TWI695731B - Method and device of automatic nucleic acid extraction - Google Patents

Abstract

The disclosure provides an automatic nucleic acid extraction device which includes a base, a cassette, a driving unit, a moving frame and a syringe. The base has a sample accommodating area, a column accommodating area, a cassette accommodating area, and a collection tube. The sample accommodating area, the column accommodating area, the cassette accommodating area, and the collection tube are arranged in a linear direction. The cassette is disposed in the cassette accommodating area. The cassette includes two parallel walls and at least two vertical walls. The parallel walls and the vertical walls form a lysis buffer well, at least one wash buffer well and an elution buffer well together. The vertical walls which form the lysis buffer well, the wash buffer wells and the elution buffer well include an engaging portion, respectively. And the lysis buffer well, the wash buffer wells and the elution buffer well are arranged in the linear direction. The engaging portion has an arcuate wall or a polygonal wall. The driving unit and the moving frame are disposed on the base. The moving frame drives by the driving unit to move in the linear direction repeatedly. The syringe is disposed on the moving frame and moved with the moving frame.

Description

Method and device for automated nucleic acid extraction

The invention relates to a device and a method, in particular to an automatic nucleic acid extraction device and an automatic nucleic acid extraction method.

With the innovation of human genome sequencing technology, the advancement of biomedical analysis technology and the emergence of big data analysis tools, the era of Precision Medicine has arrived. Precision medicine is a customized medical model. It is based on human genome information, combined with proteome, metabolome and other related internal environment information, to formulate the best treatment plan for patients, in order to maximize the therapeutic effect and minimize the side effects.

Blood is a red opaque viscous liquid flowing in the blood vessels and heart of the human body. The main components are plasma, blood cells, and genetic material (chromosomes and genes). Among them, cell-free DNA (cell free DNA, cfDNA) is a free DNA fragment found in plasma, and circulating tumor DNA (circulating tumor DNA, ctDNA) fragment refers to a DNA fragment released from tumor in cfDNA.

Almost everyone's blood contains free DNA fragments (that is, cfDNA). They may be derived from apoptosis or necrosis of cells, or they may enter the blood through active release (violent exercise). However, the content of cfDNA in blood is very low. In 1 mL of plasma, the content of cfDNA is about 1 ng to 100 ng, and the content of ctDNA is lower, accounting for only 0.1% to 5% of cfDNA.

Studies have confirmed that the total amount of cfDNA in the peripheral blood of cancer patients is higher than that of healthy people. By this point, if the content of cfDNA increases, it can play a good role as a hint for tumor screening. Therefore, purification of a very small amount of cfDNA (a type of nucleic acid) from Liquid Biopsy is the first step in precision medicine.

Researchers in the field have been devoted to the development of devices and methods for extracting nucleic acids such as cfDNA or ctDNA. For example, CN 101684463 A discloses a method for quickly extracting nucleic acids from various micro clinical samples, which includes the following steps: (a) adding clinical samples to lysate and mixing; (b) aspirating clinical samples and flowing through filter membranes, The nucleic acid components in the sample are adsorbed on the filter membrane due to the specific adsorption of the membrane. The filtrate is a waste liquid with cell debris and protein; (c) Aspirate the cleaning solution, flow through the filter membrane, and wash the remaining protein on the filter membrane Or other components, discard the filtrate; (d) Aspirate the eluent and flow through the filter membrane to elute the nucleic acid components adsorbed on the membrane to obtain a nucleic acid aqueous solution containing no other impurities for nucleic acid amplification.

TW M477925 U discloses a sample extraction device including a suction part including a first upper part, an intermediate part communicating below the first upper part and having an inner diameter smaller than the inner diameter of the first upper part, and communicating under the intermediate part And a suction portion having an inner diameter smaller than the inner diameter of the middle portion, whereby the first upper portion or the middle portion cooperatively abuts against the outer periphery of the column member, and sucks a specific liquid to the column member through the suction portion; the The sample extraction device further includes: a column component; and a connecting component, thereby extracting the expected material from the sample.

TW M536238 U discloses a machine for automatically extracting nucleic acids, including: a machine bottom plate, a tray fixing frame on the plate, the tray fixing frame can move along a horizontal track; a bracket is vertically arranged above the machine bottom plate, And a vertical track; a vertical movement unit including a base plate, a base plate track is arranged above the base plate, a movement block is arranged along the base plate track, and a block is located under the movement block and locked One syringe fixing unit of the base plate is provided with a syringe erection on it; a piston pusher fixing unit is provided with a piston pusher erection, so that when the moving block moves up and down along the track of the substrate, the piston pusher is driven to make the needle The barrel generates positive pressure or negative pressure, and the base plate is arranged along the vertical track to drive the moving block and the syringe fixing unit to move up and down corresponding to the support.

TW 201412981 A discloses a method and device for extracting nucleic acid, which is a method of extracting nucleic acid by air pressure, and is connected to a device capable of applying positive or negative gas pressure at the upper end of the purification tube, which can be sucked/exhausted from the lower tip of the purification tube The body, cleaning solution and eluent can achieve the effect of easily extracting nucleic acid without centrifuge.

Although the aforementioned prior arts already exist, these prior arts still have the disadvantage that cfDNA or ctDNA cannot be extracted in large quantities. Therefore, if an automated nucleic acid extraction device capable of extracting a large amount of cfDNA or ctDNA to increase the yield can be developed, it will bring a breakthrough to precision medicine.

The object of the present invention is to provide an automated nucleic acid extraction device and automated nucleic acid extraction method. Compared with the prior art, the automated nucleic acid extraction device and method of the present invention can quickly and conveniently extract higher yields and higher concentrations of nucleic acids, such as cfDNA and ctDNA, from the specimen.

Therefore, the present invention provides an automated nucleic acid extraction device, which is characterized in that the automated nucleic acid extraction device includes a base, a cassette, a driving unit, a moving frame and a syringe. The seat body has a sample accommodating area, a column accommodating area, a cartridge accommodating area and a collection tube, and the sample accommodating area, the column accommodating area, the cartridge accommodating area and the collection tube are along a line Sexual orientation. The cassette is arranged in the cassette accommodating area. The cassette includes two parallel walls and at least two vertical walls. The parallel walls and the vertical walls together form a dissolution liquid tank, at least one washing liquid tank and an elution liquid tank, in which the dissolving liquid is formed The vertical walls of the tank, the washing liquid tank and the eluting liquid tank are respectively provided with an abutting portion, and the dissolving liquid tank, the washing liquid tank and the eluting liquid tank are also arranged in a linear direction, wherein the abutting portion has an arc-shaped wall Or a polygonal wall. The driving unit is disposed on the base. The moving frame is vertically arranged on the seat body, and driven by the driving unit to reciprocate in a linear direction. The syringe is set on the moving frame and moves with the moving frame.

In one embodiment, the sample containing area has a sample containing space and a binding buffer containing space.

In an embodiment, the column accommodation area further includes a sample row pipette accommodation space and a column row pipette accommodation space.

In one embodiment, the automated nucleic acid extraction device further includes a sample discharge pipette, which is movably disposed in the sample discharge pipette receiving space.

In one embodiment, the automated nucleic acid extraction device further includes a pipe column pipette, which is movably disposed in the pipe column pipette receiving space.

In one embodiment, the syringe is detachably connected to the sample discharge pipette or the column discharge pipette.

In an embodiment, the sample accommodating space is used to accommodate a biological sample and a dissolution buffer to perform a dissolution reaction, and the binding buffer accommodating space is used to accommodate a binding buffer and a dissolution product to perform a binding reaction and dissolve The liquid tank is used to contain the dissolution buffer, defoamer and reaction residue. The washing liquid tanks are used to contain the washing buffer. The elution liquid tanks are used to contain the elution buffer. The sample The suction pipe accommodating space is used for accommodating the sample discharge pipette, and the column pipe suction pipe accommodating space is used for accommodating the pipe column suction pipe.

In one embodiment, the arc angle of the arc-shaped wall is greater than or equal to 90 degrees.

In an embodiment, the vertical wall between the washing liquid tank and the eluting liquid tank has a complete hollow cylindrical structure, forming a filter material accommodating space for accommodating a filter material.

In one embodiment, the polygonal wall includes at least two support walls, and the support walls include an angle of less than 180 degrees with each other.

In one embodiment, a recess is formed at the bottom of the eluent bath.

In one embodiment, the cassette further includes a snap fastener for detachably fastening the cassette to the cassette receiving area.

The present invention also provides an automated nucleic acid extraction method, characterized in that the automated nucleic acid extraction method uses the automated nucleic acid extraction device as described above, which includes the following steps: using a syringe and a column row pipette to draw a binding buffer container After mixing the reactants in the space, the nucleic acids in the reactants are bound to a membrane in the pipette pipette, and the reaction residue is discharged to the cassette, so that the nucleic acids in the reaction residue are bound to the membrane; The cylinder is matched with the column row suction pipe to suck the washing buffer in at least one washing liquid tank through the membrane, and then the washing buffer is discharged through the membrane with the syringe and the column row pipette; and the syringe is matched with the column row pipette through the membrane The chip absorbs the elution buffer in the elution liquid tank, and then uses a syringe and a column row suction tube to discharge the elution buffer with nucleic acid to the collection tube through the membrane.

In one embodiment, the automated nucleic acid extraction method is characterized in that, in the step of discharging the reaction residue to the cassette, and the step of discharging the washing buffer with a syringe and a pipe column pipette through the membrane, the tube The abutment portion of the column exhaust pipette abuts on the abutment portion of the cartridge, and then the reaction residue or the washing buffer is discharged.

In one embodiment, the automated nucleic acid extraction method is characterized in that after the abutment portion of the column discharge pipette is abutted against the abutment portion of the cartridge, and then the reaction residue or washing buffer is discharged, The method further includes the following steps: abutting the abutment portion of the column row suction tube against the abutment portion of the cassette, and moving the syringe up and down in a direction perpendicular to the linear direction by less than or equal to 5 mm.

To sum up, the effect of the present invention is: by the arrangement of the parallel wall and the vertical wall of the cassette, the dissolution liquid tank, the washing liquid tank and the elution liquid tank formed can accommodate a large volume of solution; and by The washing liquid tank and the eluent liquid tank are respectively provided with abutting parts. After the syringe discharges a large volume of solution, the sample discharge pipe or the column discharge pipe can be prevented from falling off from the syringe and separating from the syringe. In addition, the design of the cassette and the accommodating space arranged in the linear direction and the reciprocating movement of the moving frame and the syringe in the linear direction can achieve the effect of automatically performing nucleic acid extraction in a linear direction to avoid sample contamination and improve extraction efficiency. Therefore, the automated nucleic acid extraction device of the present invention can indeed quickly and conveniently extract higher yields and higher concentrations of nucleic acids, such as cfDNA and ctDNA, from the specimen. Furthermore, compared with the conventional technology, it is linearly moved by the cassette and the accommodating space (that is, the cassette moves and the syringe does not move relative to the desktop), while the present invention is carried out by the moving frame and the syringe It moves linearly (that is, the syringe moves and the cassette does not move relative to the desktop). Therefore, the automated nucleic acid extraction device of the present invention has a small operating space, which is about twice the length of the cassette and the accommodating space. The actuating space for linear movement by the cassette and the accommodating space is about 2 to 3 times the length of the cassette and the accommodating space. Therefore, the automated nucleic acid extraction device of the present invention has a smaller volume, which can save the user's use space.

The preferred embodiments of the automated nucleic acid extraction device according to the present invention will be described below with reference to related drawings, wherein the same elements will be described with the same reference symbols.

The automated nucleic acid extraction device of the present invention can quickly and conveniently extract nucleic acids of higher yield and higher concentration, such as cfDNA and ctDNA, from the specimen. In particular, the specimen includes, but is not limited to: blood, plasma, urine, saliva, tissue fluid, and tissue. The structure and features of the automated nucleic acid extraction device will be described below with examples.

Please refer to FIGS. 1 and 2, wherein FIG. 1 is a combined schematic diagram of a preferred embodiment of the automated nucleic acid extraction device of the present invention, and FIG. 2 is a side view of a preferred embodiment of the automated nucleic acid extraction device of the present invention.

As shown in FIGS. 1 and 2, the automated nucleic acid extraction device includes a base B, a cassette 31, a driving unit M, a moving frame 4 and a syringe 5.

The seat body B has a sample accommodating area 1, a column accommodating area 2, and a cartridge accommodating area 3. The sample accommodating area 1 has a sample accommodating space 11 and a binding buffer accommodating space 12, And the sample accommodating area 1, the column accommodating area 2 and the cassette accommodating area 3 are arranged along a linear direction L1. In particular, the order in which the sample accommodating area 1, the column accommodating area 2 and the cartridge accommodating area 3 are arranged along the linear direction L1 (from right to left along the drawing direction, the same below) can be, for example, but not limited to, the first sample content Placement area 1, column accommodation area 2 and then cartridge accommodation area 3; first column accommodation area 2, cartridge accommodation area 3 and then sample accommodation area 1; first cartridge accommodation area 3 and sample accommodation Zone 1 is followed by column accommodating zone 2; or cartridge accommodating zone 3 first, column accommodating zone 2 and then sample accommodating zone 1.

Referring to FIG. 2 and FIG. 6A, the cassette 31 is disposed in the cassette accommodating area 3. The cassette 31 includes two parallel walls 31a and at least two vertical walls 31b. The parallel walls 31a and the vertical walls 31b together form a The dissolving liquid tank 311, at least one washing liquid tank 312 and an eluting liquid tank 313, wherein the vertical walls 31b forming the dissolving liquid tank 311, the washing liquid tank 312 and the eluting liquid tank 313 are respectively provided with abutting portions 314 And the dissolving liquid tank 311, the washing liquid tank 312 and the eluting liquid tank 313 are also arranged along the linear direction L1. In this embodiment, the number of vertical walls 31b is nine, and together with the parallel walls 31a, two dissolution liquid tanks 311, four washing liquid tanks 312, and two elution liquid tanks 313 are formed. However, the vertical walls 31b, dissolving The number of the liquid tank 311, the washing liquid tank 312 and the elution liquid tank 313 can be adjusted according to the actual needs of the user (for example, if the sample is a tissue that is difficult to extract, the dissolving liquid tank 311 and the elution liquid tank 313 Can be increased), the present invention is not limited. In addition, by disposing the parallel wall 31a and the vertical wall 31b of the cassette 31, the dissolution liquid tank 311, the washing liquid tank 312, and the elution liquid tank 313 formed therein can accommodate a large volume of buffer solution (dissolving buffer, washing Buffer and/or elution buffer), such as but not limited to more than 2 ml, more than 5 ml, or more than 10 ml.

1 and 2 again, in this embodiment, the driving unit M is disposed on the base B, and the moving frame 4 is vertically disposed on the base B, and is driven by the driving unit M to reciprocate along the linear direction L1. The syringe 5 is provided on the moving frame 4 and moves with the moving frame 4. In particular, although the driving unit M is shown as a transmission roller belt, the driving unit M may also be other driving devices, such as a linear module or other device that can drive the linear movement of the moving frame 4, and the present invention is not limited.

Please refer to FIGS. 1, 2 and 4. In this embodiment, the column receiving area 2 further includes a sample discharge pipe receiving space 21 and a column discharge pipe receiving space 22.

Please refer to FIG. 4. In this embodiment, the automated nucleic acid extraction device further includes a sample discharge pipette 6, which is movably disposed in the sample discharge pipette accommodating space 21. In this embodiment, the automated nucleic acid extraction device further includes a column row pipette 7, which is movably disposed in the column row pipette receiving space 22. In particular, the syringe 5 is detachably connected to the sample discharge pipette 6 or the column discharge pipette 7.

Please refer to FIG. 1 to FIG. 3A, the automated nucleic acid extraction device further includes a collection tube 8, arranged along the linear direction L1 with the cassette 31 and the accommodating regions. In a better case, the collection tube 8 may be disposed adjacent to the cassette 31 (but the invention is not limited to this), because the automated nucleic acid extraction method is to extract along the linear direction L1, and the eluate is discharged in the last step Into the collection tube 8, the design of the collection tube 8 arranged adjacent to the cassette 31 and arranged in the linear direction L1 is consistent with the moving direction of the mobile rack 4, which can avoid the mobile rack 4 in the process of automated nucleic acid extraction, reciprocating through the collection Above the tube 8, causing cross contamination.

In the present embodiment, the sample accommodating space 11 is used for accommodating a biological sample and a lysis buffer to perform a lysis reaction. In this embodiment, the biological sample includes, but is not limited to: blood, plasma, urine, saliva, tissue fluid, and tissue. The binding buffer storage space 12 is used to store a binding buffer and a lysate to perform a binding reaction. The dissolving liquid tank 311 is used for containing a dissolving buffer (lysis buffer), a defoaming agent (defoaming agent), and reaction residues. The washing liquid tanks 312 are used for containing washing buffer (wash buffer). The elution liquid tank 313 is used for accommodating elution buffer. The sample discharge pipette accommodating space 21 is used for accommodating the sample discharge pipette 6. The pipe row suction pipe accommodating space 22 is used to house the pipe row suction pipe 7. In this embodiment, the sample accommodating space 11, the binding buffer accommodating space 12, the dissolving liquid tank 311, and the washing liquid tank 312 can accommodate, for example, but not limited to, 30 mL of specimen or buffer, so compared to the prior art (Usually 2 mL) has a higher sample capacity. The increase in the volume of the dissolution buffer can improve the dissolution reaction of the sample to increase the concentration of nucleic acid; the increase in the volume of the washing buffer can wash the solution remaining in the pipette 7 To increase the concentration and purity of the nucleic acids extracted in the subsequent steps. In this embodiment, the number of the dissolving liquid tank, the washing liquid tank, and the eluting liquid tank can be adjusted according to the actual needs of the user, and the present invention is not limited.

Please refer to FIG. 3C, a diaphragm 74 is disposed inside the column row suction pipe 7. The material of the membrane 74 is, for example, but not limited to, a silicone membrane, which is positively charged, and the nucleic acid contained in the specimen may be negatively charged during the nucleic acid extraction process. The characteristic can make the nucleic acid attach to the membrane 74, and then extract the nucleic acid attached to the membrane 74 through a subsequent procedure.

In this embodiment, the seat body B may further include at least one heating element H, which is disposed below the sample accommodating space 11 and/or the column exhaust pipette accommodating space 22. Here, when the sample accommodating space 11 is provided with When heating element H, it can promote the dissolution reaction between the biological sample and the dissolution buffer, so that the biological sample can be dissolved more completely, and then increase the concentration of nucleic acid extracted by the subsequent procedure; when the column row suction tube accommodating space 22 is provided with When the heating element H is heated, it can promote the volatilization of the residual solvent on the membrane 74 of the column exhaust pipette 7, thereby increasing the concentration and purity of the nucleic acid extracted by the subsequent procedure. In particular, when the nucleic acid to be extracted is ribonucleic acid (RNA), the heating element H below the sample accommodating space 11 may not be turned on (that is, it is not heated during the dissolution reaction), so as to prevent the ribonucleic acid from being decomposed and affected The concentration of ribonucleic acid after extraction.

3A and 3B, a concave portion f is formed at the bottom of the elution liquid tank 313. With this design, the sample can use a smaller amount of elution buffer when eluting, thereby increasing the concentration of nucleic acid extraction. In particular, although the figure shows that only the eluent tank 313 adjacent to the collecting tube 8 has a recess f, however, other eluent tanks 313 may also have a recess f, and the present invention is not limited. In addition, the volume of the elution buffer is, for example but not limited to, 1 ml, 500 μl, 200 μl, 100 μl, 50 μl, 30 μl, etc., which can be adjusted according to the user's needs, and the invention is not limited .

Referring to FIGS. 6A to 6C, the cassette 31 further includes an elastic fastener 316 for detachably fastening the cassette 31 to the cassette accommodating area 3. In particular, please refer to FIGS. 8A and 8B. For ease of understanding, FIG. 8A only shows a partial structure in the automated nucleic acid extraction device of FIG. 1, and FIG. 8B is an exploded view of FIG. 8A. The automated nucleic acid extraction device of this embodiment may further include an iron frame I, which is detachably disposed on the base B. The plurality of cassettes 31 and the collection tube 8 can be set on the iron frame I by the user first, and then set on the base B together with the iron frame I, so as to facilitate the user to set up the plurality of cassettes 31 and the collection tube 8. In addition, the user can also set the iron frame I on the seat body B, and then set the cassette 31 and the collection tube 8. The present invention is not limited. Furthermore, after the nucleic acid is extracted using an automated nucleic acid extraction device, the iron frame I can be removed from the seat body B to facilitate the user to take out multiple cassettes 31 and collection tubes 8 at a time. In particular, the iron frame I may include two handles I1 to facilitate the user to take out the iron frame I from the seat body B or put the iron frame I into the seat body B.

Referring to FIG. 4, the sample discharge pipette 6 includes an assembly portion 61 and a tip portion 62 connected to the assembly portion 61. The pipe column suction pipe 7 includes an assembly portion 71, an abutment portion 72, and a tip portion 73 connected to the abutment portion 72, wherein the length D2 of the tip portion 73 is less than the depth D1 of the collection tube 8 (as shown in FIG. 3C) Show). With this design, when the tip portion 73 equipped with nucleic acid enters the collection tube 8, the force generated by the nucleic acid-containing sample after being discharged from the tip portion 73 is too large to cause the sample to splash out of the collection tube 8 To increase the yield of nucleic acid extraction. In the preferred embodiment, the assembling portion 61 and the tip portion 62 are integrally formed; the assembling portion 71, the abutting portion 72 and the tip portion 73 are integrally formed. Alternatively, the assembling portion 61 and the tip portion 62 can also be detached from each other, and the assembling portion 71, the abutting portion 72 and the tip portion 73 can also be detached from each other, and the present invention is not limited. In particular, the sample discharge pipette 6 may have the same configuration as the column discharge pipette 7, the only difference between the two is that the column discharge pipette 7 has a diaphragm 74, and the sample discharge pipette 6 does not have the membrane 74 (ie, The sample discharge pipette 6 can also have an assembly part, an abutment part and a tip part, and only a single mold can be used to manufacture the sample discharge pipette 6 and the column discharge pipette 7, and each sample is prepared by taking out or putting in the diaphragm 74 Discharge suction tube 6 and column discharge suction tube 7).

Please refer to FIG. 3B to FIG. 5B, the bottom of the syringe 5 further includes a connector 51, wherein the syringe 5 is connected to the assembling parts 61, 71 through the connector 51. In detail, the joint 51 cooperates with the structure of the fitting portion 61 of the sample discharge pipette 6 or the fitting portion 71 of the column discharge pipette 7 to tightly connect the syringe 5 and the fitting portions 61 and 71. In particular, the syringe 5 and the connector 51 may be integrally formed.

Please refer to FIGS. 1 and 7A to 7B. FIG. 7A is an enlarged view of the area A in FIG. 1. The moving frame 4 includes a ejection plate 41, and a spring mechanism 42 is provided on both sides of the moving frame 4 respectively. The spring mechanism 42 drives the ejection plate 41 to control the assembling parts 61 and 71 to be detached from the bottom of the syringe 5. In detail, as shown in FIG. 7B, the spring mechanism 42 moves in the L3 direction and drives the ejector plate 41 to move in the L4 direction to separate the sample discharge pipette 6 or the column discharge pipette 7 from the syringe 5.

Please refer to FIG. 7C in conjunction with FIG. 7A, the mobile rack 4 may further include at least one syringe fixing member 43 to fix the syringe 5 on the mobile rack 4. The syringe fixing member 43 may further include a syringe locking groove 431 and at least one concave hole 432, and an elastic member R may be disposed in the concave hole 432. As shown in FIG. 7A, the syringe 5 is locked in the syringe locking grooves 431 of the two syringe fixing members 43, and the elastic member R provided in the concave hole 432 fixes the syringe 5 better in Syringe clamping groove 431. In particular, the number of the syringe fixing member 43, the concave hole 432 and the elastic member R can be adjusted according to the needs of the user, as long as the syringe 5 can be firmly fixed in the syringe clamping groove 431, the present invention does not No restrictions.

Please refer to FIG. 1, FIG. 5A, FIG. 5B and FIG. 6A, wherein FIG. 5A and FIG. 5B are partial schematic diagrams of a preferred embodiment of the automated nucleic acid extraction device of the present invention, and FIG. 6A is a cassette of the automated nucleic acid extraction device of the present invention. A top view of a preferred embodiment. As shown in FIGS. 5A, 5B, and 6A, the abutting portion 314 has an arc-shaped wall 3141 and the arc angle θ of the arc-shaped wall 3141 is greater than or equal to 90 degrees. Preferably, the arc angle θ may be 90 degrees; preferably, the arc angle θ may be 120 degrees; preferably, the arc angle θ may be 180 degrees; preferably, the arc angle θ may be 270 degrees, as long as it can The suction pipe 7 for supplying the pipe column only needs to abut. Specifically, as shown in FIG. 6A, the vertical wall 31b has an arc-shaped wall 3141, and the vertical wall 31b is connected to the parallel wall 31a, that is, both ends of the arc-shaped wall 3141 are not directly connected to the parallel wall 31a.

Please refer to FIGS. 1, 5C and 6C, wherein FIG. 5C is a partial schematic view of a preferred embodiment of the automated nucleic acid extraction device of the present invention, and FIG. 6C is another preferred embodiment of the cassette of the automated nucleic acid extraction device of the present invention. Top view. As shown in FIGS. 5C and 6C, the abutment portion 314 has a polygonal wall. The polygonal wall may include at least two support walls. Here, a structure including a first support wall 3142 and a second support wall 3143 will be used for explanation. The first supporting wall 3142 and the second supporting wall 3143 have an included angle e smaller than 180 degrees. Preferably, the included angle e may be 150 degrees; preferably, the included angle e may be 120 degrees; preferably, the included angle e may be 90 degrees; preferably, the included angle e may be 45 degrees, as long as the pipe row can be supplied The straw 7 only needs to abut and stand. In particular, please refer to FIGS. 6D to 6H to illustrate different embodiments of the abutment portion of the cassette of the automated nucleic acid extraction device of the present invention. The abutment portion 314 is, for example but not limited to, an arc-shaped wall 3141 (as shown in FIG. 6E) or a polygonal wall (as shown in FIGS. 6D, 6F to 6H), and the polygonal wall has, for example but not limited to, two support walls and three supports Wall, four support walls, five support walls, six support walls or a structure larger than six support walls, each support wall has an angle between them, the number of support walls and the angle of the included angle are not limited, as long as it can supply the pipe row The straw 7 only needs to abut and stand. In particular, as shown in FIG. 6D, both ends of the polygonal wall of the abutment portion 314 may directly contact the parallel wall 31a, however, as shown in FIGS. 6F to 6H, the polygonal wall of the abutment portion 314 of the vertical wall 31b The two ends are not directly connected to the parallel wall 31a, but are connected by the vertical wall 31b and the parallel wall 31a, which is not limited herein.

With the design of the abutment portion 314 described above, the abutment portion 72 of the column discharge pipette 7 abuts on the arc-shaped wall 3141 of the abutment portion 314 during the discharge of liquid (eg, reactants, washing buffer, etc.) Or between the first support wall 3142 and the second support wall 3143, so that when the syringe 5 discharges the liquid, the column suction pipe 7 will not fall off and separate from the syringe 5 due to excessive pressure. Even if the column discharge pipe 7 is released when the syringe 5 is discharged after the liquid is discharged, the abutment portion 72 of the column discharge pipe 7 can be made to stand against the stand by the design of the abutment portion 314 On the abutment portion 314 without being completely separated from the syringe 5, and then the syringe 5 and the column row suction tube 7 can be tightly closed again by a subsequent step of moving the syringe 5 up and down in a direction L2 perpendicular to the linear direction L1 Cooperate to facilitate subsequent extraction steps.

Please refer to FIGS. 1, 6A and 6B, wherein FIG. 6B is a top view of another preferred embodiment of the cassette of the automated nucleic acid extraction device of the present invention. The vertical wall 31b of the cartridge of FIG. 6B between the washing liquid tank 312 and the eluting liquid tank 313 has a complete hollow cylindrical structure, forming a filter material accommodating space 315 for accommodating a filter material. In detail, when the biological sample is tissue or other samples with impurities, the filter material is used to filter the lysate after dissolution to fix the tissue fragments or impurities on the outside of the filter material, to clarify the dissolution (without tissue fragments or impurities) The product can be sucked into the sample discharge pipette 6 to prevent tissue fragments or impurities from plugging the tip portion 62 of the sample discharge pipette 6. With this design, tissue samples can be directly loaded onto the machine without the need to centrifuge outside the machine to obtain a clear lysate and then be loaded onto the machine. Therefore, the step of lysing the tissue sample can be completed on the automated nucleic acid extraction device of the present invention, so that the tissue sample can also achieve the effect of fully automatic nucleic acid purification. In particular, when ribonucleic acid (RNA) in the specimen is to be extracted, the filter material accommodating space 315 can also accommodate enzymes required in the middle of the extraction process, such as deoxyribonucleic acid degrading enzyme (DNase), during the extraction process The DNA in the sample is removed from the sample, and the DNA-decomposing enzyme (DNase) can also be removed by subsequent washing steps, so that DNA can be obtained after the extraction is completed. Ribonucleic acid (RNA).

Please refer to FIG. 1 and FIG. 2, in this experimental example, the moving frame 4 and the syringe 5 move linearly L1 (that is, the syringe moves relative to the desktop and the cassette does not move), so the depth of the machine is only the length of the base B D3 plus the thickness D4 of the mobile rack 4 (D3 + D4). However, the conventional technique is that the seat body B moves linearly. In order for the syringe to absorb the solution in each storage space, the depth of the machine must be at least twice the length D3 of the seat body B plus the moving frame 4 The thickness of D4 (D3 × 2 + D4). For example, the length D3 of the base B of the automated nucleic acid extraction device of this experimental example is, for example, but not limited to 30 cm, the thickness D4 of the moving frame 4 is, for example, but not limited to 20 cm, and the depth of the machine, for example, but not limited to about 50 cm, Therefore, the use space of the user can be saved.

Please refer to FIG. 9 together with FIGS. 1 to 3C, which is a preferred embodiment of the automated nucleic acid extraction method of the present invention. The automated nucleic acid extraction method of the present invention is performed using the automated nucleic acid extraction device as described above. The bottom of the is provided with a driving roller belt, which will drive the moving frame 4 to move in the linear direction L1. In the preferred embodiment, the automated nucleic acid extraction method includes the following steps: Step S05 is to use the syringe 5 and the column pipette 7 to suck the mixed reactant in the binding buffer storage space to bind the nucleic acid in the reactant A membrane 74 in the pipette pipette 7 is discharged, and the reaction residue is discharged to the cassette 31, so that the nucleic acid in the reaction residue is bound to the membrane 74. Then, proceed to step S06, use the syringe 5 and the column row suction pipe 7 to suck the washing buffer in at least one washing liquid tank through the membrane 74, and then use the syringe 5 and the column row suction pipe 7 to pass the membrane 74 through the membrane 74 discharge. Next, proceed to step S08 to draw the elution buffer in the eluent bath through the syringe 74 with the syringe 5 and the column exhaust pipette 7, and then use the syringe 5 with the column exhaust pipette 7 to pass the nucleic acid The elution buffer is drained to the collection tube 8. Among them, a step S07 may be arranged between steps S06 and S08 to move the column discharge pipette 7 to the column discharge pipette accommodating space 22 for heating, so as to minimize the residual amount of the washing buffer on the membrane 74. The above steps S05 to S08 are performed by the automated nucleic acid extraction device. The dotted line parts shown in FIG. 9 (steps S01 to S04) can be performed manually by the user outside the automated nucleic acid extraction device, or all by the automated nucleic acid Extraction device. For example, the user can first dissolve and centrifuge the sample, then mix the supernatant with the binding buffer, and then add the binding buffer to the storage space, and the automated nucleic acid extraction device can perform the subsequent steps; or, the user can first After the sample is dissolved and centrifuged, the supernatant is added to the binding buffer storage space and mixed with the binding buffer, and the automated nucleic acid extraction device performs the next steps; or, the user can first dissolve the sample and centrifuge, then add the dissolved product to the dissolving solution In the tank, subsequent steps are performed by an automated nucleic acid extraction device, and the invention is not limited.

This embodiment is used to explain the situation where steps S01 to S08 are all performed by an automated nucleic acid extraction device. In steps S01 to S08, the automated nucleic acid extraction device and the moving frame 4 (with the syringe 5) move to a specific direction along the linear direction L1 Perform the preceding steps at the location, described in detail as follows: before step S01, the syringe 5 provided on the moving rack 4 is moved to the sample discharge pipette accommodating space 21 of the column accommodating area 2 in the linear direction L1, so that the syringe 5 is The sample discharge pipette 6 accommodated in the sample discharge pipette storage space 21 is connected to take out the sample discharge pipette 6 from the sample discharge pipette storage space 21. Next, the sample discharge pipette 6 connected to the syringe 5 moves to the cassette 31 of the cassette accommodating area 3 in the linear direction L1. Then, in step S01, the dissolution buffer solution is sucked from the dissolution solution tank 311 of the cassette 31, and then moved to the sample storage area 1. Next, step S02 is performed to discharge the dissolution buffer to the sample accommodating space 11 of the sample accommodating area 1 and perform mixing. Then, step S03 is performed, the mixed dissolution product in the sample accommodation space 11 is sucked, and moved to the binding buffer accommodation space 12 of the sample accommodation area 1. Then, step S04 is performed, and the dissolved product is discharged to the binding buffer storage space 12 and mixed with the binding buffer to perform a binding reaction. Next, before performing step S05, the syringe 5 and the sample discharge pipette 6 are moved to the sample discharge pipette accommodating space 21, and the ejector plate 41 is driven by the spring mechanism 42 to move the sample discharge pipette 6 from the syringe 5 to the sample The suction tube accommodating space 21, and then move the syringe 5 in the linear direction L1 to the column excretion tube accommodating space 22 of the column accommodating area 2, so that the syringe 5 and the The pipe column suction pipe 7 is connected to take out the pipe column suction pipe 7 provided with the diaphragm 74 from the pipe column accommodating area 2. After that, the pipe column suction pipe 7 connected to the syringe 5 moves to the binding buffer storage space 12 in the linear direction L1. Next, step S05 is performed, and the formed reactant is sucked by the syringe 5 and the column exhaust pipette 7 so that the reactant passes through the membrane 74 and the nucleic acid contained in the reactant is attached to the membrane 74. Next, the syringe 5 moves to the cassette 31 in the linear direction L1. Then, the reactant is passed through the membrane 74 again in the direction of gravity to attach the contained nucleic acid to the membrane 74, and the reaction residue is discharged to the cassette 31 in the direction of gravity, and the reaction residue is passed through the membrane twice to ensure the reaction The nucleic acid in the substance can be attached to the membrane 74. Next, in step S06, the column discharge pipette 7 with nucleic acid moves with the syringe 5 and the moving rack 4 in the linear direction L1 to the washing liquid tank 312 containing the washing buffer, and then by sucking and discharging the washing buffer For washing, the washing buffer is passed through the membrane 74 twice to wash away the reaction residues remaining in the membrane 74. After washing, the column discharge pipette 7 with nucleic acid can be moved to the column discharge pipette accommodating space 22 along the linear direction L1 along with the syringe 5 and the moving frame 4 to stay the column discharge pipette 7 in the column row The pipette accommodating space 22 is heated by the heating element H to volatilize the liquid (for example, washing buffer) remaining on the membrane 74 of the pipette pipette 7 to fully dry it (ie, step S07). Finally, the column discharge pipette 7 with nucleic acid is moved along with the syringe 5 and the moving frame 4 in the linear direction L1 to the elution liquid tank 313 containing elution buffer and proceeds to step S08, by eluting and discharging the elution The buffer is used for elution, thereby obtaining an eluate (elution buffer with nucleic acid). After that, the column row suction pipe 7 sucks the obtained eluate, and then moves to the collection tube 8 in the linear direction L1, and then discharges the eluate to the collection tube 8. In particular, the heating time of the heating element H is, for example but not limited to, 30 seconds, 1 minute, 3 minutes, and 5 minutes, as long as the liquid remaining on the membrane 74 of the column exhaust pipette 7 is volatilized and sufficiently dried, the present invention does not No restrictions.

In the preferred embodiment, in the step of discharging the reaction residue to the cassette 31 (step S05), and the step of discharging the washing buffer with the syringe 5 and the column exhaust pipette 7 through the diaphragm 74 (step S06), the abutment portion 72 of the column discharge pipette 7 is abutted on the abutment portion 314 of the cassette 31, and then the reaction residue or the washing buffer is discharged. With the design of the abutment portion 314, when the syringe 5 discharges the liquid, the pipe column suction pipe 7 is abutted against the abutment portion 314, thereby preventing the pipe column suction pipe 7 from discharging during the process of discharging the liquid When the pressure is too large, it comes off the syringe 5 and separates. Even if the column discharge pipe 7 is released when the syringe 5 is discharged after the liquid is discharged, the abutment section 72 of the column discharge pipe 7 can be made to stand against the abutment section 314 by the design of the abutment section 314 On the other hand, without being completely separated from the syringe 5, the syringe 5 and the column suction tube 7 can be tightly matched again through the subsequent steps.

In the preferred embodiment, the abutment portion 72 of the column discharge pipette 7 is abutted on the abutment portion 314 of the cassette 31, and then the reaction residue or the washing buffer is discharged (step S05, S06) After that, it further includes the following steps: abutting the abutting portion 72 of the column row suction tube 7 against the abutting portion 314 of the cassette 31, and moving the syringe 5 up and down in a direction L2 perpendicular to the linear direction L1 by less than Or equal to 5 mm. By this step, the column row suction tube 7 and the syringe 5 can be tightly connected to avoid the column row suction tube 7 from falling off. Preferably, the syringe 5 can move up and down along the direction L2 by 5 mm; preferably, the syringe 5 can move up and down along the direction L2 by 4.5 mm; preferably, the syringe 5 can move up and down along the direction L2 by 3 mm to move The column row suction pipe 7 and the syringe 5 are tightly connected again.

In this preferred embodiment, the number of washing liquid tanks 312 can be adjusted according to the actual needs of users, and the invention is not limited. In detail, if the number of washing liquid tanks 312 is greater than one, before moving the column row suction pipe 7 to the column row suction pipe accommodating space 22 for heating, the syringe 5 and the column row suction pipe 7 may be repeated through the membrane at least once The step of sucking the washing buffer solution in the at least one washing solution tank by the sheet 74, and then discharging the washing buffer solution through the membrane 74 with the syringe 5 and the column exhaust pipette 7. That is, the column discharge pipette 7 with nucleic acid moves along the syringe 5 and the moving rack 4 in the linear direction L1 to the washing liquid tank 312 containing the washing buffer, and the washing buffer is repeatedly sucked and discharged for washing The steps may be performed in different washing liquid tanks 312, and the number of washing steps is determined according to the number of washing liquid tanks 312.

In the preferred embodiment, the steps of discharging the dissolution buffer to the sample accommodation space 11 and mixing (step S02), and the step of sucking the mixed dissolution product in the sample accommodation space 11 (step S03) are It also includes the steps of moving to the filter material accommodating space 315, combining the sample discharge pipe 6 matched with the syringe 5 with the filter material in the filter material accommodating space 315; and dissolving the mixed product in the suction sample accommodating space 11 After the step (step S03), it further includes the steps of moving the sample discharge pipe 6 and the filter material to the dissolving liquid tank 311 and reciprocating in the linear direction in the dissolving liquid tank 311. In detail, when the biological sample is tissue or other samples with impurities, the filter material is used to filter the dissolved lysate to fix the tissue fragments or impurities on the outside of the filter material, and then proceed to step S03 to clarify (without tissue Fragments or impurities) are dissolved into the syringe 5. Then, the sample discharge pipe 6 and the filter material are moved to the dissolving liquid tank 311 and reciprocate in the linear direction in the dissolving liquid tank 311 to clean the impurities stuck on the outside of the filter material, and then the dissolved product in the syringe 5 is discharged To the binding buffer storage space 12 to perform the binding reaction, to prevent tissue fragments or impurities from affecting the effect of the binding reaction and thus affecting the extraction efficiency. That is, when the biological sample is a tissue or other sample with impurities, the foregoing steps may be added.

In the preferred embodiment, the sample accommodating space 11, the combined buffer accommodating space 12, the sample exhaust pipette accommodating space 21, the column exhaust pipette accommodating space 22, the dissolution liquid tank 311, the washing liquid tank 312, the washing The number of the liquid extraction tank 313, the abutment portion 314, the syringe 5, the sample discharge pipette 6, the column discharge pipette 7, the collection pipe 8 and the joint 51 can be adjusted according to the actual needs of the user, and the invention is not limited. In particular, the cassette 31, the sample accommodation space 11, the combined buffer accommodation space 12, the sample discharge pipette accommodation space 21, the column discharge pipette accommodation space 22, the syringe 5 and the collection tube 8 are along the linear direction L1 Arrangement, the order of arrangement is not limited here.

In summary, the automatic nucleic acid extraction device of the present invention is designed with abutment portions 314 for each dissolution liquid tank 311, washing liquid tank 312, and adjacent elution liquid tank 313. When the user performs automated nucleic acid extraction It can prevent the reactants from splashing out of the dissolving liquid tank 311, the washing liquid tank 312 or the eluting liquid tank 313, and can also prevent the column row suction pipe 7 from loosening and falling off. In addition, by designing that the cassette 31 and the accommodating space are arranged in the linear direction L1 and the moving frame 4 and the syringe 5 are repeatedly moved in the linear direction L1, the effect of automatically performing nucleic acid extraction in a linear direction L1 can be achieved to avoid specimens Pollution and increase extraction efficiency. Therefore, the automated nucleic acid extraction device of the present invention can indeed quickly and conveniently extract higher yields and higher concentrations of nucleic acids, such as cfDNA and ctDNA, from the specimen.

The above is only exemplary, and not restrictive. Any equivalent modifications or changes made without departing from the spirit and scope of the present invention should be included in the appended patent application.

1: Sample storage area 11: Sample storage space 12: Binding buffer storage space 2: Column storage area 21: Sample discharge pipette storage space 22: Column discharge pipette storage space 3: Cassette storage Zone 31: Cartridge 31a: Parallel wall 31b: Vertical wall 311: Dissolving liquid tank 312: Washing liquid tank 313: Elution liquid tank 314: Abutment 3141: Arc-shaped wall 3142: First support wall 3143: Second Support wall 315: filter material accommodating space 316: elastic fastener 4: moving frame 41: ejection plate 42: spring mechanism 43: syringe fixing piece 431: syringe locking groove 432: concave hole 5: syringe 51: joint 6: Sample discharge pipettes 61, 71: Assembly parts 62, 73: Tip part 7: Pipe column discharge pipette 72: Abutment part 74: Diaphragm 8: Collection tube A: Area B: Seat body D1: Depth D2, D3: Length D4: Thickness e: Angle f: Recess H: Heating element I: Iron frame I1: Handle L1, L2, L3, L4: Linear direction M: Drive unit R: Elastic element θ: Arc angle S01～S08: Step

FIG. 1 is a schematic assembly view of a preferred embodiment of an automated nucleic acid extraction device of the present invention. 2 is a side view of a preferred embodiment of the automated nucleic acid extraction device of the present invention. 3A is a partial schematic diagram of a preferred embodiment of an automated nucleic acid extraction device of the present invention. 3B is a partial schematic diagram of a preferred embodiment of the automated nucleic acid extraction device of the present invention. 3C is a partial schematic diagram of a preferred embodiment of the automated nucleic acid extraction device of the present invention. 4 is a partial schematic diagram of a preferred embodiment of the automated nucleic acid extraction device of the present invention. 5A is a partial schematic diagram of a preferred embodiment of an automated nucleic acid extraction device of the present invention. 5B is a partial schematic view of a preferred embodiment of the automated nucleic acid extraction device of the present invention. 5C is a partial schematic diagram of a preferred embodiment of the automated nucleic acid extraction device of the present invention. 6A is a top view of a preferred embodiment of the cassette of the automated nucleic acid extraction device of the present invention. 6B is a top view of another preferred embodiment of the cassette of the automated nucleic acid extraction device of the present invention. 6C is a top view of another preferred embodiment of the cassette of the automated nucleic acid extraction device of the present invention. 6D to 6H are schematic diagrams of different embodiments of the abutment portion of the cassette of the automated nucleic acid extraction device of the present invention. 7A is a partial enlarged view of the automated nucleic acid extraction device of FIG. 7B is a schematic diagram of the operation of the automated nucleic acid extraction device of FIG. 7A. 7C is another schematic diagram of the automated nucleic acid extraction device of FIG. 7A. 8A is a partial schematic diagram of the automated nucleic acid extraction device of FIG. 1A. 8B is an exploded schematic view of the automated nucleic acid extraction device of FIG. 8A. 9 is a flowchart of a preferred embodiment of the automated nucleic acid extraction method of the present invention.

1: Sample storage area 11: Sample storage space 12: Binding buffer storage space 2: Column storage area 21: Sample discharge pipette storage space 22: Column discharge pipette storage space 3: Cassette storage Zone 31: Cartridge 311: Dissolving liquid tank 312: Washing liquid tank 313: Elution liquid tank 314: Abutment 4: Moving frame 41: Rejecting plate 42: Spring mechanism 43: Syringe holder 5: Syringe 6 : Sample discharge pipette 8: Collection tube A: Area B: Seat body M: Drive unit

Claims (10)

An automated nucleic acid extraction device, characterized in that the automated nucleic acid extraction device comprises: a body having a sample containing area, a column containing area, a cartridge containing area, and a collecting tube, and The sample accommodating area, the column accommodating area, the cassette accommodating area and the collection tube are arranged in a linear direction; a cassette is disposed in the cassette accommodating area, and the cassette includes two parallel walls And at least two vertical walls, the parallel walls and the vertical walls together form a dissolving liquid tank, at least one washing liquid tank and an eluting liquid tank, wherein the dissolving liquid tank, the washing liquid tank and the eluting liquid are formed The vertical walls of the tank are respectively provided with abutting parts, and the dissolving liquid tank, the washing liquid tank and the eluting liquid tank are also arranged along the linear direction, wherein the abutting part has an arc-shaped wall or a A polygonal wall; a driving unit, which is arranged on the base; a moving frame, which is vertically arranged on the base, and is driven by the driving unit to reciprocate in the linear direction; and a syringe, which is arranged on the moving frame and Move with the mobile frame. The automatic nucleic acid extraction device as described in item 1 of the patent application scope, wherein the sample containing area has a sample containing space and a binding buffer containing space. The automatic nucleic acid extraction device as described in item 1 of the patent application scope, wherein the column storage area further includes a sample row pipette storage space and a column row pipette storage space, and the automated nucleic acid extraction apparatus further includes the same A product row suction tube and a column row suction tube, and the sample row suction tube and the column row suction tube are movably arranged in the sample row suction tube accommodation space and the column row suction tube accommodation space. An automatic nucleic acid extraction device as described in item 3 of the patent application range, wherein the syringe is detachably connected to the sample discharge pipette or the column discharge pipette. The automatic nucleic acid extraction device according to item 1 of the patent application range, wherein when the abutting portion has the arc-shaped wall, the arc angle of the arc-shaped wall is greater than or equal to 90 degrees; when the abutting portion has the polygon In the case of a wall, the polygonal wall includes at least two supporting walls, and the supporting walls form an angle of less than 180 degrees with each other. The automatic nucleic acid extraction device as described in item 1 of the patent application range, wherein the vertical wall between the washing liquid tank and the eluting liquid tank has a complete hollow cylindrical structure, forming a filter material accommodating space. An automatic nucleic acid extraction device as described in item 1 of the patent application range, wherein a recess is formed at the bottom of the eluent bath, and the cassette further includes a snap fastener. An automated nucleic acid extraction method is applied to the automated nucleic acid extraction device according to any one of claims 1 to 7, wherein the automated nucleic acid extraction method includes the following steps: using the syringe with a tube The column exhaust pipette absorbs a mixed reactant in the binding buffer storage space, so that the nucleic acid in the reactant binds to a membrane in the column exhaust pipette, and discharges the reaction residue to the cassette to cause the reaction The nucleic acid in the residue is bound to the membrane; the syringe and the column exhaust pipette are used to suck the washing buffer in the at least one washing solution tank through the membrane, and then the washing buffer is matched with the syringe and the tube The column row suction pipe is discharged through the diaphragm; and the syringe is matched with the column row suction pipe to suck the elution buffer in the eluent bath through the membrane, and then the syringe is matched with the column row suction pipe to pass through the The membrane drains the elution buffer with nucleic acids to the collection tube. The automated nucleic acid extraction method as described in item 8 of the patent application scope, in which the step of discharging the reaction residue to the cartridge, and the washing buffer is discharged through the membrane with the syringe and the column discharge pipette In the step, the abutment portion of the pipe column suction pipe is abutted on the abutment portion of the cartridge, and then the reaction residue or the washing buffer is discharged. The automated nucleic acid extraction method as described in item 9 of the patent application scope, wherein the abutting portion of the column discharge pipette is abutted against the abutting portion of the cartridge, and then the reaction residue or washing buffer After the discharging step, the following steps are further included: the abutting portion of the column discharge straw is abutted against the abutting portion of the cartridge, and the syringe is moved up and down in a direction perpendicular to the linear direction by less than Or equal to 5 mm.

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