CN117070314B - Nucleic acid extraction device, method of using the same, and application thereof - Google Patents
Nucleic acid extraction device, method of using the same, and application thereof Download PDFInfo
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- CN117070314B CN117070314B CN202311311373.6A CN202311311373A CN117070314B CN 117070314 B CN117070314 B CN 117070314B CN 202311311373 A CN202311311373 A CN 202311311373A CN 117070314 B CN117070314 B CN 117070314B
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Classifications
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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
Abstract
The invention discloses a nucleic acid extraction device, a use method and application thereof. The device comprises a first pipe body, a second pipe body, a fluid channel, a first piston, a second piston and the like. The first and second tube bodies are respectively provided with a first liquid storage cavity and a second liquid storage cavity, and the bottom end of the second tube body is provided with a conical tube structure communicated with the second liquid storage cavity; the fluid inlet and the fluid outlet of the fluid channel are respectively exposed in the first liquid storage cavity and the second liquid storage cavity; the first piston and the second piston are respectively movably arranged in the first liquid storage cavity and the second liquid storage cavity and can apply pressure to liquid in the first liquid storage cavity and the second liquid storage cavity; when the second piston is at a first station, the second piston seals the fluid outlet of the fluid channel, and when the second piston is at a second station, the fluid channel is communicated and the first liquid storage cavity and the second liquid storage cavity are communicated. The nucleic acid extraction device has the advantages of simple structure, low cost and easy use, can rapidly and accurately realize the quantitative distribution of micro-liter level samples, and avoids the pollution of external environment.
Description
Technical Field
The invention particularly relates to a nucleic acid extraction device, and belongs to the technical field of biology.
Background
Nucleic acid detection is mainly performed by detecting genetic materials (DNA or RNA) of organisms to qualitatively or quantitatively determine relevant information of the organisms, such as the types of the organisms, the properties of the organisms, the concentration of the organisms and the like, and has important application in various fields such as disease diagnosis, epidemic prevention and control, health monitoring and the like.
The nucleic acid detection process generally comprises the steps of: sample pretreatment, nucleic acid extraction, nucleic acid amplification and detection of amplification results. The conventional nucleic acid extraction method requires a multi-step process, which comprises disrupting biological sample materials such as cells, tissue materials, etc., inactivating nucleases, releasing nucleic acids, and removing other tissues or cell components such as proteins, polysaccharides, lipids, etc., thereby obtaining high-quality nucleic acids. Because traditional nucleic acid extraction methods are cumbersome, time consuming, and often require the use of toxic reagents, researchers have proposed various improvements. Currently, the techniques for extracting nucleic acids are classified into liquid phase extraction and solid phase extraction, which have advantages and disadvantages, but in general, both methods can be performed by means of complicated and expensive extraction equipment. For this reason, efforts have been made in the industry to develop a nucleic acid extraction apparatus that is simple in structure, convenient to use, and inexpensive, and this has become a problem to be solved in the art.
CN114989971B proposes a cartridge device for extracting nucleic acid and automatically separating liquid, which comprises a nucleic acid extracting structure and a liquid separating mechanism, wherein the liquid separating mechanism comprises an executing mechanism and a nucleic acid guiding-out structure, and the nucleic acid extracting structure comprises a lysis liquid pipe, a magnetic bead pipe, a first cleaning liquid pipe, a second cleaning liquid pipe, a third cleaning liquid pipe, an eluent pipe and a sample pipe which are sequentially and independently connected; the nucleic acid guiding structure is arranged on one side of the nucleic acid extracting structure and is clamped with the nucleic acid extracting structure; a liquid storage tank and a liquid circulation channel are formed in the nucleic acid guiding-out structure, a conical tube is formed at the bottom end of the nucleic acid guiding-out structure, the liquid storage tank is communicated with the conical tube through the liquid circulation channel, and the liquid storage tank is communicated with the sample tube through the liquid circulation channel; the actuating mechanism controls the opening and blocking of the liquid circulation channel. Although the cartridge device is improved compared with other existing nucleic acid extraction equipment, the structure is still relatively complex, the use method is also relatively complex, and interference of aerosol and the like existing in the environment cannot be avoided, so that false positive of detection results can be possibly caused, and therefore, the cartridge device still needs to be improved.
Disclosure of Invention
The invention mainly aims to provide a nucleic acid extraction device, a using method and application thereof, so as to overcome the defects in the prior art.
In order to achieve the purpose of the invention, the technical scheme adopted by the invention comprises the following steps:
one aspect of the present invention provides a nucleic acid extraction apparatus comprising:
the first tube body is internally provided with a first liquid storage cavity for accommodating a liquid nucleic acid extraction system;
the second pipe body is internally provided with a second liquid storage cavity, a conical pipe structure is formed at the bottom end of the second pipe body, and the second liquid storage cavity is communicated with the conical pipe structure;
the fluid inlet and the fluid outlet of the fluid channel are respectively exposed in the first liquid storage cavity and the second liquid storage cavity;
a sealing mechanism in sealing engagement with the first tubular body and at least for isolating the liquid nucleic acid extraction system from the external environment;
the second piston can move between the first station and the second station at least in the second liquid storage cavity and is at least used for separating a liquid storage space isolated from the external environment in the second liquid storage cavity, the liquid storage space is used for receiving liquid input through the fluid channel, and the liquid storage space is communicated with the conical pipe structure;
when the second piston is in the first station, the second piston blocks the fluid outlet of the fluid channel, so that the fluid channel is blocked, and when the second piston is in the second station, the second piston moves away from the fluid outlet of the fluid channel, so that the fluid channel is communicated.
In one embodiment, the sealing mechanism comprises a first piston movably arranged in the first liquid storage cavity and at least used for isolating the liquid nucleic acid extraction system from the external environment, and an opening part at least used for allowing external substances and/or the first piston to enter the first liquid storage cavity is arranged on the first pipe body.
In one embodiment, the fluid inlet of the fluid channel is formed on the inner wall of the upper portion or the middle portion of the first liquid storage cavity.
In one embodiment, a first limiting mechanism is disposed in the first tube body, and the first limiting mechanism is used for limiting the movement range of the first piston to be above the fluid inlet of the fluid channel.
In one embodiment, the fluid inlet of the fluid channel is formed on the inner wall of the upper portion or the middle portion of the first liquid storage cavity.
In one embodiment, the ratio of the depth to the diameter of the first reservoir may be greater than 1, and may be, for example, any number in the range of 1-100, 10-50, 1-20, 1-10, or 1-5, and may be, for example, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 5, 2, 1.5, etc.
The liquid nucleic acid extraction system is a mixture at least comprising a biological sample to be detected, a lysate and a nucleic acid extraction proteolytic enzyme.
In one embodiment, the first tube body is provided with an opening for at least external substances and/or the first piston to enter the first liquid storage cavity.
In one embodiment, a second limiting mechanism is further arranged in the second pipe body, the second limiting mechanism is used for limiting the moving range of the second piston in the second liquid storage cavity to be between a second station and a third station, the first station is arranged between the second station and the third station, and when the second piston moves from the second station to the third station, the volume of the liquid storage space is reduced; and, the length of the second piston satisfies the following requirements: and in the process of moving the second piston from the first station to the third station, the second piston always seals the fluid outlet of the fluid channel.
In one embodiment, a first through hole and a second through hole are formed in the pipe walls of the first pipe body and the second pipe body respectively, and when the second pipe body is combined with the first pipe body, the first through hole and the second through hole are connected with each other and form the fluid channel.
In one embodiment, the nucleic acid extraction apparatus further comprises a first fluid valve, so that the first fluid valve is connected to the first tube and/or the second tube and is at least used to control the communication or blocking of the fluid channel.
In one embodiment, the nucleic acid isolation apparatus further comprises a second fluid valve coupled to the second tube and configured to control at least one of the communication or blocking of a small fluid path coupled between the fluid storage space and the conical tube structure, or the communication or blocking of a connection path between the conical tube structure and a space outside the second tube.
Wherein the first and second fluid valves may be various types of fluid valves known in the art. The first fluid valve may be a one-way valve, so that gas, liquid, etc. flow in one direction from the first liquid storage cavity to the second liquid storage cavity or the liquid storage space through the fluid channel, and the phenomenon that aerosol, etc. in the liquid storage space enters the first liquid storage cavity in a countercurrent manner is avoided. Or, the first fluid valve may also be a common valve with a conducting/blocking function, which maintains a long-closing state, especially in the process of transporting the nucleic acid extraction device, so as to avoid the problem that the second piston is moved along with external vibration to cause the fluid channel to be erroneously conducted so as to generate liquid leakage.
Preferably, the second fluid valve may also adopt a one-way valve (or a check valve), so as to enable the liquid and the like to flow unidirectionally from the liquid storage space to the conical tube structure through the narrow liquid channel and/or enable the liquid to flow unidirectionally from the conical tube structure to the outside of the second tube body, so as to avoid the problems of pollution and the like caused by that external gas or aerosol and the like flow countercurrent from the outside space of the second tube body into the conical tube structure and/or the liquid storage space.
In one embodiment, the second pipe body is disposed at one side of the first pipe body and can be clamped with the first pipe body.
In one embodiment, the nucleic acid isolation apparatus further comprises a first piston push rod and a second piston push rod, one end of the first piston push rod is arranged outside the first pipe body, the other end of the first piston push rod is connected with the first piston, one end of the second piston push rod is arranged outside the second pipe body, and the other end of the second piston push rod is connected with the second piston.
In one embodiment, the liquid nucleic acid extraction system is a mixture comprising at least a biological sample to be detected, a lysate, and a nucleic acid extraction proteolytic enzyme.
Wherein the lysate can be strong acid or strong alkaline lysate, or neutral or weak alkaline strong salt lysate with pH value of 7-8. Illustratively, the liquid nucleic acid extraction system may comprise ionic or nonionic surfactants (e.g., ethylphenyl polyethylene glycol, etc.), proteinase K, buffer salts (e.g., tris buffer salts), etc., and have a pH of 7.7-8. If strong acid or strong alkaline lysate is adopted, high-temperature heating and the like are not needed in the process of extracting nucleic acid, and the cracking time is controlled within 20 min. If neutral or weak alkaline strong salt lysate is used, the process of extracting nucleic acid generally needs to be heated to boiling, and the lysis time is controlled to be about 10 min.
Another aspect of the present invention provides a method of using the nucleic acid extraction apparatus, comprising:
s1, placing a second piston into a second liquid storage cavity, and enabling the second piston to move to a first station so as to block a fluid channel;
s2, adding a liquid nucleic acid extraction system into the first liquid storage cavity, and sealing and combining the sealing mechanism with the first pipe body to isolate the liquid nucleic acid extraction system from the external environment;
s3, moving the second piston to a second station along the first direction so as to conduct the fluid channel, and separating a liquid storage space isolated from the external environment in the second liquid storage cavity;
s4, applying pressure to the liquid nucleic acid extraction system by using a first piston and/or forming negative pressure in the liquid storage space so that at least part of liquid in the liquid nucleic acid extraction system is injected into the liquid storage space through a fluid channel;
and S5, moving the second piston along a second direction, so that the second piston applies pressure to the liquid contained in the liquid storage space, and at least part of the liquid enters the conical tube structure, wherein the first direction is opposite to the second direction.
In yet another aspect, the invention provides the use of the nucleic acid extraction device for performing nucleic acid detection.
For example, in one embodiment of the invention, a method of nucleic acid detection is provided, the method being for non-therapeutic and diagnostic purposes, and the method comprising:
extracting nucleic acid from a biological sample to be detected by using the nucleic acid extraction device;
amplifying and detecting the extracted nucleic acid.
Compared with the prior art, the technical scheme of the invention has at least the following beneficial effects:
(1) The nucleic acid extraction device has the advantages of simple structure, easy manufacture and assembly and low cost.
(2) When the nucleic acid extraction device is used for extracting nucleic acid, the operation is simple and convenient, the control is easy, the quantitative distribution of micro liter level samples can be realized quickly and accurately, the whole nucleic acid extraction process is carried out in a closed environment, and the problems of interference of the external environment to the nucleic acid extraction process, false positive in Du Juehou continuous nucleic acid detection process and the like can be avoided.
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the invention.
FIG. 1 is a schematic diagram showing the structure of a nucleic acid isolation apparatus according to example 1 of the present invention;
FIG. 2 is a flow chart of a nucleic acid extraction method based on the nucleic acid extraction apparatus shown in FIG. 1;
FIG. 3 is a schematic diagram showing the structure of a nucleic acid isolation apparatus according to example 2 of the present invention;
FIG. 4 is a schematic diagram showing the structure of a nucleic acid isolation apparatus according to example 3 of the present invention;
FIG. 5 is a schematic diagram showing the structure of a nucleic acid isolation apparatus according to example 4 of the present invention.
Detailed Description
Other advantages and effects of the present invention will become apparent to those skilled in the art from the following disclosure, which describes the embodiments of the present invention with reference to specific examples. The invention may be practiced or carried out in other embodiments that depart from the specific details, and the details of the present description may be modified or varied from the spirit and scope of the present invention.
As described in detail in the embodiments of the present invention, the cross-sectional view of the device structure is not partially enlarged to a general scale for convenience of explanation, and the schematic drawings are only examples, which should not limit the scope of the present invention. In addition, the three-dimensional dimensions of length, width and depth should be included in actual fabrication.
For ease of description, spatially relative terms such as "under", "below", "beneath", "above", "upper" and the like may be used herein to describe one element or feature's relationship to another element or feature as illustrated in the figures. It will be understood that these spatially relative terms are intended to encompass other orientations of the device in use or operation in addition to the orientation depicted in the figures. Furthermore, when a layer is referred to as being "between" two layers, it can be the only layer between the two layers or one or more intervening layers may also be present.
In the context of the present invention, a structure described as a first feature being "on" a second feature may include embodiments where the first and second features are formed in direct contact, as well as embodiments where additional features are formed between the first and second features, such that the first and second features may not be in direct contact.
It should be noted that, the illustrations provided in the present embodiment merely illustrate the basic concept of the present invention by way of illustration, and only the components related to the present invention are shown in the drawings rather than the number, shape and size of the components in actual implementation, and the form, number and proportion of each component in actual implementation may be arbitrarily changed, and the layout of the components may be more complex.
Example 1
Referring to fig. 1 and 2, the nucleic acid extracting apparatus provided in the present embodiment includes a first tube 1, a second tube 2, a first piston mechanism 3, a second piston mechanism 4, and so on.
The inner cavities of the first and second tubes 1 and 2 are used for storing liquid nucleic acid extraction system and nucleic acid extraction solution separated from the liquid nucleic acid extraction system, respectively, and thus can be defined as a first liquid storage cavity 11 and a second liquid storage cavity 21, respectively, and a conical tube structure 22 is formed at the bottom end of the second tube 2, and is communicated with the second liquid storage cavity 21 by forming a narrow liquid channel 25 in the second tube 2. The diameter of the small liquid passage 25 may be in the order of millimeters or micrometers, depending on the actual needs. For example, if a liquid with micro-upgrades to be dispensed is desired, the diameter of the small liquid channel is preferably in the order of microns.
The first piston mechanism 3 mainly comprises a first piston 31 and a first piston push rod 32, the second piston mechanism 4 mainly comprises a second piston 41 and a second piston push rod 42, and the first piston 31 and the second piston 41 respectively enter the first liquid storage cavity 11 and the second liquid storage cavity 21 through an opening 12 at the upper end of the first pipe body 1 and an opening 23 at the upper end of the second pipe body 2 and can freely move in the first liquid storage cavity 11 and the second liquid storage cavity 21 along the vertical direction. Obviously, the first piston 31 and the second piston 41 are in sliding sealing fit with the inner walls of the first liquid storage cavity 11 and the second liquid storage cavity 21 respectively. One end of the first piston push rod 32 is fixedly connected with the first piston 31, and the other end is exposed outside the first pipe body 1. Similarly, one end of the second piston push rod 42 is fixedly connected to the second piston 41, and the other end is exposed outside the second pipe body 2. When the liquid is stored in the first liquid storage cavity 11 and the second liquid storage cavity 21 and the first piston 31 and the second piston 41 are assembled into the first liquid storage cavity 11 and the second liquid storage cavity 21, the first piston 31 and the second piston 41 can seal and package the liquid, and the contact between the liquid and the external environment is avoided, so that the pollution to the liquid in the first liquid storage cavity 11 and the second liquid storage cavity 21 caused by external aerosol and the like is prevented.
Meanwhile, a through hole is formed on the walls of the first tube body 1 and the second tube body 2 respectively, when the first tube body 1 is clamped and combined with the second tube body 2, the two through holes are connected in a sealing way to form a fluid channel 5, the fluid channel 5 is used for communicating the first liquid storage cavity 11 with the second liquid storage cavity 21, namely, a fluid inlet and a fluid outlet of the fluid channel 5 are respectively positioned on the inner walls of the first tube body 1 and the second tube body 2 and are respectively exposed in the first liquid storage cavity 11 and the second liquid storage cavity 21. The diameter of the fluid channel 5 may be in the order of millimeters or micrometers, depending on the actual needs. The clamping mechanism for combining the first pipe body 1 and the second pipe body 2 may be various, for example, refer to the clamping structure in CN114989971B, but is not limited thereto. The aperture of the two aforementioned through holes may be according to practical requirements, for example, in the order of millimeters or micrometers, preferably micrometers, so as to more precisely control the flow rate of the liquid transferred from the first liquid storage chamber 11 into the second liquid storage chamber 21, and in particular to precisely control the quantitative transfer of the micro-scaled liquid.
In some cases, the first tube body 1 and the second tube body 2 may be fixedly connected by an adhesive or the like, and the two through holes may be sealed and connected to form a fluid channel. Alternatively, the first tube 1 and the second tube 2 may be integrally formed by injection molding or other processes.
Preferably, the first tube 1 has a certain depth-to-diameter ratio, and the fluid inlet of the fluid channel 5 is disposed on the inner wall of the upper portion or the middle portion of the first liquid storage chamber 11, so that after the biological sample to be detected, the lysis solution, and the nucleic acid extracting protease are fully mixed in the first liquid storage chamber 11 to form a liquid nucleic acid extraction system, and the liquid nucleic acid extraction system is layered by fully standing or centrifuging, the supernatant containing fewer impurities (i.e., the nucleic acid extraction solution) can be introduced into the second liquid storage chamber 21 through the fluid channel 5, and the lower liquid containing more impurities is prevented from being introduced into the second liquid storage chamber 21. In some cases, the fluid inlet of the fluid channel 5 may be provided on the inner wall of the upper part of the first reservoir 11, so as to avoid, on the one hand, the lower liquid entering the second reservoir 21 and, on the other hand, the supernatant liquid being transferred into the second reservoir 21 as much as possible.
Further, the second piston 41 is movable at least between the first station a1 and the second station a2 in the second liquid storage chamber 21, and is capable of dividing a liquid storage space isolated from the external environment in the second liquid storage chamber 21. Wherein, at the first station, the second piston 41 seals off the fluid outlet of the fluid channel 5, so that the fluid channel 5 is blocked, and at the second station, the fluid channel 5 is communicated and the liquid storage space is communicated with the first liquid storage cavity 11. In this embodiment, the second station a2 is located above the first station a 1.
In this embodiment, the first tube 1 and the second tube 2 may be made of plastic such as polyethylene, and the first piston 31 and the second piston 41 may be made of rubber, but are not limited thereto.
With continued reference to fig. 2, a method for extracting nucleic acid from a biological sample using the nucleic acid extraction apparatus comprises the steps of:
s1, the first pipe body 1 is clamped with the second pipe body 2, the second piston 41 is placed into the second liquid storage cavity 21, the second piston 41 moves down to the first station to block the fluid channel 5, and at the moment, the second piston 41 also separates a liquid storage space 24 isolated from the external environment in the second liquid storage cavity 21. Obviously, the liquid storage space 24 is a space with a variable volume, and the volume size of the space varies with the position of the second piston 41 in the second liquid storage cavity 21.
S2, adding at least lysate, biological sample to be detected, nucleic acid extraction proteolytic enzyme and the like into the first liquid storage cavity 11 to form a mixture, arranging the first piston 31 into the first liquid storage cavity 11 to isolate the mixture from the external environment, fully and uniformly mixing the mixture through oscillation or other modes to form a liquid nucleic acid extraction system 6, and fully standing to enable the liquid nucleic acid extraction system to be layered, wherein solids are settled at the lower part, and the extracted nucleic acid is mainly distributed in supernatant. The lysate may be stored in the first liquid storage chamber 11 in advance, and the biological sample to be detected, the nucleic acid extraction proteolytic enzyme and the like may be added into the first liquid storage chamber 11 when nucleic acid extraction is performed, so as to be mixed with the lysate to form a liquid nucleic acid extraction system.
S3, the second piston 41 is moved upwards (i.e. in the first direction) to the second station, so that the fluid channel 5 is communicated. At this time, the volume of the liquid storage space 24 becomes large, and a certain negative pressure is also generated therein. The first piston 31 is then moved downward, so that the first piston 31 applies pressure to the liquid nucleic acid extraction system, whereby at least part of the supernatant liquid therein is injected into the liquid storage space 24 via the fluid channel 5. In some cases, if less supernatant is required to be diverted into the liquid storage space 24, the first piston mechanism 3 may not be operated, and the supernatant may be automatically introduced into the liquid storage space 24 only by using the negative pressure in the liquid storage space 24.
And S4, the second piston 41 is moved downwards (namely along the second direction), so that the second piston 41 applies pressure to the supernatant liquid contained in the liquid storage space 24, and at least part of the supernatant liquid enters the conical tube structure 22, and the liquid separation operation is completed.
In fig. 2, the dashed arrows show the movement directions of the first piston, the second piston, and the solid arrows show the liquid flow directions.
The positions of the first piston 31 and the second piston 41 in the first pipe body 1 and the second pipe body 2 are simply adjusted, so that the nucleic acid extraction and quantitative liquid separation can be conveniently, quickly and accurately realized, the pollution of pollutants in the external environment to substances in the nucleic acid extraction device can be effectively prevented, and the leakage of the substances in the nucleic acid extraction device can be avoided in the process of extracting and separating the nucleic acid.
Example 2
Referring to fig. 3, the structure of a nucleic acid extracting apparatus according to this embodiment is substantially the same as that of embodiment 1, except that:
a first limiting mechanism 13 is further arranged in the first pipe body 1, and the first limiting mechanism 13 is positioned at the upper part of the first liquid storage cavity 11 and above the fluid inlet of the fluid channel 5. By using the first limiting mechanism 13, the movement range of the first piston 31 in the first liquid storage cavity 11 can be limited in a proper range, and the problem that the downward movement stroke is overlarge and the fluid inlet of the fluid channel 5 is exposed to the external environment can be prevented.
Optionally, a second limiting mechanism 26 may be further disposed in the second tube body 2, where the second limiting mechanism 26 includes a first portion 261 and a second portion 262 located in the second liquid storage cavity 21, and the first portion 261 and the second portion 262 are located above and below the first station, respectively, so as to limit the movement range of the second piston 41 between the second station and a third station (not shown in the figure). The third station is located below the first station, and when the second piston 41 moves to the third station, the second piston 41 contacts with the limiting mechanism and seals the fluid outlet of the fluid channel 5. By providing the second stopper mechanism 26, it is possible to prevent the second piston 41 from being moved upward or downward by an excessive stroke due to improper operation of the worker, thereby disengaging the second piston 41 from the second tube 2 or exposing the fluid outlet of the fluid passage 5 to the outside of the liquid storage space 24, eventually resulting in a situation in which the supernatant in the first tube 1 leaks into the second liquid storage chamber 21 and is exposed to the outside environment.
Preferably, the length of the second piston 41 also satisfies the following requirements: during the movement of the second piston 41 from the first station to the third station, the second piston 41 always seals the fluid outlet of the fluid channel 5, thereby better eliminating the problem of leakage of supernatant fluid in the first tube 1 through the fluid channel 5 and exposure to the external environment.
In the present embodiment, the first limiting mechanism 13 and the second limiting mechanism 26 may be a step structure integrally formed on the inner wall of the first pipe body 1 or the second pipe body 2, or may be one or more protruding portions fixed on the inner wall of the first pipe body 1 or the second pipe body 2, for example, a continuous annular protruding portion or two or more point-like protruding portions spaced apart from each other, etc., and are not limited thereto.
Example 3
Referring to fig. 4, the structure of a nucleic acid extraction apparatus provided in this embodiment is substantially the same as that of embodiment 1 or embodiment 2, except that: a first fluid valve 7 is also provided at the fluid channel 5. The first fluid valve 7 may be a one-way valve, so that fluid may flow only in one direction from the first reservoir to the second reservoir or a reservoir space therein, preventing reverse flow of gas or the like in the second reservoir or the reservoir space into the first reservoir. Alternatively, the first fluid valve 7 may be a common valve with blocking/conducting function, which keeps a blocking state when the nucleic acid extracting apparatus is transported or stored, so as to prevent the second piston from being touched by a worker by mistake or from being moved by external vibration, etc., so that the fluid channel is erroneously conducted to cause the fluid in the first fluid storage chamber to leak into the second fluid storage chamber.
Optionally, a second fluid valve 8 is further disposed at the conical tube structure 22, and the second fluid valve 8 may adopt a one-way valve or a non-return valve, so that the liquid may flow unidirectionally from the conical tube structure to the outside of the second tube, so as to avoid the problem of pollution caused by that external gas or aerosol enters the conical tube structure from the space outside the second tube in a countercurrent manner.
Example 4
Referring to fig. 5, the structure of a nucleic acid extracting apparatus according to the present embodiment is substantially the same as that of embodiment 1, except that: a sealing cap 14 is also sealingly coupled to the opening 12 of the first tubular body 1. A sealing cap 27 is also sealingly coupled to the opening 23 of the second tubular body 2. The first piston push rod 32 passes through a through hole in the sealing cover 14 and is in sliding sealing engagement with the sealing cover 14. The second piston push rod 42 passes through the through hole in the sealing cover 27 and is in sliding sealing fit with the sealing cover 27. The sealing of the inner cavities of the first pipe body 1 and the second pipe body 2 can be better realized by the sealing cover 14 and the sealing cover 27, and the problem that the first piston 31 and the second piston 41 are separated from the first pipe body 1 and the second pipe body 2 due to misoperation of staff can be effectively prevented.
The above embodiments are merely illustrative of the principles of the present invention and its effectiveness, and are not intended to limit the invention. Modifications and variations may be made to the above-described embodiments by those skilled in the art without departing from the spirit and scope of the invention. Accordingly, it is intended that all equivalent modifications and variations of the invention be covered by the claims, which are within the ordinary skill of the art, be within the spirit and scope of the present disclosure.
Claims (10)
1. A method of using a nucleic acid extraction device, the nucleic acid extraction device comprising:
the first tube body is internally provided with a first liquid storage cavity for accommodating a liquid nucleic acid extraction system;
the second pipe body is internally provided with a second liquid storage cavity, a conical pipe structure is formed at the bottom end of the second pipe body, and the second liquid storage cavity is communicated with the conical pipe structure;
the fluid inlet and the fluid outlet of the fluid channel are respectively exposed in the first liquid storage cavity and the second liquid storage cavity, wherein the fluid inlet is formed on the inner wall of the upper part of the first liquid storage cavity and is lower than the liquid level of the liquid nucleic acid extraction system;
the sealing mechanism is in sealing fit with the first pipe body and comprises a first piston, the first piston is movably arranged in the first liquid storage cavity and is at least used for isolating the liquid nucleic acid extraction system from the external environment, and an opening part at least allowing external substances and/or the first piston to enter the first liquid storage cavity is arranged on the first pipe body;
the second piston can move between the first station and the second station at least in the second liquid storage cavity and is at least used for separating a liquid storage space isolated from the external environment in the second liquid storage cavity, the liquid storage space is used for receiving liquid input through the fluid channel, and the liquid storage space is communicated with the conical pipe structure;
when the second piston is in the first station, the second piston blocks the fluid outlet of the fluid channel to block the fluid channel, and when the second piston is in the second station, the second piston moves away from the fluid outlet of the fluid channel to conduct the fluid channel, and the second station is positioned above the first station;
wherein the liquid nucleic acid extraction system is a mixture at least comprising a biological sample to be detected, a lysate and a nucleic acid extraction proteolytic enzyme;
the using method comprises the following steps:
s1, placing the second piston into a second liquid storage cavity, and enabling the second piston to move to the first station so as to block a fluid channel;
s2, adding a liquid nucleic acid extraction system into the first liquid storage cavity, and placing the first piston into the first liquid storage cavity so as to isolate the liquid nucleic acid extraction system from the external environment;
s3, the second piston is moved upwards to the second station so as to conduct a fluid channel, a liquid storage space isolated from the external environment is separated in the second liquid storage cavity, and negative pressure is generated in the liquid storage space;
s4, utilizing the action of the negative pressure and/or applying pressure to a liquid nucleic acid extraction system by the first piston, so that at least part of liquid in the liquid nucleic acid extraction system is injected into the liquid storage space through the fluid channel;
and S5, the second piston is moved downwards, so that the second piston applies pressure to the liquid contained in the liquid storage space, and at least part of the liquid enters the conical tube structure.
2. The method of use according to claim 1, wherein: the sealing mechanism comprises a sealing cover, the sealing cover is combined with an opening part on the first pipe body in a sealing way, and the opening part is communicated with the first liquid storage cavity.
3. The method of use according to claim 1, wherein: the first pipe body is internally provided with a first limiting mechanism, and the first limiting mechanism is used for limiting the moving range of the first piston to be above the fluid inlet of the fluid channel.
4. The method of use according to claim 1, wherein: the ratio of the depth to the diameter of the first liquid storage cavity is more than 1.
5. The method of use according to claim 1, wherein: the second pipe body is internally provided with a second limiting mechanism which is used for limiting the movable range of the second piston in the second liquid storage cavity between a second station and a third station, the first station is arranged between the second station and the third station, and when the second piston moves from the second station to the third station, the volume of the liquid storage space is reduced; and, the length of the second piston satisfies the following requirements: and in the process of moving the second piston from the first station to the third station, the second piston always seals the fluid outlet of the fluid channel.
6. The method of use according to claim 1, wherein: and when the second pipe body is combined with the first pipe body, the first through hole and the second through hole are connected with each other and form the fluid channel.
7. The method of use of claim 1, further comprising:
a first fluid valve for unidirectional flow of gas or liquid from the first reservoir to the second reservoir or the reservoir space via the fluid passage;
and/or a second fluid valve for unidirectional flow of liquid from the liquid storage space to the conical tube structure via a narrow liquid passage or unidirectional flow of liquid from the conical tube structure to the outside of the second tube body;
the first fluid valve and the second fluid valve are both check valves.
8. The method of use according to claim 1, wherein: the second pipe body is arranged on one side of the first pipe body and is clamped with the first pipe body.
9. The method of claim 1, further comprising a first piston push rod and a second piston push rod, wherein one end of the first piston push rod is arranged outside the first pipe body, the other end of the first piston push rod is connected with the first piston, one end of the second piston push rod is arranged outside the second pipe body, and the other end of the second piston push rod is connected with the second piston.
10. A method of nucleic acid detection for non-therapeutic and diagnostic purposes, comprising: extracting nucleic acid from a biological sample to be detected by the use of the nucleic acid extraction device according to any one of claims 1 to 9, and amplifying and detecting the extracted nucleic acid.
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| CN202311311373.6A CN117070314B (en) | 2023-10-11 | 2023-10-11 | Nucleic acid extraction device, method of using the same, and application thereof |
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| CN202311311373.6A CN117070314B (en) | 2023-10-11 | 2023-10-11 | Nucleic acid extraction device, method of using the same, and application thereof |
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| CN108660074A (en) * | 2018-05-28 | 2018-10-16 | 北京贝泰科技有限公司 | Nucleic acid extraction PCR amplification detects integrative solution |
| CN113278512A (en) * | 2021-05-31 | 2021-08-20 | 广州源起健康科技有限公司 | Integrated sample treatment consumable for automatic nucleic acid detection |
| CN114317208A (en) * | 2021-12-21 | 2022-04-12 | 江苏宏微特斯生物科技有限公司 | Nucleic acid extraction kit |
| CN217265711U (en) * | 2021-12-27 | 2022-08-23 | 宁波爱基因科技有限公司 | Nucleic acid extraction tube structure and bar magnet cover |
| CN114989971A (en) * | 2022-08-08 | 2022-09-02 | 上海科源电子科技有限公司 | Card box device for nucleic acid extraction and automatic liquid separation and analysis method thereof |
| CN116606849A (en) * | 2023-05-05 | 2023-08-18 | 浙江大学医学院附属第一医院 | Method and matched device for rapidly extracting nucleic acid from biological sample by one-step method |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108660074A (en) * | 2018-05-28 | 2018-10-16 | 北京贝泰科技有限公司 | Nucleic acid extraction PCR amplification detects integrative solution |
| CN113278512A (en) * | 2021-05-31 | 2021-08-20 | 广州源起健康科技有限公司 | Integrated sample treatment consumable for automatic nucleic acid detection |
| CN114317208A (en) * | 2021-12-21 | 2022-04-12 | 江苏宏微特斯生物科技有限公司 | Nucleic acid extraction kit |
| CN217265711U (en) * | 2021-12-27 | 2022-08-23 | 宁波爱基因科技有限公司 | Nucleic acid extraction tube structure and bar magnet cover |
| CN114989971A (en) * | 2022-08-08 | 2022-09-02 | 上海科源电子科技有限公司 | Card box device for nucleic acid extraction and automatic liquid separation and analysis method thereof |
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| CN117070314A (en) | 2023-11-17 |
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