PCR reaction liquid drops with uniform size;
(d) the PCR reaction liquid drops sequentially flow through the amplified serpentine pipeline (6), and the amplification reaction is completed after 40 cycles;
(e) and after all the liquid drops enter the static detection position (8), taking a picture by using a CCD (charge coupled device), counting positive results by using image analysis, and calculating the absolute content of the template DNA.
The drawings and examples are only for the purpose of illustrating the invention and it is within the scope of the invention that the invention is not limited thereto but may be modified slightly within the spirit and scope of the invention as defined by the appended claims. Such as the material, shape and size of the microfluidic chip, the shape and size of the chamber, the shape and size of various functional and connective channels, etc. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention.
Claims (10)
1. A liquid drop digital PCR chip is characterized in that three functional units of liquid drop formation, amplification and detection are integrated;
the chip liquid drop forming unit comprises an oil hole, a PCR reagent hole and corresponding pipelines, and the oil hole pipeline and the PCR reagent hole pipeline form a cross;
the chip amplification unit is a serpentine pipeline;
the chip detection unit comprises a dynamic detection position and a static detection position, the dynamic detection position is a pipeline, and the static detection position is a square bin;
the chip contains a vent hole.
2. The digital PCR chip for liquid drops according to claim 1, wherein the chip comprises a cover plate and a substrate, the cover plate comprises at least 1 PCR reagent hole and 1 oil hole, and the substrate comprises a chamber and a pipeline.
3. The digital PCR chip for liquid drop of claim 1, wherein the amplification unit of the chip is coupled to an external temperature control system to form a temperature gradient zone.
4. The digital droplet PCR chip of claim 1, wherein PCR droplets are formed and then sequentially flow through the serpentine channel of the amplification region to achieve amplification by a flow-based amplification method.
5. The digital droplet PCR chip of claim 4, wherein the amplification process comprises isothermal amplification and temperature-variable amplification.
6. The digital PCR chip of claim 1, wherein the chip is transparent and made of one or more of PS, PC, COC, COP, PMMA, PTFE, and PDMS.
7. A droplet digital PCR chip according to claim 1, wherein 2 to 2 million PCR droplets are formed, and the droplet size is 2 picoliters to 0.2 ml.
8. The digital droplet PCR chip of claim 1, wherein the amplification result can be detected by two methods, including dynamic detection and static detection.
9. The digital PCR chip of claim 8, wherein the dynamic detection means detects the amplified droplets in real time by an optical detection module at a dynamic detection position, and the static detection means collects all the amplified droplets at a static detection position and detects them simultaneously by a CCD camera.
10. A method of using a droplet digital PCR chip according to any one of claims 1 to 9,
(1) injecting corresponding reagents into the PCR reagent hole and the oil hole;
(2) sealing gaskets are covered on the PCR reagent hole and the oil hole;
(3) driving the liquid in the reagent hole and the oil hole to flow to the cross to form PCR reaction liquid drops with uniform size;
(4) the PCR reaction liquid drops sequentially flow through the amplified serpentine pipeline to finish the amplification reaction;
(5) and detecting the positive liquid drops at the dynamic detection position in real time, or simultaneously detecting the positive liquid drops at the static detection position after the amplification reaction is completely finished.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810970247.4A CN110616144A (en) | 2018-08-24 | 2018-08-24 | Liquid drop digital PCR chip and use method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810970247.4A CN110616144A (en) | 2018-08-24 | 2018-08-24 | Liquid drop digital PCR chip and use method thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN110616144A true CN110616144A (en) | 2019-12-27 |
Family
ID=68921038
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810970247.4A Pending CN110616144A (en) | 2018-08-24 | 2018-08-24 | Liquid drop digital PCR chip and use method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN110616144A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111729700A (en) * | 2020-07-09 | 2020-10-02 | 墨卓生物科技(上海)有限公司 | Liquid drop detection method adopting dPCR integrated chip |
| CN112553063A (en) * | 2020-12-22 | 2021-03-26 | 苏州缔因安生物科技有限公司 | Integrated digital nucleic acid amplification chip based on micro-droplets and use method and application thereof |
| CN112630148A (en) * | 2020-12-16 | 2021-04-09 | 海南大学 | Integrated liquid drop digital PCR detection platform |
Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20080166720A1 (en) * | 2006-10-06 | 2008-07-10 | The Regents Of The University Of California | Method and apparatus for rapid nucleic acid analysis |
| CN102527454A (en) * | 2012-01-31 | 2012-07-04 | 复旦大学 | Micro-fluid control drop concentration device for sample enrichment |
| KR20130079799A (en) * | 2012-01-03 | 2013-07-11 | 한국과학기술원 | Fabrication method of uniform submicron droplets and polymeric monodiperse particles using microfluidic flow-focusing devices with three-dimensional topography |
| CN103343092A (en) * | 2013-07-19 | 2013-10-09 | 中国科学院上海微系统与信息技术研究所 | Method for manufacturing digital PCR (polymerase chain reaction) chip based on mineral-oil saturated PDMS (polydimethylsiloxane) material |
| US20140127290A1 (en) * | 2012-11-08 | 2014-05-08 | Ohio State Innovation Foundation | Microcapsules Encapsulating Living Cells |
| CN105505761A (en) * | 2015-12-21 | 2016-04-20 | 中国科学院苏州生物医学工程技术研究所 | Digital isothermal nucleic acid detecting device and detecting method thereof |
| CN105765055A (en) * | 2013-08-27 | 2016-07-13 | 基纽拜奥股份有限公司 | Microfluidic devices and methods of their use |
| CN107478629A (en) * | 2017-09-04 | 2017-12-15 | 中国科学院苏州生物医学工程技术研究所 | A kind of large area digital pcr droplet fluorescence high pass amount detecting device and method |
| CN107583692A (en) * | 2017-05-23 | 2018-01-16 | 深圳市博瑞生物科技有限公司 | Drop micro-fluidic chip and preparation method thereof |
-
2018
- 2018-08-24 CN CN201810970247.4A patent/CN110616144A/en active Pending
Patent Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20080166720A1 (en) * | 2006-10-06 | 2008-07-10 | The Regents Of The University Of California | Method and apparatus for rapid nucleic acid analysis |
| KR20130079799A (en) * | 2012-01-03 | 2013-07-11 | 한국과학기술원 | Fabrication method of uniform submicron droplets and polymeric monodiperse particles using microfluidic flow-focusing devices with three-dimensional topography |
| CN102527454A (en) * | 2012-01-31 | 2012-07-04 | 复旦大学 | Micro-fluid control drop concentration device for sample enrichment |
| US20140127290A1 (en) * | 2012-11-08 | 2014-05-08 | Ohio State Innovation Foundation | Microcapsules Encapsulating Living Cells |
| CN103343092A (en) * | 2013-07-19 | 2013-10-09 | 中国科学院上海微系统与信息技术研究所 | Method for manufacturing digital PCR (polymerase chain reaction) chip based on mineral-oil saturated PDMS (polydimethylsiloxane) material |
| CN105765055A (en) * | 2013-08-27 | 2016-07-13 | 基纽拜奥股份有限公司 | Microfluidic devices and methods of their use |
| CN105505761A (en) * | 2015-12-21 | 2016-04-20 | 中国科学院苏州生物医学工程技术研究所 | Digital isothermal nucleic acid detecting device and detecting method thereof |
| CN107583692A (en) * | 2017-05-23 | 2018-01-16 | 深圳市博瑞生物科技有限公司 | Drop micro-fluidic chip and preparation method thereof |
| CN107478629A (en) * | 2017-09-04 | 2017-12-15 | 中国科学院苏州生物医学工程技术研究所 | A kind of large area digital pcr droplet fluorescence high pass amount detecting device and method |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111729700A (en) * | 2020-07-09 | 2020-10-02 | 墨卓生物科技(上海)有限公司 | Liquid drop detection method adopting dPCR integrated chip |
| CN112630148A (en) * | 2020-12-16 | 2021-04-09 | 海南大学 | Integrated liquid drop digital PCR detection platform |
| CN112553063A (en) * | 2020-12-22 | 2021-03-26 | 苏州缔因安生物科技有限公司 | Integrated digital nucleic acid amplification chip based on micro-droplets and use method and application thereof |
| CN112553063B (en) * | 2020-12-22 | 2024-03-01 | 苏州缔因安生物科技有限公司 | Micro-droplet-based integrated digital nucleic acid amplification chip and use method and application thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Rane et al. | Microfluidic continuous flow digital loop-mediated isothermal amplification (LAMP) | |
| Shang et al. | Loop-mediated isothermal amplification-based microfluidic chip for pathogen detection | |
| Rödiger et al. | Nucleic acid detection based on the use of microbeads: a review | |
| US11125752B2 (en) | Method for rapid accurate dispensing, visualization and analysis of single cells | |
| Sato et al. | Microbead-based rolling circle amplification in a microchip for sensitive DNA detection | |
| KR102502083B1 (en) | Portable nucleic acid analysis system and high-performance microfluidic electroactive polymer actuators | |
| WO2018099420A1 (en) | Droplet digital pcr chip | |
| CN110616144A (en) | Liquid drop digital PCR chip and use method thereof | |
| Zhang et al. | Nanolitre droplet array for real time reverse transcription polymerase chain reaction | |
| US20240033739A1 (en) | Fluidic devices with bead well geometries with spatially separated bead retention and signal detection segments and related methods | |
| US10684212B2 (en) | Method and system for reference-assisted droplet detection, indexing and sorting for assays and diagnostics | |
| Shi et al. | Real-time PCR of single bacterial cells on an array of adhering droplets | |
| CN101472940A (en) | Droplet-based biochemistry | |
| CN104894106A (en) | High-integration equidistance equipartition nucleic acid amplification micro-fluidic chip and application | |
| US20200276582A1 (en) | Methods and systems for automated sample processing | |
| JP2018531369A6 (en) | Fluidic device having a bead well shape and a signal detection segment to hold the beads spatially separated and associated method | |
| Jagannath et al. | Pathogen detection on microfluidic platforms: Recent advances, challenges, and prospects | |
| Perez-Toralla et al. | FISH in chips: turning microfluidic fluorescence in situ hybridization into a quantitative and clinically reliable molecular diagnosis tool | |
| CN106854674A (en) | A kind of nucleic acid high flux method for quick based on capillary microarray | |
| Wei et al. | A droplet-based multivolume microfluidic device for digital polymerase chain reaction | |
| Cai et al. | Droplet Encoding‐Pairing Enabled Multiplexed Digital Loop‐Mediated Isothermal Amplification for Simultaneous Quantitative Detection of Multiple Pathogens | |
| CN209276542U (en) | A kind of micro-fluidic XOR operation system based on molecular beacon DNA | |
| CN115651807B (en) | Nucleic acid detection chip and nucleic acid detection method | |
| CN116497095A (en) | Multiple nucleic acid quantification method, device and medium based on sediment bright field image processing | |
| US20240123447A1 (en) | Methods and related aspects for multiplexed analyte detection using sequential magnetic particle elution |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| EE01 | Entry into force of recordation of patent licensing contract |
Application publication date: 20191227 Assignee: Beijing Linke Biotechnology Co.,Ltd. Assignor: BEIJING INTEGRATED BIOSYSTEMS Co.,Ltd. Contract record no.: X2022990000932 Denomination of invention: A Droplet Digital PCR Chip and Its Application License type: Common License Record date: 20221114 |
|
| EE01 | Entry into force of recordation of patent licensing contract | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20191227 |
|
| RJ01 | Rejection of invention patent application after publication |