WO2021099852A1

WO2021099852A1 - Bioreactor and stirring device thereof and extraction method

Info

Publication number: WO2021099852A1
Application number: PCT/IB2020/058549
Authority: WO
Inventors: Wen-Shan Yang; Ching-Ko Lin; Fu-chun LI; Pin-Hsing Chou; Yun-Lung Tsai; Pei-Yu Lee; Hsiao-Fen Chang
Original assignee: Genereach Biotechnology Corporation
Priority date: 2019-11-18
Filing date: 2020-09-15
Publication date: 2021-05-27
Also published as: TWI756585B; TW202120679A

Abstract

A bioreactor(100) includes a machine frame unit(3), a stirring device(20) disposed on the machine frame unit(3), a carrier(30) movably disposed on the machine frame unit(3) along a conveying direction, and a carrier driving mechanism(40) for driving movement of the carrier(30) along the conveying direction. The stirring device(20) includes a lifting module(50) configured to move up and down in a longitudinal direction, a connector(70) rotatably disposed on the lifting module(50) and having a threaded hole(71), and a magnetic rod assembly(60) that includes a threaded rod(61) connected to threaded hole(71), and a magnetic member(63) disposed on the bottom end of the threaded rod(61).

Description

BIOREACTOR AND STIRRING DEVICE THEREOF AND

EXTRACTION ME THOD

FIELD

The disclosure relates to an equipment and a method for extracting nucleic acid, more particularly to a bioreactor and a stirring device thereof and an extraction method.

BACKGROUND

Referring to FIG. 1, a conventional nucleic acid purification equipment, as disclosed in Chinese Patent No. CN206692665U, includes a machine frame 1, a magnet cover holder 2 movably disposed on the machine frame 1 in an up-down direction, a plurality of magnet covers 3 disposed on the magnet cover holder 2, a first lifting mechanism 4 for driving up and down movement of the magnet cover holder 2, a magnetic rod holder 5 movably disposed on the machine frame 1 in the up-down direction, a plurality of magnetic rods 6 disposed on the magnetic rod holder 5, a second lifting mechanism 7 for driving up and down movement of the magnetic rod holder 5, and a liquid holding plate 8 disposed on the machine frame

1 and formed with a plurality of tubular grooves 801 arranged in a matrix.

Through the aforesaid configuration, the first lifting mechanism 4 can drive the magnet cover holder

2 to move and extend the magnet covers 3 into the corresponding tubular grooves 801, and then up and down reciprocating vibrations are performed so that reagents, a biological sample and magnetic beads in the corresponding tubular grooves 801 can be mixed with each other to extract nucleic acid from the biological sample. However, the magnet covers 3 can only vibrate up and down relative to the corresponding tubular grooves 801, and cannot rotate and stir the reagents, the biological sample and the magnetic beads in the corresponding tubular grooves 801, so that the mixing effect still needs to be improved.

SUMMARY

Therefore, an object of the present disclosure is to provide a stirring device that can alleviate at least one of the drawbacks of the prior art.

According to this disclosure, a stirring device suitable for driving a stirring sleeve includes a lifting module, a connector, and a magnetic rod assembly. The lifting module is configured to move up and down in a longitudinal direction. The connector is rotatably disposed on the lifting module, has a threaded hole, and is configured to connect with the stirring sleeve. The magnetic rod assembly includes a threaded rod threadedly connected to the threaded hole, and a magnetic member disposed on a bottom end of the threaded rod. When the lifting module is actuated, the connector is driven by the lifting module to move up and down along a length of the threaded rod, and is simultaneously driven to rotate relative to the threaded rod. The connector is configured to bring the stirring sleeve to move up and down and rotate therealong.

Another object of of the present disclosure is to provide a bioreactor that can alleviate at least one of the drawbacks of the prior art.

According to this disclosure, a bioreactor includes a machine frame unit, the above stirring device, a carrier, and a carrier driving mechanism. The stirring device is disposed on the machine frame unit.The carrier is movably disposed on the machine frame unit along a conveying direction, and is configured for mounting thereon at least one reagent cassette which is formed with a plurality of tubular grooves respectively containing different reagents. The carrier driving mechanism is provided for driving movement of the carrier along the conveying direction.

Still another object of the present disclosure is to provide an extraction method that can alleviate at least one of the drawbacks of the prior art.

According to this disclosure, an extraction method includes the following steps:

(A) moving a stirring sleeve up and down relative to a first tubular groove of a reagent cassette along a longitudinal direction and simultaneously rotating the stirring sleeve relative to the first tubular groove, the reagent cassette being formed with a plurality of tubular grooves respectively containing different reagents, the first tubular groove further containing a plurality of magnetic beads and a biological sample;

(B) moving upwardly the stirring sleeve along the longitudinal direction relative to a threaded rod,which is coaxial with the stirring sleeve and which has a bottom end connected with a magnetic member, until a bottom end of the stirring sleeve is adjacent to the bottom end of the threaded rod;

(C) simultaneously lowering the stirring sleeve and the threaded rod until the bottom end of the stirring sleeve and the bottom end of the threaded rod are adjacent to a bottom end of the first tubular groove;

(D) simultaneously raising the stirring sleeve and the threaded rod until the stirring sleeve and the threaded rod are moved out of the first tubular groove, the bottom end of the stirring sleeve being attached with the magnetic beads, the magnetic beads being attached with nucleic acid;

(E) repeating steps (A) to (D) but in the other tubular grooves of the reagent cassette for washing the magnetic beads; and

(F) separating the nucleic acid from the magnetic beads by elution and removing the magnetic beads from the last tubular groove. BRIEF DESCRIPTION OF THE DRAWINGS

Other features and advantages of the disclosure will become apparent in the following detailed description of the embodiment with reference to the accompanying drawings, in which:

FIG. 1 is a schematic side view of a conventional nucleic acid purification equipment disclosed in Chinese Patent No. CN206692665U;

FIG.2 is a perspective view of a bioreactor according to an embodiment of the present disclosure with a side plate of a straddling frame being removed for illustrating that this embodiment can be used in conjunction with a plurality of stirring sleeves and reagent cassettes;

FIG. 3 is a schematic side view of the embodiment;

FIG. 4 is an exploded perspective view of a lifting module, a magnetic rod assembly, a connector and a stirring sleeve;

FIG. 5 is a sectional view of the embodiment;

FIG. 6 is a sectional view taken along line VI-VI of FIG. 5;

FIG. 7 is a view similar to FIG. 5, but with a reagent cassette being moved until a first tubular groove thereof is located below the stirring sleeve;

FIG. 8 is a view similar to FIG. 7, but with the stirring sleeve and a threaded rod of the magnetic rod assembly being moved downward into the first tubular groove ;

FIG. 9 is a view similar to FIG. 8, but with the connector being moved up and down reciprocally along the threaded rod and simultaneously rotating relative to the threaded rod and bringing the stirring sleeve to move up and down and rotate therealong;

FIG. 10 is a view similar to FIG. 9, but with the connector and the stirring sleeve being moved up until a bottom end of the stirring sleeve is proximate to a bottom end of the threaded rod;

FIG. 11 is a view similar to FIG. 10, but with the connector, the stirring sleeve and the threaded rod being moved down until the bottom ends of the stirring sleeve and the threaded rod are adjacent to a bottom end of the first tubular groove;

FIG. 12 is a view similar to FIG. 11, but with the connector, the stirring sleeve and the threaded rod being moved up until the bottom ends of the stirring sleeve and the threaded rod are moved out of a top end of the first tubular groove and are located above the reagent cassette; and

FIG. 13 is a flow chart, illustrating the steps involved in an extraction method using the bioreactor of the embodiment.

DE TAILED DESCRIPTION

Referring to FIGS.2 to 6, a bioreactor 100 according to an embodiment of the present disclosure can be used in conjunction with four stirring sleeves 200 and four reagent cassettes 300. Each stirring sleeve 200 has an upper engaging end 210 formed with four spaced-apart axial slits 211 and four circumferentially spaced-apart engaging grooves 212 (only three are visible in FIG. 4). Each reagent cassette 300 is formed with a plurality of tubular grooves 310. However, in other variations of this embodiment, the number of each of the stirring sleeve 200 and the reagent cassette 300 may be one, two, three or more than four. The bioreactor 100 of this embodiment includes a machine frame unit 3, a stirring device 20, a carrier 30 and a carrier driving mechanism 40.

The machine frame unit 3 includes a base frame 11, and a straddling frame 12 disposed on the base frame 11 and having an inverted U-shaped structure.

The stirring device 20 is disposed on the straddling frame 12.

The carrier 30 is movably disposed on the base frame 11 along a conveying direction (X), and is configured for removably mounting thereon the reagent cassettes 300 The tubular grooves 310 of each reagent cassette 300 are arranged along the conveying direction (X), and respectively contain different reagents 320. The first tubular groove 310 of each reagent cassette 300 further contains a plurality of magnetic beads 330 and a biological sample 340.

The carrier driving mechanism 40 is used to drive movement of the carrier 30 along the conveying direction (X). In this embodiment, the carrier driving mechanism 40 includes a gear rack 41 disposed on a bottom side of the carrier 30 and extending along the conveying direction (X), a pinion 42 meshing with the gear rack 41, and a carrier driving motor 43 disposed on a bottom side of the base frame 11 for driving rotation of the pinion 42.

The stirring device 20 is suitable for driving the stirring sleeves 200, and includes a lifting module 50, four magnetic rod assemblies 60, four connectors 70, a first driving mechanism 80 and a second driving mechanism 90. In this embodiment, the number of the magnetic rod assemblies 60 and the connectors 70 corresponds to the number of the stirring sleeves 200.

The lifting module 50 of this embodiment is configured to move up and down in a longitudinal direction (Z) transverse to the conveying direction (X), and includes a lifting base 51 having a plurality of bearing holes 53 (see FIGS. 4 and 6), and four bearings 52 respectively disposed in the bearing holes 53. The bearing holes 53 are spatially arranged in a horizontal direction (Y) transverse to the conveying direction (X) and the longitudinal direction (Z). The number of the bearings 52 corresponds to the number of the connectors 70 .

Each of the magnetic rod assemblies 60 includes an elongated threaded rod 61, an adapter sleeve 62 disposed on a bottom end of the threaded rod 61, and a magnetic member 63 disposed on the bottom end of the threaded rod 61 and extending outwardly from the adapter sleeve 62. In this embodiment, the magnetic member 63 is a magnet.

Each of the connectors 70 is rotatably secured to the lifting base 51 by means of a respective one of the bearings 52 which permits relative rotational motion therebetween. Each connector 70 is fitted in the respective bearing 52, and has an axially extending threaded hole 71 threadedly engaged with the threaded rod 61 of a respective one of the magnetic rod assemblies 60, and an annular engaging flange 72 protruding outwardly and radially from a bottom portion thereof. In this embodiment, each connector 70 is suitable to be connected with a respective one of the stirring sleeves 200. When each stirring sleeve 200 is connected to the respective connector 70, the annular engaging flange 72 of each connector 70 is engaged with the engaging grooves 212 in the upper engaging end 210 of the respective stirring sleeve 200, so that each stirring sleeve 200 is positioned on the respective connector 70.

The first driving mechanism 80 includes a first nut 81 fixedly disposed on the lifting base 51, a first threaded shaft 82 threadedly engaged with the first nut 81 and parallel to the threaded rods 61 of the magnetic rod assemblies 60, and a first driving motor 83 disposed on the straddling frame 12 for driving rotation of the first threaded shaft 82. In this embodiment, when the first driving motor 83 is activated and drives the first threaded shaft 82 to rotate, the first nut 81 and the lifting base 51 are driven to move up and down along a length of the first threaded shaft 82, and the lifting base 51, in turn, drives each connector 70 to move up and down along a length of the threaded rod 61 of the respective magnetic rod assembly 60 and to simultaneously rotate relative to the threaded rod 61. Each connector 70 is configured to bring each stirring sleeve 200 to move up and down and rotate therealong.

The second driving mechanism 90 includes a mounting plate 91, a second nut 92 fixedly disposed on the mounting plate 91, a second threaded shaft 93 threadedly engaged with the second nut 92 and parallel to the threaded rods 61, a driven gear 94 coaxially fixed to a top end of the second threaded shaft 93, a driving gear 95 meshing with the driven gear 94, and a second driving motor 96 for driving rotation of the driving gear 95. In this embodiment, top ends of the threaded rods 61 are fixed to the mounting plate 91, the mounting plate 91 is higher than the lifting base 51 in the longitudinal direction (Z), and the second threaded shaft 93 is rotatably disposed on an intermediate portion of the straddling frame 12. When the second driving motor 96 is activated and drives the second threaded shaft 93 to rotate, the second nut 92 and the mounting plate 91 are driven to move up and down along a length of the second threaded shaft 93, thereby bringing the threaded rods 61 to move up and down along the longitudinal direction (Z).

It should be noted that when the bioreactor 100 is to be used for nucleic acid extraction operation, the below two pre-processing steps may first be performed. Moreover, only one magnetic rod assembly 60, one connector 70, one reagent cartridge 300 and one stirring sleeve 200 that work together with each other will be described and exemplified herein.

In a first pre-processing step, as shown in FIGS. 3 and 7, the carrier driving motor 43 is activated to drive the pinion 42 to rotate, so that the gear rack 41 can drive the carrier 30 to move along the conveying direction (X) until a first tubular groove 310 of the reagent cassette 300 is located below the stirring sleeve 200, but is not limited thereto. That is, the carrier 30 may be moved until one of the tubular grooves 310 of the reagent cassette 300 is located below the stirring sleeve 200. In this embodiment, the tubular grooves 310 of the reagent cassette 300 contain reagents 320 having different functions. However, only the first tubular groove 310 further contains magnetic beads 330 and a biological sample 340.

In a second pre-processing step, as shown in FIG. 8, the first and second driving motors 83, 96 are simultaneously activated to drive the first and second threaded shafts 82, 93 to rotate, so that the first and second nuts 81, 92 can respectively drive the lifting base 51 and the mounting plate 91 to simultaneously move down, thereby simultaneously driving the connector 70, the stirring sleeve 200 attached to the connector 70, and the magnetic rod assembly 60 to move down until the stirring sleeve 200 enters the first tubular groove 310.

After completing the first and second pre-processing steps, referring to FIG.13, an extraction method of this disclosure using the bioreactor 100 includes the following steps:

Step 410: As shown in FIG. 9, moving the stirring sleeve 200 up and down relative to the first tubular groove 310 and simultaneously rotating the stirring sleeve 200 relative to the same.

In this step, the first driving motor 83 is activated to drive the first threaded shaft 82 to rotate, so that the first nut 81 can drive the lifting base 51 to move up and down therealong, thereby simultaneously driving the connector 70 and the stirring sleeve 200 to repeatedly move up and down relative to the threaded rod 61. During this process, since the threaded hole 71 in the connector 70 is threadedly connected to the threaded rod 61, apart from driving the stirring sleeve 200 to move up and down, the connector 70 can also rotate relative to the threaded rod 61, thereby bringing the stirring sleeve 200 to rotate therealong. As such, through the repeated up and down movement and rotation of the stirring sleeve 200, the stirring sleeve 200 can stir the reagent 320 having the effect of cell lysis, the magnetic beads 330 and the biological sample 340 contained in the first tubular groove 310, so that the reagent 320, the magnetic beads 330 and the biological sample 340 can be mixed thoroughly and uniformly to achieve homogenization. Further, cell wall and cell membrane of the biological sample 340 can be disrupted and disintegrated to release the nucleic acid therein, and some biological residues and the nucleic acid will attach to the magnetic beads 330.

Step 420: As shown in FIG. 10, moving upwardly the stirring sleeve 200 relative to the coaxial threaded rod 61 along the longitudinal direction (Z) until a bottom end of the stirring sleeve 200 is adjacent to a bottom end of the threaded rod 61.

In this step, the first driving motor 83 is activated to drive the first threaded shaft 82 to rotate, so that the first nut 81 can drive the lifting base 51 to move upward therealong, thereby simultaneously driving the connector 70 and the stirring sleeve 200 to move up relative to the threaded rod 61 along the longitudinal direction (Z)until the bottom end of the stirring sleeve 200 is adjacent to the magnetic member 63 disposed on the bottom end of the threaded rod 61.At this time, the stirring sleeve 200 is moved away from a liquid surface of the reagent 320 in the first tubular groove 310.

Step 430: As shown in FIG. 11, simultaneously lowering the stirring sleeve 200 and the magnetic rod assembly 60 along the longitudinal direction (Z) until the bottom end of the stirring sleeve 200 and the bottom end of the threaded rod 61 are adjacent to the bottom end of the first tubular groove 310.

In this step, the first and second driving motors 83, 96 are simultaneously activated to drive rotation of the first and second threaded shafts 82, 93, respectively, so that the first and second nuts 81, 92 can respectively drive the lifting base 51 and the mounting plate 91 to move downward therealong, thereby simultaneously driving the connector 70, the stirring sleeve 200 and the magnetic rod assembly 60 to move downward until the bottom end of the stirring sleeve 200 and the magnetic member 63 are adjacent to the bottom end of the first tubular groove 310.As such, the magnetic member 63 can magnetically attract the magnetic beads 330 to attach to an outer surface of the stirring sleeve 200. In this process, since the connector 70 and the magnetic rod assembly 60 move down simultaneously, the connector 70 will not drive the stirring sleeve 200 to rotate relative to the threaded rod 61, so that when the stirring sleeve 200 moves down into the first tubular groove 310, it will not rotate and stir the reagent 320 therein.

Step 440:As shown in FIG. 12, simultaneously moving upward the stirring sleeve 200 and the magnetic rod assembly 60 until the stirring sleeve 200 and the magnetic rod assembly 60 are moved out of the first tubular groove 310.

In this step, the first and second driving motors 83, 96 are simultaneously activated to drive rotation of the first and second threaded shafts 82, 93, respectively, so that the first and second nuts 81, 92 can respectively drive the lifting base 51 and the mounting plate 91 to move upward therealong, thereby simultaneously driving the connector 70, the stirring sleeve 200 and the magnetic rod assembly 60 to move upward until the bottom end of the stirring sleeve 200 and the magnetic member 63 of the magnetic rod assembly 60 are moved out of the first tubular groove 310 to be located above the reagent cassette 300. In this process, since the connector 70 and the magnetic rod assembly 60 move up simultaneously, the connector 70 will not drive the stirring sleeve 200 to rotate relative to the threaded rod 61, so that the magnetic member 63 can attract the magnetic beads 330 to attach stably to the outer surface of the stirring sleeve 200.As such, the magnetic beads 330 can be moved out of the first tubular groove 310 along with the stirring sleeve 200.

Step 450: Repeating steps 410 to 440 but in the other tubular grooves 310 of the reagent cassette 300 for washing the magnetic beads 330.

In this step,the aforesaid first pre-processing step is performed again, and the other tubes 310 of the reagent cassette 300 are sequentially move to be located below the stirring sleeve 200.After the magnetic beads 330 are released into the other tubes 310, the aforesaid second pre-processing step and steps 410 to 440 can be repeatedly performed, so that the magnetic beads 330 are sequentially washed in different reagents 320, which have the effect of removing non-nucleic acid substances, contained in the other tubes 310, thereby achieving the purpose of washing the magnetic beads 330.

Step 460: Separating the nucleic acid from the magnetic beads 330 by elution and removing the magnetic beads 330 from the last tubular groove 310. In this step, the elution is performed using an elution buffer.

From the aforesaid description, the advantages of the present disclosure can be summarized as: In comparison with the prior art, through the threaded connection between the connector 70 and the threaded rod 61 of the magnetic rod assembly 60 of this disclosure, when the first driving mechanism 80 is activated, the lifting module 50 can be driven to repeatedly move the connector 70 and the stirring sleeve 200 up and down relative to the threaded rod 61, apart from driving the stirring sleeve 200 to repeatedly move up and down therealong, the connector 70 can further drive the stirring sleeve 200 to rotate therealong. As such, the stirring sleeve 200 can stir the reagent 320, the magnetic beads 330 and the biological sample 340 in the tubular grooves 310 through its repeated up, down and rotation movement. Through this, the reagent 320, the magnetic beads 330 and the biological sample 340 can be thoroughly and uniformly mixed to achieve homogenization.

It is worth to mention herein that, with reference to FIGS. 2, 3 and 5, the carrier 30 is provided with a plurality of detachable temporary storage tubes 31. The stirring sleeves 200 are respectively placed in the temporary storage tubes 31 before being used. To use the stirring sleeves 200, the carrier driving mechanism 40 is first activated to move the carrier 30 until the stirring sleeves 200 are located below the connectors 70 and the magnetic rod assemblies 60. Next, the connectors 70 and the magnetic rods 60 are simultaneous lowered until the stirring sleeves 200 are respectively engaged with the connectors 70, after which the connectors 70 and the magnetic rod assemblies 60 are simultaneously moved upward to remove the stirring sleeves 200 from the respective temporary storage tubes 31, thereby facilitating the above-mentioned nucleic acid extraction operation. After the completion of the nucleic acid extraction, the carrier driving mechanism 40 is operated to drive the carrier 30 to move until the temporary storage tubes 31 are located below the respective stirring sleeves 200, after which the magnetic rod assemblies 60 are moved downward relative to the connectors 70 to push the stirring sleeves 200 so as to be disengaged from the respective connectors 70 and dropped into the respective temporary storage tubes 31, thereby facilitating removal of the stirring sleeves 200.

While the disclosure hasbeen described in connection with what is considered the exemplary embodiment, it is understood that this disclosure is not limited to the disclosed embodiment but is intended to cover various arrangements included within the spirit and scope of the broadest interpretation so as to encompass all such modifications and equivalent arrangements.

Claims

What is claimed is:

1 .A stirring device suitable for driving a stirring sleeve, comprising: a lifting module configured to move up and down in a longitudinal direction; a connector rotatably disposed on said lifting module and having a threaded hole, said connector being configured to connect with the stirring sleeve; and a magnetic rod assembly including a threaded rod threadedly connected to said threaded hole, and a magnetic member disposed on a bottom end of said threaded rod; wherein, when said lifting module is actuated, said connector is driven by said lifting module to move up and down along a length of said threaded rod, and is simultaneously driven to rotate relative to said threaded rod, said connector being configured to bring the stirring sleeve to move up and down and rotate therealong.

2. The stirring device as claimed in Claim 1, wherein said lifting module includes a lifting base having a bearing hole, and a bearing disposed in said bearing hole, said connector being fitted in said bearing.

3. The stirring device as claimed in Claim 1, wherein said magnetic rod assembly further includes an adapter sleeve disposed on said bottom end of said threaded rod such that said magnetic member extends outwardly from said adapter sleeve, said magnetic member being a magnet.

4. The stirring device as claimed in Claim 1, further comprising a first driving mechanism which includes a first nut disposed on said lifting module, and a first threaded shaft threadedly engaged with said first nut and parallel to said threaded rod.

5. The stirring device as claimed in Claim 4, wherein said first driving mechanism further includes a first driving motor for driving rotation of said first threaded shaft.

6. The stirring device as claimed in Claim 4, further comprising a second driving mechanism which includes a mounting plate, a second nut disposed on said mounting plate, and a second threaded shaft threadedly engaged with said second nut and parallel to said threaded rod, a top end of said threaded rod being fixed to said mounting plate, said mounting plate being higher than said lifting module in the longitudinal direction.

7. The stirring device as claimed in Claim 6, wherein said second driving mechanism further includes a driven gear coaxially fixed to a top end of said second threaded shaft, a driving gear meshing with said driven gear, and a second driving motor for driving rotation of said second threaded shaft.

8.A bioreactor comprising: a machine frame unit; a stirring device, as claimed in any one of Claims 1 to 7, disposed on said machine frame unit; a carrier movably disposed on said machine frame unit along a conveying direction and configured for mounting thereon at least one reagent cassette which is formed with a plurality of tubular grooves arranged along the conveying direction and respectively containing different reagents; and a carrier driving mechanism for driving movement of said carrier along the conveying direction.

9. The bioreactor as claimed in Claim 8, wherein said carrier driving mechanism includes a gear rack disposed on said carrier and extending along the conveying direction, a pinion meshing with said gear rack, and a carrier driving motor disposed on said machine frame unit for driving rotation of said pinion .

10.An extraction method comprising:

(A) moving a stirring sleeve up and down relative to a first tubular groove of a reagent cassette along a longitudinal direction and simultaneously rotating the stirring sleeve relative to the first tubular groove, the reagent cassette being formed with a plurality of tubular grooves respectively containing different reagents, the first tubular groove further containing a plurality of magnetic beads and a biological samp 1e;

(B) moving upwardly the stirring sleeve along the longitudinal direction relative to a threaded rod, which is coaxial with the stirring sleeve and which has a bottom end connected with a magnetic member, until a bottom end of the stirring sleeve is adjacent to the bottom end of the threaded rod;

(C) simultaneously lowering the stirring sleeve and the threaded rod until the bottom end of the stirring sleeve and the bottom end of the threaded rod are adjacent to a bottom end of the first tubular groove ;

(D) simultaneously raising the stirring sleeve and the threaded rod until the stirring sleeve and the threaded rod aremoved out of the first tubular groove, the bottom end of the stirring sleeve being attached with the magnetic beads, the magnetic beads being attached with nucleic acid; (E) repeating steps (A) to (D) but in the other tubular grooves of the reagent cassette for washing the magnetic beads; and

(F) separating the nucleic acid from the magnetic beads by elution and removing the magnetic beads from the last tubular groove.