CN117363451A - Nucleic acid extraction device and method - Google Patents
Nucleic acid extraction device and method Download PDFInfo
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- CN117363451A CN117363451A CN202311092669.3A CN202311092669A CN117363451A CN 117363451 A CN117363451 A CN 117363451A CN 202311092669 A CN202311092669 A CN 202311092669A CN 117363451 A CN117363451 A CN 117363451A
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- 238000000034 method Methods 0.000 title abstract description 9
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- 238000004140 cleaning Methods 0.000 claims abstract description 27
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- 230000001681 protective effect Effects 0.000 claims description 14
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- 238000009434 installation Methods 0.000 claims description 10
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- 238000011901 isothermal amplification Methods 0.000 claims description 8
- 239000000758 substrate Substances 0.000 claims description 7
- 238000012408 PCR amplification Methods 0.000 claims description 6
- 239000007853 buffer solution Substances 0.000 claims description 6
- 238000006243 chemical reaction Methods 0.000 claims description 6
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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
- C12N15/1017—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by filtration, e.g. using filters, frits, membranes
Abstract
The application provides a nucleic acid extraction device and a method, relates to the technical field of nucleic acid extraction, and is used for solving the technical problem of poor applicability of the current nucleic acid extraction device, wherein the nucleic acid extraction device comprises a main body, a nucleic acid extraction membrane, a puncture needle assembly and a sample collector; a drainage cavity and at least three independent accommodating cavities communicated with the drainage cavity are arranged in the main body, and an opening at the bottom of each accommodating cavity is sealed by a first sealing film; a drainage channel is arranged between the sample collector and the drainage cavity, and openings at two ends of the drainage channel are respectively sealed by a nucleic acid extraction film and a second sealing film; the three accommodating cavities are respectively filled with nucleic acid lysate, cleaning liquid and nucleic acid eluent, the lancets of the lancet assembly are inserted into the accommodating cavities, and the tips of the lancets can puncture the first sealing film; and the first sealing film, the nucleic acid extraction film and the second sealing film can be sequentially punctured by a puncture needle in the accommodating cavity containing the nucleic acid eluent. The nucleic acid extraction device provided by the application extracts and purifies nucleic acid.
Description
Technical Field
The present application relates to the field of nucleic acid extraction technology, and in particular, to a nucleic acid extraction apparatus and method.
Background
Nucleic acid is a biological macromolecular compound polymerized by a plurality of polynucleotides, is a carrier of organism genetic information and is one of the most basic substances of life; therefore, whether the research of nucleic acid sequence and structure is carried out or the research of gene functions, expression and the like is carried out, the nucleic acid needs to be separated and purified firstly; at the same time, the quality of the nucleic acid extract product will also be directly related to the success or failure of the subsequent experiments. Therefore, the extraction and purification of nucleic acids are particularly important in molecular biology experimental techniques.
The current methods for extracting and purifying nucleic acid are various, and can relate to various treatment modes such as biology, chemistry, machinery and the like. However, whatever the manner of treatment, it basically includes the major steps of releasing the cell contents, separating and purifying the nucleic acid, precipitating or adsorbing the nucleic acid, and almost every step requires the cooperation of different reagents, and the sample reacts differently between the various reagents, so that the final separation and purification of the nucleic acid is achieved.
However, the extraction and purification of nucleic acids are performed by professionals using nucleic acid extraction equipment in biological laboratories, so that the reagents necessary for nucleic acid extraction are packaged, integrated, and integrated in a portable extraction device, respectively, which not only simplifies the nucleic acid extraction and purification, but also improves the applicability thereof.
Disclosure of Invention
In view of the above, embodiments of the present application provide a nucleic acid extraction apparatus and method, which can simplify the nucleic acid extraction and purification process and improve the applicability thereof.
In order to achieve the above purpose, the embodiment of the present application provides the following technical solutions:
a first aspect of embodiments of the present application provides a nucleic acid extraction device comprising a body, a nucleic acid extraction membrane, a first sealing membrane, a second sealing membrane, a spike assembly, and a sample collector; the drainage device comprises a main body, at least three independent accommodating cavities and a sealing membrane, wherein the main body is internally provided with a drainage cavity and at least three independent accommodating cavities, each accommodating cavity is arranged above the drainage cavity along a first direction, the bottom of each accommodating cavity is provided with a first opening communicated with the drainage cavity, and the first opening is sealed by the sealing membrane; the sample collector is detachably arranged below the drainage cavity, a drainage channel is arranged between the sample collector and the drainage cavity, the drainage channel is provided with a second opening communicated with the sample collector, and the second opening is sealed by the second sealing film; the drainage channel has a third opening in communication with the drainage lumen, the third opening being sealed by the nucleic acid extraction membrane; the three accommodating cavities are respectively filled with nucleic acid lysate, cleaning liquid and nucleic acid eluent, the puncture needle assembly comprises puncture needles corresponding to the accommodating cavities, and the puncture needles are inserted into the accommodating cavities and kept sealed with the accommodating cavities; the lancet is configured to press the lancet in a first direction, a tip of which pierces the first sealing membrane; and the puncture needles corresponding to the accommodation cavity containing the nucleic acid eluent can puncture the first sealing membrane, the nucleic acid extraction membrane and the second sealing membrane in sequence.
In an alternative embodiment, the nucleic acid extraction apparatus further comprises a waste collection chamber and a waste diversion channel; the waste liquid collecting cavity is communicated with the first end of the waste liquid diversion channel, and the second end of the waste liquid diversion channel extends to the drainage channel and is communicated with a space between the nucleic acid extraction membrane and the second sealing membrane.
In an alternative embodiment, a first profile and a second profile are arranged in the main body at intervals along a first direction; the first section bar and the second section bar are respectively connected with the inner wall of the main body in a sealing way, and the first section bar is positioned above the second section bar, and the drainage cavity is formed between the first section bar and the second section bar; and the drainage channel penetrates through the second section bar; each accommodating cavity is arranged between the first section bar and the top of the main body, and the first opening penetrates through the first section bar.
In an alternative embodiment, the opening end face of the third opening is located at a level with the surface of the second profile surrounding it; or the opening end face of the third opening is positioned at a height lower than the surface of the second section bar around the opening end face.
In an alternative embodiment, the second profile is arranged obliquely in the main body so as to have a guide surface; the third opening is positioned at the joint of the lower side of the flow guiding surface and the inner wall of the main body.
In an alternative embodiment, the device further comprises a protective shell; the protective housing detachably connects in the below of main part to cover establishes the sample collector.
In an alternative embodiment, the device further comprises a waste collection tube detachably mounted to the body, the waste collection tube having the waste collection chamber; the first section bar is provided with a first installation position and a second installation position, the sample collector is detachably installed at the first installation position, and the waste liquid collecting pipe is installed at the second installation position.
In an alternative embodiment, the sample collector contains a PCR amplification reaction solution or a nucleic acid isothermal amplification detection solution or a nucleic acid preservation solution; the protective shell is configured as a transparent shell, and the sample collector is configured as a transparent sample collection tube.
In an alternative embodiment, the PCR amplification reaction solution, the nucleic acid isothermal amplification detection solution each comprise an enzyme, a substrate, and a buffer; the sample collector further comprises a third sealing membrane positioned below the second sealing membrane, and a storage cavity is formed between the third sealing membrane and the second sealing membrane; the enzyme and the substrate are frozen at the bottom of the sample collector, the buffer solution is sealed in the storage cavity, and the puncture needle corresponding to the containing cavity containing the nucleic acid eluent can puncture the third sealing film.
In an alternative embodiment, four independent accommodating cavities are arranged in the main body, and the four accommodating cavities are a first accommodating cavity, a second accommodating cavity, a third accommodating cavity and a fourth accommodating cavity respectively; the first accommodating cavity is used for accommodating nucleic acid lysate, the second accommodating cavity and the third accommodating cavity are used for accommodating cleaning liquid, and the fourth accommodating cavity is used for accommodating nucleic acid eluent; the puncture needle assembly comprises a first puncture needle, a second puncture needle, a third puncture needle and a fourth puncture needle, and the first puncture needle is inserted in the first accommodating cavity in a sealing way and can puncture the first sealing film; the second puncture needle is inserted into the second accommodating cavity in a sealing way and can puncture the first sealing film; the third puncture needle is inserted into the third accommodating cavity in a sealing way and can puncture the first sealing film; the fourth puncture needle is inserted in the fourth accommodating cavity in a sealing manner and can puncture the first sealing membrane, the nucleic acid extraction membrane and the second sealing membrane.
In an alternative embodiment, the device further comprises a locking structure; the locking structure comprises a locking ring and a locking groove matched with the locking ring, and the locking groove is arranged at a position, close to the locking ring, of the puncture needle assembly; the side wall of the main body is provided with an inserting port, and the locking ring is inserted into the main body through the inserting port and can lock the first puncture needle, the second puncture needle, the third puncture needle and the fourth puncture needle.
In an alternative embodiment, the nucleic acid extraction membrane is configured as a silica gel membrane or a cellulose membrane.
A second aspect of embodiments of the present application provides a nucleic acid extraction method based on the nucleic acid extraction apparatus described in the first aspect, comprising the steps of:
adding a sample to be detected into a containing cavity containing nucleic acid lysate for nucleic acid lysis;
after the lysis, sequentially pressing a puncture needle with a holding cavity for nucleic acid lysate and a puncture needle with a holding cavity for cleaning liquid to enrich and clean nucleic acid on the nucleic acid extraction membrane;
a spike having a containment chamber for nucleic acid eluting solution is pressed, the spike puncturing the first sealing membrane, the nucleic acid extraction membrane and the second sealing membrane to collect nucleic acid to the sample collector.
Compared with the related art, the nucleic acid extraction device and the extraction method provided by the embodiment of the application have the following advantages:
according to the nucleic acid extraction device and the nucleic acid extraction method, at least three mutually independent accommodating cavities are integrated in the nucleic acid extraction device, the drainage cavities are communicated with the accommodating cavities, the sample collector is communicated with the drainage cavities, the nucleic acid lysate, the cleaning liquid and the nucleic acid eluent are respectively contained in each accommodating cavity, the corresponding needles are respectively arranged in each accommodating cavity, the needles can be sequentially pushed to pierce the first sealing film at the bottom of the accommodating cavity according to the sequence, so that the nucleic acid lysate and the cleaning liquid sequentially flow into the drainage cavities through the nucleic acid eluent, and nucleic acid adsorbed on the nucleic acid extraction film is pushed to the sample collector after being cleaned and eluted under the action of the needles.
When the nucleic acid extraction device provided by the embodiment of the application is used for extracting nucleic acid, a sample is placed in a containing cavity with a nucleic acid lysate for lysis; after the lysis, the puncture needle in the holding cavity containing the nucleic acid lysate is pressed, so that the puncture needle punctures the first sealing membrane, the nucleic acid lysate with the nucleic acid flows into the drainage cavity and is drained to the nucleic acid extraction membrane, and the nucleic acid is specifically captured by the nucleic acid extraction membrane and is adsorbed on the nucleic acid extraction membrane.
Then, pressing a puncture needle in the accommodating cavity containing the cleaning liquid, and puncturing the first sealing film by the puncture needle so as to enable the cleaning liquid to flow into the drainage cavity and clean impurities on the nucleic acid extraction film; finally, the puncture needle of the containing cavity containing the nucleic acid eluent is pressed, so that the puncture needle sequentially punctures the first sealing film, the nucleic acid extraction film and the second sealing film, the nucleic acid eluent enters the drainage cavity, nucleic acid adsorbed on the nucleic acid extraction film is eluted, and a nucleic acid sample is further collected into the sample collector, so that the cracking, cleaning, extracting and enriching of the nucleic acid sample are completed.
Compared with the scheme that the extraction and purification of nucleic acid in the related art are carried out by professionals by utilizing nucleic acid extraction equipment in a biological laboratory, the nucleic acid extraction device provided by the embodiment of the application is integrated with a plurality of accommodating cavities and drainage cavities, and a sample collector, wherein each accommodating cavity respectively accommodates nucleic acid lysate, cleaning liquid and nucleic acid eluent, namely reagents necessary for extracting and purifying nucleic acid can be respectively packaged, integrated and integrated in a portable extraction device so as to realize the extraction and purification of nucleic acid.
Furthermore, the nucleic acid extraction device has the characteristics of simple structure, low cost, simplified operation steps, realization of nucleic acid extraction and purification, small volume, light weight and portability, and improves the applicability and flexibility.
In addition to the technical problems, features, and advantages of the technical solutions described above, the nucleic acid extraction apparatus and method according to the embodiments of the present disclosure, other technical problems, features, and advantages of the technical features that may be solved by the embodiments of the present disclosure, further detailed description will be given in the detailed description of the embodiments.
Drawings
In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, a brief description will be given below of the drawings that are needed in the embodiments or the prior art descriptions, and it is obvious that the drawings in the following description are some embodiments of the present application, and that other drawings can be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a schematic diagram showing the overall structure of a nucleic acid isolation apparatus according to an embodiment of the present application;
fig. 2 is a schematic diagram showing an internal structure of a nucleic acid isolation apparatus according to an embodiment of the present application.
Reference numerals illustrate:
10-a body; 101-a first part; 102-a second part;
11-a receiving cavity; 12-drainage cavity; 13-elution chamber; 14-a waste liquid collection chamber; 15-a waste liquid diversion channel;
20-lancet assembly;
21-a first lancet; 22-a second lancet; 23-third lancet; 24-fourth lancet;
30-a first sealing film;
40-nucleic acid extraction membrane;
50-a second sealing film;
60-sample collector;
70-locking structure;
71-locking ring; 72-locking grooves;
80-protective shell.
Detailed Description
As described in the background art, the nucleic acid extraction apparatus in the related art is generally installed in a biological laboratory, and the extraction, purification, etc. of nucleic acids are performed by a professional, so that the current nucleic acid extraction device has a limited application range and poor flexibility.
To the above technical problems, the embodiments of the present application provide a nucleic acid extraction device, which is integrated with a plurality of accommodating cavities, drainage cavities, and a sample collector, wherein each accommodating cavity respectively accommodates a nucleic acid lysate, a cleaning solution, and a nucleic acid eluent, i.e. reagents necessary for extracting and purifying nucleic acids, and respectively encapsulates, integrates, and integrates in a portable extraction device, so as to achieve nucleic acid extraction and purification.
Furthermore, the nucleic acid extraction device has the characteristics of simple structure, low cost, simplified operation steps, realization of nucleic acid extraction and purification, small volume, light weight and portability, and improves the applicability and flexibility.
In order to make the above objects, features and advantages of the embodiments of the present application more comprehensible, the following description will make the technical solutions of the embodiments of the present application clear and complete with reference to the accompanying drawings in the embodiments of the present application. It will be apparent that the described embodiments are only some, but not all, of the embodiments of the present application. All other embodiments, based on the embodiments herein, which are within the scope of the protection of the present application, will be within the purview of one of ordinary skill in the art without the exercise of inventive faculty.
For the convenience of describing the embodiments of the present application, a coordinate system referred to in the drawings is first described, where a Z-axis direction is a first direction, and the first direction may be an axial direction of the main body, and an X-axis direction is a second direction perpendicular to the first direction, and the second direction may be a radial direction of the main body.
As shown in fig. 1 and 2, the embodiment of the present application provides a nucleic acid extraction device that extracts nucleic acids based on a nucleic acid filter method, wherein the nucleic acids include DNA or RNA not limited to viruses, bacteria, or other microorganisms.
The nucleic acid extraction device includes a main body 10, a nucleic acid extraction membrane 40, a first sealing membrane 30, a second sealing membrane 50, and a lancet assembly 20; wherein the main body 10 is cylindrical, and along a first direction, the main body 10 comprises a first portion 101 and a second portion 102, wherein the first portion 101 is provided with at least three accommodating chambers 11 independent from each other, and the three accommodating chambers 11 may be cylindrical chambers, and axes of the accommodating chambers 11 are arranged in parallel.
The second portion 102 also provides a drainage lumen 12, and the drainage lumen 12 may be a cylindrical lumen. The drainage chamber 12 is located below the accommodation chambers 11 and communicates with each accommodation chamber 11. Illustratively, a first opening is provided along the bottom of the first direction accommodating chamber 11, through which each accommodating chamber 11 communicates with the drainage chamber 12, respectively. It should be noted that, in some embodiments, to facilitate processing of the main body 10, the first portion 101 and the second portion 102 are separately manufactured, and the first portion 101 and the second portion 102 are detachably and hermetically connected together.
Further, since each of the accommodation chambers 11 stores a reagent necessary for each step of nucleic acid extraction, the first sealing film 30 is provided at the first opening and seals the first opening. For example, each first opening is provided with a first sealing film 30 corresponding to the first opening, or all the first openings share one first sealing film 30 for sealing, which is not limited in the embodiment of the present application.
Preferably, in the embodiment of the present application, each accommodating cavity 11 is provided with one first sealing film 30, that is, the number of first sealing films 30 is consistent with the number of accommodating cavities 11. By means of the arrangement, each accommodating cavity 11 can be independently sealed, so that the independence and the tightness of each accommodating cavity 11 are guaranteed, and the mutual influence of adjacent accommodating cavities 11 when the first sealing film 30 is subsequently pierced is avoided.
The sample collector 60 provided by embodiments of the present application may be a sample collection tube having an elution chamber 13 and used to collect a purified nucleic acid sample. Along a first direction, the sample collector 60 is detachably mounted below the main body 10, and a drainage channel is arranged between the sample collector 60 and the drainage cavity 12 so as to realize communication between the sample collector 60 and the drainage cavity 12.
The drainage channel extends in a first direction and includes a second opening configured as a bottom opening of the drainage channel and the drainage channel communicates with the sample collector 60 through the second opening and a third opening configured as a top opening of the drainage channel and the drainage channel communicates with the drainage cavity 12 through the third opening.
Further, the second sealing film 50 is disposed at the second opening, and the second sealing film 50 can seal the second opening. The nucleic acid isolation membrane 40 is disposed at the third opening, and the nucleic acid isolation membrane 40 seals the third opening, i.e., the nucleic acid isolation membrane 40 is disposed above the second sealing membrane 50 at intervals along the first direction. Further, the nucleic acid extraction membrane 40 may be a silica gel membrane or a cellulose membrane, and the nucleic acid extraction membrane 40 is capable of specifically adsorbing a nucleic acid sample.
In the embodiment of the present application, the three accommodating chambers 11 respectively contain a nucleic acid lysate, a cleaning solution and a nucleic acid eluent, and it should be noted that, in the embodiment of the present application, the main body 10 includes at least three accommodating chambers 11 which are independently arranged, and the number of accommodating chambers 11 is related to the number of times of cleaning, for example, the main body 10 includes three accommodating chambers 11, and each accommodating chamber 11 respectively contains a nucleic acid lysate, a cleaning solution and a nucleic acid eluent; alternatively, the main body 10 includes four accommodating chambers 11, wherein two accommodating chambers 11 respectively contain the nucleic acid lysate and the nucleic acid eluent, and the remaining two accommodating chambers 11 each contain the cleaning solution, which is not limited in this embodiment.
The lancet assembly 20 comprises a plurality of lancets, the number of lancets being identical to the number of accommodating chambers 11 so that one lancet is provided in each accommodating chamber 11. The lancet can be inserted into the accommodating cavity 11 and kept sealed with the accommodating cavity 11, and it should be noted that an opening communicating with each accommodating cavity 11 is provided at the top of the main body 10, so that the lancet can be inserted into the accommodating cavity 11.
In this embodiment of the application, the spike includes the body and sets up the point on the body, and the body is provided with sealing member, and sealing member can be the cover and establish the sealing washer on the body, and sealing member and the inner wall contact of holding chamber 11 to make the body seal with holding chamber 11. The body may move in a first direction under the action of an external force, and the tip portion may pierce the first sealing film 30. And, if necessary, the lancet provided in the accommodating chamber 11 containing the nucleic acid eluent can puncture not only the first sealing membrane 30 but also the nucleic acid extraction membrane 40 and the second sealing membrane 50, and the tip can be inserted into the eluting chamber 13.
In some embodiments, the body 10 includes four receiving chambers 11 independent of each other, and the four receiving chambers 11 may be disposed in a certain order. For example, four accommodation chambers 11 define a first accommodation chamber, a second accommodation chamber, a third accommodation chamber, and a fourth accommodation chamber in this order clockwise.
Further, the corresponding reagents may be placed in the corresponding accommodating chambers 11, respectively, in the order in which the reagents are added as required for the nucleic acid extraction process. Wherein the first chamber that holds is used for holding nucleic acid lysate, and the second holds the chamber, the third holds the chamber and all is used for depositing the washing liquid, and the fourth holds the chamber and is used for depositing the nucleic acid eluent, so set up, can carry out the secondary to the nucleic acid that adsorbs on nucleic acid extraction membrane 40 to get rid of the impurity in the nucleic acid, in order to further purify the nucleic acid.
Accordingly, the spike assembly 20 comprises a first spike 21, a second spike 22, a third spike 23 and a fourth spike 24, wherein the first spike 21 is sealingly inserted in the first receiving chamber, the first spike 21 is pressed, the first spike 21 is moved in a first direction within the receiving chamber 11, and the tip of the first spike 21 pierces the first sealing membrane 30 to allow the nucleic acid lysate to enter the drainage chamber 12.
Similarly, the second lancet 22 is sealingly inserted into the second receiving chamber and is capable of puncturing the first sealing membrane 30 to allow the cleaning fluid to flow into the drainage chamber 12 and to first flush the nucleic acid adsorbed to the nucleic acid extraction membrane 40. Furthermore, the third lancet 23 is sealingly inserted into the third accommodating chamber and is capable of puncturing the first sealing membrane 30 to allow the cleaning liquid to flow into the drainage chamber 12 for a second flushing of the nucleic acid adsorbed to the nucleic acid extracting membrane 40.
Further, the fourth lancet 24 is inserted into the fourth accommodating cavity in a sealing manner, the tip of the fourth lancet 24 is pressed to puncture the first sealing membrane 30, the nucleic acid extraction membrane 40 and the second sealing membrane 50 in sequence, so that the nucleic acid eluent enters the drainage cavity 12 and elutes the nucleic acid on the nucleic acid extraction membrane 40, and as the nucleic acid extraction membrane 40 and the second sealing membrane 50 are punctured, fragments of the nucleic acid extraction membrane 40 carry the nucleic acid and collect into the elution cavity 13, and then are stored.
It should be noted that, in the embodiment of the present application, the first puncture needle 21, the second puncture needle 22, the third puncture needle 23, and the fourth puncture needle 24, where the lengths of the first puncture needle 21, the second puncture needle 22, and the third puncture needle 23 may be the same, the length of the fourth puncture needle 24 may be greater than that of the first puncture needle 21, and when each puncture needle is inserted into the corresponding accommodating cavity 11, the end of the puncture needle away from the tip portion may be kept at a height, so that the arrangement may facilitate the operation of each puncture needle.
And, each lancet has a corresponding number or mark so that a user can sequentially inject a corresponding reagent, for example, the first lancet 21, the second lancet 22, the third lancet 23, and the fourth lancet 24 are numbered in sequence, respectively, with the numbers "1-4", and the first lancet 21, the second lancet 22, the third lancet 23, and the fourth lancet 24 are arranged clockwise in the circumferential direction of the body 10.
On the basis of the above embodiments, when the nucleic acid extraction device provided in the embodiments of the present application is used for extracting nucleic acid, the extraction method includes the following steps:
the sample to be measured is added into the containing cavity 11 containing the nucleic acid lysate for nucleic acid lysis. Specifically, the obtained nucleic acid sample is placed in the first accommodation chamber, and nucleic acid cleavage is performed on the nucleic acid sample. For example, the nucleic acid sample may be a pharyngeal swab sample, blood or body fluid, or the like.
After the lysis, the lancet having the holding chamber 11 for nucleic acid lysate and the lancet having the holding chamber 11 for washing liquid are sequentially pressed to perform the enrichment and washing of nucleic acids on the nucleic acid extraction membrane 40.
Specifically, after the nucleic acid sample is lysed, the first lancet 21 may be pressed to puncture the first sealing membrane 30, so that the nucleic acid lysate enters the drainage chamber 12 and flows through the nucleic acid extraction membrane 40, so that the nucleic acid extraction membrane 40 adsorbs the nucleic acid in the nucleic acid lysate.
Next, the second lancet 22 is pressed, and the second lancet 22 pierces the first sealing film 30 so that the cleaning liquid flows into the drainage chamber 12, and the impurities on the nucleic acid extraction film 40 are cleaned for the first time; then, the third lancet 23 is pressed, and the third lancet 23 pierces the first sealing membrane 30 to allow the cleaning liquid to flow into the drainage chamber 12, and the impurities on the nucleic acid extraction membrane 40 are cleaned for the second time. Of course, in some embodiments, one rinse or more than two rinses may be performed, and this embodiment is not limited in this regard.
Finally, the lancet of the holding chamber 11 having the nucleic acid eluting solution is pressed, and the lancet pierces the first sealing membrane 30, the nucleic acid extraction membrane 40, and the second sealing membrane 50 to collect nucleic acid to the sample collector 60.
Specifically, the fourth lancet 24 can be pressed, and the tip of the fourth lancet 24 can pierce the first sealing membrane 30, the nucleic acid extraction membrane 40, and the second sealing membrane 50 in this order, so that the nucleic acid eluent enters the drainage lumen 12 and elutes the nucleic acid on the nucleic acid extraction membrane 40.
Further, as the nucleic acid extraction membrane 40 and the second sealing membrane 50 are pierced, fragments of the nucleic acid extraction membrane 40 carry the nucleic acids and are collected together in the elution chamber 13 and stored, thereby completing the lysis, washing, extraction and enrichment of the nucleic acid sample.
Compared with the scheme that the extraction and purification of nucleic acid in the related art needs to be performed by a professional using a nucleic acid extraction device in a biological laboratory, the nucleic acid extraction device provided in the embodiment of the present application is integrated with a plurality of accommodating chambers 11, a drainage chamber 12 and a sample collector 60, wherein each accommodating chamber 11 respectively accommodates a nucleic acid lysate, a cleaning solution and a nucleic acid eluent, i.e. reagents necessary for extracting and purifying nucleic acid can be respectively packaged, integrated and integrated in a portable extraction device, so as to achieve the extraction and purification of nucleic acid.
On the basis of the above embodiment, in the embodiment of the present application, after the nucleic acid lysate and the cleaning solution pass through the nucleic acid extraction membrane 40, further collection is required, so as to avoid biological contamination or virus propagation, the nucleic acid device provided in the embodiment of the present application further includes a waste liquid collection chamber 14 and a waste liquid guiding channel 15, along the first direction, the waste liquid collection chamber 14 may be disposed below the main body 10, that is, the waste liquid collection chamber 14 is disposed below the drainage chamber 12, which may be located in the same space with the sample collector 60, and the two may be disposed opposite to each other.
The waste liquid diversion channel 15 is obliquely arranged along the first direction, the waste liquid diversion channel 15 comprises a first end and a second end which are oppositely arranged, wherein the first end of the waste liquid diversion channel 15 is communicated with the waste liquid collection cavity 14, the second end of the waste liquid diversion channel 15 is communicated with the drainage channel, and the second end of the waste liquid diversion channel 15 is positioned between the nucleic acid extraction membrane 40 and the second sealing membrane 50.
After the nucleic acid lysate flows through the nucleic acid extraction membrane 40 and the nucleic acid is adsorbed to the nucleic acid extraction membrane 40, the nucleic acid lysate flows through the drainage channel to the waste liquid guide channel 15, and enters the waste liquid collection chamber 14 through the waste liquid guide channel 15. The cleaning liquid washes the impurities on the nucleic acid extraction membrane 40, and the cleaning liquid carrying the impurities flows to the waste liquid guide channel 15 through the guide channel and is further collected to the waste liquid collection chamber 14.
On the basis of the above embodiment, the first section bar and the second section bar are arranged in the main body 10 at intervals along the first direction, wherein the first section bar is located above the second section bar, the main body 10 is internally provided with a cavity for forming the drainage cavity 12, the first section bar and the second section bar can be in a circular flat plate structure, the edges of the first section bar and the second section bar are in sealing connection with the inner wall of the main body 10, and the drainage cavity 12 is formed between the first section bar and the second section bar.
Further, each accommodating chamber 11 is disposed between the first profile and the top of the main body 10, and each accommodating chamber 11 is provided with a first opening at a position opposite to the first profile, and the first opening penetrates the entire first profile. And a drainage channel formed on the second section bar and penetrating the entire second section bar, wherein the third opening of the drainage channel is opposite to the first opening of the fourth receiving cavity, and the drainage channel extends vertically along the first direction, so that the fourth lancet 24 is pressed, and the tip of the fourth lancet 24 can enter the third opening and penetrate the drainage channel into the second opening to puncture the nucleic acid extraction membrane 40 and the second sealing membrane 50.
On the basis of the embodiment, the height of the opening end face of the third opening in the embodiment of the application is flush with the surface of the surrounding second section bar. Preferably, the height of the opening end face of the third opening is lower than the surface of the second section bar around the third opening, so that the reagent flowing to the drainage cavity 12 can flow towards the position of the third opening, thereby ensuring that the nucleic acid lysate, the cleaning solution and the eluent flow to the drainage channel and improving the purification and enrichment degree of the nucleic acid.
To further increase the flow rate of each reagent in the drainage chamber 12, the flow rate into the drainage channel is increased; the second profile is obliquely arranged relative to the main body 10, so that the surface of the second profile, which is positioned in the drainage cavity 12, forms a drainage surface. The guide surface comprises a first side and a second side which are opposite, wherein the first side is located at a height greater than that of the second side, and the third opening is located at the connection part of the second side of the guide surface and the inner wall of the main body 10.
On the basis of the above embodiment, the second profile is provided with a first mounting location for detachably mounting the sample collector 60. For example, the sample collector 60 may be a sample collection tube, the first mounting location may be a first location land formed on a surface of the second section bar remote from the drainage lumen 12, the first location land having a location hole, and the location hole being opposite the drainage channel, the sample collection tube being insertable into the location hole, and the sample collection tube being in communication with the drainage channel.
Furthermore, in some embodiments, a waste collection tube is also provided below the body 10, the waste collection tube having a waste collection chamber 14, the second profile being provided with a second mounting location, the waste collection tube being removably mounted in the second mounting location. For example, the surface of the second section bar, which is away from the drainage cavity 12, is provided with a plugging groove, the waste liquid collection pipe is detachably connected into the plugging groove and is in tight fit with the plugging groove, and the waste liquid collection pipe is communicated with the waste liquid drainage channel.
So set up, this application embodiment sample collecting pipe, waste liquid collecting pipe demountable installation are in main part 10, can be convenient for shift and further detect etc. the sample to and also be convenient for carry out independent processing to the waste liquid collecting pipe, avoid biological pollution and virus transmission.
The embodiment of the application protects sample collection pipe, waste liquid collection pipe and sets up it in sealed space, and the nucleic acid extraction device that this application embodiment provided still includes protective housing 80, and protective housing 80 demountable installation is in the below of main part 10 to form sealed space, and sample collection pipe, waste liquid collection pipe hold in sealed space.
For example, the main body 10 includes a first portion 101 and a second portion 102 which are detachable, the second portion 102 is screwed with the protective housing 80, and thus, in the first direction, the first portion 101, the second portion 102 and the protective housing 80 form an upper, middle and lower three-layer structure, in which one layer has the accommodation chamber 11, the middle layer has the drainage chamber 12, and the lower layer has the waste liquid collection chamber 14 and the elution chamber 13.
Illustratively, in the first direction, the end of the protective shell 80 near the main body 10 is provided with a first threaded connection section, the first threaded connection section has an internal thread, the protective shell 80 is provided with a second threaded connection section, the second threaded connection section has an external thread, the second portion 102 and the protective shell 80 can be screwed together through the first threaded connection section and the second threaded connection section, and a sealing member can be provided between the first threaded connection section and the second threaded connection section in order to promote tightness of the two. Further, the protective housing 80, the sample collection tube and the waste collection tube may be provided to be transparent so as to facilitate observation of the nucleic acid sample collection condition as well as the waste collection condition.
In some embodiments, to prevent the malfunction of the lancet assembly 20, which may lead to failure of the nucleic acid extraction device, the nucleic acid extraction device according to the embodiments of the present application further includes a locking structure 70, where the locking structure 70 is configured to lock the lancet assembly 20, so as to prevent the first sealing film 30 from being pierced by pressing the lancet due to accidental touching of the lancet in the non-operating state.
The locking structure 70 in the embodiment of the application includes a locking ring 71 and a locking groove 72 matched with the locking ring 71, wherein the locking groove 72 is provided on each lancet, the locking ring 71 is inserted on the main body 10 along the second direction and can extend to the inside of the main body 10, and the locking ring 71 can be embedded in the locking groove 72 and limit the lancet in the first direction.
For example, the side wall of the main body 10 is provided with an insertion port that is provided in cooperation with the lock ring 71. The locking groove 72 is disposed at a position of the lancet assembly 20 near the locking ring 71, so that when the locking ring 71 is inserted into the main body 10, the locking groove 72 can be simultaneously embedded into each lancet, thereby realizing the limitation of each lancet. It will be appreciated that when nucleic acid extraction is desired by the nucleic acid extraction apparatus, the locking ring 71 can be pulled out in advance to press each lancet.
In some embodiments, to ensure the activity of the extracted nucleic acid, the sample collector 60 of the present embodiment has a nucleic acid preservation solution therein in which the extracted nucleic acid sample is preserved for transport transfer.
Further, the sample collector 60 in the embodiment of the present application further includes a PCR amplification reaction solution, a nucleic acid isothermal amplification detection solution, etc.; by the arrangement, the nucleic acid extraction device in the embodiment of the application can be used for extracting and enriching the nucleic acid sample, and can amplify the nucleic acid sample so as to facilitate subsequent detection.
For example, the nucleic acid extraction device provided in the embodiments of the present application can detect the nucleic acid sample by combining different nucleic acid amplification detection modes after the nucleic acid sample is split, enriched and extracted into the elution chamber 13, where the current nucleic acid amplification detection modes include a PCR detection mode and a isothermal amplification detection mode, and the following description will be given of the nucleic acid amplification solutions and the detection processes corresponding to the two detection modes respectively.
For example, in one embodiment, the sample collector 60 has a polymerase chain reaction (Polymerase Chain Reaction, abbreviated as PCR) amplification reaction solution therein, which includes PCR amplification primers (substrates), buffers, enzymes, etc., which can be adapted to a fluorescent quantitative PCR detector (qPCR). After sample processing is performed on one or more persons respectively, amplification detection can be performed through a qPCR detector with high throughput (96 throughput, 384 throughput, etc.) at one time.
The PCR detection mode is more suitable for application sites requiring high-flux instant detection, such as customs, and the like, the nucleic acid extraction device provided by the embodiment of the application is utilized for extracting nucleic acid from inbound passengers, the sample collection pipe with the two-dimensional code mark is unified to customs or a detection mechanism, and the customs is used for carrying out high-flux qPCR detection rapidly and uniformly on site, so that the detection time and cost are greatly saved, and the working pressure of external anti-input is effectively reduced.
In another embodiment, the nucleic acid extraction device provided in the embodiment of the present application may integrate a nucleic acid isothermal amplification detection method (such as loop-mediated isothermal amplification detection), and directly implement in-situ amplification detection and result reading after nucleic acid enrichment and extraction, in which case a heating component may be disposed outside the sample collection tube to heat the elution chamber 13 to a desired temperature. The heating element may have an open chamber in which the elution chamber 13 is housed, the elution chamber 13 being heated as a water bath by injection of hot water heating element: the elution chamber 13 is preloaded with primers, buffers, amplification enzymes, etc. required for isothermal amplification of nucleic acids.
Compared with the common PCR technology which needs variable temperature for amplification, the isothermal detection technology of nucleic acid can realize rapid amplification detection of nucleic acid under isothermal conditions. Therefore, the protection shell 80 and the sample collection tube of the nucleic acid extraction device provided by the embodiment of the application are transparent, so that the rapid detection can be realized on site, for example, in remote areas with backward conditions, after the sampling is finished, the amplification detection and the result reading can be directly carried out, and the local infectious people can be rapidly screened.
Based on the above embodiments, according to the needs of different detection scenarios, different amplification detection solutions need to be contained in the sample collector 60 in the embodiments of the present application, in order to facilitate integration of the amplification detection solutions into the nucleic acid extraction device, the enzyme (amplification enzyme) and the substrate of the amplification detection solutions are frozen at the bottom of the sample collector 60, and the buffer solution is sealed in the elution chamber 13 alone, and after the nucleic acid sample is enriched, the buffer solution can be released and mixed with the enzyme and the substrate.
For example, the nucleic acid extraction device provided in the embodiment of the present application further includes a third sealing membrane disposed at the inlet of the sample collector 60, and the second sealing membrane 50 is located below the second sealing membrane 50, which has a certain interval along the first direction, and forms a sealed storage cavity, and the buffer solution can be stored in the storage cavity. The spike (fourth spike 24) corresponding to the containing chamber 11 containing the nucleic acid eluent may pierce the third sealing membrane so that the buffer solution enters the eluting chamber 13 together with the nucleic acid sample when the nucleic acid sample is collected in the eluting chamber 13.
In this specification, each embodiment or implementation is described in a progressive manner, and each embodiment focuses on a difference from other embodiments, and identical and similar parts between the embodiments are all enough to refer to each other.
It should be noted that references in the specification to "one embodiment," "an example embodiment," "some embodiments," etc., indicate that the embodiment described may include a particular feature, structure, or characteristic, but every embodiment may not necessarily include the particular feature, structure, or characteristic. Moreover, such phrases are not necessarily referring to the same embodiment. Furthermore, when a particular feature, structure, or characteristic is described in connection with an embodiment, it is submitted that it is within the knowledge of one skilled in the art to effect such feature, structure, or characteristic in connection with other embodiments whether or not explicitly described.
Generally, terms should be understood at least in part by use in the context. For example, the term "one or more" as used herein may be used to describe any feature, structure, or characteristic in a singular sense, or may be used to describe a combination of features, structures, or characteristics in a plural sense, at least in part depending on the context. Similarly, terms such as "a" or "an" may also be understood to convey a singular usage or a plural usage, depending at least in part on the context.
It should be readily understood that the terms "on … …", "above … …" and "above … …" in this disclosure should be interpreted in the broadest sense such that "on … …" means not only "directly on something", but also includes "on something" with intermediate features or layers therebetween, and "above … …" or "above … …" includes not only the meaning "on something" or "above" but also the meaning "above something" or "above" without intermediate features or layers therebetween (i.e., directly on something).
Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present application, and not for limiting the same; although the present application has been described in detail with reference to the foregoing embodiments, it should be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some or all of the technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit of the corresponding technical solutions from the scope of the technical solutions of the embodiments of the present application.
Claims (13)
1. A nucleic acid extraction device comprising a body, a nucleic acid extraction membrane, a first sealing membrane, a second sealing membrane, a spike assembly, and a sample collector;
the drainage device comprises a main body, at least three independent accommodating cavities and a sealing membrane, wherein the main body is internally provided with a drainage cavity and at least three independent accommodating cavities, each accommodating cavity is arranged above the drainage cavity along a first direction, the bottom of each accommodating cavity is provided with a first opening communicated with the drainage cavity, and the first opening is sealed by the sealing membrane;
the sample collector is detachably arranged below the drainage cavity, a drainage channel is arranged between the sample collector and the drainage cavity, the drainage channel is provided with a second opening communicated with the sample collector, and the second opening is sealed by the second sealing film; the drainage channel has a third opening in communication with the drainage lumen, the third opening being sealed by the nucleic acid extraction membrane;
the three accommodating cavities are respectively filled with nucleic acid lysate, cleaning liquid and nucleic acid eluent, the puncture needle assembly comprises puncture needles corresponding to the accommodating cavities, and the puncture needles are inserted into the accommodating cavities and kept sealed with the accommodating cavities;
the lancet is configured to press the lancet in a first direction, a tip of which pierces the first sealing membrane; and the puncture needles corresponding to the accommodation cavity containing the nucleic acid eluent can puncture the first sealing membrane, the nucleic acid extraction membrane and the second sealing membrane in sequence.
2. The nucleic acid extraction apparatus of claim 1, further comprising a waste collection chamber and a waste diversion channel;
the waste liquid collecting cavity is communicated with the first end of the waste liquid diversion channel, and the second end of the waste liquid diversion channel extends to the drainage channel and is communicated with a space between the nucleic acid extraction membrane and the second sealing membrane.
3. The nucleic acid isolation device of claim 2, wherein a first profile and a second profile are disposed within the body at intervals along a first direction;
the first section bar and the second section bar are respectively connected with the inner wall of the main body in a sealing way, the first section bar is positioned above the second section bar, the drainage cavity is formed between the first section bar and the second section bar, and the drainage channel penetrates through the second section bar;
each accommodating cavity is arranged between the first section bar and the top of the main body, and the first opening penetrates through the first section bar.
4. The nucleic acid isolation apparatus according to claim 3, wherein the opening end face of the third opening is positioned at a level with a surface of the second profile around the opening end face; or,
the opening end face of the third opening is located at a height lower than the surface of the second section bar around the opening end face.
5. The nucleic acid isolation device according to claim 3, wherein the second profile is disposed in the main body so as to be inclined to have a guide surface;
the third opening is positioned at the joint of the lower side of the flow guiding surface and the inner wall of the main body.
6. The nucleic acid isolation device of claim 3, further comprising a protective shell;
the protective housing detachably connects in the below of main part to cover establishes the sample collector.
7. The nucleic acid extraction apparatus according to claim 6, further comprising a waste collection tube detachably attached to the main body, the waste collection tube having the waste collection chamber;
the first section bar is provided with a first installation position and a second installation position, the sample collector is detachably installed at the first installation position, and the waste liquid collecting pipe is installed at the second installation position.
8. The nucleic acid extraction device according to claim 7, wherein the sample collector contains a PCR amplification reaction solution or a nucleic acid isothermal amplification detection solution or a nucleic acid preservation solution;
the protective shell is configured as a transparent shell, and the sample collector is configured as a transparent sample collection tube.
9. The nucleic acid extraction device according to claim 8, wherein the PCR amplification reaction solution and the nucleic acid isothermal amplification detection solution each comprise an enzyme, a substrate, and a buffer;
the sample collector further comprises a third sealing membrane positioned below the second sealing membrane, and a storage cavity is formed between the third sealing membrane and the second sealing membrane;
the enzyme and the substrate are frozen at the bottom of the sample collector, the buffer solution is sealed in the storage cavity, and the puncture needle corresponding to the containing cavity containing the nucleic acid eluent can puncture the third sealing film.
10. The nucleic acid isolation device of any one of claims 1 to 9, wherein four accommodating chambers independent of each other are provided in the main body, the four accommodating chambers being a first accommodating chamber, a second accommodating chamber, a third accommodating chamber, and a fourth accommodating chamber, respectively;
the first accommodating cavity is used for accommodating nucleic acid lysate, the second accommodating cavity and the third accommodating cavity are used for accommodating cleaning liquid, and the fourth accommodating cavity is used for accommodating nucleic acid eluent;
the puncture needle assembly comprises a first puncture needle, a second puncture needle, a third puncture needle and a fourth puncture needle, and the first puncture needle is inserted in the first accommodating cavity in a sealing way and can puncture the first sealing film;
the second puncture needle is inserted into the second accommodating cavity in a sealing way and can puncture the first sealing film; the third puncture needle is inserted into the third accommodating cavity in a sealing way and can puncture the first sealing film;
the fourth puncture needle is inserted in the fourth accommodating cavity in a sealing manner and can puncture the first sealing membrane, the nucleic acid extraction membrane and the second sealing membrane.
11. The nucleic acid extraction apparatus of claim 10, further comprising a locking structure;
the locking structure comprises a locking ring and a locking groove matched with the locking ring, and the locking groove is arranged at a position, close to the locking ring, of the puncture needle assembly;
the side wall of the main body is provided with an inserting port, and the locking ring is inserted into the main body through the inserting port and can lock the first puncture needle, the second puncture needle, the third puncture needle and the fourth puncture needle.
12. The nucleic acid extraction apparatus according to claim 1, wherein the nucleic acid extraction membrane is configured as a silica gel membrane or a cellulose membrane.
13. A nucleic acid extraction method based on the nucleic acid extraction apparatus according to any one of claims 1 to 12, characterized by comprising the steps of:
adding a sample to be detected into a containing cavity containing nucleic acid lysate for nucleic acid lysis;
after the lysis, sequentially pressing a puncture needle with a holding cavity for nucleic acid lysate and a puncture needle with a holding cavity for cleaning liquid to enrich and clean nucleic acid on the nucleic acid extraction membrane;
a spike having a containment chamber for nucleic acid eluting solution is pressed, the spike puncturing the first sealing membrane, the nucleic acid extraction membrane and the second sealing membrane to collect nucleic acid to the sample collector.
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