TWI731566B - 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 filter material. The base has a sample accommodating area, a cassette accommodating area, and a collection tube. The sample 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 a plurality of 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 lysis buffer well, the wash buffer wells and the elution buffer well are arranged in the linear direction. One of the vertical walls has a filter material accommodating area. 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 filter material is disposed in the filter material accommodating area.

Description

Method and device for automatic nucleic acid extraction

The present invention relates to a device and 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 relevant internal environment information, to tailor the best treatment plan for the patient, in order to maximize the treatment 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 (cfDNA) is the free DNA fragment found in plasma, and circulating tumor DNA (ctDNA) fragment refers to the DNA fragment released from the tumor in cfDNA.

Almost everyone's blood contains free DNA fragments (ie cfDNA), which may come from cell apoptosis and necrosis, or they may enter the blood through active release (strength exercise). However, the content of cfDNA in blood is very low. In 1 mL of plasma, the content of cfDNA is between 1 ng and 100 ng, and the content of ctDNA is even lower, accounting for only between 0.1% and 5% of cfDNA.

Studies have confirmed that the total amount of cfDNA in the peripheral blood of tumor patients is higher than that of healthy people. Through this, if the content of cfDNA increases, it can play a good prompting role and use it as a means of initial tumor screening. Therefore, purifying a very small amount of cfDNA (a type of nucleic acid) from Liquid Biopsy is the first step in precision medicine.

At present, researchers in the field have devoted themselves to developing devices and methods for extracting nucleic acids such as cfDNA or ctDNA. For example, CN 101684463 A discloses a method for rapidly extracting nucleic acid from various micro-clinical samples, which includes the following steps: (a) adding the clinical sample to the lysis solution and mixing; (b) aspirating the clinical sample and flowing through the filter membrane, The nucleic acid components in the sample are adsorbed on the filter membrane due to the specific adsorption of the membrane, and the filtrate is a waste liquid containing cell debris and protein; (c) aspirate the cleaning solution, flow through the filter membrane, and clean 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 an aqueous nucleic acid solution free of other impurities for nucleic acid amplification.

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

TW M536238 U discloses a machine for automatic nucleic acid extraction, comprising: a machine bottom plate, on which a tray holder is mounted, the tray holder can move along a horizontal track; a support is vertically arranged above the machine bottom plate, And there is a vertical track thereon; a vertical movement unit, the vertical movement unit includes a base plate, a base plate track is arranged above the base plate, a moving block is arranged along the base plate track, and a moving block is located below the moving block and fixedly locked in A syringe fixing unit of the base plate is provided for a syringe to be erected; a piston thruster fixing unit is provided for a piston thruster to be erected, so that when the moving block moves up and down along the base plate track, the piston thruster 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 bracket.

TW 201412981 A discloses a method and device for extracting nucleic acid, which uses air pressure to extract nucleic acid. The upper end of the purification tube is connected with a device that can be applied with positive or negative gas pressure, which can be sucked/discharged from the lower tip of the purification tube. Body, cleaning fluid and extracting fluid, to achieve the effect of simply extracting nucleic acid without the need for a centrifuge.

Although the above-mentioned prior art has existed, these prior art still have the disadvantage of not being able to extract cfDNA or ctDNA in large quantities. Therefore, if an automated nucleic acid extraction device that can extract a large amount of cfDNA or ctDNA to increase the yield can be developed, it will bring a breakthrough in precision medicine.

The purpose of the present invention is to provide an automated nucleic acid extraction device and an 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 nucleic acids with higher yield and higher concentration from the sample, such as cfDNA and ctDNA.

Therefore, the present invention provides an automatic nucleic acid extraction device, which includes a base, a cassette, a driving unit, a moving frame, and a filter material. The base has a sample accommodating area, a cassette accommodating area and a collection tube, and the sample accommodating area, the cassette accommodating area and the collection tube are arranged along a linear direction. The cassette is arranged in the cassette accommodating area. The cassette includes two parallel walls and a plurality of vertical walls. The parallel walls and the vertical walls together form a dissolving liquid tank, at least one washing liquid tank, and an eluent tank, wherein the dissolving liquid tank The washing liquid tank and the eluting liquid tank are also arranged in a linear direction, and one of the vertical walls has a filter material accommodating space. The driving unit is arranged on the base body. The moving frame is vertically arranged on the base and is driven by the driving unit to move back and forth in a linear direction. The filter material is movably arranged in the filter material accommodating space.

In one embodiment, the automated nucleic acid extraction device further includes a syringe, which is arranged on the movable frame and moves with the movable frame. The sample accommodating area has a sample accommodating space and a binding buffer accommodating space.

In one embodiment, the base body further includes a column accommodating area, and the sample accommodating area, the column accommodating area, the cassette accommodating area, and the collection tube are arranged in a linear direction.

In one embodiment, the pipe string accommodating area further includes a sample suction pipe accommodating space and a pipe string suction pipe accommodating space.

In one embodiment, the automated nucleic acid extraction device further includes a sample suction tube, which is movably arranged in the sample suction tube accommodating space.

In one embodiment, the automated nucleic acid extraction device further includes a pipe string suction pipe, which is movably arranged in the pipe string suction pipe accommodating space.

In one embodiment, the base body further includes at least one heating element, which is disposed under the sample accommodating space and/or the pipe string accommodating space.

In one embodiment, the filter material accommodating space is formed on the vertical wall between the washing liquid tank and the eluent tank, and is a complete hollow cylindrical structure. In the accommodating space of the filter material, an enzyme is further accommodated, and the enzyme is a DNA deoxyribonuclease (DNase).

In one embodiment, the automatic nucleic acid extraction device further includes an iron frame, which is detachably arranged on the base.

The present invention also provides an automated nucleic acid extraction method, using the aforementioned automated nucleic acid extraction device, which includes the following steps: using a syringe with a sample suction tube and a filter material to absorb the mixed lysate in the sample accommodating space; and removing the needle The dissolved product in the cartridge is discharged to the binding buffer containing space and mixed with the binding buffer.

In one embodiment, before the step of using the syringe with the sample suction tube and the filter material to absorb the dissolved product mixed in the sample accommodating space, the following step is further included: matching the syringe with the sample suction tube and the filter material in the accommodating space Filter material combination.

In one embodiment, after the step of discharging the lysate in the syringe to the binding buffer accommodating space and mixing it with the binding buffer, the method further includes the following step: using the syringe to match the column drain pipe to suck the binding buffer and contain it After mixing the reactants in the space, the nucleic acid in the reactant is bound to the membrane in the pipe column suction pipe, and the reaction residue is discharged to the cassette, so that the nucleic acid in the reaction residue is bound to the membrane; Match the column drain pipe to suck the washing buffer in at least one washing liquid tank through the membrane, and then discharge the washing buffer through the membrane with a syringe and column drain tube; move the syringe with the column drain tube to the column Heat the accommodating space of the suction tube; and use the syringe with the column suction tube to suck the elution buffer in the eluent tank through the diaphragm, and then use the syringe with the column suction tube to elute the nucleic acid through the diaphragm. Drain the buffer to the collection tube.

In one embodiment, the step of using a syringe with a sample suction tube and a filter material to suck the lysate mixed in the sample accommodating space is the same as discharging the lysate in the syringe to the binding buffer accommodating space and interacting with the binding buffer. The mixing steps further include the following steps: moving the syringe with the sample suction tube and the filter material to the dissolving liquid tank and moving back and forth in the dissolving liquid tank in a linear direction.

To sum up, the effect of the present invention is that by the arrangement of the parallel walls and vertical walls of the cassette, the dissolving liquid tank, the washing liquid tank, and the eluent tank formed by the cassette can contain a large volume of solution; The washing liquid tank and the eluent tank are respectively provided with abutment parts, which can prevent the sample suction tube or the column suction tube from falling off the syringe and being separated from the syringe after the syringe has discharged a large volume of solution. In addition, by the design of the cassettes and the accommodating space arranged in a linear direction and the moving frame and the syringe reciprocating in the linear direction, the effect of automatic nucleic acid extraction in a linear direction can be achieved, thereby avoiding sample contamination and improving extraction efficiency. Therefore, the automated nucleic acid extraction device of the present invention can indeed quickly and conveniently extract nucleic acids with a higher yield and a higher concentration from the sample, such as cfDNA and ctDNA. Moreover, compared with the conventional technology, it is linearly moved by the cassette and the accommodating space (that is, relative to the desktop, the cassette moves while the syringe does not move), while the present invention is performed by the moving frame and the syringe Linear movement (that is, relative to the desktop, the syringe moves but the cassette does not move), so the automatic 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, and the prior art The operating space for the linear movement of the cassette and the accommodating space is about 2 to 3 times the length of the cassette and the accommodating space. Therefore, the volume of the automatic nucleic acid extraction device of the present invention is small, and the user's use space can be saved.

Hereinafter, a preferred embodiment of the automatic nucleic acid extraction device according to the present invention will be described with reference to related drawings, in which the same elements will be described with the same reference symbols.

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

Please refer to FIGS. 1 and 2. FIG. 1 is a schematic diagram of a combination 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 FIG. 1 and FIG. 2, the automatic nucleic acid extraction device includes a base B, a cassette 31, a driving unit M, a moving frame 4 and a syringe 5.

The base body B has a sample accommodating area 1, a column accommodating area 2, and a cassette accommodating area 3. The sample accommodating area 1 has a sample accommodating space 11 and a binding buffer accommodating space 12, In addition, the sample containing area 1, the column containing area 2 and the cassette containing area 3 are arranged along a linear direction L1. Particularly, the order of the sample containing area 1, the column containing area 2 and the cassette containing area 3 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 container. Placement area 1, pipe column housing area 2, then cassette housing area 3; first pipe column housing area 2, cassette housing area 3, then sample housing area 1, first cassette housing area 3, sample housing Zone 1 and then column accommodating zone 2; or cassette accommodating zone 3, column accommodating zone 2 and sample accommodating zone 1 first.

Please refer to FIG. 2 in conjunction with 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 jointly form a A dissolving liquid tank 311, at least one washing liquid tank 312, and an eluent tank 313, wherein the vertical walls 31b forming the dissolving liquid tank 311, the washing liquid tank 312, and the eluent tank 313 are respectively provided with an abutting portion 314 , And the dissolving liquid tank 311, the washing liquid tank 312, and the eluent tank 313 are also arranged along the linear direction L1. In this embodiment, the number of vertical walls 31b is 9, and together with the parallel walls 31a, 2 dissolving liquid tanks 311, 4 washing liquid tanks 312, and 2 eluent tanks 313 are formed. However, the vertical wall 31b, dissolving The number of liquid tank 311, washing liquid tank 312, and eluent 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 eluent tank 313 Can be added), the present invention is not limited. In addition, by the arrangement of the parallel walls 31a and vertical walls 31b of the cassette 31, the dissolving solution tank 311, the washing solution tank 312, and the eluent tank 313 formed can accommodate a large volume of buffer (dissolving buffer, washing Buffer and/or elution buffer), for example but not limited to 2 ml or more, 5 ml or more, or 10 ml or more.

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 arranged on the movable frame 4 and moves with the movable frame 4. In particular, although the figure shows that the driving unit M is a transmission roller belt, the driving unit M can also be other driving devices, such as a linear module or other devices that can drive the movable frame 4 to move linearly. The present invention is not limited.

Please refer to FIGS. 1, 2 and 4, in this embodiment, the pipe string accommodating area 2 further includes a sample suction pipe accommodating space 21 and a pipe string suction pipe accommodating space 22.

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

1 to 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 containing areas. In a better case, the collection tube 8 can be arranged adjacent to the cassette 31 (but the present invention is not limited to this), because the automatic nucleic acid extraction method is to extract along the linear direction L1, and then discharge the eluate in the final step To the collection tube 8, the collection tube 8 is arranged adjacent to the cassette 31 and arranged along the linear direction L1, which is consistent with the moving direction of the mobile rack 4, which can prevent the mobile rack 4 from reciprocating through the collection during the automated nucleic acid extraction process. Above the pipe 8, causing cross-contamination.

In this 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 accommodating space 12 is used for accommodating a binding buffer and a lysate to perform a binding reaction. The dissolving solution tank 311 is used to hold a lysis buffer, a defoaming agent, and reaction residues. The washing liquid tanks 312 are used to contain wash buffers. The eluent tanks 313 are used to contain elution buffer. The sample suction pipe accommodating space 21 is used for accommodating the sample suction pipe 6. The pipe string suction pipe accommodating space 22 is used for accommodating the pipe string suction pipe 7. In this embodiment, the sample accommodating space 11, the binding buffer accommodating space 12, the dissolving solution tank 311, and the washing solution tank 312 can accommodate, for example, but not limited to, 30 mL of sample or buffer. Therefore, compared with 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 clean the solution remaining in the column drain pipe 7 , In order to improve the concentration and purity of the nucleic acid extracted in the subsequent steps. In this embodiment, the number of dissolving liquid tanks, washing liquid tanks, and eluent tanks 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 provided inside the pipe string suction pipe 7. The material of the membrane 74 is, for example, but not limited to, a silica membrane, and it is positively charged. During the nucleic acid extraction process of the sample, the nucleic acid contained in it can be negatively charged, and the positive and negative charges are used to attract each other. With the characteristic of, the nucleic acid can be attached to the membrane 74, and then the nucleic acid attached to the membrane 74 can be extracted through subsequent procedures.

In this embodiment, the base body B may further include at least one heating element H, which is disposed under the sample accommodating space 11 and/or the pipe string accommodating space 22. Here, when the sample accommodating space 11 is provided below 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, thereby increasing the concentration of nucleic acid extracted by subsequent procedures; when the pipe column suction pipe accommodating space 22 is provided with When heating the element H, it can promote the volatilization of the residual solvent on the membrane 74 of the pipe column suction pipe 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 under the sample containing space 11 may not be turned on (that is, it is not heated during the dissolution reaction) to prevent the ribonucleic acid from being decomposed and affecting The concentration of ribonucleic acid after extraction.

Referring to FIGS. 3A and 3B, a recess f is formed at the bottom of the eluent tanks 313. With this design, a smaller amount of elution buffer can be used when the sample is eluted, thereby increasing the concentration of nucleic acid extraction. In particular, although the figure shows that only the eluent tank 313 adjacent to the collection 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, such as but not limited to 1 ml, 500 μl, 200 μl, 100 μl, 50 μl, 30 μl, etc., can be adjusted according to the needs of the user, and the present invention is not limited. .

6A to 6C, the cassette 31 further includes an elastic fastener 316 for detachably buckling 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 part of the structure of the automated nucleic acid extraction device of FIG. 1, and FIG. 8B is an exploded view of FIG. 8A. The automatic nucleic acid extraction device of this embodiment may also include an iron frame I, which is detachably disposed on the base B. The multiple cassettes 31 and the collection tube 8 can be set on the iron frame 1 by the user first, and then set on the base B together with the iron frame I, so that the user can set the multiple cassettes 31 and the collection tube 8 conveniently. In addition, the user can also set the iron frame I on the base body B first, and then set the cassette 31 and the collection tube 8. The present invention is not limited. Furthermore, after the nucleic acid is extracted by the automatic nucleic acid extraction device, the iron frame 1 can be taken out from the base body B, so that the user can take out multiple cassettes 31 and collection tubes 8 at a time. In particular, the iron frame I may include two handles I1, which is convenient for the user to take out the iron frame I from the base body B or put the iron frame I into the base body B.

Please refer to FIG. 4, the sample suction pipe 6 includes an assembling part 61 and a tip part 62 connected with the assembling part 61. The pipe string suction tube 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, it is possible to prevent the sample with nucleic acid from generating too much force after being discharged from the tip portion 73, causing the sample to splash out of the collection tube 8. , In order to improve the yield of nucleic acid extraction. In this preferred embodiment, the assembling part 61 and the tip part 62 are integrally formed; the assembling part 71, the abutting part 72 and the tip part 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 suction pipe 6 and the column suction pipe 7 may have the same configuration, and the difference between the two is only that the column suction pipe 7 has a diaphragm 74, while the sample suction pipe 6 does not have a diaphragm 74 (that is, The sample suction tube 6 can also have an assembling part, abutting part and a tip part. The sample suction tube 6 and the column suction tube 7 can be manufactured by using only one mold, and the sample is made by taking out or putting in the diaphragm 74. Suction pipe 6 and pipe string suction pipe 7).

Please refer to FIGS. 3B to 5B, the bottom of the syringe 5 also includes a connector 51, wherein the syringe 5 is connected to the assembly parts 61 and 71 through the connector 51. In detail, the joint 51 matches the structure of the assembling part 61 of the sample suction tube 6 or the assembling part 71 of the column suction tube 7 to tightly connect the syringe 5 with the assembling parts 61 and 71. In particular, the syringe 5 and the connector 51 may be integrally formed.

Please refer to FIG. 1 and FIG. 7A to FIG. 7B. FIG. 7A is a partial enlarged view of area A in FIG. 1. The moving frame 4 includes a stripping plate 41, and two sides of the moving frame 4 are respectively provided with a spring mechanism 42, and the spring mechanism 42 drives the stripping plate 41 to act to control the assembly parts 61, 71 to be disassembled from the bottom of the syringe 5. In detail, as shown in FIG. 7B, the spring mechanism 42 moves in the L3 direction to drive the ejector plate 41 to move in the L4 direction to separate the sample suction tube 6 or the column suction tube 7 from the syringe 5.

Please refer to FIG. 7C in conjunction with FIG. 7A, the movable frame 4 may further include at least one syringe fixing member 43 to fix the syringe 5 on the movable frame 4. The syringe fixing member 43 may further include a syringe locking groove 431 and at least one recessed hole 432, and an elastic member R can be disposed in the recessed hole 432. As shown in FIG. 7A, the needle cylinder 5 is clamped in the needle cylinder fixing grooves 431 of the two needle cylinder fixing members 43, and the needle cylinder 5 is better fixed in the needle cylinder through the elastic member R arranged in the recessed hole 432 The needle cylinder locking groove 431. In particular, the number of the syringe fixing member 43, the recessed holes 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 locking groove 431, and the present invention does not Unlimited.

Please refer to Figure 1, Figure 5A, Figure 5B and Figure 6A, where Figure 5A and Figure 5B are partial schematic diagrams of a preferred embodiment of the automated nucleic acid extraction device of the present invention, and Figure 6A is the 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 can be 90 degrees; preferably, the arc angle θ can be 120 degrees; preferably, the arc angle θ can be 180 degrees; preferably, the arc angle θ can be 270 degrees, as long as it can The suction pipe 7 of the supply pipe string can be pressed against. In particular, as shown in FIG. 6A, the vertical wall 31b has an arc-shaped wall 3141, which is connected to the parallel wall 31a by the vertical wall 31b, that is, both ends of the arc-shaped wall 3141 are not directly connected to the parallel wall 31a.

Please refer to Figure 1, Figure 5C and Figure 6C, where Figure 5C is a partial schematic diagram of a preferred embodiment of the automated nucleic acid extraction device of the present invention, and Figure 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 abutting portion 314 has a polygonal wall. The polygonal wall may include at least two supporting walls. Here, a structure including a first supporting wall 3142 and a second supporting wall 3143 is used for illustration. , The first supporting wall 3142 and the second supporting wall 3143 have an included angle e that is less than 180 degrees. Preferably, the included angle e can be 150 degrees; preferably, the included angle e can be 120 degrees; preferably, the included angle e can be 90 degrees; preferably, the included angle e can be 45 degrees, as long as the pipe string can be used The straw 7 can be leaned against and stand. In particular, please refer to FIGS. 6D to 6H to illustrate different implementation aspects of the abutting portion of the cassette of the automated nucleic acid extraction device of the present invention. The abutting 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 FIG. 6D, FIGS. 6F to 6H). The polygonal wall is, for example, but not limited to, having two supporting walls and three supporting walls. Walls, four supporting walls, five supporting walls, six supporting walls or a structure greater than six supporting walls, each supporting wall has an included angle, the number of supporting walls and the angle of the included angle are not limited, as long as the pipe string can be used The straw 7 can be leaned against and stood up. In particular, as shown in FIG. 6D, both ends of the polygonal wall of the abutting portion 314 may directly meet the parallel wall 31a, however, as shown in FIGS. 6F to 6H, the polygonal wall of the abutting portion 314 of the vertical wall 31b The two ends are not directly connected to the parallel wall 31a, but are connected to the parallel wall 31a by the vertical wall 31b, which is not limited here.

With the design of the abutment portion 314 described above, the abutment portion 72 of the column suction pipe 7 abuts against the arc-shaped wall 3141 of the abutment portion 314 during the process of discharging liquid (for example, reactant, washing buffer, etc.) Or between the first supporting wall 3142 and the second supporting wall 3143, so that when the syringe 5 discharges liquid, the pipe string suction tube 7 will not fall off and separate from the syringe 5 due to excessive pressure. Even if the pipe string suction tube 7 is loosened when the syringe 5 rises after the syringe 5 is discharged, the design of the abutment portion 314 can make the abutment portion 72 of the pipe string suction tube 7 abut and stand on the abutment. On the leaning part 314 without being completely separated from the syringe 5, then the syringe 5 and the pipe string suction tube 7 can be tightened again by the subsequent step of moving the syringe 5 up and down in the direction L2 perpendicular to the linear direction L1 Cooperate to facilitate subsequent extraction steps.

Please refer to FIG. 1, FIG. 6A and FIG. 6B, where 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 cassette shown in FIG. 6B between the washing liquid tank 312 and the eluent tank 313 has a complete hollow cylindrical structure to form 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 dissolved lysate, so as 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 suction tube 6 to prevent tissue fragments or impurities from clogging the tip portion 62 of the sample suction tube 6. With this design, the tissue samples can be directly put on the machine without using a centrifuge outside the machine to obtain the clear lysate and then put on the machine. Therefore, the step of lysing the tissue sample can be completed on the automatic nucleic acid extraction device of the present invention, so that the tissue sample can also achieve the effect of automatic nucleic acid purification. In particular, when the ribonucleic acid (RNA) in the sample is to be extracted, the filter accommodating space 315 can further accommodate the enzymes required in the extraction process, such as deoxyribonucleic acid (DNase), to facilitate the extraction process. Remove the deoxyribonucleic acid (DNA) in the sample, and the deoxyribonucleic acid (DNase) can also be washed and removed by the subsequent washing steps, so after the extraction is completed, you can get no deoxyribonucleic acid (DNA) Ribonucleic acid (RNA).

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

Please refer to Figure 9 in conjunction with Figures 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 by using the aforementioned automated nucleic acid extraction device. A transmission roller belt is provided at the bottom of the, which will drive the movable 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 suction tube 7 to suck the mixed reactants in the binding buffer holding space to bind the nucleic acids in the reactants. To a membrane 74 in the column drain pipe 7, and discharge the reaction residue 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 with the column suction tube 7 to suck the washing buffer in at least one washing liquid tank through the membrane 74, and then pass the washing buffer through the syringe 5 with the column suction tube 7 through the membrane 74 discharge. Then proceed to step S08. Use the syringe 5 with the column drain tube 7 to suck the elution buffer in the eluent tank through the membrane 74, and then use the syringe 5 with the column drain tube 7 to transfer the nucleic acid through the membrane 74. The elution buffer is discharged to the collection tube 8. Among them, a step S07 can be arranged between steps S06 and S08 to move the column suction pipe 7 to the column suction pipe accommodating space 22 for heating, so as to minimize the residual amount of washing buffer on the membrane 74. The above steps S05 to S08 are performed by an automated nucleic acid extraction device. The dashed part 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. The extraction device is performed. For example, the user can first dissolve the sample and centrifuge, then mix the supernatant with the binding buffer, and then add the binding buffer accommodating space to the automated nucleic acid extraction device for subsequent steps; or, the user can first After the sample is dissolved and centrifuged, the supernatant is added to the binding buffer holding space and mixed with the binding buffer, and the subsequent steps are performed by the automatic nucleic acid extraction device; or, the user can first dissolve the sample and centrifuge, and then add the lysate to the lysis solution In the tank, the subsequent steps are performed by the automatic nucleic acid extraction device, and the present invention is not limited.

This embodiment is used to illustrate the situation that 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 (driving the syringe 5) move to a specific position along the linear direction L1. Perform the foregoing steps, which are described in detail as follows: before step S01, the syringe 5 set in the moving frame 4 is moved to the sample suction tube accommodating space 21 of the column accommodating area 2 in the linear direction L1, so that the syringe 5 and The sample suction tube 6 accommodated in the sample suction tube accommodating space 21 is connected, and the sample suction tube 6 is taken out from the sample suction tube accommodating space 21. Then, the sample suction tube 6 connected to the syringe 5 moves to the cassette 31 of the cassette accommodating area 3 along the linear direction L1. Then, step S01 is performed to suck the dissolution buffer from the dissolving liquid tank 311 of the cassette 31, and then move to the sample holding area 1. Next, step S02 is performed to discharge the dissolution buffer into the sample accommodating space 11 of the sample accommodating area 1 and mix. Then, step S03 is performed to suck the mixed dissolved product in the sample containing space 11 and move to the binding buffer containing space 12 of the sample containing area 1. Then, step S04 is performed to discharge the lysate into the binding buffer containing space 12 and mix with the binding buffer to perform the binding reaction. Next, before proceeding to step S05, move the syringe 5 and the sample suction tube 6 to the sample suction tube accommodating space 21, and the ejector plate 41 is driven by the spring mechanism 42 to move the sample suction tube 6 from the syringe 5 to the sample The suction pipe accommodating space 21, and then move the syringe 5 along the linear direction L1 to the pipe string accommodating space 22 of the pipe string accommodating area 2, so that the syringe 5 and the pipe string accommodating space 22 The pipe string suction pipe 7 is connected to take out the pipe string suction pipe 7 with the diaphragm 74 inside from the pipe string accommodating area 2. After that, the pipe string suction tube 7 connected to the syringe 5 moves to the binding buffer accommodating space 12 along the linear direction L1. Then, step S05 is performed to suck the formed reactant with the syringe 5 and the column suction tube 7 so that the reactant passes through the membrane 74 and the nucleic acid contained in the reactant is attached to the membrane 74. Then, the syringe 5 moves to the cassette 31 in the linear direction L1. Then the reactants are passed through the diaphragm 74 again in the direction of gravity to attach the contained nucleic acid to the diaphragm 74. As for the reaction residues will be discharged to the cassette 31 in the direction of gravity, and the reaction residues are passed through the diaphragm twice to ensure the reaction The nucleic acid in the substance can be attached to the diaphragm 74. Next, proceed to step S06, the pipe column suction tube 7 with nucleic acid moves along the linear direction L1 with the syringe 5 and the moving rack 4 to the washing liquid tank 312 containing washing buffer, and then the washing buffer is sucked and discharged. For washing, the washing buffer is passed through the membrane 74 twice to wash away the reaction residue remaining on the membrane 74. After washing, the pipe string suction tube 7 with nucleic acid can be moved along the linear direction L1 with the syringe 5 and the moving rack 4 to the pipe string suction pipe accommodating space 22, and the pipe string suction pipe 7 can be stayed in the pipe column row. The straw accommodating space 22 is heated by the heating element H to volatilize the remaining liquid (for example, the washing buffer) on the membrane 74 of the column suction tube 7 to make it sufficiently dry (ie, step S07). Finally, move the tube column drain tube 7 with nucleic acid along the linear direction L1 with the syringe 5 and the moving rack 4 to the eluent tank 313 containing the elution buffer, and proceed to step S08, by sucking and discharging the elution The buffer is used for elution, thereby obtaining an eluate (eluate, elution buffer with nucleic acid). After that, the column suction pipe 7 sucks the obtained eluate, then moves to the collection pipe 8 along the linear direction L1, and then discharges the eluate to the collection pipe 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 suction pipe 7 is volatilized and sufficiently dried. The present invention is not limited to 30 seconds, 1 minute, 3 minutes, and 5 minutes. Unlimited.

In this preferred embodiment, in the step of discharging the reaction residue to the cassette 31 (step S05), and the step of discharging the washing buffer through the membrane 74 with the syringe 5 and the column drain tube 7 (step S05) S06), the abutment part 72 of the pipe string suction pipe 7 is abutted against the abutment part 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 is discharging liquid, the pipe string suction tube 7 abuts against the abutment portion 314, thereby preventing the pipe string suction pipe 7 from being damaged during the process of discharging liquid. The pressure is too high and falls off and separates from the syringe 5. Even if the pipe string suction tube 7 is loosened when the syringe 5 is discharged after liquid is discharged, the design of the abutment portion 314 can make the abutment portion 72 of the pipe string suction pipe 7 abut and stand on the abutment portion 314 Without being completely separated from the syringe 5, the syringe 5 can be closely matched with the pipe string suction tube 7 through subsequent steps.

In this preferred embodiment, in the step of pressing the abutting portion 72 of the pipe string suction pipe 7 against the abutting portion 314 of the cassette 31, and then discharging the reaction residue or washing buffer solution (step S05, S06) Afterwards, it further includes the following steps: abut the abutment portion 72 of the pipe string suction tube 7 against the abutment portion 314 of the cassette 31, and move the syringe 5 up and down in the direction L2 perpendicular to the linear direction L1 by less than Or equal to 5 mm. Through this step, the pipe string suction tube 7 can be tightly connected with the syringe 5 to prevent the pipe string suction pipe 7 from falling off. Preferably, the needle cylinder 5 can move up and down by 5 mm in the direction L2; preferably, the needle cylinder 5 can move up and down by 4.5 mm in the direction L2; preferably, the needle cylinder 5 can be moved up and down by 3 mm in the direction L2. The column suction tube 7 and the syringe 5 are closely connected again.

In this preferred embodiment, the number of washing liquid tanks 312 can be adjusted according to the actual needs of the user, and the present invention is not limited. In detail, if the number of washing liquid tanks 312 is greater than one, before the pipe string suction pipe 7 is moved to the pipe string suction pipe accommodating space 22 for heating, the syringe 5 and the pipe string suction pipe 7 can be used to pass through the membrane at least once. The sheet 74 absorbs the washing buffer in at least one washing liquid tank, and then the washing buffer is discharged through the membrane 74 with the syringe 5 and the column suction tube 7. That is, the column suction tube 7 with nucleic acid moves along the linear direction L1 with the syringe 5 and the moving rack 4 to the washing liquid tank 312 containing washing buffer, and the washing buffer is repeatedly sucked and discharged to perform washing. The steps can be performed in different washing liquid tanks 312, and the number of washing steps is determined by the number of washing liquid tanks 312.

In this preferred embodiment, one of the steps of discharging and mixing the dissolution buffer into the sample containing space 11 (step S02) and the step of sucking the mixed dissolved product in the sample containing space 11 (step S03) It further includes the steps of moving to the filter accommodating space 315, combining the sample suction tube 6 with the syringe 5 and the filter material in the filter accommodating space 315; and sucking the dissolved product mixed in the sample accommodating space 11 After the step (step S03), it further includes the step of moving the sample suction tube 6 and the filter material to the dissolving liquid tank 311 and reciprocating in the dissolving liquid tank 311 in a linear direction. In detail, when the biological sample is tissue or other samples with impurities, the filter material is used to filter the dissolved lysate, so as to fix the tissue fragments or impurities on the outside of the filter material, and then proceed to step S03 to clarify (no tissue The dissolved product of fragments or impurities) is sucked into the syringe 5. Then move the sample drain tube 6 and the filter material to the dissolving liquid tank 311 and reciprocate in a linear direction in the dissolving liquid tank 311 to clean the impurities stuck on the outside of the filter material, and then discharge the dissolved product in the syringe 5 To the binding buffer accommodating space 12 to perform the binding reaction, so as 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 tissue or other samples with impurities, the aforementioned steps can be added.

In this preferred embodiment, the sample accommodating space 11, the binding buffer accommodating space 12, the sample suction pipe accommodating space 21, the column suction pipe accommodating space 22, the dissolving solution tank 311, the washing solution tank 312, the washing solution The number of the liquid extraction tank 313, the abutment portion 314, the syringe 5, the sample drain tube 6, the column drain tube 7, the collection tube 8 and the joint 51 can be adjusted according to the actual needs of the user, and the present invention is not limited. In particular, the cassette 31, the sample accommodating space 11, the binding buffer accommodating space 12, the sample suction pipe accommodating space 21, the column suction pipe accommodating 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 dissolving solution tank 311, washing solution tank 312, and adjacent eluent tank 313, so that the user can perform automatic nucleic acid extraction. It can prevent the reactants from splashing out of the dissolving liquid tank 311, the washing liquid tank 312, or the eluent tank 313, and it can also prevent the pipe string suction pipe 7 from falling off. In addition, by the design that the cassette 31 and the accommodating space are arranged along the linear direction L1, and the movable frame 4 and the syringe 5 move repeatedly along the linear direction L1, the effect of automatic nucleic acid extraction in a linear direction L1 can be achieved and the sample is avoided. Pollution and improve extraction efficiency. Therefore, the automated nucleic acid extraction device of the present invention can indeed quickly and conveniently extract nucleic acids with a higher yield and a higher concentration from the sample, such as cfDNA and ctDNA.

The above descriptions are merely illustrative and not restrictive. Any equivalent modifications or alterations that do not depart from the spirit and scope of the present invention should be included in the scope of the appended patent application.

1: Sample holding area 11: Sample holding space 12: Combined buffer storage space 2: pipe column accommodating area 21: Sample suction pipe accommodation space 22: The accommodating space of the pipe string drain pipe 3: Cassette accommodating area 31: Cassette 31a: parallel walls 31b: vertical wall 311: Dissolving Liquid Tank 312: Washing liquid tank 313: eluent tank 314: Abutment Department 3141: arc-shaped wall 3142: The first support wall 3143: second support wall 315: filter holding space 316: elastic fastener 4: Mobile frame 41: Return plate 42: Spring mechanism 43: Syringe holder 431: Syringe Clamping Slot 432: Hole 5: Syringe 51: Connector 6: Sample drain pipe 61: Assembly Department 71: Assembly Department 62: Tip 73: Tip 7: String drain pipe 72: abutment 74: diaphragm 8: Collection tube A: area B: Seat D1: depth D2: length D3: length D4: thickness e: included angle f: recess H: heating element I: Iron frame I1: Handle L1: Linear direction L2: Linear direction L3: Linear direction L4: Linear direction M: drive unit R: Elastic θ: arc angle S01~S08: steps

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

1: Sample holding area

11: Sample holding space

12: Combined buffer storage space

2: pipe column accommodating area

21: Sample suction pipe accommodation space

22: The accommodating space of the pipe string drain pipe

3: Cassette accommodating area

31: Cassette

311: Dissolving Liquid Tank

312: Washing liquid tank

313: eluent tank

314: Abutment Department

4: Mobile frame

41: Return plate

42: Spring mechanism

43: Syringe holder

5: Syringe

6: Sample drain pipe

8: Collection tube

A: area

B: Seat

M: drive unit

Claims (10)

An automatic nucleic acid extraction device, comprising: a base having a sample accommodating area, a cassette accommodating area, and a collection tube, and the sample accommodating area, the cassette accommodating area, and the collection tube Is arranged along a linear direction; a cassette is arranged in the cassette accommodating area, the cassette includes two parallel walls and a plurality of vertical walls, the two parallel walls and the vertical walls together form a solution tank, at least A washing liquid tank and an eluent tank, wherein the dissolving liquid tank, the at least one washing liquid tank, and the eluent tank are also arranged along the linear direction, and one of the vertical walls has a filter material accommodating space ; A driving unit, arranged on the base; a moving frame, vertically arranged on the base, and driven by the driving unit to reciprocate along the linear direction; and a filter material, movably arranged in the filter accommodating space . The automated nucleic acid extraction device according to claim 1, further comprising a syringe arranged on the moving rack and moving with the moving rack, wherein the sample accommodating area has a sample accommodating space and a binding buffer accommodating space . The automated nucleic acid extraction device according to claim 2, wherein the base body further includes a tube column accommodating area, the sample accommodating region, the tube column accommodating region, the cassette accommodating region, and the collection tube are along Arranged in the linear direction, the column accommodating area further includes a sample drain pipe accommodating space and a column drain pipe accommodating space, and the automated nucleic acid extraction device further includes a sample drain pipe and a column drain pipe, and The sample suction pipe and the pipe string suction pipe are movably arranged in the sample suction pipe accommodating space and the pipe string suction pipe accommodating space. The automated nucleic acid extraction device according to claim 3, wherein the base body further includes at least one heating element disposed under the sample accommodating space and/or the pipe string accommodating space. The automated nucleic acid extraction device according to claim 2, wherein the filter accommodating space is formed on the vertical wall between the at least one washing liquid tank and the eluent tank, and is a complete hollow cylindrical structure. In the accommodating space of the filter material, an enzyme is further accommodated, and the enzyme is a deoxyribonuclease (DNase). The automatic nucleic acid extraction device according to claim 2, further comprising: an iron frame, which is detachably arranged on the base body. An automated nucleic acid extraction method is applied to the automated nucleic acid extraction device described in any one of items 3 to 6 in the scope of patent application. The automated nucleic acid extraction method includes the following steps: the syringe is matched with the sample suction tube and the The filter material absorbs the mixed lysate in the sample accommodating space; and discharges the lysate in the syringe to the binding buffer accommodating space and mixes with the binding buffer. The automated nucleic acid extraction method according to claim 7, wherein before the step of using the syringe with the sample suction tube and the filter material to suck the lysate mixed in the sample containing space, the method further comprises the following steps: The cartridge is matched with the sample suction pipe and the filter material combination in the filter material accommodating space. The automated nucleic acid extraction method according to claim 7, wherein after the step of discharging the lysate in the syringe into the binding buffer accommodating space and mixing with the binding buffer, the method further comprises the following steps: matching the syringe The column suction pipe sucks the mixed reactants in the binding buffer accommodating space, binds the nucleic acid in the reactants to a diaphragm in the pipe column suction pipe, and discharges the reaction residues to the cassette, The nucleic acid in the reaction residue is bound to the membrane; the syringe is matched with the column suction tube 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 The pipe string suction tube is discharged through the diaphragm; the needle cylinder is matched with the pipe string suction pipe to the accommodating space of the pipe string suction pipe for heating; and the needle cylinder is matched with the pipe string suction pipe to suck the pipe through the diaphragm. The elution buffer in the elution tank is then used with the syringe and the column suction tube to drain the elution buffer with nucleic acid to the collection tube through the membrane. The automated nucleic acid extraction method according to claim 7, wherein the step of using the syringe with the sample suction tube and the filter material to suck the lysate mixed in the sample accommodating space is the same as discharging the lysate in the syringe To the step of mixing the binding buffer solution accommodating space and mixing with the binding buffer solution, it further includes the following steps: moving the syringe with the sample suction tube and the filter material to the dissolving solution tank and along the line in the dissolving solution tank The sexual direction moves back and forth.

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