A nucleic acid purification kit for batch silica bead or magnetic bead based experiments

Abstract

The utility model provides a nucleic acid purification kit for batch silica bead method or magnetic bead method experiments, including a deep well plate, a sleeve rack and a centrifugal inner sleeve, the top of the deep well plate is provided with a plurality of deep holes, the side of the deep well plate is provided with a card slot, the sleeve rack is provided with a plurality of through holes, the positions of the through holes correspond to the deep holes, and the side of the sleeve rack is provided with a tenon, when the nucleic acid purification kit is in an assembled state, the centrifugal inner sleeve passes through the through hole and is inserted into the deep hole. The nucleic acid purification kit of the utility model provides a standardized, high-throughput nucleic acid purification solution, and improves the extraction efficiency of nucleic acids.

Description

Technical Field

[0001] The utility model belongs to the technical field of medical testing equipment, and in particular relates to a nucleic acid purification kit for separating and purifying nucleic acids. Background Art

[0002] The separation and purification of nucleic acids is a common operation in medical testing, scientific research experiments, and other work. Methods for nucleic acid separation include phenol-chloroform extraction, organic solvent method, chelex-100 method, salting-out method, magnetic bead method, silica bead method, etc. The silica bead method has low pollution and low cost, and is gradually being promoted in the industry. The basic process of DNA extraction by the silica bead method is that the silica particles specifically adsorb DNA in the adsorption liquid environment, and after impurities are removed and rinsed, the elution liquid is added to release the DNA from the silica beads.

[0003] The application of centrifugal cannula in silica bead DNA extraction shows good results, because the cannula can reduce the pipetting operation in the extraction process, and the filter mechanism inside the cannula can also effectively remove water-insoluble impurities such as proteins, so the DNA extraction efficiency is high and the quality is good. However, the use of centrifugal cannula currently requires manual operation by experimenters, and each sample is operated in a single tube, and each centrifugation is a single tube centrifugation. The work efficiency is not high and it is difficult to meet the needs of processing a large number of samples. Therefore, it is necessary to develop new equipment to meet the needs of automated and high-throughput sample testing. Utility Model Content

[0004] The utility model aims to provide a nucleic acid purification kit to solve the problems in the prior art, so as to facilitate large-scale high-quality extraction of nucleic acids.

[0005] To achieve the above-mentioned purpose, the utility model provides a nucleic acid purification kit, characterized in that it includes a deep well plate, a sleeve rack and a centrifugal inner sleeve;

[0006] The top of the deep-well plate is vertically downwardly provided with a plurality of deep holes with top openings and bottom closings, and the side of the deep-well plate is provided with a card slot;

[0007] The body of the casing rack is in the shape of a plate, and a plurality of circular through holes are formed on the plate-shaped body. The positions of the through holes correspond to the deep holes. An upper riser is vertically arranged upward along the circumference of the through holes. A plug cover matching the inner diameter of the upper riser is connected to the side of the upper riser through a connecting belt.

[0008] A tenon is provided on the side of the sleeve rack, and the position of the tenon corresponds to the slot;

[0009] The centrifugal inner sleeve comprises an upper sleeve and a lower sleeve, wherein the lower portion of the outer wall of the upper sleeve is clamped with the upper portion of the inner wall of the lower sleeve.

[0010] The top of the upper sleeve is provided with a flange, the outer diameter of the flange is larger than the inner diameter of the upper riser, the inner diameter of the flange matches the plug cover, and a first center hole is opened in the center of the bottom wall of the upper sleeve.

[0011] A filter is provided in the lumen of the lower sleeve, and a second central hole is opened in the center of the bottom wall of the lower sleeve;

[0012] When the nucleic acid purification kit is in an assembled state, the sleeve rack covers the top of the deep-well plate, and the centrifugal inner sleeve passes through the through hole and is inserted into the deep well.

[0013] In a nucleic acid purification kit provided in a further embodiment of the utility model, the upper portion of the deep hole is cylindrical and the lower portion gradually narrows, the first side wall of the lower portion of the deep hole extends vertically downward, the second side wall is opposite to the first side wall, and the second side wall is inclined.

[0014] In a nucleic acid purification kit provided in a further embodiment of the present invention, the ratio of the total depth of the deep hole to the top cross-sectional diameter of the deep hole is 2:1-5:1.

[0015] In a nucleic acid purification kit provided in a further embodiment of the present invention, the top of the deep well has a circle of protrusions that are higher than the plane of the deep well plate.

[0016] In a nucleic acid purification kit provided in a further embodiment of the utility model, a side panel extending vertically downward is provided along the edge of the plate-like body of the sleeve rack, and when the sleeve rack is covered on the deep-well plate, the side panel is matched with the side clearance of the deep-well plate.

[0017] In a nucleic acid purification kit provided in a further embodiment of the utility model, a lower riser is provided vertically downward along the circumference of the through hole, and when the sleeve rack is covered on the deep well plate, the outer surface of the lower portion of the lower riser is gap-matched with the inner surface of the upper portion of the deep well.

[0018] In a nucleic acid purification kit provided in a further embodiment of the utility model, the filter includes a first filter membrane and a second filter membrane stacked up and down, a spacer is provided between the first filter membrane and the second filter membrane, and the spacer is provided with one or more liquid guide holes near the edge.

[0019] In a nucleic acid purification kit provided in a further embodiment of the utility model, the centrifugal inner sleeve is fixedly connected to the sleeve rack.

[0020] In a nucleic acid purification kit provided in a further embodiment of the present invention, the centrifugal inner sleeve and the sleeve rack are integrally formed.

[0021] Compared with the prior art, the utility model has one or more of the following beneficial effects:

[0022] The nucleic acid purification kit of the utility model provides a standardized, high-throughput nucleic acid purification solution. The various components of the kit are suitable for being clamped and moved by a robotic arm, and the injection and aspiration of liquid can be completed by a multi-channel pipette, which greatly improves the efficiency of nucleic acid extraction and is particularly suitable for nucleic acid purification of large quantities of trace samples. The nucleic acid purification kit of the utility model also provides a feasible technical solution for the realization of an automated silica bead method / magnetic bead method nucleic acid purification platform. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 A stereoscopic view of an exemplary deep well plate provided by the present invention.

[0024] Figure 2 A perspective view of an exemplary deep well plate provided by the present invention.

[0025] Figure 3 A three-dimensional diagram of an exemplary deep hole provided by the utility model.

[0026] Figure 4 A three-dimensional diagram of an exemplary casing rack provided by the utility model.

[0027] Figure 5 The present invention is provided as a three-dimensional view of an exemplary casing rack viewed from a bottom angle.

[0028] Figure 6 A cross-sectional view of an exemplary centrifugal inner casing provided by the utility model.

[0029] Figure 7 A three-dimensional diagram of an exemplary nucleic acid purification kit assembly state provided by the present invention.

[0030] Figure 8 The utility model is a perspective view of a centrifugal inner sleeve provided by the utility model being installed on a sleeve rack and covered with a plug cover.

[0031] Fig. 9 The utility model provides a perspective view of a casing rack equipped with a centrifugal inner casing and covered on a deep-well plate.

[0032] Fig.10 The utility model is a partial cross-sectional view of a casing frame equipped with a centrifugal inner casing in a state where the casing frame is covered with a deep-well plate.

[0033] Fig.11 The utility model is a perspective view of a sleeve rack equipped with a centrifugal inner sleeve in a state of being removed from a deep well plate. DETAILED DESCRIPTION

[0034] The specific implementation scheme of the utility model is further described below with reference to examples. Based on the embodiments described in the utility model, all other embodiments obtained by ordinary technicians in the field without creative work are within the scope of protection of the utility model.

[0035] like Figure 1-11 As shown, an exemplary nucleic acid purification kit provided by the present invention includes a deep well plate 1, a sleeve rack 2 and a centrifugal inner sleeve 3.

[0036] Deep well plate

[0037] like Figure 1 , 2 As shown, the top of the deep-well plate 1 is vertically downwardly provided with a plurality of concave tubular deep holes 11 with an open top and a closed bottom, as a mechanism for accommodating the filtrate from the inner casing. The upper part of the deep hole 11 is cylindrical, and the lower part gradually narrows. The ratio of the total depth of the deep hole 11 to the top cross-sectional diameter should be greater than 1:1, preferably 2:1~5:1, thereby ensuring that the deep hole 11 has sufficient depth to prevent the liquid from being thrown out during centrifugation. Preferably, the top of the deep hole 11 is slightly higher than the top plane of the deep-well plate, that is, the top of the deep hole 11 has a circle of protrusions 12 that are higher than the plane of the deep-well plate, which is convenient for sealing the deep-well plate 1 during use. The sealing film can achieve a tight fit with the help of the protrusions 12 at the top of the deep hole 11 to seal each deep hole.

[0038] The multiple deep wells 11 on the deep well plate 1 are arranged in an array, for example, in the form of 4×3, 6×4, 8×6, 12×8, etc. The deep wells are preferably arranged in a 6×4 array. When the volume of a single deep well meets most experimental requirements, the size of the 24-well deep well plate can match other commonly used laboratory instruments and equipment (such as a plate centrifuge), which helps to improve the versatility of the nucleic acid purification kit.

[0039] A clamping groove 13 is provided on the side of the deep hole plate 1 for clamping with the casing rack 2 .

[0040] In a preferred embodiment, the deep hole 11 has an asymmetric structure. Specifically, Figure 3As shown, the way in which the lower part of the deep hole 11 gradually narrows is that the first side wall 111 on one side of the lower part of the deep hole 11 extends vertically downward, while the second side wall 112 opposite thereto is inclined. During use, the deep well plate 1 containing liquid and silicon beads is centrifuged with a flat centrifuge. During centrifugation, the position of the deep well plate 1 changes from horizontal to vertical. During the centrifugation, the silicon beads will gather at the inclined second side wall 112 instead of at the bottom of the deep hole 11. When the liquid is subsequently removed, the tip of the pipette is inserted into the bottom of the deep hole 11 to absorb the liquid, and it is not easy to touch the silicon beads at the inclined second side wall 112, which can effectively prevent the loss of nucleic acid template caused by sucking away (mis-absorption) of silicon beads. After centrifugation of a conventional symmetrical centrifuge tube, the silicon beads gather at the bottom of the tube. When the pipette tip is inserted into the bottom of the tube during aspiration, it is easy to mistakenly suck out some of the silicon beads. The preferred asymmetric deep hole structure of the utility model can avoid the loss of silicon beads caused by mis-absorption, that is, avoid the loss of nucleic acid samples adsorbed on the silicon beads. This is very beneficial for the successful inspection of trace samples.

[0041] Casing rack

[0042] The casing rack 2 is a component that carries the centrifugal inner casing. Figure 4 As shown, the main body of the sleeve rack 2 is in the shape of a plate, and a plurality of circular through holes 21 are formed on the plate. The positions of the through holes 21 correspond to the deep holes 11 of the deep well plate 1, that is, when the sleeve rack 2 is covered on the deep well plate 1, the through holes 21 on the sleeve rack 2 are aligned with the deep holes 11 on the deep well plate 1, and when the nucleic acid purification kit is in the assembled state, the centrifugal inner sleeve 3 passes through the through holes 21 on the sleeve rack 2 and is inserted into the deep hole 11 on the deep well plate.

[0043] A tenon 23 is provided on the side of the casing rack 2, and the position of the tenon 23 corresponds to the slot 13 of the deep well plate 1. When the casing rack 2 is covered on the deep well plate 1, the casing rack 2 and the deep well plate 1 are firmly connected by the tenon 23 and the slot 13.

[0044] A hollow cylindrical upper stand pipe 22 is vertically arranged along the circumference of the through hole 21 to carry the centrifugal inner sleeve 3. A plug cover 24 matching the inner diameter of the upper stand pipe is connected to the side of the upper stand pipe 22 through a connecting belt.

[0045] Preferably, if Figure 5 As shown, a side panel 25 extending vertically downward is provided along the edge of the plate-shaped body of the casing rack, which is used to guide the casing rack 2 to accurately position and cover the deep-well plate 1. The enclosed size of the side panel 25 matches the outer periphery of the deep-well plate 1. When the casing rack covers the deep-well plate, the side clearance of the side panel 25 and the deep-well plate 1 is matched. In addition, the side panel 25 can also provide sufficient area for the robot arm to clamp. The robot arm clamps the side panel 25 on the side of the casing rack 2, presses the tenon 23 on the side of the casing rack 2, and can release the clamping state between the casing rack 2 and the deep-well plate 1, and then lifts the casing rack 2 to complete the removal of the casing rack 2.

[0046] Preferably, a hollow cylindrical lower riser 26 is provided vertically downward along the circumference of the through hole 21, and the outer diameter of the lower riser 26 matches the inner diameter of the top of the deep hole 11 of the deep hole plate 1. When the casing rack 2 is covered on the top of the deep hole plate 1, the lower riser 26 can be inserted into the top of the deep hole 11 of the deep hole plate 1, and the outer surface of the lower part of the lower riser 26 forms a clearance fit with the inner surface of the upper part of the deep hole 11, which is convenient for guiding the correct positioning of the casing rack 2 and the deep hole plate 1, and can also enhance the stability of the casing rack 2 covering the deep hole plate 1.

[0047] Centrifugal inner casing

[0048] like Figure 6 As shown, the centrifugal inner sleeve 3 includes an upper sleeve 31 and a lower sleeve 32. The lower portion of the outer wall of the upper sleeve 31 is clamped with the upper portion of the inner wall of the lower sleeve 32. The clamping form is not limited. For example, it can be clamped by the cooperation of a clamping block and a groove, or by the cooperation of a concave ring and a convex ring.

[0049] The upper sleeve 31 is a bottomed cylindrical shape, and is used to hold the lysate of the sample to be extracted with nucleic acid. A flange 311 is provided on the top of the upper sleeve, and the outer diameter of the flange 311 is larger than the inner diameter of the upper vertical tube 22 of the sleeve rack 2, so that the centrifugal inner sleeve 3 can be reliably installed on the sleeve rack 2. The inner diameter of the flange 311 matches the plug cover 24 on the aforementioned sleeve rack 2, so that the plug cover 24 can cover the upper sleeve 31 tightly.

[0050] A first central hole 312 is formed in the center of the bottom wall of the upper sleeve 31 . During centrifugation, the lysate flows into the lower sleeve 32 from the first central hole 312 .

[0051] The lower sleeve 32 is a bottomed cylindrical shape, and a second center hole 322 is opened in the center of the bottom wall. A filter is arranged in the lumen of the lower sleeve 32, and when the lysate flows through the filter, impurities such as proteins are filtered out. An exemplary filter structure includes a first filter membrane 323 and a second filter membrane 324 stacked up and down, a spacer 325 is arranged between the first filter membrane and the second filter membrane, and one or more liquid guide holes 326 are opened near the edge of the spacer 325. The lysate from the first center hole 312 of the upper sleeve flows through the first filter membrane 323 and is filtered once, and then flows to the second filter membrane 324 through the liquid guide hole 326 of the spacer for secondary filtration, and the filtrate flows out from the second center hole 322.

[0052] When in use, the centrifugal inner sleeve 3 is passed through the through hole 21 on the sleeve rack 2 and installed on the sleeve rack 2. The upper vertical tube 22 of the sleeve rack 2 supports the flange 311 of the upper sleeve 31 of the centrifugal inner sleeve 3, and the lower part of the centrifugal inner sleeve 3 is inserted into the deep hole 11 of the deep well plate 1. There are multiple holes on the sleeve rack 2. If the actual number of samples is less than the total number of holes on the sleeve rack 2, a corresponding number of centrifugal inner sleeves 3 can be taken according to the number of samples and installed on the sleeve rack 2. The combination state of the complete nucleic acid purification kit is as follows Figure 7 shown.

[0053] In another embodiment of the present invention, the centrifugal inner sleeve 3 is pre-fixedly connected to the sleeve rack 2, or further, the centrifugal inner sleeve 3 is integrally formed with the sleeve rack 2, and the centrifugal inner sleeve 3 and the sleeve rack 2 form a whole. When in use, there is no need to install the centrifugal inner sleeve 3 on the sleeve rack 2, and it can be used immediately, which is suitable for application scenarios where the sample volume is large and experiments need to be performed quickly.

[0054] Application Example 1

[0055] An exemplary use process of the nucleic acid purification kit of the present invention is as follows:

[0056] Take the required number of centrifugal inner sleeves 3, pass them through the through holes 21 on the sleeve rack 2 and install them on the sleeve rack 2 (if the centrifugal inner sleeve 3 and the sleeve rack 2 are integrated components, this step is not required). Add the sample to be tested and the lysate into the upper sleeve 31 of the centrifugal inner sleeve 3, and cover the centrifugal inner sleeve 3 with the plug cover 24 (such as Figure 8 As shown), the sample is heated and lysed. The process of nucleic acid sample lysis and nucleic acid release is completed in the centrifugal inner sleeve. After the lysis is completed, the sleeve rack 2 is transferred to the top of the deep well plate 1, aligned, and the sleeve rack 2 is covered on the deep well plate 1, and the tenon 23 on the side of the sleeve rack 2 is engaged with the slot 13 on the side of the deep well plate 1. At this time, the centrifugal inner sleeve 3 is inserted into the deep hole 11 of the deep well plate 1 (as shown in FIG. Fig. 9 , 10 As shown). The kit is sent to a flat centrifuge for centrifugation. Under the action of centrifugal force, the water-soluble nucleic acid is filtered through the filter in the centrifugal inner sleeve 3 and centrifuged to the lower part of the deep well 11 of the deep well plate 1 along with the lysate. After the washing process is completed, the latch 23 is pressed to release the engagement between the sleeve rack 2 and the deep well plate 1, and the sleeve rack 2 is removed, and the centrifugal inner sleeve 3 is removed together with the sleeve rack 2 (as shown). Fig.11 As shown). Add adsorption liquid and silica beads to each deep well 11 of the deep well plate 1, let it stand for 15 minutes, and adsorb the water-soluble nucleic acid on the silica beads. Seal the deep well plate 1, and then perform the steps of centrifugation, removal of adsorption liquid, washing, elution, etc.

[0057] Application Example 2

[0058] The basic steps of Application Example 2 are the same as those of Application Example 1, except that: after the lysis is completed, the nucleic acid sample is filtered through the inner sleeve and enters the deep hole 11. After the centrifugal inner sleeve 3 is removed together with the sleeve rack 2, adsorption liquid and magnetic beads are added to each deep hole 11 of the deep well plate 1, and the mixture is allowed to stand to adsorb the water-soluble nucleic acid on the magnetic beads. The deep well plate 1 is placed on the magnetic rack, the magnetic beads are sucked to the desired position, and then the residual liquid is removed, and washing, elution and other steps are performed. This application example shows that the nucleic acid purification kit of the utility model can also support automated and efficient magnetic bead-based nucleic acid purification, and has a wide range of applicable scenarios.

[0059] The picking up and moving of the various components of the nucleic acid purification kit of the utility model can be completed by a robotic arm, multiple centrifugal inner sleeves and deep holes are arranged in an array, and the injection and aspiration of liquid can be completed by a multi-channel pipette, which greatly improves the efficiency of nucleic acid extraction and provides a feasible technical solution for the realization of an automated silica bead method / magnetic bead method nucleic acid purification platform.

[0060] The above examples are only used to illustrate the technical concept and features of the utility model, and their purpose is to enable people in the field to understand the content of the utility model and implement it accordingly, but they cannot be used to limit the protection scope of the utility model. Any equivalent changes or modifications made according to the spirit of the utility model should be included in the protection scope of the utility model.

Claims

1. A nucleic acid purification kit, characterized in that: It comprises a deep well plate (1), a casing rack (2) and a centrifugal inner casing (3); Wherein, a plurality of deep holes (11) with top openings and bottom closings are vertically arranged at the top of the deep hole plate (1), and a slot (13) is arranged at the side of the deep hole plate (1); The body of the casing rack (2) is plate-shaped, and a plurality of circular through holes (21) are formed on the plate-shaped body. The positions of the through holes (21) correspond to the deep holes (11). An upper riser (22) is vertically arranged upward along the circumference of the through holes (21). A plug cover (24) matching the inner diameter of the upper riser (22) is connected to the side of the upper riser (22) via a connecting belt. A latch (23) is provided on the side of the sleeve rack (2), and the position of the latch (23) corresponds to the latch groove (13); The centrifugal inner casing (3) comprises an upper casing (31) and a lower casing (32), wherein the lower portion of the outer wall of the upper casing (31) is clamped with the upper portion of the inner wall of the lower casing (32). A flange (311) is provided at the top of the upper sleeve (31), the outer diameter of the flange (311) is larger than the inner diameter of the upper riser (22), the inner diameter of the flange (311) matches the plug cover (24), and a first center hole (312) is provided at the center of the bottom wall of the upper sleeve (31). A filter is provided in the lumen of the lower sleeve (32), and a second central hole (322) is opened in the center of the bottom wall of the lower sleeve (32); When the nucleic acid purification kit is in an assembled state, the sleeve rack (2) covers the top of the deep-well plate (1), and the centrifugal inner sleeve (3) passes through the through hole (21) and is inserted into the deep hole (11).

2. The nucleic acid purification kit according to claim 1, characterized in that: The upper portion of the deep hole (11) is cylindrical, and the lower portion is gradually narrowed; a first side wall (111) at the lower portion of the deep hole (11) extends vertically downward, and a second side wall (112) is opposite to the first side wall (111), and the second side wall (112) is inclined.

3. The nucleic acid purification kit according to claim 1, characterized in that: The ratio of the total depth of the deep hole (11) to the top cross-sectional diameter of the deep hole (11) is 2:1-5:

1.

4. The nucleic acid purification kit according to claim 1, characterized in that: The top of the deep hole (11) has a circle of protrusions (12) that are higher than the plane of the deep hole plate.

5. The nucleic acid purification kit according to claim 1, characterized in that: A side panel (25) extending vertically downward is provided along the edge of the plate-like body of the sleeve rack (2); when the sleeve rack (2) is covered on the deep-hole plate (1), the side panel (25) is gap-matched with the side surface of the deep-hole plate (1).

6. The nucleic acid purification kit according to claim 1, characterized in that: A lower riser (26) is provided vertically downward along the circumference of the through hole (21); when the sleeve rack (2) is covered on the deep hole plate (1), the outer surface of the lower portion of the lower riser (26) is clearance-matched with the inner surface of the upper portion of the deep hole (11).

7. The nucleic acid purification kit according to claim 1, characterized in that: The filter comprises a first filter membrane (323) and a second filter membrane (324) stacked one above the other, a spacer (325) being provided between the first filter membrane (323) and the second filter membrane (324), and one or more liquid guide holes (326) being provided near the edge of the spacer (325).

8. The nucleic acid purification kit according to any one of claims 1 to 7, characterized in that: The centrifugal inner casing (3) is fixedly connected to the casing frame (2).

9. The nucleic acid purification kit according to claim 8, characterized in that: The centrifugal inner sleeve (3) and the sleeve frame (2) are integrally formed.

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