CN111534428A - Integrated nucleic acid extraction and amplification detection system based on PCR amplification - Google Patents
Integrated nucleic acid extraction and amplification detection system based on PCR amplification Download PDFInfo
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- CN111534428A CN111534428A CN202010441656.2A CN202010441656A CN111534428A CN 111534428 A CN111534428 A CN 111534428A CN 202010441656 A CN202010441656 A CN 202010441656A CN 111534428 A CN111534428 A CN 111534428A
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- 150000007523 nucleic acids Chemical class 0.000 title claims abstract description 96
- 102000039446 nucleic acids Human genes 0.000 title claims abstract description 96
- 108020004707 nucleic acids Proteins 0.000 title claims abstract description 96
- 238000000605 extraction Methods 0.000 title claims abstract description 73
- 238000001514 detection method Methods 0.000 title claims abstract description 39
- 230000003321 amplification Effects 0.000 title claims abstract description 21
- 238000003199 nucleic acid amplification method Methods 0.000 title claims abstract description 21
- 238000012408 PCR amplification Methods 0.000 title claims abstract description 13
- 239000007788 liquid Substances 0.000 claims abstract description 44
- 238000004140 cleaning Methods 0.000 claims abstract description 24
- 238000006243 chemical reaction Methods 0.000 claims abstract description 20
- 239000011324 bead Substances 0.000 claims abstract description 12
- 239000012295 chemical reaction liquid Substances 0.000 claims abstract description 8
- 238000010828 elution Methods 0.000 claims abstract description 7
- 238000003756 stirring Methods 0.000 claims abstract description 7
- 239000002699 waste material Substances 0.000 claims abstract description 7
- 238000005406 washing Methods 0.000 claims abstract 2
- 239000012528 membrane Substances 0.000 claims description 6
- 238000003825 pressing Methods 0.000 claims description 5
- 230000005284 excitation Effects 0.000 claims description 4
- 238000011049 filling Methods 0.000 claims description 3
- 229920001296 polysiloxane Polymers 0.000 claims description 3
- 238000000018 DNA microarray Methods 0.000 claims description 2
- 238000000504 luminescence detection Methods 0.000 claims description 2
- 238000002156 mixing Methods 0.000 claims description 2
- 239000000758 substrate Substances 0.000 claims 1
- 238000012864 cross contamination Methods 0.000 abstract description 5
- 238000003752 polymerase chain reaction Methods 0.000 description 34
- 238000005516 engineering process Methods 0.000 description 5
- 241000700605 Viruses Species 0.000 description 3
- 230000009286 beneficial effect Effects 0.000 description 2
- 239000003480 eluent Substances 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 1
- 244000052616 bacterial pathogen Species 0.000 description 1
- 238000005336 cracking Methods 0.000 description 1
- 230000009089 cytolysis Effects 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 230000010354 integration Effects 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000000034 method Methods 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 241000894007 species Species 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
-
- 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
- C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
-
- 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
- C12Q1/686—Polymerase chain reaction [PCR]
Abstract
The invention discloses an integrated nucleic acid extraction and amplification detection system based on PCR amplification, which comprises a bag-type nucleic acid extraction chip, a runner-type PCR chip and a control detector. The bag type nucleic acid extraction chip comprises a nucleic acid extraction cavity, a first cleaning liquid cavity, a second cleaning liquid cavity, a third cleaning liquid cavity, an elution liquid cavity and a waste liquid cavity, and liquids required by nucleic acid extraction are respectively stored in the washing liquid cavity, the elution liquid cavity and the waste liquid cavity. The flow channel type PCR chip comprises a PCR pretreatment cavity, a multi-temperature-zone PCR reaction flow channel and a reaction liquid collecting cavity. The control detector comprises a push rod controller, a controllable alternating electromagnet, a temperature controller and a fluorescence detector, wherein the push rod controller controls the flow direction of liquid, the controllable alternating electromagnet stirs and adsorbs magnetic beads, the temperature controller controls the temperature required by nucleic acid extraction and reaction, and the fluorescence detector detects fluorescence after PCR reaction. The invention realizes the integrated nucleic acid extraction and amplification detection based on PCR amplification, can avoid the cross contamination of nucleic acid and is convenient for quickly replacing the detected sample.
Description
Technical Field
The invention belongs to the field of nucleic acid detection, and particularly relates to an integrated nucleic acid extraction and amplification detection system based on a PCR technology.
Background
Nucleic acid detection is a gold standard for identifying biological species, and is an important means for detecting and identifying microorganisms such as viruses and bacteria. PCR (Polymerase Chain Reaction) is one of the most commonly used nucleic acid amplification techniques, and one PCR cycle includes three steps of denaturation-annealing-extension to double the nucleic acid amplification. Exponential nucleic acid multiplication can then be achieved through several cycles of PCR reactions.
At present, commercial nucleic acid extraction and detection equipment is open, nucleic acid cross contamination is easily caused, and safety risks to people exist during detection of pathogenic bacteria or viruses. Because open nucleic acid extraction easily causes cross contamination, for this reason, the common solution is that nucleic acid extraction and PCR amplification are strictly carried out in different regions, and integration of nucleic acid extraction and amplification cannot be achieved.
Closed type and full-automatic nucleic acid detection, sample in and result out type detection has great market demand, can greatly reduce the requirements of nucleic acid detection on fields and personnel, and can greatly expand the application range of nucleic acid detection.
Disclosure of Invention
In order to solve the problems, the invention provides an integrated nucleic acid extraction and amplification detection system based on a PCR technology.
The invention provides an integrated nucleic acid extraction and amplification detection system based on a PCR technology, which comprises a bag-type nucleic acid extraction chip, a flow channel-type PCR chip and a control detector. The bag type nucleic acid extraction chip comprises a nucleic acid extraction cavity 1, a first cleaning liquid cavity 2, a second cleaning liquid cavity 3, a third cleaning liquid cavity 4, an elution liquid cavity 5 and a waste liquid cavity 6, which are respectively used for storing various liquids required by nucleic acid extraction, and the cavities are connected through micro-channels. The flow channel type PCR chip comprises a PCR pretreatment cavity 7, a multi-temperature-zone PCR reaction flow channel 9 and a reaction liquid collecting cavity 8. The control detector comprises a first push rod controller 10, a second push rod controller 11, a third push rod controller 12, a fourth push rod controller 13, a fifth push rod controller 14, a sixth push rod controller 15, a seventh push rod controller 16, an eighth push rod controller 17, a first controllable alternating electromagnet 18, a second controllable alternating electromagnet 19, a first temperature controller 21, a second temperature controller 22, a third temperature controller 23, a fourth temperature controller 24 and a fluorescence detector 20. The bag-type nucleic acid extraction chip and the flow channel PCR chip are connected through a silicone tube. During detection, the bag-type nucleic acid extraction chip and the flow-channel PCR chip filled with the sample are placed in a control detector, and the push rod controller controls the pressing and the bouncing of the elastic membrane to change the volume of the microcavity, so that the liquid is controlled to flow in and out. The controllable alternating electromagnet stirs and adsorbs magnetic beads, the temperature controller controls the temperature required by nucleic acid extraction and reaction, and the fluorescence detector is used for luminescence detection of nucleic acid.
The bag type nucleic acid extraction chip consists of a plurality of mutually communicated micro-cavities processed on a hard base material and an elastic membrane bonded on the micro-cavities.
The bag-type nucleic acid extraction chip is pre-filled with required liquid through a filling port, then is closed, and blocks liquid mixing under an uncontrolled condition through flow resistance of a flow channel.
The bag-type nucleic acid extraction chip is connected with the runner-type PCR chip through a silicone tube; the bag type nucleic acid extraction chip, the flow channel type PCR chip and the control detector are relatively separated, push rods of the control detector respectively correspond to the elastic membranes of the micro-cavities, and liquid is controlled to flow among the cavities through the sequential pressing and lifting of the push rods.
The controllable alternating electromagnet is aligned to the nucleic acid extraction cavity and is respectively arranged on two sides of the nucleic acid extraction cavity, the stirring function is realized through the alternating magnetic field, and meanwhile, the electromagnet adsorbs magnetic beads when the cleaning and the elution are completed.
The temperature controllers respectively control the temperature of the nucleic acid extraction cavity and the flow channel type PCR chip, and the temperature required by nucleic acid extraction and amplification reaction.
The fluorescence detector consists of excitation light and a fluorescence detector, and the excitation light and the fluorescence detector are matched with a fluorescence waveband after PCR reaction.
The invention has the beneficial effects that: the invention realizes the integrated, closed and full-automatic rapid nucleic acid extraction and PCR amplification detection, can rapidly and accurately realize the nucleic acid detection, can avoid the nucleic acid cross contamination during the detection and reduce the biological safety risk, and in addition, the bag-type nucleic acid extraction chip, the flow channel-type PCR chip and the control detector are relatively separated, so that the detection sample can be rapidly replaced, and the practical detection application is convenient.
Drawings
FIG. 1 is a schematic diagram of an integrated nucleic acid extraction and amplification detection system based on PCR technology according to an embodiment of the present invention.
Description of reference numerals: 1. a nucleic acid extraction chamber; 2. a first cleaning liquid cavity; 3. a second cleaning liquid cavity; 4. a third cleaning liquid cavity; 5. an eluate chamber; 6. a waste fluid chamber; 7, a PCR pretreatment cavity; 8. a reaction liquid collection chamber; 9. a multi-temperature zone PCR reaction flow channel; 10. a first push rod controller; 11. a second push rod controller; 12. a third push rod controller; 13. a fourth push rod controller; 14. a fifth push rod controller; 15. a sixth push rod controller; 16. a seventh push rod controller; 17. a eighth push rod controller; 18. a first controllable alternating electromagnet; 19. a second controllable alternating electromagnet; 20. a fluorescence detector; 21. a first temperature controller; 22. a second temperature controller; 23. a third temperature controller; 24. and a fourth temperature controller.
Detailed Description
The present invention will be described in further detail with reference to the following detailed description and accompanying drawings, it being emphasized that the following description is illustrative only and is not intended to limit the scope and application of the present invention.
The embodiment provides an integrated nucleic acid extraction and amplification detection system based on a PCR technology, which comprises the following steps: the lysis solution and the magnetic beads are pre-loaded in the nucleic acid extraction cavity; pre-filling the first cleaning liquid, the second cleaning liquid and the third cleaning liquid in the first cleaning liquid cavity, the second cleaning liquid cavity and the third cleaning liquid cavity respectively; pre-loading the eluent in an eluent cavity; the PCR reaction solution is pre-loaded in a PCR pretreatment cavity to construct a kit for extracting and detecting nucleic acid.
Firstly, adding a target sample into a nucleic acid extraction cavity, and putting a bag type biochip into a control detector, wherein a push rod controller I, a push rod controller II, a push rod controller III, a push rod controller IV and a push rod controller IV respectively correspond to the nucleic acid extraction cavity; the controllable alternating electromagnets are arranged at two sides of the nucleic acid extraction cavity; the fluorescence detector corresponds to the reaction liquid collecting cavity; the first temperature controller, the second temperature controller, the third temperature controller and the fourth temperature controller respectively correspond to the nucleic acid extraction cavity and the three temperature zones of the multi-temperature-zone PCR reaction flow channel.
When the detection is started, the temperature controller controls the temperature of the nucleic acid extraction cavity to 50 ℃, and the alternating electromagnets on the left side and the right side of the nucleic acid extraction cavity are controlled to make the magnetic beads reciprocate in the cavity to play a role in stirring. After the cracking is finished, controlling a magnet on one side to absorb the magnetic beads in the cavity, then controlling the first push rod controller to extrude the bag of the nucleic acid extraction cavity, and simultaneously lifting the sixth push rod controller to enable liquid in the nucleic acid extraction cavity to flow to the waste liquid cavity, and leaving the nucleic acid and the magnetic beads in the nucleic acid extraction cavity.
Then controlling a second push rod controller to press down, simultaneously lifting the first push rod controller, enabling the first cleaning solution to enter the nucleic acid extraction cavity, controlling an alternating electromagnet, and stirring magnetic beads; after cleaning, controlling a magnet on one side to absorb magnetic beads in the cavity, then pressing down the first push rod controller, and simultaneously lifting the sixth push rod controller to enable liquid in the nucleic acid extraction cavity to flow to the waste liquid cavity, and leaving nucleic acid and magnetic beads in the nucleic acid extraction cavity to finish one-time cleaning.
Sequentially controlling a third push rod controller, a first push rod controller and a sixth push rod controller according to the steps to finish secondary cleaning; controlling a fourth push rod controller, a first push rod controller and a sixth push rod controller to finish three times of cleaning; and controlling the fifth push rod controller, the first push rod controller and the seventh push rod controller to complete nucleic acid elution and make the nucleic acid enter the PCR pretreatment cavity.
After nucleic acid enters the PCR pretreatment cavity, the temperature controllers II, III and IV control the three temperature regions of the multi-temperature-region PCR reaction flow passage to be 95 ℃, 55 ℃ and 72 ℃ respectively. When the three temperature zones reach corresponding temperatures, the seventh push rod controller and the eighth push rod controller are controlled to transfer the liquid in the PCR pretreatment cavity to the reaction liquid collection cavity after flowing through the multi-temperature-zone PCR reaction flow channel. When the nucleic acid reaction solution flows through the multi-temperature-zone PCR reaction flow channel, multiple rounds of PCR reactions can be completed.
And when the amplified nucleic acid enters the reaction liquid collecting cavity, starting the fluorescence detector, detecting a fluorescence signal of the reaction liquid collecting cavity, and judging the negativity or the positivity of the nucleic acid detection.
After the detection is finished, a new group of bag-type nucleic acid extraction chips and flow channel PCR chips are put into a control detector, and the detection of a new group of target samples can be started according to the steps. The invention has the main advantages that the nucleic acid extraction and detection are totally closed and integrated, thereby reducing the safety risk and the cross contamination risk and being beneficial to realizing field inspection; the bag-type nucleic acid extraction chip, the flow channel PCR chip and the control detector are relatively separated, so that not only is the detection sample convenient to replace, but also the nucleic acid extraction and detection of different viruses can be realized by replacing the detection kit.
Claims (7)
1. An integrated nucleic acid extraction and amplification detection system based on PCR amplification comprises a bag-type nucleic acid extraction chip, a flow channel-type PCR chip and a control detector, and is characterized in that,
the bag type nucleic acid extraction chip comprises a nucleic acid extraction cavity (1), a first cleaning liquid cavity (2), a second cleaning liquid cavity (3), a third cleaning liquid cavity (4), an elution liquid cavity (5) and a waste liquid cavity (6), which are respectively used for storing various liquids required by nucleic acid extraction, and the cavities are connected through micro-channels;
the flow channel type PCR chip comprises a PCR pretreatment cavity (7), a multi-temperature-zone PCR reaction flow channel (9) and a reaction liquid collecting cavity (8);
the control detector comprises a first push rod controller (10), a second push rod controller (11), a third push rod controller (12), a fourth push rod controller (13), a fifth push rod controller (14), a sixth push rod controller (15), a seventh push rod controller (16), an eighth push rod controller (17), a first controllable alternating electromagnet (18), a second controllable alternating electromagnet (19), a first temperature controller (21), a second temperature controller (22), a third temperature controller (23), a fourth temperature controller (24) and a fluorescence detector (20);
the bag-type nucleic acid extraction chip and the flow-channel PCR chip are connected through a silicone tube, and during detection, the bag-type nucleic acid extraction chip and the flow-channel PCR chip filled with a sample are placed in a control detector, and the push rod controller controls the pressing down and the bouncing of the elastic membrane to change the volume of the microcavity, so that the inflow and the outflow of liquid are controlled. The controllable alternating electromagnet stirs and adsorbs magnetic beads, the temperature controller controls the temperature required by nucleic acid extraction and reaction, and the fluorescence detector is used for luminescence detection of nucleic acid.
2. The integrated nucleic acid extraction and amplification detection system based on PCR amplification of claim 1, wherein the bag-type nucleic acid extraction chip comprises a plurality of interconnected micro-cavities processed on a rigid substrate and an elastic membrane bonded thereon.
3. The integrated nucleic acid extraction and amplification detection system based on PCR amplification of claim 1, wherein the bag-type nucleic acid extraction chip is pre-filled with a desired liquid through a filling port, then closed, and the liquid mixing under uncontrolled conditions is blocked by a flow resistance of a flow channel.
4. The integrated nucleic acid extraction and amplification detection system based on PCR amplification of claim 1, wherein the bag-type biochip is separated from the control detector, push rods of the control detector are respectively corresponding to the elastic membranes of the micro-cavities, and the liquid is controlled to flow between the cavities by sequentially pressing down and lifting up the push rods.
5. The integrated nucleic acid extraction and amplification detection system based on PCR amplification of claim 1, wherein the controllable alternating electromagnet is aligned with the nucleic acid extraction chamber and is respectively arranged at two sides of the nucleic acid extraction chamber, and the stirring function is realized by the alternating magnetic field, and the magnetic beads are adsorbed by the electromagnet when the washing is completed and the elution is carried out.
6. The integrated nucleic acid extraction and amplification detection system based on PCR amplification of claim 1, wherein the temperature controller controls the temperature of the nucleic acid extraction chamber and the flow-channel PCR chip, respectively, and the temperature required by the nucleic acid extraction and amplification reaction.
7. The integrated nucleic acid extraction and amplification detection system based on PCR amplification of claim 1, wherein the fluorescence detector comprises an excitation light and a fluorescence detector, and the excitation light and the fluorescence detector are matched with the fluorescence band after PCR reaction.
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| Application Number | Priority Date | Filing Date | Title |
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| CN202010441656.2A CN111534428A (en) | 2020-05-22 | 2020-05-22 | Integrated nucleic acid extraction and amplification detection system based on PCR amplification |
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| CN202010441656.2A CN111534428A (en) | 2020-05-22 | 2020-05-22 | Integrated nucleic acid extraction and amplification detection system based on PCR amplification |
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Citations (6)
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| JP2015050968A (en) * | 2013-09-09 | 2015-03-19 | セイコーエプソン株式会社 | Nucleic acid amplification system |
| CN206375900U (en) * | 2016-12-14 | 2017-08-04 | 深圳市埃克特生物科技有限公司 | A kind of miniflow bag for nucleic acid integration detection |
| WO2018137513A1 (en) * | 2017-01-24 | 2018-08-02 | 北京万泰生物药业股份有限公司 | System for detecting convective pcr amplification and method for detecting convective pcr amplification |
| CN110331089A (en) * | 2019-05-21 | 2019-10-15 | 宁波迪亚生物科技有限公司 | A kind of full-automatic nucleic acid extraction augmentation detection micro-fluidic chip box and its application |
| CN111073810A (en) * | 2019-12-20 | 2020-04-28 | 深圳市华迈生物医疗科技有限公司 | Microfluidic chip, system and method integrating nucleic acid extraction, amplification and detection |
-
2020
- 2020-05-22 CN CN202010441656.2A patent/CN111534428A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104254595A (en) * | 2012-02-13 | 2014-12-31 | 纽莫德克斯莫勒库拉尔公司 | Microfluidic cartridge for processing and detecting nucleic acids |
| JP2015050968A (en) * | 2013-09-09 | 2015-03-19 | セイコーエプソン株式会社 | Nucleic acid amplification system |
| CN206375900U (en) * | 2016-12-14 | 2017-08-04 | 深圳市埃克特生物科技有限公司 | A kind of miniflow bag for nucleic acid integration detection |
| WO2018137513A1 (en) * | 2017-01-24 | 2018-08-02 | 北京万泰生物药业股份有限公司 | System for detecting convective pcr amplification and method for detecting convective pcr amplification |
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