Automatic extraction device for large-volume free nucleic acid by centrifugal column method

Abstract

An automatic extraction device for large-volume free nucleic acid through a centrifugal column method comprises a back plate, a shifting mechanism and a reagent filling module, the shifting mechanism and the reagent filling module are sequentially and fixedly arranged on the back plate, and a centrifugal column filtering module with a negative-pressure liquid pumping module is arranged at the bottom of the reagent filling module. Through a centrifugal column specifically combined with nucleic acid and a special reagent filling module, after a sample is subjected to cracking and protease K digestion treatment, nucleic acid in the sample is rapidly adsorbed and rapidly washed through a filter membrane, so that protein, pigments, lipids and other impurities are removed to the maximum extent; the obtained total nucleic acid can be stored in an eluent for a long time to be used for a next experiment. The whole device can realize centrifugal column method large-volume free nucleic acid automatic extraction for extracting, enriching and purifying the free total nucleic acid from fresh or frozen serum, plasma, lymph and other cell-free body fluids.

Description

Technical Field

[0001] The present invention relates to a technology in the field of bioengineering, in particular to an automatic device for extracting large-volume free nucleic acids using a centrifugal column method. Background Art

[0002] Current nucleic acid extraction processes are lengthy and complex, and the associated equipment is bulky and expensive, limiting its application scenarios. Some equipment still requires extensive manual operation, increasing the risk of operational errors and resulting in low and unstable recovery rates. Furthermore, the relatively high price of high-quality nucleic acid extraction equipment limits its widespread adoption. Summary of the Invention

[0003] The present invention addresses the problems that existing technologies are unable to process large-volume samples, cannot extract, enrich, and purify free total nucleic acids from fresh or frozen serum, plasma, lymph, and other cell-free body fluids, and face the problems of being unable to remove impurities, low recovery rate and stability. The present invention proposes a centrifugal column method for the automated extraction of large-volume free nucleic acids. By using a centrifugal column that specifically binds to nucleic acids and a special reagent injection module, after the sample is lysed and digested with proteinase K, the nucleic acids in the sample are quickly adsorbed and quickly washed through a filter membrane to remove proteins, pigments, lipids and other impurities to the maximum extent. The obtained total nucleic acid can be stored in an eluent for a long time for the next experiment. The present invention as a whole can realize the automated extraction of large-volume free nucleic acids by the centrifugal column method for extracting, enriching, and purifying free total nucleic acids from fresh or frozen serum, plasma, lymph, and other cell-free body fluids.

[0004] The present invention is achieved through the following technical solutions:

[0005] The present invention relates to an automatic extraction device for large-volume free nucleic acid using a centrifugal column method, comprising a back plate, a shift mechanism and a reagent filling module fixedly arranged thereon in sequence, wherein a centrifugal column filtration module with a negative pressure liquid extraction module is provided at the bottom of the reagent filling module.

[0006] The shift mechanism includes: a transverse moving motor, a transverse slider, a longitudinal moving motor, a longitudinal slider and a pressure rod, wherein: the transverse slider is arranged on the transverse moving motor and fixedly connected to the reagent filling module, and the transverse moving motor drives the slider to move so that different syringes in the reagent filling module are coaxial with the centrifugal column in sequence; the longitudinal slider is arranged on the longitudinal moving motor and fixedly connected to the pressure rod, and the longitudinal moving motor drives the slider to move so that the pressure rod presses the push rod of the syringe downward to inject liquid into the centrifugal column.

[0007] The centrifugal column filtration module comprises a centrifugal column, a filter port with a filter element arranged inside the centrifugal column, and a heating ring arranged at the bottom of the centrifugal column.

[0008] The negative pressure liquid extraction module includes: a diaphragm pump and a waste liquid bag, wherein: the fluid inlet of the diaphragm pump is connected to the filter port via a hose, and the fluid outlet is connected to the fluid inlet of the waste liquid bag via a hose.

[0009] The reagent filling module includes: a syringe bracket arranged on a horizontal slider, a buckle locking piece arranged on the syringe bracket, several groups of syringes and their buckles, wherein: the buckles are in contact with the outer wall of the syringe, the tail of each buckle is movably arranged on the buckle locking piece, and the push rod of the syringe is located below the pressure rod.

[0010] Technical Effects

[0011] The present invention uses an improved reagent filling module, which can accurately inject different reagents into the centrifugal column through the cooperation of a snap lock, a syringe and its snap, combined with a shift mechanism; in the centrifugal column filtration module, the centrifugal column has a combination of a filter port with a filter element inside and a bottom heating ring and its application in the nucleic acid extraction process; each module is integrated on the backplane as a whole, realizing a system architecture of closed, automated, and integrated operation of liquid addition, reaction, and filtration, which is different from the existing equipment with single functions or cumbersome operation. Compared with the existing technology, the present invention facilitates the realization of integrated and automated operation, efficient impurity removal and high recovery rate, closed operation and biosafety, and miniaturization and low cost of equipment. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 、 Figure 2 It is a schematic diagram of the overall structure of the present invention;

[0013] Figure 3 is a schematic diagram of the shift structure;

[0014] Figure 4 Schematic diagram of the centrifugal column filtration module and the negative pressure liquid extraction module;

[0015] Figure 5 、 Figure 6 This is a schematic diagram of the reagent filling module;

[0016] In the figure: reagent filling module 1, horizontal slider 2, centrifugal column 3, heating ring 4, movable door 5, diaphragm pump 6, waste liquid bag 7, horizontal moving motor 8, left side plate 9, top cover 10, longitudinal moving motor 11, back plate 12, right side plate 13, longitudinal slider 14, pressure rod 15, syringe bracket 101, snap lock 102, medium volume syringe 103, micro syringe 104, 105, 106, 108, 109, 110, medium volume syringe push rod 107, snaps 111, 112, 113, 114, filter port with filter element 301, fluid inlet 601, fluid outlet 602, fluid inlet 701, fluid outlet 702. DETAILED DESCRIPTION

[0017] like Figure 1 As shown, this embodiment relates to an automated extraction device for large-volume free nucleic acid using a centrifugal column method, comprising: a back plate 12 and a shift mechanism and a reagent filling module 1 fixed thereon in sequence, wherein: a centrifugal column filtration module with a negative pressure liquid extraction module is provided at the bottom of the reagent filling module 1.

[0018] like Figure 3 As shown, the shifting mechanism includes: a transverse moving motor 8, a transverse slider 2, a longitudinal moving motor 11, a longitudinal slider 14 and a pressure rod 15, wherein: the transverse slider 2 is arranged on the transverse moving motor 8 and is fixedly connected to the reagent filling module 1, and the transverse moving motor 8 drives the slider 2 to move so that the different syringes in the reagent filling module 1 are coaxial with the centrifugal column 3 in sequence; the longitudinal slider 14 is arranged on the longitudinal moving motor 11 and is fixedly connected to the pressure rod 15, and the longitudinal moving motor 11 drives the slider 14 to move so that the pressure rod presses the push rod of the syringe downward to inject liquid into the centrifugal column.

[0019] like Figure 4 As shown, the centrifugal column filtration module includes: a centrifugal column 3, a filter port 301 with a filter element arranged inside the centrifugal column, and a heating ring 4 arranged at the bottom thereof, wherein: the heating ring 4 is a C-shaped aluminum ring with a heating film on the outside, which can achieve heating.

[0020] like Figure 4 As shown, the negative pressure liquid extraction module includes: a diaphragm pump 6 and a waste liquid bag 7, wherein: the fluid inlet 601 of the diaphragm pump 6 is connected to the filter port 301 via a hose, the fluid outlet 602 is connected to the fluid inlet 701 of the waste liquid bag via a hose, and the air outlet 702 of the waste liquid bag is used to balance the air pressure.

[0021] The diaphragm pump can extract the liquid from the centrifugal column 3 through the filter port 301 into the waste liquid bag 7 under airtight conditions to achieve filtration.

[0022] like Figure 5 and Figure 6 As shown, the reagent filling module includes: a syringe bracket 101 arranged on the horizontal slider 2 and a snap lock 102 arranged on the syringe bracket, several groups of syringes and their snaps, wherein: the snaps are in contact with the outer wall of the syringe, the tail of each snap is movably arranged on the snap lock 102, and the push rod of the syringe is located below the pressure rod 15.

[0023] This embodiment relates to an automated nucleic acid extraction method based on the above-mentioned device, comprising:

[0024] Step 1: Take 300 μL of proteinase K solution, 3 mL of sample (cell-free body fluid such as serum, plasma, lymph), and 2.4 mL of lysate, shake and mix well, and then add to the centrifugal column 3. Close the movable door 5.

[0025] Step 2: Incubate at 60°C for 30 minutes.

[0026] Step 3: The motor 8 starts to align the 5 mL injection tube 103 with the centrifugal column 3.

[0027] Step 4: The motor 11 starts working, the pressure rod 15 presses down the push rod 107, and after 5 mL of the binding solution in the syringe is added to the centrifugal column and mixed, the motor 11 starts working and the pressure rod 15 moves up to the standby position.

[0028] Step 5: Let stand for 5 minutes.

[0029] Step 6: The motor 8 starts to align the 1 mL injection tube 104 with the centrifugal column 3 .

[0030] Step 7: The motor 11 starts working, the pressure rod 15 presses down the push rod 108, and after 600 μl of the first rinsing solution in the syringe is added to the centrifugal column, the motor 11 starts working, and the pressure rod 15 moves up to the standby position.

[0031] Step 8: The diaphragm pump starts working to perform filtering operation.

[0032] Step 9: The motor 8 starts to align the 1 mL injection tube 105 with the centrifugal column 3.

[0033] Step 10: The motor 11 starts working, the pressure rod 15 presses down the push rod 109, and after 750 μl of the second rinsing solution in the syringe is added to the centrifugal column, the motor 11 starts working, and the pressure rod 15 moves up to the standby position.

[0034] Step 11: The diaphragm pump starts working to perform filtering operation.

[0035] Step 12: The motor 8 starts to align the 1 mL injection tube 106 with the centrifugal column 3.

[0036] Step 13: The motor 11 starts working, the pressure rod 15 presses down the push rod 110, and after 750 μl of anhydrous ethanol in the syringe is added to the centrifugal column, the motor 11 starts working and the pressure rod 15 moves up to the standby position.

[0037] Step 14: The diaphragm pump starts working to perform filtering operation.

[0038] Step 15: Open the movable door 5, take out the centrifugal column 3 to obtain the required sample, and at the same time take out the diaphragm pump 6, the waste liquid bag 7 and the four syringes and discard them.

[0039] Compared to existing technologies, the present invention's centrifugal column-based automated large-volume free nucleic acid extraction method and device achieves a closed, automated, and integrated process for liquid addition, reaction, and filtration, eliminating the time-consuming and labor-intensive manual labor and the risk of infection, while also simplifying waste liquid disposal. Furthermore, the device is low-cost and compact, making it suitable for batch production and large-scale applications.

[0040] The above-mentioned specific implementation can be partially adjusted in different ways by those skilled in the art without departing from the principles and purpose of the present invention. The scope of protection of the present invention shall be based on the claims and shall not be limited by the above-mentioned specific implementation. All implementation schemes within its scope shall be subject to the constraints of the present invention.

Claims

1. A centrifugal column method for the automated extraction of large-volume free nucleic acids, characterized in that: include: A back plate and a shifting mechanism and a reagent filling module fixed thereon in sequence, wherein: a centrifugal column filtration module with a negative pressure liquid extraction module is provided at the bottom of the reagent filling module; The shift mechanism includes: a transverse moving motor, a transverse slider, a longitudinal moving motor, a longitudinal slider and a pressure rod, wherein: the transverse slider is arranged on the transverse moving motor and fixedly connected to the reagent filling module, and the transverse moving motor drives the slider to move so that different syringes in the reagent filling module are coaxial with the centrifugal column in sequence; the longitudinal slider is arranged on the longitudinal moving motor and fixedly connected to the pressure rod, and the longitudinal moving motor drives the slider to move so that the pressure rod presses the push rod of the syringe downward to inject liquid into the centrifugal column.

2. The automatic extraction device for large-volume free nucleic acid using a centrifugal column method according to claim 1, wherein: The centrifugal column filtration module comprises a centrifugal column, a filter port with a filter element arranged inside the centrifugal column, and a heating ring arranged at the bottom of the centrifugal column.

3. The automatic extraction device for large-volume free nucleic acid using a centrifugal column method according to claim 1, wherein: The negative pressure liquid extraction module includes: a diaphragm pump and a waste liquid bag, wherein: the fluid inlet of the diaphragm pump is connected to the filter port via a hose, the fluid outlet is connected to the fluid inlet of the waste liquid bag via a hose, and the air outlet of the waste liquid bag is used to balance the air pressure.

4. The automatic extraction device for large-volume free nucleic acid using a centrifugal column method according to claim 1, wherein: The reagent filling module includes: a syringe bracket arranged on a horizontal slider, a buckle locking piece arranged on the syringe bracket, several groups of syringes and their buckles, wherein: the buckles are in contact with the outer wall of the syringe, the tail of each buckle is movably arranged on the buckle locking piece, and the push rod of the syringe is located below the pressure rod.

5. A method for automatically extracting nucleic acid based on the device according to any one of claims 1 to 4, characterized in that: include: Step 1: Take proteinase K solution, sample and lysate, shake and mix, then add to the centrifuge column and close the movable door; Step 2: Incubate at 60°C for 30 minutes; Step 3: Start the motor and align the injection tube with the centrifugal column; Step 4: Start the motor, press the push rod downward, add the binding liquid in the syringe to the centrifuge column and mix it, then start the motor and move the push rod upward to the standby position; Step 5: Let stand for 5 minutes; Step 6: Start the motor and align the injection tube with the centrifugal column; Step 7: Start the motor, press the push rod downward, add the first rinse solution in the syringe into the centrifuge column, start the motor, and move the push rod upward to the standby position; Step 8: The diaphragm pump starts working to perform filtering operation; Step 9: Start the motor and align the injection tube with the centrifugal column; Step 10: Start the motor, press the push rod downward, add the second rinse solution in the syringe into the centrifuge column, start the motor, and move the push rod upward to the standby position; Step 11: The diaphragm pump starts working to perform filtering operation; Step 12: Start the motor and align the injection tube with the centrifugal column; Step 13: Start the motor, press the push rod downward, add the anhydrous ethanol in the syringe into the centrifugal column, start the motor, and move the push rod upward to the standby position; Step 14: The diaphragm pump starts working to perform filtering operation; Step 15: Open the movable door, remove the centrifugal column to obtain the required sample, and remove the diaphragm pump, waste liquid bag and syringe and discard them.

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