CN111575176A - Closed full-automatic nucleic acid extraction and detection system based on CRISPR technology - Google Patents
Closed full-automatic nucleic acid extraction and detection system based on CRISPR technology Download PDFInfo
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- 150000007523 nucleic acids Chemical class 0.000 title claims abstract description 77
- 108020004707 nucleic acids Proteins 0.000 title claims abstract description 76
- 102000039446 nucleic acids Human genes 0.000 title claims abstract description 76
- 238000001514 detection method Methods 0.000 title claims abstract description 66
- 238000000605 extraction Methods 0.000 title claims abstract description 45
- 238000005516 engineering process Methods 0.000 title claims abstract description 13
- 108091033409 CRISPR Proteins 0.000 title claims abstract 12
- 238000010354 CRISPR gene editing Methods 0.000 title claims abstract 12
- 239000007788 liquid Substances 0.000 claims abstract description 44
- 238000004140 cleaning Methods 0.000 claims abstract description 27
- 238000000018 DNA microarray Methods 0.000 claims abstract description 19
- 230000003321 amplification Effects 0.000 claims abstract description 19
- 238000003199 nucleic acid amplification method Methods 0.000 claims abstract description 19
- 239000011324 bead Substances 0.000 claims abstract description 12
- 238000003756 stirring Methods 0.000 claims abstract description 7
- 239000002699 waste material Substances 0.000 claims abstract description 7
- 238000010828 elution Methods 0.000 claims abstract description 5
- 239000012528 membrane Substances 0.000 claims description 6
- 239000003480 eluent Substances 0.000 claims description 4
- 230000005284 excitation Effects 0.000 claims description 4
- 238000003825 pressing Methods 0.000 claims description 4
- 238000011049 filling Methods 0.000 claims description 3
- 238000002156 mixing Methods 0.000 claims description 2
- 239000000758 substrate Substances 0.000 claims 1
- 238000005406 washing Methods 0.000 claims 1
- 238000012864 cross contamination Methods 0.000 abstract description 4
- 238000000504 luminescence detection Methods 0.000 abstract 1
- 230000001276 controlling effect Effects 0.000 description 10
- 241000700605 Viruses Species 0.000 description 3
- 238000011901 isothermal amplification Methods 0.000 description 3
- 230000009286 beneficial effect Effects 0.000 description 2
- 238000011068 loading method Methods 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 1
- 238000012408 PCR amplification Methods 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
- 238000010362 genome editing Methods 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
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- 244000005700 microbiome Species 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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Abstract
The invention discloses a closed full-automatic nucleic acid extraction detection system based on CRISPR technology, which comprises a bag type biochip and a control detector. The bag type biochip 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, a waste liquid cavity, an amplification cavity and a CRISPR detection cavity, which are respectively used for storing various liquids required by nucleic acid extraction and detection, and the cavities are connected through micro-channels. The control detector comprises a push rod controller, a controllable alternating electromagnet and a fluorescence detector, wherein the push rod controller is used for controlling the flow direction of liquid, the controllable alternating electromagnet is used for stirring and adsorbing magnetic beads, and the fluorescence detector is used for CRISPR luminescence detection of nucleic acid. The invention realizes a closed full-automatic nucleic acid extraction detection system based on CRISPR technology, can avoid nucleic acid cross contamination during detection, and can quickly replace a detection sample by relatively separating a bag type biochip and a control detector, thereby facilitating practical detection application.
Description
Technical Field
The invention belongs to the field of nucleic acid detection, and particularly relates to a closed full-automatic nucleic acid extraction detection system based on a CRISPR 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. CRISPR (clustered regulated Short Palindromic repeats) technology is used as a gene editing technology, can accurately identify nucleic acid fragments, and can be widely used for nucleic acid detection. In nucleic acid detection, nucleic acid extraction and concentration are usually required, and nucleic acid detection is performed, which contributes to improvement of the sensitivity of nucleic acid detection.
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. On the other hand, the nucleic acid detection methods commonly used at present are based on PCR amplification, isothermal amplification or gene sequencing, but the detection methods require a long time. With the increasing demand for rapid nucleic acid detection, nucleic acid detection based on CRISPR technology is receiving more and more attention.
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 a closed full-automatic nucleic acid extraction and detection system based on CRISPR technology.
The invention provides a closed full-automatic nucleic acid extraction detection system based on CRISPR technology, which comprises a bag type biochip and a control detector. The bag type biochip 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, a waste liquid cavity 6, an amplification cavity 7 and a CRISPR detection cavity 8, which are respectively used for storing various liquids required by nucleic acid extraction and detection, and the cavities are connected through a micro-channel. The control detector comprises a first push rod controller 9, a second push rod controller 10, a third push rod controller 11, a fourth push rod controller 12, a fifth push rod controller 13, a sixth push rod controller 14, a seventh push rod controller 15, an eighth push rod controller 16, a first controllable alternating electromagnet 17, a second controllable alternating electromagnet 18, a first temperature controller 20, a second temperature controller 21, a third temperature controller 22 and a fluorescence detector (19). During detection, a bag type biochip filled with a sample is placed in a control detector, the push rod controller controls the pressing and bouncing of the elastic membrane to change the volume of the microcavity, so that the inflow and outflow of liquid are controlled, the controllable alternating electromagnet stirs and adsorbs magnetic beads, the temperature controller respectively controls the cavity temperature during nucleic acid extraction, amplification and detection, and the fluorescence detector detects fluorescence emitted during CRISPR detection.
The bag type biochip is composed 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 biochip is pre-filled with required liquid through a filling port, then is closed, and blocks liquid mixing under an uncontrolled condition through the flow resistance of a flow channel.
The bag type biochip is relatively separated from the control detector, 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, the temperature of the amplification cavity and the temperature required by the nucleic acid extraction, amplification and CRISPR 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 CRISPR reaction.
The invention has the beneficial effects that: the invention realizes the closed full-automatic nucleic acid detection based on the CRISPR, can quickly 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 biochip is relatively separated from the control detector, so that the detection sample can be quickly replaced, and the actual detection application is convenient.
Drawings
Fig. 1 is a schematic composition diagram of a closed full-automatic nucleic acid extraction and detection system based on CRISPR 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. an amplification chamber; 8, CRISPR detection cavity; 9. 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. a eighth push rod controller; 17. a first controllable alternating electromagnet; 18. a second controllable alternating electromagnet; 19. a fluorescence detector; 20. a first temperature controller; 21. a second temperature controller; 22. and a third 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 a closed full-automatic nucleic acid extraction and detection system based on a CRISPR 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; pre-loading the amplification solution in the amplification chamber; the CRISPR reaction solution is pre-packaged in a CRISPR detection cavity to construct a nucleic acid extraction and detection kit.
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; the controllable alternating electromagnets are arranged at two sides of the nucleic acid extraction cavity; the fluorescence detector corresponds to a CRISPR detection cavity; the first temperature controller, the second temperature controller and the third temperature controller respectively correspond to the nucleic acid extraction cavity, the amplification cavity and the CRISPR detection cavity.
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 amplification cavity.
When the nucleic acid enters the amplification cavity, the temperature controller II controls the temperature of the amplification cavity to 39 ℃ and starts isothermal amplification reaction. And after the isothermal amplification is finished, controlling the No. seven push rod controller and the No. eight push rod controller, and transferring the liquid in the amplification cavity to the CRISPR detection cavity.
And after the nucleic acid enters the CRISPR detection cavity, controlling the temperature of the amplification cavity to 37 ℃ by using the third temperature controller, starting the CRISPR reaction, starting the fluorescence detector after the CRISPR reaction is finished, detecting a fluorescence signal in the CRISPR detection cavity, and judging the negativity or the positivity of the nucleic acid detection.
After the detection is completed, a new set of bag-type biochips is placed in the control detector, and the detection of a new set 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 biochip is relatively separated from the control detector, 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. A closed full-automatic nucleic acid extraction detection system based on CRISPR technology comprises a bag type biochip and a control detector,
the bag type biochip 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 eluent cavity (5), a waste liquid cavity (6), an amplification cavity (7) and a CRISPR detection cavity (8), wherein the nucleic acid extraction cavity, the first cleaning liquid cavity, the second cleaning liquid cavity, the third cleaning liquid cavity, the eluent cavity, the waste liquid cavity, the amplification cavity and the CRISPR detection cavity are respectively used for storing various liquids required by nucleic acid extraction and detection, and the cavities are connected through;
the control detector comprises a first push rod controller (9), a second push rod controller (10), a third push rod controller (11), a fourth push rod controller (12), a fifth push rod controller (13), a sixth push rod controller (14), a seventh push rod controller (15), an eighth push rod controller (16), a first controllable alternating electromagnet (17), a second controllable alternating electromagnet (18), a first temperature controller (20), a second temperature controller (21), a third temperature controller (22) and a fluorescence detector (19);
during detection, a bag type biochip filled with a sample is placed in a control detector, the push rod controller controls the pressing and bouncing of the elastic membrane to change the volume of the microcavity, so that the inflow and outflow of liquid are controlled, the controllable alternating electromagnet stirs and adsorbs magnetic beads, the temperature controller respectively controls the cavity temperature during nucleic acid extraction, amplification and detection, and the fluorescence detector detects fluorescence emitted during CRISPR detection.
2. The automatic nucleic acid extracting and detecting system as claimed in claim 1, wherein the bag-type biochip is composed of several interconnected micro-cavities processed on a rigid substrate and an elastic membrane bonded thereon.
3. The system for automatically extracting and detecting nucleic acid according to claim 1, wherein the bag-type biochip is filled with a desired liquid through the filling port, is closed, and blocks the mixing of the liquids under uncontrolled conditions by the flow resistance of the flow channel.
4. The automatic nucleic acid extracting and detecting system as claimed in 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 the push rods being pressed and lifted in sequence.
5. The automatic nucleic acid extracting and detecting system as claimed in claim 1, wherein the controllable alternating electromagnet is aligned with the nucleic acid extracting chamber and separately arranged at two sides of the nucleic acid extracting chamber, and the stirring function is realized by the alternating magnetic field, and the magnetic beads are adsorbed by the electromagnet when the washing and the elution are completed.
6. The fully automatic nucleic acid extraction and detection system of claim 1, wherein the temperature controller controls the temperature of the nucleic acid extraction chamber, the amplification chamber and the CRISPR detection chamber, respectively, using the temperature required for nucleic acid extraction, amplification and CRISPR reaction.
7. The fully automatic nucleic acid extraction and detection system 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 a fluorescence band after CRISPR reaction.
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| CN202010441655.8A CN111575176A (en) | 2020-05-22 | 2020-05-22 | Closed full-automatic nucleic acid extraction and detection system based on CRISPR technology |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112126587A (en) * | 2020-09-03 | 2020-12-25 | 华东理工大学 | Nucleic acid detection chip device, nucleic acid detection chip and preparation method thereof |
| CN112791754A (en) * | 2020-12-31 | 2021-05-14 | 苏阳 | Digital microfluidic polymerase chain reaction multi-connection detection system |
| CN113866401A (en) * | 2021-10-09 | 2021-12-31 | 广东粤港澳大湾区国家纳米科技创新研究院 | One minute quick poison detection device |
| CN114107035A (en) * | 2021-11-30 | 2022-03-01 | 珠海黑马生物科技有限公司 | Closed full-automatic nucleic acid extraction kit |
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| CN114107035A (en) * | 2021-11-30 | 2022-03-01 | 珠海黑马生物科技有限公司 | Closed full-automatic nucleic acid extraction kit |
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