CN112760194A - Closed simple RNA nucleic acid extraction device and preparation method and use method thereof - Google Patents
Closed simple RNA nucleic acid extraction device and preparation method and use method thereof Download PDFInfo
- Publication number
- CN112760194A CN112760194A CN202110013429.4A CN202110013429A CN112760194A CN 112760194 A CN112760194 A CN 112760194A CN 202110013429 A CN202110013429 A CN 202110013429A CN 112760194 A CN112760194 A CN 112760194A
- Authority
- CN
- China
- Prior art keywords
- extraction
- way
- way liquid
- bin
- nucleic acid
- 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.)
- Granted
Links
Images
Classifications
-
- 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
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Analytical Chemistry (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biophysics (AREA)
- Immunology (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
- Physics & Mathematics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Apparatus Associated With Microorganisms And Enzymes (AREA)
Abstract
The invention provides a closed simple RNA nucleic acid extraction device which comprises an inner layer structure and an outer layer structure, wherein the inner layer structure comprises an extraction bin, a one-way liquid inlet pipe, a one-way liquid discharge pipe, a plurality of reagent storage pipes and magnetic beads placed in the extraction bin, and the outer layer structure comprises a device shell. The invention designs a closed simple nucleic acid extraction device, so that an extraction reaction system is completely isolated from the external environment. The invention also provides a preparation method and a using method of the closed simple RNA nucleic acid extraction device. The invention leads the nucleic acid extraction to be completely separated from large-scale instruments and equipment by using the closed nucleic acid extraction equipment, and realizes the field detection target of sampling and extracting simultaneously.
Description
Technical Field
The invention relates to the field of RNA nucleic acid extraction and field detection in environment, in particular to a closed simple RNA nucleic acid extraction device. The invention also relates to a preparation method of the closed simple RNA nucleic acid extraction device. The invention also relates to a use method of the closed simple RNA nucleic acid extraction device.
Technical Field
Because of the high infectivity of the novel coronavirus, the environment is extremely easily polluted in the processes of sample collection, transportation, pretreatment, extraction and nucleic acid detection, and workers are infected, so that the epidemic situation prevention and control and the detection work of the novel coronavirus are carried out according to the three-level protection standard. Relevant specifications and implementation rules are established for sample collection, transportation, extraction and detection. Therefore, the requirement of the new crown nucleic acid detection on a laboratory is higher, and a full-automatic instrument is required to be adopted for extraction, so that accidental infection caused by artificial extraction is avoided. The target gene detected by PCR is ORF1ab gene and nucleocapsid protein N of new coronavirus, and when double-gene positive appears, the sample is judged to detect the new coronavirus. However, the disease course of the patient is unpredictable in the actual detection process, and the virus sample can not be collected once the sampling time period is not in the virus elimination period of the patient. In addition, the quality of the sample collection tube will affect the protective effect of the buffer solution of the virus sampling tube on viruses, resulting in partial degradation during the transportation of the viruses. Meanwhile, the sample transportation conditions are harsh, and the virus collection tube needs to be placed at a lower temperature so as to ensure the integrity of the virus in the sample. Finally, the detection result is directly influenced by the irregular operation and unstable reagents in the sample extraction and detection processes.
In summary, the conventional PCR method for detecting the novel coronavirus is limited by a large number of objective factors, and the new corona must be diagnosed by repeated retesting and antibody detection. However, the sensitivity of antibody detection is low, and only the antibody with higher concentration can be combined with the detection reagent, so that a positive reaction occurs. In contrast, nucleic acid detection has the characteristic of high sensitivity, but the conventional PCR detection method is highly dependent on large-scale laboratory instruments and equipment, and high-purity extraction of nucleic acid is required, so that the requirement of field detection cannot be met.
When the existing nucleic acid detection is used for extracting nucleic acid, equipment is complex, and the biological safety in the nucleic acid extraction process cannot be ensured.
Disclosure of Invention
Aiming at the prior technical problems, the invention hopes to solve the technical difficulty of the RPA field detection of the novel coronavirus. A closed simple nucleic acid extraction device, a preparation method and a use method thereof are designed, so that an extraction reaction system is completely isolated from the external environment, and meanwhile, the device is simple in structure and convenient to carry, and can be used for sampling and extracting anywhere.
The invention provides a closed simple RNA nucleic acid extraction device, which comprises an extraction structure, a storage structure, a magnetic structure and a driving structure,
the extraction structure comprises an extraction bin, a one-way liquid inlet pipe communicated with the extraction bin, a one-way liquid outlet pipe communicated with the extraction bin, and suction ports communicated with the one-way liquid inlet pipe and one end of the one-way liquid outlet pipe, which is far away from the extraction bin;
the storage structure comprises a plurality of storage tubes which are communicated with the extraction bin and used for storing reaction reagents;
the magnetic structure comprises a magnetic bead arranged in the extraction bin and a magnetic ring used for controlling the magnetic bead;
the driving structure comprises a first driving component for driving the extracting structure to extract liquid or discharge the liquid through the suction port, and a second driving component for driving the storage structure to convey a reaction reagent into the extracting bin;
the diameter of the magnetic beads is larger than that of the one-way liquid inlet pipe and that of the one-way liquid outlet pipe, a sealing element is arranged at a suction port of the extraction structure, and the closed simple RNA nucleic acid extraction device is in a syringe shape.
Further, the closed simple RNA nucleic acid extraction device comprises an outer layer structure and an inner layer structure;
the inner layer structure comprises the extracting structure, the first driving assembly arranged at the tail end of the extracting bin, a plurality of storage tubes arranged outside the extracting bin in a surrounding mode, and the second driving assembly arranged at the tail end of each storage tube;
outer shell, be used for dismantling the setting and be in outer device shell of inner structure, be used for the setting to be in the sealing member and the slidable cover of suction port department are established be used for control on the device shell extract in the storehouse the magnetic bead the magnetism ring, wherein, the lower extreme of device shell with extract structure fixed connection.
Further, it is the U type to draw the storehouse, one-way fluid-discharge tube is located draw the storehouse below and communicate rather than the bottom, the liquid outlet of one-way fluid-discharge tube with the inlet of one-way fluid feed pipe communicates, one-way fluid feed pipe slope is located one side of one-way fluid-discharge tube, just the liquid outlet of one-way fluid feed pipe extends to draw in the storehouse and be higher than the inlet of one-way fluid-discharge tube.
Further, the extraction structure is still including the parcel one-way fluid-discharge tube with the outer back taper of one-way liquid inlet pipe, the wide end of back taper with the bottom surface connection who draws the storehouse, the narrow end of back taper with absorb a mouthful fixed connection.
Furthermore, three one-way valves are respectively arranged in the one-way liquid inlet pipe and the one-way liquid discharge pipe.
Furthermore, the extraction structure further comprises a locking structure which is arranged at the liquid inlet of the one-way liquid discharge pipe and is used for controlling the liquid inlet to be opened or closed.
Furthermore, the hasp structure includes center pin, valve and pull ring, the valve is disk-shaped structure, and its size matches with one-way flowing back intraductal diameter, one end of center pin passes one-way flowing back intraductal valve fixed connection of one-way flowing back, and the pull ring outside the one-way flowing back pipe of other end fixed connection, and rotatory pull ring passes through the center pin and drives the valve rotation, realizes opening and close of one-way flowing back pipe.
Further, the storage structure comprises four storage tubes which are axially parallel to and communicated with the extraction bin, namely a lysis solution storage tube, an RNA protective solution storage tube, a neutralization solution storage tube and an eluent storage tube, wherein a one-way silica gel valve is arranged at an outlet of each storage tube; the second driving assembly comprises four storage pipe pistons which are arranged in one-to-one correspondence with the storage pipes, and the storage pipes discharge the stored reagents into the extraction bin under the action of the storage pipe pistons.
Further, the magnetic structure further comprises a guide structure for guiding the sliding of the magnetic ring; the guide structure comprises at least two movable guide rails uniformly distributed on the outer surface of the device shell, and the length direction of the movable guide rails is consistent with the axial direction of the device shell.
Furthermore, the first driving assembly comprises a main piston, a piston rod and a piston cap, wherein one end of the piston rod is fixedly connected with the main piston, and the other end of the piston rod is fixedly connected with the piston cap.
The invention also provides a use method of the closed simple RNA nucleic acid extraction device, which comprises the following steps:
a1, taking down the sealing element, and sealing the suction opening by the sealing element after the sample is extracted from the extraction bin through the suction opening under the action of the first driving assembly;
a2, removing the magnetic bead adsorption state by moving the magnetic ring, depositing the magnetic bead on the bottom of the extraction bin, sending the lysis solution and the RNA protection solution in the storage tube into the extraction bin through a second driving assembly, fully oscillating, and vertically standing for lysis;
a3, adsorbing magnetic beads by a movable magnetic ring, placing the magnetic beads in the middle of an extraction bin, sending the neutralization solution in a storage tube into the extraction bin by a second driving assembly, and uniformly mixing;
a4, removing a sealing element, discharging the mixed liquid from the extraction bin through the suction port under the action of the first driving assembly, sucking 70% ethanol from the extraction bin through the suction port under the action of the first driving assembly until the ethanol submerges the magnetic beads, and shaking or shaking the extraction bin after the sealing element seals the suction port;
a5, taking down a sealing element, discharging ethanol washing liquid from the extraction bin through a suction port under the action of a first driving assembly, and sealing the suction port by the sealing element;
a6, removing the magnetic bead adsorption state by moving the magnetic ring, depositing the magnetic bead at the bottom of the extraction bin, sending the eluent in the storage tube into the extraction bin through the second driving component, shaking or shaking and standing to complete the nucleic acid extraction.
Further, the time of full oscillation in the step A2 is 1-3 minutes, and the time of vertical standing is 10 min; shaking or shaking time in the step A4 is 1-3 minutes; in the step A5, repeated operation is needed to ensure that the ethanol washing liquid is drained completely, and the time interval between each operation is 5 minutes; in the step A6, the shaking or shaking time is 1-3 minutes, and the standing time is 1 minute.
The invention also provides a preparation method of the closed simple RNA nucleic acid extraction device, which comprises the following steps:
s1, preparing two glass tubes which are respectively a one-way liquid inlet tube and a one-way liquid outlet tube, wherein the one-way liquid inlet tube is 3cm longer than the one-way liquid outlet tube, 3 one-way valves are respectively arranged in the one-way liquid inlet tube and the one-way liquid outlet tube, and a lock catch structure is arranged on the one-way liquid outlet tube;
s2, preparing an extraction bin, manufacturing the cylindrical extraction bin, installing a one-way liquid inlet pipe and a one-way liquid discharge pipe, wherein one end of the one-way liquid inlet pipe and one-way liquid discharge pipe is communicated with the lower end of the extraction bin, the other end of the one-way liquid inlet pipe and the other end of the one-way liquid discharge pipe are converged and communicated with an absorption port, the one-way liquid inlet pipe extends into the cylindrical extraction bin by 3cm and is tightly attached to the bottle wall of the cylindrical bottle, the opening direction of a one-way valve in the one-way liquid inlet pipe is from outside to inside the extraction bin, the opening direction of the one-way valve in the one-way liquid discharge pipe is from inside to outside the extraction bin, 4 storage pipes for storing reaction reagents are prepared outside the cylindrical bottle, the lower end of each storage pipe is bent and is fired and communicated with the cylindrical extraction bin, 4 storage pipes are respectively:
lysis solution: 0.1M NaOH, 50. mu.L CHCl3, 50. mu.L guanidinium isothiocyanate, total volume 500. mu.L;
RNA protective solution: 200 microliter of RNAscope LD with the purity of 100%;
neutralizing liquid: 500. mu.L of 0.1M NaAC;
eluent: 50 mu L of ultrapure water containing 1-3U/mu L of RNase inhibitor Murine;
s3, mounting magnetic beads, a first driving assembly and a storage tube piston, wherein the magnetic beads are placed in the extraction bin, the storage tube piston is mounted at the upper end of the storage tube, and the first driving assembly is mounted at the upper end of the extraction bin and comprises a main piston, a piston rod connected with the center of the main piston and a piston cap connected with the piston rod;
s4, preparing a device shell and installing a magnetic ring, splicing the device shell according to an upper cylindrical shape and a lower conical shape, wrapping an extraction bin, a storage pipe, a one-way liquid inlet pipe and a one-way liquid outlet pipe, fixing the upper end of the device shell and the upper edge of the extraction bin, punching according to a first driving assembly and the position of a storage pipe piston, sleeving the circular magnetic ring outside the device shell in a sliding mode, sleeving a sealing element on an absorption opening, and wrapping the circular magnetic ring completely to guarantee system sealing.
The invention has the beneficial effects that:
1. the extraction device has the advantages of simple structure, simple use and no pollution by the arrangement of the extraction structure, the storage structure, the magnetic structure and the driving structure, can be effectively combined with three-level biosafety protection management, can simplify the RNA nucleic acid extraction method, can avoid biosafety problems by the arrangement of mutual sealing connection, realizes the purpose of extraction at any time, and can meet the field extraction work of an isolation area environment or a field sample without the support of large-scale equipment.
2. The invention can ensure the sealing performance of the extraction device and the safety in the extraction process by arranging the inner layer structure and the outer layer structure.
3. The one-way liquid inlet pipe is higher than the one-way liquid outlet pipe, so that the biological pollution of washing liquid in the extraction process is avoided; the sealing performance of the device can be ensured by arranging the one-way valve; the safety of the reaction reagent can be ensured through a plurality of storage tubes which are separately arranged; through the setting of guide structure, can make things convenient for the slip of magnetic ring for it is more convenient to draw the process.
Drawings
FIG. 1 is a perspective view of the simple RNA nucleic acid extraction device of the present invention.
FIG. 2 is a schematic view of the storage tube installation of the present invention.
FIG. 3 is a schematic view of the downward sliding motion of the storage tube piston according to the present invention.
FIG. 4 is a schematic view of the locking mechanism of the present invention for opening and closing the one-way drain.
FIG. 5 is a cross-sectional view of the one-way drain pipe and the one-way valve in the one-way liquid inlet pipe of the present invention.
Fig. 6 is a top view of fig. 1 without the piston cap.
Fig. 7 is a schematic structural view of a guide structure of the present invention.
Reference numerals: 1. a suction port; 2. a device housing; 3. a one-way liquid inlet pipe; 4. a one-way drain pipe; 5. a one-way valve; 6. a locking structure; 7. an extraction bin; 8. magnetic beads; 9. a storage tube; 10. a primary piston; 11. a magnetic ring; 12. a reservoir tube piston; 13. a piston rod; 14. a piston cap; 15. a moving guide rail; 16. a magnet bayonet; 17. fixing the coil; 18. a housing slot.
Detailed Description
The technical solutions in the embodiments of the present invention are 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 only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the scope of protection of the present invention.
Example 1
The invention provides a closed simple RNA nucleic acid extraction device, as shown in figure 1, so the device is designed into a multilayer structure and comprises an inner layer structure and an outer layer structure, wherein the inner layer structure is divided into an upper part, a middle part and a lower part: the middle layer is a cylindrical extraction bin 7 and 4 small cylindrical storage tubes 9 are designed outside the extraction bin 7, the 4 storage tubes 9 are sequentially marked as A, B, C, D in the clockwise direction when viewed from top to bottom, as shown in fig. 6, wherein a lysis solution is placed in the storage tube A9, an RNA protective solution is placed in the storage tube B9, a neutralizing solution is placed in the storage tube C9, an eluent is placed in the storage tube D9, a smooth neck is designed at the lower edge of the storage tube 9, a one-way silica gel valve is arranged at the narrow part of the smooth neck to ensure that a reagent cannot naturally flow, and the lower end of the storage tube 9 is directly communicated with the extraction bin 7; the upper layer is a first driving component and a second driving component which are positioned in the center, the second driving component comprises storage tube pistons 12 which are dispersed in 4 directions around, the first driving component is slidably mounted at the upper end of the extraction bin 7, the first driving component freely moves along the inner wall of the extraction bin 7, the storage tube pistons 12 are slidably mounted at the upper end of the storage tubes 9 and freely move along the inner wall of the storage tubes 9, and the discharge of liquid in the extraction bin 7 and the addition of reagents in the storage tubes 9 can be controlled by sliding the first driving component and the storage tube pistons 12 downwards, as shown in fig. 2 and 3; the lower layer is 2 glass tubes for absorbing and discharging liquid and other switches, the 2 glass tubes are a one-way liquid inlet tube 3 and a one-way liquid outlet tube 4, one end of the one-way liquid inlet tube 4 and one end of the one-way liquid inlet tube 3 are communicated with the extraction bin 7, the other end of the one-way liquid inlet tube 3 are communicated with the extraction bin 1, specifically, a liquid inlet of the one-way liquid inlet tube 4 is communicated with the extraction bin 7, a liquid outlet of the one-way liquid inlet tube 3 is communicated with the extraction bin 1, the liquid outlet of the one-way liquid inlet tube is communicated with the extraction bin 7, a sealing element is sleeved on the extraction bin 1, when the sealing element is installed, the liquid in the extraction bin 7 cannot be discharged, otherwise, 2 independent pipelines are arranged to help to avoid biological pollution of washing liquid in the extraction process, 3 simple one-way valves 5 are arranged in the one-way liquid inlet tube 3 and the one-way liquid outlet tube 4, the one, the liquid of the one-way liquid discharge pipe 4 can only flow outwards from the extraction bin 7, so that the sealing performance of the device is ensured, as shown in fig. 5. In addition, a locking structure 6 for opening and closing the one-way liquid discharge pipe 4 is further designed at the liquid inlet of the one-way liquid discharge pipe 4, as shown in fig. 4. The outer shell structure is device shell 2, and the main component is polycarbonate PC, and thickness 0.15cm, device shell 2 parcel draws storehouse 7, storage tube 9, one-way feed liquor pipe 3 and one-way fluid-discharge tube 4, device shell 2 upper end is equipped with the hole that is used for storage tube piston 12 and first drive assembly to pass through. As shown in fig. 7, a guiding structure is arranged outside the device housing 2, the guiding structure comprises at least two moving guide rails 15 uniformly distributed on the outer surface of the device housing 2, the length direction of the moving guide rail 15 is consistent with the axial direction of the device shell 2, the guide structure further comprises a shell clamping groove 18, a magnet clamping opening 16 and a fixed coil 17, the movable guide rail 15 is arranged in the shell clamping groove 18, the fixed coil 17 is arranged at the upper end of the movable guide rail 15, the magnet bayonet 16 is arranged on the movable guide rail 15, the magnetic ring 11 is sleeved on the movable guide rail 15 in a sliding manner, the magnetic ring 11 can be controlled to move up and down along the central axis of the extraction bin 7 through the fixed coil 17, the sliding position of the magnetic ring 11 can be limited through the magnet bayonet 16, so that the extraction bin 7 can be fixed and stopped at different height positions to meet the experimental requirements, and the magnetic ring 11 is preferably a circular magnet.
In the embodiment, the RNA nucleic acid extraction method can be simplified as much as possible by virtue of a closed extraction device, and meanwhile, the biological safety problem can be effectively avoided; in addition, the aim of extracting samples at the same time of sampling can be realized, and the field extraction work of isolated area environments or field samples without the support of large-scale equipment can be met.
In this embodiment, preferably, as shown in fig. 4, the latch structure 6 includes a central shaft, a valve and a pull ring, the valve is a disk-shaped structure, the size of the valve is matched with the inner diameter of the one-way liquid discharge pipe 4, one end of the central shaft passes through the one-way liquid discharge pipe 4 and is fixedly connected with the valve inside the one-way liquid discharge pipe 4, the other end of the central shaft is fixedly connected with the pull ring outside the one-way liquid discharge pipe 4, the valve is driven to rotate by rotating the pull ring through the central shaft, so as to open and close the one-way liquid discharge pipe 4, specifically, when the pull ring is in a horizontal state, the one-way liquid discharge pipe 4.
In this embodiment, preferably, as shown in fig. 1, the first driving assembly includes a main piston 10, a piston rod 13 and a piston cap 14, one end of the piston rod 13 is fixedly connected to the main piston 10, and the other end is fixedly connected to the piston cap 14, and the arrangement of the piston rod 13 of the piston cap 14 can facilitate the suction and discharge of the liquid in the extraction chamber 7.
Example 2
The invention also provides a preparation method of the closed simple RNA nucleic acid extraction device, which comprises the following steps as shown in figures 1 to 6:
s1, preparing two glass tubes which are respectively a one-way liquid inlet tube 3 and a one-way liquid outlet tube 4, wherein the materials are polypropylene PP, the diameters of the two glass tubes are 0.5cm, the length of the one-way liquid inlet tube 3 is 10cm, the length of the one-way liquid outlet tube 4 is 7cm, 3 one-way valves 5 are respectively arranged in the one-way liquid inlet tube 3 and the one-way liquid outlet tube 4, and a lock catch structure 6 is arranged at the liquid inlet of the one-way liquid outlet tube 4; the lock catch structure 6 consists of a central shaft, a lock ring and a disk-shaped valve, and the rotary lock ring can drive the valve to rotate through the central shaft to achieve the effect of opening and closing the one-way liquid discharge pipe 4;
s2, preparing an extraction bin 7, manufacturing the cylindrical extraction bin 7, wherein the material is polypropylene PP, the diameter is 3.0cm, the height is 8.0cm, installing a one-way liquid inlet pipe 3 and a one-way liquid outlet pipe 4, one end of the one-way liquid inlet pipe 3 and one-way liquid outlet pipe 4 is communicated with the lower end of the extraction bin 7, the other end of the one-way liquid inlet pipe 3 and one-way liquid outlet pipe 4 are converged and communicated with a suction port 1, the one-way liquid inlet pipe 3 extends into the cylindrical extraction bin 73 cm and is tightly attached to the bottle wall of the cylindrical bottle, the opening direction of a one-way valve 5 in the one-way liquid inlet pipe 3 is ensured to be from the outside to the extraction bin 7 during installation, the opening direction of a one-way valve 5 in the one-way liquid outlet pipe 4 is from the inside to the outside of the extraction bin 7, 4 storage pipes 9 are prepared outside the cylindrical bottle, the material is polypropylene, the diameter is 1.5cm, the height is 5cm, the lower port is slightly contracted to be 1cm, respectively placing lysis solution, RNA protective solution, neutralizing solution and eluent, wherein the reagent parameters are as follows:
lysis solution: 0.1M NaOH, 50. mu.L CHCl3, 50. mu.L guanidinium isothiocyanate, total volume 500. mu.L;
RNA protective solution: 200 μ L of RNAscope LD (100%);
neutralizing liquid: 500. mu.L of 0.1M NaAC;
eluent: 50 muL of ultrapure water containing 1-3U/muL of RNase inhibitor Murine;
s3, mounting a first driving assembly and a storage tube piston 12, wherein the storage tube piston 12 is mounted at the upper end of a storage tube 9, the first driving assembly is mounted at the upper end of the extraction bin 7, the first driving assembly comprises a main piston 10, a piston rod 13 connected with the center of the main piston 10 and a piston cap 14 connected with the piston rod 13, the main piston 10 is made of cylindrical rubber, the diameter of the main piston is 2.8cm, and the height of the main piston is 1.5 cm; the piston rod 13 is 6cm long; the reservoir piston 12 is also made of cylindrical rubber, and has a diameter of 1.3cm and a height of 1 cm.
S4, preparing a device shell 2 and installing a magnetic ring 11, wherein the magnetic ring 11 can be a circular magnet, the device shell 2 is made of PC, and the device shell 2 is spliced according to an upper cylindrical part and a lower conical part, wherein the cylindrical part is 4.8cm in diameter, 5.2cm in height and 0.15cm in thickness; conical bottom diameter is 4.8cm, height is 7.5cm, will draw storehouse 7, storage tube 9, one-way feed liquor pipe 3 and one-way fluid-discharge tube 4 parcel, device shell 2 upper end is fixed with drawing storehouse 7 upper reason, punch according to first drive assembly and storage tube piston 12 position, set up shell draw-in groove 18, fixed coil 17, magnet bayonet 16 and removal guide rail 15 isotructure, install removal guide rail 15 in shell draw-in groove 18, the annular magnet slip cup joints on removal guide rail 15, and through the magnet bayonet 16 restriction position on the removal guide rail 15, wherein the annular external diameter is 5.5cm, the internal diameter is 5.2cm, height is 1.0cm, sheathe sealing member on the absorption mouth 1 of device lower extreme, the sealing member can select the closing cap of silica gel system, it is most advanced to wrap up completely, in order to guarantee the system is sealed.
In the embodiment, the extraction device prepared by the preparation method can simplify the RNA nucleic acid extraction method as much as possible, and can effectively avoid the biological safety problem; in addition, the aim of extracting samples at the same time of sampling can be realized, and the field extraction work of isolated area environments or field samples without the support of large-scale equipment can be met.
Example 3
The invention also provides a use method of the closed simple RNA nucleic acid extraction device, which comprises the following steps as shown in figures 1 to 6:
a1, taking down a sealing element, and sealing the suction port 1 by the sealing element after the sample is extracted from the extraction bin 7 through the suction port 1 under the action of the first driving assembly;
a2, removing the adsorption state of the magnetic beads 8 by moving the magnetic ring 11, depositing the magnetic beads 8 at the bottom of the extraction bin 7, sending the lysis solution and the RNA protection solution in the storage tube 9 into the extraction bin 7 through a second driving assembly, fully oscillating, and vertically standing for lysis;
a3, adsorbing magnetic beads 8 by a movable magnetic ring 11, placing the magnetic beads in the middle of an extraction bin 7, sending the neutralization solution in a storage tube 9 into the extraction bin 7 through a second driving assembly, and uniformly mixing;
a4, taking down a sealing element, discharging a mixed solution from an extraction bin 7 through a suction port 1 under the action of a first driving assembly, sucking 70% ethanol (without specific volume requirement) from the extraction bin 7 through the suction port 1 under the action of the first driving assembly until the ethanol submerges the magnetic beads 8, and shaking or shaking after the sealing element seals the suction port 1;
a5, taking down a sealing element, discharging ethanol washing liquid from the extraction bin 7 through the suction port 1 under the action of the first driving assembly, and sealing the suction port 1 by the sealing element;
a6, removing the adsorption state of the magnetic beads 8 by moving the magnetic ring 11, depositing the magnetic beads 8 at the bottom of the extraction bin 7, sending the eluent in the storage tube 9 into the extraction bin 7 through the second driving component, shaking or shaking and then standing, and finishing the nucleic acid extraction work.
In the embodiment, when the extraction device is used for extracting nucleic acid, the RNA nucleic acid extraction method can be simplified as much as possible, and meanwhile, the biological safety problem can be effectively avoided; in addition, the aim of extracting samples at the same time of sampling can be realized, and the field extraction work of isolated area environments or field samples without the support of large-scale equipment can be met.
Finally, the above embodiments are only used for illustrating the technical solutions of the present invention and not for limiting, and other modifications or equivalent substitutions made by the technical solutions of the present invention by those of ordinary skill in the art should be covered within the scope of the claims of the present invention as long as they do not depart from the spirit and scope of the technical solutions of the present invention.
Claims (12)
1. The utility model provides a closed simple and easy RNA nucleic acid extraction element, includes extraction structure, storage structure, magnetism structure and drive structure, its characterized in that:
the extraction structure comprises an extraction bin (7), a one-way liquid inlet pipe (3) communicated with the extraction bin (7), a one-way liquid discharge pipe (4) communicated with the extraction bin (7), and a suction port (1) communicated with one ends of the one-way liquid inlet pipe (3) and the one-way liquid discharge pipe (4) far away from the extraction bin (7);
the storage structure comprises a plurality of storage tubes (9) which are communicated with the extraction bin (7) and are used for storing reaction reagents;
the magnetic structure comprises magnetic beads (8) arranged in the extraction bin (7) and a magnetic ring (11) used for controlling the magnetic beads (8);
the driving structure comprises a first driving component for driving the extracting structure to extract or discharge liquid through the suction port (1) and a second driving component for driving the storage structure to send reaction reagents into the extracting bin (7);
the diameter of the magnetic bead (8) is larger than that of the one-way liquid inlet pipe (3) and that of the one-way liquid outlet pipe (4), a sealing element is arranged at the suction port (1) of the extraction structure, and the closed simple RNA nucleic acid extraction device is in a syringe shape.
2. The closed simple RNA nucleic acid extraction device of claim 1, wherein: the closed simple RNA nucleic acid extraction device comprises an outer layer structure and an inner layer structure;
the inner layer structure comprises the extraction structure, the first driving assembly arranged at the tail end of the extraction bin (7), a plurality of storage pipes (9) arranged outside the extraction bin (7) in a surrounding mode, and the second driving assembly arranged at the tail end of each storage pipe (9);
outer shell (2), be used for dismantling the setting and be in outer device shell (2), the slidable cover of inner structure are established be used for control on device shell (2) draw in storehouse (7) magnetic bead (8) magnetism ring (11), wherein, the lower extreme of device shell (2) with draw structure fixed connection.
3. The closed simple RNA nucleic acid extraction device according to claim 1 or 2, wherein: draw storehouse (7) and be the U type, one-way fluid-discharge tube (4) are located draw storehouse (7) below and communicate rather than the bottom, the liquid outlet of one-way fluid-discharge tube (4) with the inlet intercommunication of one-way fluid-discharge tube (3), one-way fluid-discharge tube (3) slope is located one-way fluid-discharge tube (4) one side, just the liquid outlet of one-way fluid-discharge tube (3) extends to draw in the storehouse (7) and be higher than the inlet of one-way fluid-discharge tube (4).
4. The closed simple RNA nucleic acid extraction device of claim 3, wherein: the extraction structure further comprises an inverted cone wrapped outside the one-way liquid discharge pipe (4) and the one-way liquid inlet pipe (3), the wide end of the inverted cone is connected with the outer surface of the bottom end of the extraction bin (7), and the narrow end of the inverted cone is fixedly connected with the suction port (1).
5. The closed simple RNA nucleic acid extraction device of claim 1, wherein: three one-way valves (5) are respectively arranged in the one-way liquid inlet pipe (3) and the one-way liquid outlet pipe (4).
6. The closed simple RNA nucleic acid extraction device of claim 1, wherein: the extraction structure also comprises a lock catch structure (6) which is arranged at the liquid inlet of the one-way liquid discharge pipe (4) and is used for controlling the liquid inlet to be opened or closed.
7. The closed simple RNA nucleic acid extraction device of claim 6, wherein: the locking structure (6) comprises a central shaft, a valve and a pull ring, the valve is of a disc-shaped structure, the size of the valve is matched with the inner diameter of the one-way liquid discharge pipe (4), one end of the central shaft penetrates through the one-way liquid discharge pipe (4) and is fixedly connected with the valve in the one-way liquid discharge pipe (4), the other end of the central shaft is fixedly connected with the pull ring outside the one-way liquid discharge pipe (4), and the rotary pull ring drives the valve to rotate through the central shaft, so that the one-way liquid discharge pipe (4.
8. The closed simple RNA nucleic acid extraction device according to claim 1 or 2, wherein: the storage structure comprises four storage tubes (9) which are axially parallel to and communicated with the extraction bin (7), namely a lysis solution storage tube, an RNA protective solution storage tube, a neutralization solution storage tube and an eluent storage tube, wherein a one-way silica gel valve is arranged at an outlet of each storage tube; the second driving assembly comprises four storage pipe pistons (12) which are arranged in one-to-one correspondence with the storage pipes (9), and the storage pipes (9) discharge the stored reagents into the extraction bin (7) under the action of the storage pipe pistons (12).
9. The closed simple RNA nucleic acid extraction device of claim 2, wherein: the magnetic structure further comprises a guide structure for guiding the sliding of the magnetic ring (11); the guide structure comprises at least two moving guide rails (15) which are uniformly distributed on the outer surface of the device shell (2), and the length direction of the moving guide rails (15) is consistent with the axial direction of the device shell (2).
10. A use method of a closed simple RNA nucleic acid extraction device is characterized in that: the method comprises the following steps:
a1, removing a sealing element, and sealing the suction port (1) by the sealing element after the sample is extracted from the extraction bin (7) through the suction port (1) under the action of the first driving assembly;
a2, removing the adsorption state of the magnetic beads (8) by moving the magnetic ring (11), depositing the magnetic beads (8) at the bottom of the extraction bin (7), sending the lysate and the RNA protection solution in the storage tube (9) into the extraction bin (7) through a second driving assembly, fully oscillating, and vertically standing for lysis;
a3, adsorbing magnetic beads (8) by a movable magnetic ring (11), placing the magnetic beads in the middle of an extraction bin (7), sending a neutralization solution in a storage tube (9) into the extraction bin (7) by a second driving assembly, and uniformly mixing;
a4, removing a sealing element, discharging mixed liquor from an extraction bin (7) through a suction port (1) under the action of a first driving assembly, sucking 70% ethanol from the extraction bin (7) through the suction port (1) under the action of the first driving assembly until the ethanol submerges magnetic beads (8), and shaking or shaking the extraction bin after the sealing element seals the suction port (1);
a5, removing a sealing element, discharging ethanol washing liquid from the extraction bin (7) through the suction port (1) under the action of the first driving assembly, and sealing the suction port (1) by the sealing element;
a6, removing the adsorption state of the magnetic beads (8) by moving the magnetic ring (11), depositing the magnetic beads (8) at the bottom of the extraction bin (7), sending the eluent in the storage tube (9) into the extraction bin (7) through the second driving component, shaking or shaking and then standing to finish the nucleic acid extraction work.
11. The use method of the closed simple RNA nucleic acid extraction device according to claim 10, wherein: in the step A2, the full oscillation time is 1-3 minutes, and the vertical standing time is 10 minutes; shaking or shaking time in the step A4 is 1-3 minutes; in the step A5, repeated operation is needed to ensure that the ethanol washing liquid is drained completely, and the time interval between each operation is 5 minutes; in the step A6, the shaking or shaking time is 1-3 minutes, and the standing time is 1 minute.
12. A preparation method of a closed simple RNA nucleic acid extraction device is characterized by comprising the following steps: the method comprises the following steps:
s1, preparing two glass tubes which are respectively a one-way liquid inlet tube (3) and a one-way liquid outlet tube (4), wherein the one-way liquid inlet tube (3) is 3cm longer than the one-way liquid outlet tube (4), 3 one-way valves (5) are respectively arranged in the one-way liquid inlet tube (3) and the one-way liquid outlet tube (4), and a lock catch structure (6) is arranged on the one-way liquid outlet tube (4);
s2, preparing an extraction bin (7), manufacturing the cylindrical extraction bin (7), installing a one-way liquid inlet pipe (3) and a one-way liquid discharge pipe (4), wherein one end of the one-way liquid inlet pipe (3) and one end of the one-way liquid discharge pipe (4) are communicated with the lower end of the extraction bin (7), the other end of the one-way liquid inlet pipe and the other end of the one-way liquid discharge pipe are converged and communicated with a suction port (1), the one-way liquid inlet pipe (3) extends into the cylindrical extraction bin (7) by 3cm and is tightly attached to the cylindrical bottle wall, the opening direction of a one-way valve (5) in the one-way liquid inlet pipe (3) is ensured to be from outside to inside the extraction bin (7) during installation, the opening direction of the one-way valve (5) in the one-way liquid discharge pipe (4) is from inside to outside the extraction bin (7), 4 storage pipes (9) for storing reaction reagents are prepared outside the cylindrical bottle, the lower end of each storage pipe (9) is bent and communicated with the, RNA protective solution, neutralizing solution and eluent, and the reagent parameters are as follows:
lysis solution: 0.1M NaOH, 50. mu.L CHCl3, 50. mu.L guanidinium isothiocyanate, total volume 500. mu.L;
RNA protective solution: 200 microliter of RNAscope LD with the purity of 100%;
neutralizing liquid: 500. mu.L of 0.1M NaAC;
eluent: 50 mu L of ultrapure water containing 1-3U/mu L of RNase inhibitor Murine;
s3, mounting magnetic beads (8), a first driving assembly and a storage tube piston (12), wherein the magnetic beads (8) are placed in an extraction bin (7), the storage tube piston (12) is mounted at the upper end of a storage tube (9), the first driving assembly is mounted at the upper end of the extraction bin (7), and the first driving assembly comprises a main piston (10), a piston rod (13) connected with the center of the main piston (10) and a piston cap (14) connected with the piston rod (13);
s4, preparing a device shell (2) and installing a magnetic ring (11), splicing the device shell (2) according to an upper cylindrical shape and a lower conical shape, wrapping an extraction bin (7), a storage pipe (9), a one-way liquid inlet pipe (3) and a one-way liquid discharge pipe (4), fixing the upper end of the device shell (2) with the upper edge of the extraction bin (7), punching according to a first driving assembly and a storage pipe piston (12), slidably sleeving the circular magnetic ring (11) on the outer side of the device shell (2), sleeving a sealing element on a suction port (1), and completely wrapping the tip end to ensure that a system is sealed.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110013429.4A CN112760194B (en) | 2021-01-06 | 2021-01-06 | Closed simple RNA nucleic acid extraction device and preparation method and use method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110013429.4A CN112760194B (en) | 2021-01-06 | 2021-01-06 | Closed simple RNA nucleic acid extraction device and preparation method and use method thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN112760194A true CN112760194A (en) | 2021-05-07 |
| CN112760194B CN112760194B (en) | 2022-11-25 |
Family
ID=75700203
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202110013429.4A Active CN112760194B (en) | 2021-01-06 | 2021-01-06 | Closed simple RNA nucleic acid extraction device and preparation method and use method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN112760194B (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113308364A (en) * | 2021-06-09 | 2021-08-27 | 北京京东方技术开发有限公司 | Nucleic acid extraction substrate, nucleic acid extraction apparatus, and nucleic acid extraction method |
| CN114032173A (en) * | 2022-01-11 | 2022-02-11 | 至美时代生物智能科技(北京)有限公司 | Closed air sampling bottle |
| CN117000324A (en) * | 2023-06-30 | 2023-11-07 | 上海金鑫生物科技有限公司 | Pipetting element |
Citations (15)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104046555A (en) * | 2013-03-13 | 2014-09-17 | 精工爱普生株式会社 | Cartridge For Nucleic Acid Amplification Reaction |
| US9145581B1 (en) * | 2014-10-17 | 2015-09-29 | Daniel Lai | Rapid nucleic acid extraction method and apparatus |
| WO2016051795A1 (en) * | 2014-09-30 | 2016-04-07 | Seiko Epson Corporation | Biological substance extraction device and biological substance extraction apparatus |
| CN205672925U (en) * | 2016-06-07 | 2016-11-09 | 东莞市中鼎检测技术有限公司 | A kind of laboratory reextraction separatory funnel |
| WO2017067093A1 (en) * | 2015-10-21 | 2017-04-27 | 陈辉 | Nucleic acid extraction apparatus and extraction method |
| CN108220125A (en) * | 2018-03-21 | 2018-06-29 | 中国检验检疫科学研究院 | A kind of nucleic acid rapid extraction device |
| CN208121116U (en) * | 2017-12-20 | 2018-11-20 | 中国农业科学院兰州兽医研究所 | A kind of device of paramagnetic particle method rapidly extracting nucleic acid |
| CN108865658A (en) * | 2018-08-27 | 2018-11-23 | 苏州绘真医学检验有限公司 | A kind of tubular type plasma free method for extracting nucleic acid and system |
| CN109456880A (en) * | 2018-12-20 | 2019-03-12 | 中国检验检疫科学研究院 | Nucleic acid on-site rapidly extracting pipe and its application method |
| CN111607485A (en) * | 2020-05-27 | 2020-09-01 | 邵建永 | Simple nucleic acid extraction device and nucleic acid extraction method |
| CN212292766U (en) * | 2020-04-22 | 2021-01-05 | 南京南大药业有限责任公司 | Be applied to branch liquid cover of pharmacy industry |
| CN112358965A (en) * | 2020-11-09 | 2021-02-12 | 中国计量大学 | Magnetic bead method nucleic acid extraction equipment |
| CN112779355A (en) * | 2021-01-06 | 2021-05-11 | 常州市疾病预防控制中心 | Self-heating amplification detection device and preparation method and use method thereof |
| CN113249188A (en) * | 2021-06-03 | 2021-08-13 | 宁波康程德诺生物医药有限公司 | Rapid nucleic acid extraction device and method for extracting nucleic acid |
| US20210363514A1 (en) * | 2020-05-22 | 2021-11-25 | Wizbiosolutions lnc. | Syringe-type nucleic acid extraction apparatus, nucleic acid extraction kit and nucleic acid extraction method therewith |
-
2021
- 2021-01-06 CN CN202110013429.4A patent/CN112760194B/en active Active
Patent Citations (15)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104046555A (en) * | 2013-03-13 | 2014-09-17 | 精工爱普生株式会社 | Cartridge For Nucleic Acid Amplification Reaction |
| WO2016051795A1 (en) * | 2014-09-30 | 2016-04-07 | Seiko Epson Corporation | Biological substance extraction device and biological substance extraction apparatus |
| US9145581B1 (en) * | 2014-10-17 | 2015-09-29 | Daniel Lai | Rapid nucleic acid extraction method and apparatus |
| WO2017067093A1 (en) * | 2015-10-21 | 2017-04-27 | 陈辉 | Nucleic acid extraction apparatus and extraction method |
| CN205672925U (en) * | 2016-06-07 | 2016-11-09 | 东莞市中鼎检测技术有限公司 | A kind of laboratory reextraction separatory funnel |
| CN208121116U (en) * | 2017-12-20 | 2018-11-20 | 中国农业科学院兰州兽医研究所 | A kind of device of paramagnetic particle method rapidly extracting nucleic acid |
| CN108220125A (en) * | 2018-03-21 | 2018-06-29 | 中国检验检疫科学研究院 | A kind of nucleic acid rapid extraction device |
| CN108865658A (en) * | 2018-08-27 | 2018-11-23 | 苏州绘真医学检验有限公司 | A kind of tubular type plasma free method for extracting nucleic acid and system |
| CN109456880A (en) * | 2018-12-20 | 2019-03-12 | 中国检验检疫科学研究院 | Nucleic acid on-site rapidly extracting pipe and its application method |
| CN212292766U (en) * | 2020-04-22 | 2021-01-05 | 南京南大药业有限责任公司 | Be applied to branch liquid cover of pharmacy industry |
| US20210363514A1 (en) * | 2020-05-22 | 2021-11-25 | Wizbiosolutions lnc. | Syringe-type nucleic acid extraction apparatus, nucleic acid extraction kit and nucleic acid extraction method therewith |
| CN111607485A (en) * | 2020-05-27 | 2020-09-01 | 邵建永 | Simple nucleic acid extraction device and nucleic acid extraction method |
| CN112358965A (en) * | 2020-11-09 | 2021-02-12 | 中国计量大学 | Magnetic bead method nucleic acid extraction equipment |
| CN112779355A (en) * | 2021-01-06 | 2021-05-11 | 常州市疾病预防控制中心 | Self-heating amplification detection device and preparation method and use method thereof |
| CN113249188A (en) * | 2021-06-03 | 2021-08-13 | 宁波康程德诺生物医药有限公司 | Rapid nucleic acid extraction device and method for extracting nucleic acid |
Non-Patent Citations (1)
| Title |
|---|
| 臧志和等: "《药学基础化学实验》", 31 May 2014, 西南交通大学出版社 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113308364A (en) * | 2021-06-09 | 2021-08-27 | 北京京东方技术开发有限公司 | Nucleic acid extraction substrate, nucleic acid extraction apparatus, and nucleic acid extraction method |
| CN114032173A (en) * | 2022-01-11 | 2022-02-11 | 至美时代生物智能科技(北京)有限公司 | Closed air sampling bottle |
| CN114032173B (en) * | 2022-01-11 | 2022-03-15 | 至美时代生物智能科技(北京)有限公司 | Closed air sampling bottle |
| CN117000324A (en) * | 2023-06-30 | 2023-11-07 | 上海金鑫生物科技有限公司 | Pipetting element |
Also Published As
| Publication number | Publication date |
|---|---|
| CN112760194B (en) | 2022-11-25 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN112760194B (en) | Closed simple RNA nucleic acid extraction device and preparation method and use method thereof | |
| US20130252796A1 (en) | Device for insertion into a rotor of a centrifuge, centrifuge and method for the fluidic coupling of cavities | |
| KR101964131B1 (en) | Closed extraction and purification apparatus | |
| CN102788724A (en) | Device for safely processing biochemical samples | |
| WO2022041402A1 (en) | Container and method of using the same | |
| CN113817600A (en) | Sample processing and detecting device and application thereof | |
| CN112094729A (en) | Rotary liquid-transferring type totally-enclosed nucleic acid detection card box | |
| CN105784454A (en) | Combined water sample filter | |
| US7534397B2 (en) | Sample preparation device | |
| CN218491744U (en) | Sample detection pretreatment equipment and reagent card box therein | |
| CN219641333U (en) | Liquid medicine sampling device | |
| CN109459281B (en) | Apparatus for processing biochemical samples | |
| CN206321612U (en) | A kind of filter-type chromatogram bottle and chromatograph | |
| CN214422599U (en) | Rotary liquid-transferring type totally-enclosed nucleic acid detection card box | |
| CN209865685U (en) | Auxiliary device for elution of adsorption column | |
| CN111841679B (en) | Centrifuge tube with double-layer structure | |
| CN210954062U (en) | Reaction box and reaction unit for extracting and detecting biomacromolecules | |
| CN1384347A (en) | Vacuum tube and microscope inspection, chemical treatment and microbe determination method of urine precipitate | |
| CN218202803U (en) | Exempt from to uncap liquid-transfering virus sampling pipe | |
| CN217359144U (en) | Nucleic acid release sampling device | |
| CN204255863U (en) | A kind of integrated chemical reaction device for fast detecting | |
| CN220364542U (en) | Nucleic acid extraction device | |
| CN114907962A (en) | Sample detection pretreatment equipment and reagent card box therein | |
| CN219502784U (en) | Liquid separation equipment and nucleic acid extraction system | |
| CN221046102U (en) | Breathable pipe cover with good antibacterial effect |
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 | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |