CN212610607U

CN212610607U - Trace biological sample purifies preceding processing apparatus

Info

Publication number: CN212610607U
Application number: CN202020462092.6U
Authority: CN
Inventors: 王德明; 周杰锋
Original assignee: Ningbo Ajcore Biotechnology Co ltd
Current assignee: Ningbo Ajcore Biotechnology Co ltd
Priority date: 2020-04-01
Filing date: 2020-04-01
Publication date: 2021-02-26
Anticipated expiration: 2030-04-01

Abstract

The application relates to the field of biological sample pretreatment, in particular to a trace biological sample purification pretreatment device. The device includes centrifugal column and collecting pipe, centrifugal column set up in the collecting pipe, centrifugal column bottom is provided with the surface tension layer, the surface tension layer enable the surface tension layer top liquid have surface tension on the surface tension layer, this surface tension can overcome liquid gravity itself and make liquid can't pass through from the surface tension layer, when the centrifugal column receives centrifugal force to act on, surface tension can be overcome to liquid and the collecting pipe is instiled into from the centrifugal column through the surface tension layer. The device adopts the structure of integration, can directly drip into the collecting pipe through the liquid that the centrifugal action was handled in directly will centrifuging tube and collect, and is quick, has made things convenient for the operation.

Description

Trace biological sample purifies preceding processing apparatus

Technical Field

The application relates to the field of biological sample pretreatment, in particular to a trace biological sample purification pretreatment device.

Background

The real-time fluorescent quantitative PCR technology is widely applied to a plurality of fields such as genetic disease molecular diagnosis, clinical examination, animal and plant import and export quarantine, food safety monitoring, soil microorganism detection, paternity test and the like. Because samples such as blood, food, soil and the like contain a large amount of inhibiting factors such as hemoglobin, methemoglobin, lactoferrin, humic acid and the like, the Taq DNA polymerase has obvious inhibiting effect on the conventional Taq DNA polymerase. Therefore, nucleic acids must be isolated from these test samples and then used for PCR amplification. Nucleic acid extraction is the first step of nucleic acid detection, and is also one of the key methods in molecular biology. Provides a basis for downstream nucleic acid detection, and the quality and integrity of extraction directly influence clinical research or diagnosis.

The general nucleic acid extraction process comprises three steps: lysis, binding and purification of the sample. The traditional extraction reagent has relatively low extraction rate and complex steps, thus bringing certain influence on extraction work, and particularly being difficult to obtain qualified nucleic acid when the sample amount is rare. In addition, the traditional method has the disadvantages of multiple operation steps, high cost, large sample requirement, easy cross contamination and inconvenience for customers to quickly extract and purify nucleic acid samples.

In addition, in the current real-time fluorescence PCR technology with the widest application of nucleic acid detection, most reagents are not ready-to-use, so that the problems of complicated operation, high labor and cost and the like of PCR detection generally exist, complicated configuration work is required before use, and errors and even failures of tests are easily caused. Experimental errors also arise from different personnel operations; the rapid extraction of nucleic acid by one-step method cannot be realized; PCR detection reagents need to be manually configured on site, multiple steps need to be manually marked, detection instruments at all stages are independent, and full-automatic treatment of a large number of test samples is not facilitated.

Disclosure of Invention

In order to solve the technical problem, the application aims to provide a pretreatment device for purifying a trace biological sample, which adopts an integrated structure, can directly collect the treated liquid in a centrifugal column into a collecting pipe through a centrifugal effect, is quick and is convenient to operate.

In order to achieve the above object, the present application adopts the following technical solutions:

the utility model provides a processing apparatus before trace biological sample purifies, the device includes centrifugal column and collecting pipe, centrifugal column set up in the collecting pipe, centrifugal column bottom is provided with the surface tension layer, the surface tension layer enable the liquid of surface tension layer top have surface tension on the surface tension layer, this surface tension can overcome liquid gravity itself and make liquid can't pass through from the surface tension layer, when centrifugal column received centrifugal force or passed through gas pressure effect, liquid can overcome surface tension and instil into the collecting pipe through the surface tension layer from the centrifugal column.

As a further improvement, the upper part and/or the lower part of the surface tension layer can be provided with a common filter layer.

As a further improvement, the surface tension layer adopts a hydrophobic sieve plate, the hydrophobic sieve plate is formed by sintering ultrahigh molecular weight polyethylene (UHMW-PE), the filtration precision of the hydrophobic sieve plate is 10-100 microns, and the sieve plate can block the infiltration of blood which takes water as a main component and can block denatured plasma protein. The surface tension layer can also adopt other non-woven fabric materials for the surface tension of the liquid.

As a further improvement, the upper part of the centrifugal column is provided with a convex ring, the centrifugal column is arranged on the upper end surface of the collecting pipe through the convex ring, and a liquid collecting cavity is formed at the lower part of the centrifugal column. The centrifugal column is arranged on the upper end surface of the collecting pipe through the convex ring so that the centrifugal column is hung on the collecting pipe and can be locked through arranging the test tube cover.

As a further improvement, the device also comprises a test tube cover, wherein the test tube cover is arranged at the upper end of the collecting tube to seal the collecting tube, the test tube cover comprises a cover body, a storage tube is arranged on the cover body, the bottom of the storage tube is sealed and extends into the centrifugal column to be arranged, an additive is arranged in the storage tube, a puncturing device capable of puncturing the bottom of the storage tube is arranged on the test tube cover, and after the bottom of the storage tube is punctured, the additive in the storage tube falls into the centrifugal column. The scheme is to preset the additive in the storage pipe, so that the additive can be preset in the whole device, the situation that the additive needs to be additionally added and prepared in field use is avoided, the operation is rapid, and the convenience is brought to the operation.

As a further improvement, the device also comprises a reagent solution which is arranged in the centrifugal column and is positioned above the surface tension layer. Furthermore, the reagent solution is preset in the test tube, so that the field configuration work is avoided, and the extracted sample can be extracted under the action of the reagent solution by adding or not adding additives.

As a still further improvement, the reagent solution comprises one or a mixture of two of a precipitation solution and a lysis solution. The reagent solution can be selected according to the needs, the precipitation solution can be selected, then the lysis solution can be added as an additive in the follow-up process, or the lysis solution, then the additive can be the precipitation solution or other extraction reagents.

As a further improvement, the additive is one or more of lysis solution, precipitation solution, protein adsorption material and protein precipitation material. If the inside of the centrifugal column is the precipitation solution, the additive can be lysis solution; if the column contains a lysis solution, the additive may be one or more of a precipitation solution, a protein adsorption material and a protein precipitation material, so that the lysis and purification are completed in one step. Because samples such as blood, food, soil and the like contain a large amount of hemoglobin, methemoglobin, lactoferrin, humic acid and the like, the additives are preferably protein adsorbing materials and protein precipitating materials; particularly, the additive is most preferably hemoglobin adsorbing material, so that the influence of hemoglobin on the fluorescence PCR can be avoided.

As a further improvement, the test tube cover is connected with the collecting tube through threads, and the bottom sealing material of the storage tube is aluminum foil or tin foil. Of course, the bottom of the storage tube may be flaked and sealed.

After the breaking device breaks through the bottom of the storage pipe, due to air pressure difference or surface tension, the additive in the storage pipe may not fall into the centrifugal column completely. For this purpose, as a specific embodiment, the puncturing device comprises a puncturing rod, a pressing head and a sealing rubber piston, wherein the storage tube is arranged in the inner cavity of the cover body and is connected into a whole through a connecting part; the breaking and penetrating rod is connected with the pressing head, the lower part of the breaking and penetrating rod is positioned in the storage pipe, the sealing rubber piston is fixedly arranged on the breaking and penetrating rod, the outer wall of the sealing rubber piston is attached to the inner wall of the storage pipe, the pressing head is pressed downwards to enable the breaking and penetrating rod to break the bottom of the storage pipe, and the sealing rubber piston pushes pretreatment additives in the storage pipe to flow out of the bottom of the storage pipe.

As a further improvement, the cover body also comprises a spiral pipe and a pressing stroke pipe, wherein the spiral pipe is internally provided with threads, and the pressing stroke pipe is positioned at the upper part of the spiral pipe; the pressing head is arranged on the pressing stroke pipe, the upper part of the breaking-through rod is a guide rod, the guide rod is matched with the inner wall of the storage pipe, and the upper end of the guide rod is fixedly connected with the pressing head.

In the application, when in storage and transportation, due to the problems of misoperation or air pressure difference, the pressing head can be pressed downwards to cause the bottom of the storage tube to be broken, therefore, as an improvement, the pressing head is provided with a downward annular sleeve, the lower part of the annular sleeve is provided with a convex point protruding out of the annular sleeve, the inner side of the upper part of the pressing stroke tube is provided with a notch matched with the convex point, and the convex point is clamped in the notch to form positioning; therefore, a limit is formed, and the pressing head cannot be pressed down due to misoperation when normal operation is not performed. Preferably, the two sides of the salient point are respectively provided with a strip-shaped groove, and the two strip-shaped grooves enable the salient point to be positioned on one elastic sheet, so that the elastic sheet has better elasticity and is convenient for the pressing head to press down to break through limit; preferably, the bottom of the breaking rod is a pointed end, a liquid guide groove is axially arranged above the pointed end, and the phenomenon that the breaking rod is tightly attached to the bottom broken position to cause the additive to fall and be blocked in the liquid guide groove is avoided.

As another specific embodiment, the puncturing device comprises a puncturing rod and a pressing head, the pressing head adopts a rubber ball head or a plastic folding head, and the storage tubes are arranged in the inner cavity of the test tube cover and connected into a whole through a connecting part; the pressing head is positioned above the storage pipe, the upper part of the breaking rod is fixedly connected with the upper wall inside the pressing head, and the breaking rod is pressed downwards to penetrate through the bottom of the storage pipe by pressing the pressing head.

As another specific implementation mode, the puncturing device comprises a puncturing rod, a lifting platform and a rotating body, wherein an opening is formed in the middle of the test tube cover, the storage tube is fixedly arranged below the opening, the rotating body is located on the opening, the rotating body is rotationally matched with the opening, the lifting platform is arranged in the rotating body, the lifting platform is connected with the inner wall of the rotating body through threads, a reverse rotation guide convex strip and a groove are arranged between the lower portion of the lifting platform and the opening, the puncturing rod is arranged below the lifting platform, the lifting platform is driven to move downwards through rotation of the rotating body, and the puncturing rod downwards penetrates through the bottom of the storage tube.

In addition, the application also discloses a micro-biological sample purification pretreatment method, which adopts the device and comprises the following steps:

1) adding the reagent solution into a centrifugal column, or presetting the reagent solution in the centrifugal column;

2) adding the extracted sample into a centrifugal column, and treating the extracted sample under the action of a reagent solution;

3) with or without the addition of additives;

4) the collecting tube with centrifugal column is placed in a centrifugal device for centrifugation, and the liquid containing nucleic acid is dropped into the collecting tube from the centrifugal column by overcoming the surface tension through the action of centrifugal force.

Preferably, the application can be applied to nucleic acid purification pretreatment, and applicable samples comprise various samples such as blood, serum, plasma, saliva, urine, tissue fluid, semen, secretion, pus, respiratory fluid and mucus.

Adopt the structure of integration, can directly drip into the collecting pipe through the liquid that the centrifugal action was handled in the direct centrifugal column and collect, it is quick, made things convenient for the operation. Furthermore, the reagent solution is preset in the centrifugal column, so that the field configuration work is avoided, and the extracted sample can be extracted under the action of the reagent solution by adding or not adding an additive. Taking the purification pretreatment of nucleic acid as an example, the nucleic acid precipitation is not needed after the cracking, and the cracked product can be directly detected, so the method is rapid and convenient to operate.

Drawings

Fig. 1 is a schematic structural diagram of the present application.

Fig. 2 is a schematic diagram of the explosive structure of the present application.

FIG. 3 is a schematic view of the structure of the centrifuge tube.

FIG. 4 is a schematic view showing the structure of a test tube cover in example 2.

FIG. 5 is a schematic view showing the structure of a lid for a test tube in example 3.

Detailed Description

The following detailed description of embodiments of the present application will be made with reference to the accompanying drawings.

Example 1

The device comprises a collecting pipe 1, a centrifugal column 2, a test tube cover 7 and a reagent solution 3. The centrifugal column is characterized in that the test tube cover 7 is connected with the collecting pipe 1 through threads, the centrifugal column 2 is arranged in the collecting pipe 1, a convex ring 6 is arranged on the upper portion of the centrifugal column 2, the centrifugal column 2 is arranged on the upper end face of the collecting pipe 1 through the convex ring 6, and a liquid collecting cavity is formed in the lower portion of the centrifugal column 2. The centrifugal column 2 is arranged on the upper end surface of the collecting pipe 1 through a convex ring 6, so that the centrifugal column 2 is hung on the collecting pipe 1, and the centrifugal column 2 can be locked by arranging a test tube cover 7.

As shown in fig. 1, the test tube cover 7 is arranged at the upper end of the collecting tube 1 to seal the collecting tube 1, the test tube cover 7 comprises a cover body and a puncturing device, the cover body comprises a spiral tube, a storage tube 8 and a pressing stroke tube, threads are arranged in the spiral tube, the pressing stroke tube is positioned at the upper part of the spiral tube, the storage tube 8 is arranged in the inner cavity of the cover body and is connected with the cover body into a whole through a connecting part, and the bottom end of the storage tube 8 is sealed; as shown in fig. 2, the pressing head 13 is provided with a downward annular sleeve, the lower part of the annular sleeve is provided with a protruding point 21 protruding out of the annular sleeve, the inner side of the upper part of the pressing stroke tube is provided with a notch 22 matched with the protruding point 21, the protruding point 21 is clamped in the notch 22 to form a positioning, and two sides of the protruding point 21 are respectively provided with a strip-shaped groove 23.

As shown in fig. 2, the puncturing device comprises a puncturing rod 12, a pressing head 13 and a sealing rubber piston 14, wherein the upper part of the puncturing rod 12 is a guide rod 11, the guide rod 11 is matched with the inner wall of the storage tube 8, and the upper end of the guide rod 11 is fixedly connected with the pressing head 13. The lower part of the puncturing rod 12 is positioned in the storage pipe 8, the bottom of the puncturing rod 12 is a tip, and a liquid guide groove 24 is axially arranged above the tip. The pressing head 13 is arranged on the pressing stroke pipe, the sealing rubber piston 14 is fixedly arranged on the piercing rod 12, the outer wall of the sealing rubber piston 14 is attached to the inner wall of the storage pipe 8, the piercing rod 12 can pierce the bottom of the storage pipe 8 by pressing the pressing head 13 downwards, and the sealing rubber piston 14 pushes the pretreatment additive in the storage pipe 8 to flow out of the bottom of the storage pipe 8 and fall into the centrifugal column 2.

As shown in FIG. 3, the bottom of the centrifugal column 2 is provided with a surface tension layer 4, and the upper part of the surface tension layer 4 is provided with a common filter layer 5. The surface tension layer 4 is made of a hydrophobic sieve plate which is formed by sintering ultra-high molecular weight polyethylene (UHMW-PE), and the filtering precision of the hydrophobic sieve plate is 10-100 microns. The reagent solution 3 is arranged in the centrifugal column 2 and is positioned above the surface tension layer 4, and the liquid of the reagent solution 3 has surface tension on the surface tension layer 4 so as to overcome the gravity of the liquid and prevent the liquid from passing through the surface tension layer 4. When the experiment is carried out, the whole set of device can be placed in a centrifuge, and when the centrifugal column 2 is acted by centrifugal force, liquid overcomes surface tension and drops into the collecting pipe 1 from the centrifugal column 2. Alternatively, the vial cap 7 may be opened and the liquid pushed into the collection tube 1 by providing an injection plunger rod. Of course, it is also possible to push liquid into the collection tube 1 by breaking the rubber sealing piston 14 at the upper end of the device, while ensuring a sufficient seal between the cuvette lid 7, the spin column 2 and the collection tube 1.

Taking the extraction of nucleic acid from blood as an example, the reagent solution 3 in this embodiment is a lysis solution, and the additive 9 is a hemoglobin-adsorbing material.

The method for pretreatment of nucleic acid for purification of the present embodiment comprises the steps of:

1) adding the reagent solution 3 into the centrifugal column 2, or presetting the reagent solution 3 in the centrifugal column 2;

2) adding the extracted sample into a centrifugal column 2, and cracking the extracted sample under the action of a reagent solution 3;

3) with or without the addition of additive 9;

4) the collecting tube 1 with the centrifugal column 2 is placed in a centrifugal device for centrifugation, and liquid containing nucleic acid overcomes surface tension and drops into the collecting tube 1 from the centrifugal column 2 under the action of centrifugal force.

Example 2

As shown in fig. 4, the puncturing device comprises a puncturing rod 12 and a pressing head 15, the pressing head 15 is a rubber ball head or a plastic folding head, an opening is arranged in the middle of the test tube cover 7, and the storage tubes 8 are arranged in the inner cavity of the test tube cover 7 and connected into a whole through a connecting part; the pressing head 15 is positioned above the storage pipe 8, the upper part of the breaking rod 12 is fixedly connected with the inner upper wall of the pressing head 15, the breaking rod 12 downwards presses and breaks the bottom of the storage pipe 8 by pressing the pressing head 15, and meanwhile, the additive 9 is pushed into the centrifugal column 2 by the air pressure change of the pressing head 15. The other structure of this embodiment is as shown in embodiment 1.

Example 3

As shown in fig. 5, the puncturing device comprises a puncturing rod 12, a rotating body 16 and a guide table 17, an opening is formed in the middle of the test tube cover 7, the storage tube 8 is fixedly arranged below the opening, the rotating body 16 is positioned on the opening, the rotating body 16 is in rotating fit with the opening, the guide table 17 is arranged in the rotating body 16, the guide table 17 is in threaded connection with the inner wall of the rotating body 16, a reverse rotation guide convex strip 19 and a groove 18 are arranged between the lower portion of the guide table 17 and the opening, the puncturing rod 12 is arranged below the guide table 17, the guide table 17 is driven to move downwards through rotation of the rotating body 16, and the puncturing rod 12 downwards punctures the bottom of the storage tube 8. The guide table 17 may be provided with ventilation channels 20 on its inside or side walls, through which the additive falls by gravity into the spin column 2, in other configurations according to the embodiment shown in example 1.

The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention, including any reference to the above-mentioned embodiments. Various modifications to these embodiments will be readily apparent to those skilled in the art. The general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. The utility model provides a processing apparatus before trace biological sample purifies, its characterized in that, the device includes collecting pipe (1) and centrifugal column (2), centrifugal column (2) set up in collecting pipe (1), centrifugal column (2) bottom is provided with surface tension layer (4), surface tension layer (4) enable surface tension layer (4) top liquid have surface tension on surface tension layer (4), this surface tension can overcome liquid gravity and make liquid can't pass through from surface tension layer (4), when centrifugal column (2) received centrifugal force or passed through gas pressure effect, liquid can overcome surface tension and drip into collecting pipe (1) from centrifugal column (2) through surface tension layer (4).

2. The pretreatment device for purification of a micro biological sample according to claim 1, wherein the surface tension layer (4) is a hydrophobic sieve plate, and the filtration precision of the hydrophobic sieve plate is 10-100 μm.

3. The pretreatment device for purification of a micro biological sample according to claim 1 or 2, wherein a common filter layer (5) is provided on the upper and/or lower part of the surface tension layer (4).

4. The pretreatment device for purification of a micro biological sample according to claim 1, further comprising a reagent solution (3), wherein the reagent solution (3) is disposed in the centrifugal column (2) above the surface tension layer (4).

5. The pretreatment device for purification of a micro biological sample according to claim 4, wherein the reagent solution (3) comprises one of a precipitation solution and a lysis solution.

6. The pretreatment device for purifying a micro biological sample according to claim 1, wherein a convex ring (6) is arranged at the upper part of the centrifugal column (2), the centrifugal column (2) is arranged on the upper end surface of the collecting pipe (1) through the convex ring (6), and a liquid collecting cavity is formed at the lower part of the centrifugal column (2).

7. The pretreatment device for purification of the micro biological sample according to claim 1, further comprising a test tube cover (7), wherein the test tube cover (7) is disposed at the upper end of the collection tube (1) to seal the collection tube (1), the test tube cover (7) comprises a cover body, a storage tube (8) is disposed on the cover body, the bottom of the storage tube (8) is sealed and extends into the centrifugal column (2), an additive (9) is disposed in the storage tube (8), a piercing device capable of piercing the bottom of the storage tube (8) is disposed on the test tube cover (7), and after the bottom of the storage tube (8) is pierced, the additive (9) in the storage tube (8) falls into the centrifugal column (2).

8. The pretreatment device for purification of a micro biological sample according to claim 7, wherein the additive (9) is one of a lysis solution, a precipitation solution, a protein adsorption material and a protein precipitation material.

9. The pretreatment device for purification of a micro biological sample according to claim 7, wherein the test tube cover (7) is connected to the collection tube (1) by a screw thread, and the bottom sealing material (10) of the storage tube (8) is aluminum foil or tin foil.

10. The pretreatment device for purification of a micro biological sample according to claim 7, wherein the puncture device comprises a puncture rod (12), a first pressing head (13) and a sealing rubber piston (14), the storage tube (8) is arranged in the inner cavity of the cover body and is connected into a whole through a connecting part; the breaking and penetrating rod (12) and the first pressing head (13) are connected with each other, the lower portion of the breaking and penetrating rod (12) is located in the storage pipe (8), the sealing rubber piston (14) is fixedly arranged on the breaking and penetrating rod (12), the outer wall of the sealing rubber piston (14) is attached to the inner wall of the storage pipe (8), the first pressing head (13) is pressed downwards to enable the breaking and penetrating rod (12) to penetrate the bottom of the storage pipe (8), and pretreatment additives in the storage pipe (8) are pushed to flow out of the bottom of the storage pipe (8) through the sealing rubber piston (14).

11. The pretreatment device for purifying a micro biological sample according to claim 10, wherein the cover further comprises a spiral tube and a pressing stroke tube, wherein the spiral tube is internally provided with threads, and the pressing stroke tube is positioned at the upper part of the spiral tube; the first pressing head (13) is arranged on the pressing stroke pipe, the upper part of the penetrating rod (12) is provided with a guide rod (11), the guide rod (11) is matched with the inner wall of the storage pipe (8), and the upper end of the guide rod (11) is fixedly connected with the first pressing head (13).

12. The pretreatment device for purifying a micro biological sample according to claim 11, wherein the first pressing head (13) is provided with a downward annular sleeve, the lower part of the annular sleeve is provided with a convex point (21) protruding out of the annular sleeve, the inner side of the upper part of the pressing stroke tube is provided with a notch (22) matched with the convex point (21), and the convex point (21) is clamped in the notch (22) to form positioning.

13. The pretreatment device for purifying a micro biological sample according to claim 11, wherein a strip-shaped groove (23) is provided on each side of the protruding point (21).

14. The pretreatment device for purification of a micro biological sample according to claim 11, wherein the bottom of the piercing rod (12) is a tip, and a liquid guide groove (24) is axially disposed above the tip.

15. The pretreatment device for purification of a micro biological sample according to claim 10, wherein the puncture device comprises a puncture rod (12) and a second pressing head (15), the second pressing head (15) adopts a rubber ball head or a plastic folding head, and the storage tube (8) is arranged in the inner cavity of the test tube cover (7) and is connected into a whole through a connecting part; the second pressing head (15) is positioned above the storage pipe (8), and the upper part of the breaking rod (12) is fixedly connected with the inner upper wall of the second pressing head (15).

16. The pretreatment device for purification of a micro biological sample according to claim 10, wherein the puncture device comprises a puncture rod (12), a rotator (16) and a guide platform (17), an opening is formed in the middle of the test tube cover (7), the storage tube (8) is fixedly arranged below the opening, the rotator (16) is positioned on the opening, the rotator (16) is rotatably matched with the opening, the guide platform (17) is arranged in the rotator (16), the guide platform (17) is in threaded connection with the inner wall of the rotator (16), a reverse guide convex strip (19) and a groove (18) are arranged between the lower part of the guide platform (17) and the opening, the puncture rod (12) is arranged below the guide platform (17), and the guide platform (17) is driven to move downwards by the rotation of the rotator (16).