Nucleic acid extraction and methylation all-in-one machine

Abstract

The invention discloses a nucleic acid extraction and methylation all-in-one machine which comprises a methylation station mechanism, a vacuum chuck arm and a nucleic acid extraction arm. In the application, the active guide seat moves forwards and drives the guide shaft and the pressing plate to move downwards until the pressing plate compresses the silica gel pad, so that the cover is automatically closed, then after the corresponding time is maintained, the motor drives the push plate to move backwards, the push plate moves backwards and the guide connecting plate does not move due to the action of the spring, and the active guide seat drives the guide shaft and the pressing plate to move upwards; and finally, a solution in the PCR tube is sucked to a nucleic acid extraction station to be purified again, so that the nucleic acid extraction efficiency is improved by automatically opening and closing the cover while the check calculation extraction operation is completed, and the operation is simple and convenient. And as manual stations do not need to be additionally arranged for opening and closing the cover, the occupied area of the accounting and extraction methylation all-in-one machine can be effectively reduced.

Description

Technical Field

[0001] The present invention relates to the technical field of nucleic acid extraction, and in particular to an all-in-one machine for nucleic acid extraction and methylation. Background Art

[0002] In addition to detecting methylation in tumor tissues, a large number of studies have used DNA methylation detection in tumor cells in the serum and other body fluids of tumor patients to assist in the diagnosis of tumors and the judgment of prognosis, including sputum and bronchial lavage fluid of lung cancer patients, nipple aspirates of breast cancer patients, feces of colorectal cancer patients, urine of bladder cancer and prostate cancer patients, and plasma and serum of tumor patients. Therefore, methylation can be used as a relatively ideal biomarker and prognostic evaluation indicator for early diagnosis of tumors, which is of great significance for tumor screening and risk assessment, early diagnosis, staging, prognosis judgment and treatment monitoring. With the study of tumor pathogenesis, gene methylation has become more and more prominent and is a very promising molecular marker for the diagnosis of cancer. The application of DNA methylation detection technology in the field of cancer is still in a relatively early stage. At present, there are very few products approved in China. The technology based on the analysis of cfDNA and ctDNA methylation provides a new way for the early diagnosis of various cancers, and DNA methylation characteristics are very likely to become a routine clinical tumor marker due to their sensitivity, specificity and easy analysis.

[0003] The fully automatic nucleic acid extraction and methylation all-in-one machine is a small automated nucleic acid extraction and conversion instrument that integrates DNA extraction and purification with methylation pre-treatment steps. Based on magnetic bead separation technology, it can efficiently extract, purify and sulfite convert the nucleic acid of the sample. The processed product can be used for genetic testing. The whole process from extraction to conversion is automated, reducing manual operations, avoiding human contamination, and saving experimental time. It is specially used for nucleic acid extraction and methylation.

[0004] Currently, the size of 32-throughput nucleic acid extraction and methylation integrated machines is relatively large, basically around 1000mm*750mm*900mm. They are too large for hospitals where every inch of land is valuable, and they cannot solve the problem of opening and closing the lid of PCR tubes. Therefore, it is urgent to propose a corresponding nucleic acid extraction and methylation integrated machine to solve the above problems. Summary of the invention

[0005] The purpose of the present invention is to propose a nucleic acid extraction and methylation integrated machine in order to solve the above-mentioned problem.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] The nucleic acid extraction and methylation integrated machine comprises a methylation station mechanism, a vacuum suction cup arm and a nucleic acid extraction arm. The method for using the nucleic acid extraction and methylation integrated machine comprises the following steps:

[0008] S1, combining, combining the magnetic beads with the nucleic acid in the sample in the PCR tube to form a nucleic acid and magnetic bead complex;

[0009] S2, washing, using the differences in physical and chemical properties between nucleic acids and impurities such as proteins and sugars to remove impurities;

[0010] S3, elution, washing the DNA from the magnetic beads at 50℃-60℃;

[0011] S4, methylation, transferring the PCR tube to the methylation site for demethylation, including the following steps:

[0012] S41, 120ul sulfite solvent is added to the PCR tube;

[0013] S42, closing the lid of the PCR tube after adding liquid;

[0014] S43, temperature control at 98°C for 8 minutes;

[0015] S44, temperature control 54℃ for 60 minutes;

[0016] S45, keep warm at 4℃ for 10-15 minutes;

[0017] S46. Open the lid and pipette the solution in the PCR tube to the nucleic acid extraction station for further purification.

[0018] Preferably, the methylation station mechanism includes a motor, a heating module, a push plate, a silicone pad, a guide shaft, a guide connecting plate, a pushing component for vertically lifting the guide shaft, and an elastic component for positioning the guide connecting plate. The bottom of the guide shaft is fixedly connected to a pressure plate through a support, the inner wall of the guide connecting plate is slidably connected to the outer wall of the guide shaft, and the output end of the motor is fixedly connected to the outer wall of the push plate.

[0019] Preferably, the pushing assembly comprises an active guide seat fixedly connected to the push plate, and the outer wall of the guide shaft is slidably connected to the inner wall of the active guide seat via an inclined sliding groove.

[0020] Preferably, both sides of the horizontal end of the guide shaft are fixedly connected to stoppers, and the outer wall of the stopper is slidably connected to the outer wall of the guide connecting plate.

[0021] Preferably, the elastic component comprises a spring, and the spring is fixedly connected between the push plate and the guide connecting plate.

[0022] Preferably, two groups of bearings are fixedly connected to the outer surface wall of the guide shaft, and the two groups of bearings are slidably connected to the guide connecting plate and the inner surface wall of the active guide seat respectively.

[0023] Preferably, the outer wall of the guide connecting plate is fixedly connected to a slider, the outer wall of the methylation station mechanism is fixedly connected to a guide rail slidably matched with the slider, and the outer wall of the guide rail is fixedly connected to a limit screw.

[0024] Preferably, a limit pin is fixedly connected to the inner wall of the push plate, the limit pin is fixedly connected to the outer wall of the guide connecting plate, and the limit pin is penetrated into the spring.

[0025] In summary, due to the adoption of the above technical solution, the beneficial effects of the present invention are:

[0026] 1. In this application, the PCR tube is placed on the heating module. After the combination, washing and elution are completed, 120ul of sulfite solvent is added to the PCR tube. The cover is placed on the top of the PCR tube through the vacuum suction cup arm. The motor starts to move and moves the push plate forward. The push plate drives the guide connecting plate, the pressure plate and the slider forward through the spring. When the slider reaches the limit screw, it is blocked. At this time, the motor drives the push plate to continue to move forward. The spring is compressed, and the active guide seat continues to move forward, driving the guide shaft and the pressure plate to move downward until the pressure plate presses the silicone pad, thereby automatically closing the cover. After maintaining the corresponding time, the motor drives the push plate to move back. Due to the action of the spring, the push plate retreats, the guide connecting plate does not move, and the active guide seat drives the guide shaft and the pressure plate to move upward to complete the pressure plate rising action. The push plate continues to retreat to drive the pressure plate to reset, thereby completing the cover opening action, and finally aspirates the solution in the PCR tube to the nucleic acid extraction station for re-purification. In this way, the nucleic acid extraction operation is completed. At the same time, the nucleic acid extraction efficiency is improved by automatically opening and closing the lid. In addition, since there is no need to add a manual station to open and close the lid, the floor space of the nucleic acid extraction and methylation all-in-one machine can be effectively reduced.

[0027] 2. In the present application, the guide pin is slidably matched with the inner surface wall of the push plate and is fixed to the guide connecting plate, and the guide pin is passed through the inside of the spring, thereby limiting the vertical displacement of the spring and the guide connecting plate, and limiting the position of the push plate after it is reset, thereby further ensuring the stability of the use of the nucleic acid extraction and methylation integrated machine. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 It shows a schematic diagram of the overall structure provided by an embodiment of the present invention;

[0029] Figure 2 A schematic diagram of the structure of a heating module provided in an embodiment of the present invention is shown;

[0030] Figure 3 A schematic diagram of the guide pin structure provided according to an embodiment of the present invention is shown;

[0031] Figure 4 A diagram showing a layout of equipment provided according to an embodiment of the present invention is shown;

[0032] Figure 5 A schematic diagram of a workflow provided according to an embodiment of the present invention is shown.

[0033] Legend:

[0034] 1. Motor; 2. Active guide seat; 3. Spring; 4. Guide shaft; 5. Guide connecting plate; 6. Guide rail; 7. Silicone pad; 8. Pillar; 9. Slider; 10. Limit screw; 11. Mounting plate; 12. Heating module; 13. Press plate; 14. Bearing; 15. Stop block; 16. Push plate; 17. Guide pin; 18. Methylation position mechanism. DETAILED DESCRIPTION

[0035] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0036] See also Figure 1-5 , the present invention provides a technical solution:

[0037] The nucleic acid extraction and methylation integrated machine comprises a methylation station mechanism 18, a vacuum suction cup arm and a nucleic acid extraction arm. The method for using the nucleic acid extraction and methylation integrated machine comprises the following steps:

[0038] S1, combining, combining the magnetic beads with the nucleic acid in the sample in the PCR tube to form a nucleic acid and magnetic bead complex;

[0039] S2, washing, using the differences in physical and chemical properties between nucleic acids and impurities such as proteins and sugars to remove impurities;

[0040] S3, elution, washing the DNA from the magnetic beads at 50℃-60℃;

[0041] S4, methylation, transferring the PCR tube to the methylation site for demethylation, including the following steps:

[0042] S41, 120ul sulfite solvent is added to the PCR tube;

[0043] S42, closing the lid of the PCR tube after adding liquid;

[0044] S43, temperature control at 98°C for 8 minutes;

[0045] S44, temperature control 54℃ for 60 minutes;

[0046] S45, keep warm at 4℃ for 10-15 minutes;

[0047] S46. Open the lid and pipette the solution in the PCR tube to the nucleic acid extraction station for further purification.

[0048] The methylation station mechanism 18 includes a motor 1, a heating module 12, a push plate 16, a silicone pad 7, a guide shaft 4, a guide connecting plate 5, a push component for vertically lifting the guide shaft 4, and an elastic component for positioning the guide connecting plate 5. The bottom of the guide shaft 4 is fixedly connected to a pressing plate 13 through a support 8. The inner surface wall of the guide connecting plate 5 is slidably connected to the outer surface wall of the guide shaft 4. The output end of the motor 1 is fixedly connected to the outer surface wall of the push plate 16.

[0049] The PCR tube is placed on the heating module 12. After the combination, washing and elution are completed, 120ul of sulfite solvent is added to the PCR tube. The cover is placed on the top of the PCR tube through the vacuum suction cup arm. The motor 1 starts to move, and moves the push plate 16 forward. The push plate 16 drives the guide connecting plate 5, the pressure plate 13, and the slider 9 to move forward through the spring 3. The slider 9 is blocked when it reaches the limit screw 10. At this time, the motor 1 drives the push plate 16 to continue to move forward. The spring 3 is compressed, and the active guide seat 2 continues to move forward, driving the guide shaft 4 and the pressure plate 13 to move downward until the pressure plate 13 presses the silicone pad 7. The cover is automatically closed, and then the cover is closed. After maintaining the corresponding time, the motor 1 drives the push plate 16 to move back. Due to the action of the spring 3, the push plate 16 retreats, the guide connecting plate 5 does not move, and the active guide seat 2 drives the guide shaft 4 and the pressure plate 13 to move upward, completing the rising action of the pressure plate 13. The push plate 16 retreats all the way to drive the pressure plate 13 to reset, thereby completing the opening action, and finally sucking the solution in the PCR tube to the nucleic acid extraction station for re-purification. In this way, the nucleic acid extraction operation is completed. At the same time, the nucleic acid extraction efficiency is improved by automatically opening and closing the lid. In addition, since there is no need to add a manual station to open and close the lid, the floor space of the nucleic acid extraction and methylation integrated machine can be effectively reduced.

[0050] Specifically, Figure 2 and Figure 4As shown, the pushing assembly includes an active guide seat 2 fixedly connected to the push plate 16, the outer wall of the guide shaft 4 is slidably connected to the inner wall of the active guide seat 2 through an inclined sliding groove, and the vertical movement of the pressure plate 13 is achieved through the offset sliding of the inclined groove and the guide shaft 4. The two sides of the horizontal end of the guide shaft 4 are fixedly connected to the stopper 15, and the outer wall of the stopper 15 is slidably connected to the outer wall of the guide connecting plate 5 to limit the horizontal deviation of the guide shaft 4. The elastic assembly includes a spring 3, which is fixedly connected between the push plate 16 and the guide connecting plate 5. While maintaining the position of the guide connecting plate 5 and the push plate 16, it also provides a support for the push plate 16. The ability of independent movement is reserved, and the outer wall of the guide shaft 4 is fixedly connected with two sets of bearings 14, and the two sets of bearings 14 are respectively slidably connected with the guide connecting plate 5 and the inner wall of the active guide seat 2, ensuring the stability of the sliding fit between the guide connecting plate 5 and the active guide seat 2 and the guide shaft 4. The outer wall of the guide connecting plate 5 is fixedly connected with a slider 9, and the outer wall of the methylation position mechanism 18 is fixedly connected with a guide rail 6 that slidably cooperates with the slider 9, and the outer wall of the guide rail 6 is fixedly connected with a limit screw 10, which ensures the stable movement of the guide connecting plate 5. At the same time, the horizontal position of the guide connecting plate 5 can be limited by the limit screw 10.

[0051] Specifically, Figure 1 and Figure 2 As shown, the inner wall of the push plate 16 is fixedly connected to a limit pin 17, the limit pin 17 is fixedly connected to the outer wall of the guide connecting plate 5, and the limit pin 17 is passed through the inside of the spring 3. The guide pin 17 is slidably matched with the inner wall of the push plate 16 and is fixed to the guide connecting plate 5, and the guide pin 17 is passed through the inside of the spring 3, thereby limiting the vertical displacement of the spring 3 and the guide connecting plate 5, and limiting the position of the push plate 16 after it is reset, thereby further ensuring the stability of the nucleic acid extraction and methylation integrated machine.

[0052] Working principle: The PCR tube is placed on the heating module 12. After the combination, washing and elution are completed, 120ul of sulfite solvent is added to the PCR tube. The lid is placed on the top of the PCR tube through the vacuum suction cup arm, and the motor 1 starts to move, moving the push plate 16 forward. The push plate 16 drives the guide connecting plate 5, the pressure plate 13, and the slider 9 forward through the spring 3. The slider 9 is blocked when it reaches the limit screw 10. At this time, the motor 1 drives the push plate 16 to continue to move forward. The spring 3 is compressed, and the active guide seat 2 continues to move forward, driving the guide shaft 4 and the pressure plate 13 to move downward until the pressure plate 13 presses the silicone pad 7. The lid is automatically closed, and then after maintaining the corresponding time, the motor 1 drives the push plate 16 to move back. Due to the action of the spring 3, the push plate 16 retreats, the guide connecting plate 5 does not move, and the active The guide seat 2 drives the guide shaft 4 and the pressure plate 13 to move upward, completing the rising action of the pressure plate 13, and the push plate 16 retreats all the way to drive the pressure plate 13 to reset, thereby completing the opening action, and finally absorbing the solution in the PCR tube to the nucleic acid extraction station for re-purification, thereby completing the nucleic acid extraction operation, and automatically opening and closing the lid, thereby improving the efficiency of nucleic acid extraction, and since there is no need to add a manual station to open and close the lid, the floor space of the nucleic acid extraction and methylation integrated machine can be effectively reduced, and the guide pin 17 is slidably matched with the inner surface wall of the push plate 16 and fixed with the guide connecting plate 5, and the guide pin 17 is penetrated inside the spring 3, thereby limiting the vertical displacement of the spring 3 and the guide connecting plate 5, and can also limit the position of the push plate 16 after resetting, thereby further ensuring the stability of the nucleic acid extraction and methylation integrated machine.

[0053] The above description of the embodiments enables those skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but will conform to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A nucleic acid extraction and methylation integrated machine, comprising a methylation station mechanism (18), a vacuum suction cup arm and a nucleic acid extraction arm, characterized in that: The method for using the nucleic acid extraction and methylation integrated machine comprises the following steps: S1, combining, combining the magnetic beads with the nucleic acid in the sample in the PCR tube to form a nucleic acid and magnetic bead complex; S2, washing, using the differences in physical and chemical properties between nucleic acids and impurities such as proteins and sugars to remove impurities; S3, elution, washing the DNA from the magnetic beads at 50℃-60℃; S4, methylation, transferring the PCR tube to the methylation site for demethylation, including the following steps: S41, 120ul sulfite solvent is added to the PCR tube; S42, closing the lid of the PCR tube after adding liquid; S43, temperature control at 98°C for 8 minutes; S44, temperature control 54℃ for 60 minutes; S45, keep warm at 4℃ for 10-15 minutes; S46. Open the lid and pipette the solution in the PCR tube to the nucleic acid extraction station for further purification.

2. The nucleic acid extraction and methylation integrated machine according to claim 1, characterized in that: The methylation station mechanism (18) comprises a motor (1), a heating module (12), a push plate (16), a silicone pad (7), a guide shaft (4), a guide connecting plate (5), a pushing component for vertically lifting the guide shaft (4), and an elastic component for positioning the guide connecting plate (5); the bottom of the guide shaft (4) is fixedly connected to a pressing plate (13) via a support (8); the inner surface wall of the guide connecting plate (5) is slidably connected to the outer surface wall of the guide shaft (4); and the output end of the motor (1) is fixedly connected to the outer surface wall of the push plate (16).

3. The nucleic acid extraction and methylation integrated machine according to claim 2, characterized in that: The pushing assembly comprises an active guide seat (2) fixedly connected to the push plate (16), and the outer wall of the guide shaft (4) is slidably connected to the inner wall of the active guide seat (2) via an inclined sliding groove.

4. The nucleic acid extraction and methylation integrated machine according to claim 3, characterized in that: Both sides of the horizontal end of the guide shaft (4) are fixedly connected to stoppers (15), and the outer wall of the stopper (15) is slidably connected to the outer wall of the guide connecting plate (5).

5. The nucleic acid extraction and methylation integrated machine according to claim 4, characterized in that: The elastic component comprises a spring (3), and the spring (3) is fixedly connected between the push plate (16) and the guide connecting plate (5).

6. The nucleic acid extraction and methylation integrated machine according to claim 5, characterized in that: Two groups of bearings (14) are fixedly connected to the outer wall of the guide shaft (4), and the two groups of bearings (14) are slidably connected to the guide connecting plate (5) and the inner wall of the active guide seat (2) respectively.

7. The nucleic acid extraction and methylation integrated machine according to claim 6, characterized in that: The outer wall of the guide connecting plate (5) is fixedly connected to a slider (9), the outer wall of the methylation station mechanism (18) is fixedly connected to a guide rail (6) that slidably cooperates with the slider (9), and the outer wall of the guide rail (6) is fixedly connected to a limit screw (10).

8. The nucleic acid extraction and methylation integrated machine according to claim 7, characterized in that: The inner surface wall of the push plate (16) is fixedly connected to a limit pin (17), the limit pin (17) is fixedly connected to the outer surface wall of the guide connecting plate (5), and the limit pin (17) is inserted into the interior of the spring (3).

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