CN119081835A

CN119081835A - Detection device and method for extracting nucleic acid and preparing library

Info

Publication number: CN119081835A
Application number: CN202411281409.5A
Authority: CN
Inventors: 成泽欣; 徐志; 薛辰全; 孙雪光; 吴晓晖
Original assignee: Shanghai Weihe Medical Laboratory Co ltd
Current assignee: Shanghai Weihe Medical Laboratory Co ltd
Priority date: 2024-09-12
Filing date: 2024-09-12
Publication date: 2024-12-06

Abstract

The present disclosure relates to detection devices and methods for extracting nucleic acids and preparing libraries. The detection device includes a base, a tray resiliently secured to the base and configured to vibrate relative to the base under drive, a magnetic assembly supported on a side of the tray facing away from the base and configured to vibrate with the tray, and a sample container carrier disposed on a side of the magnetic assembly facing away from the tray, the sample container carrier including a magnetic force for receiving a sample container, wherein the magnetic assembly is configured to control a magnetic force to a magnetic bead in the sample container. In this way, through setting up the magnetism subassembly of inhaling that can vibrate along with the tray for the check out test set according to the embodiment of this disclosure possesses vibrations and magnetism simultaneously and inhale multiple functions, has reduced the board position and has taken up, has improved the flexibility of instrument.

Description

Detection device and method for extracting nucleic acid and preparing library

Technical Field

The present disclosure relates to the technical field of clinical medical devices, and more particularly, to detection apparatus and methods for extracting nucleic acids and preparing libraries.

Background

The nucleic acid sequences encode information necessary for the functioning and reproduction of the organism. Determining such sequences is thus a useful tool in pure research of how and where organisms survive and in application sciences such as drug development. One focus of the sequencing industry has shifted to the discovery of higher throughput and/or lower cost nucleic acid sequencing technologies, sometimes referred to as "next generation" sequencing (NGS) technologies. NGS detection typically requires first extracting nucleic acids and then preparing an NGS library.

Disclosure of Invention

In a first aspect of the present disclosure, a detection apparatus is provided. The detection device includes a base, a tray resiliently secured to the base and configured to vibrate relative to the base under drive, a magnet assembly supported on a side of the tray facing away from the base and configured to vibrate with the tray, and a sample container carrier disposed on a side of the magnet assembly facing away from the tray, the sample container carrier including a plurality of slots for receiving the sample containers, wherein the magnet assembly is configured to control magnetic force to magnetic beads in the sample containers.

In a second aspect of the present disclosure, a method for extracting nucleic acid is provided. The method comprises the steps of operating a magnetic attraction component of the detection device to attract magnetic beads added into a first mixed liquid, enabling the magnetic beads to be static relative to a sample container, enabling the first mixed liquid to be located in the sample container placed in a sample container carrier, after removing supernatant in the first mixed liquid and adding ethanol to obtain a second reaction liquid, operating the magnetic attraction component of the detection device to reduce attraction to the magnetic beads in the second reaction liquid, enabling the magnetic beads to move relative to the sample container, and operating a tray to vibrate so as to mix the second reaction liquid in the sample container. After removing the ethanol in the second reaction solution and adding the eluent, the magnetic attraction assembly is operated to attract the magnetic beads in the eluted solution, so that the eluent is extracted to obtain nucleic acids.

It should be understood that what is described in this summary is not intended to limit the critical or essential features of the embodiments of the disclosure nor to limit the scope of the disclosure. Other features of the present disclosure will become apparent from the following description.

Drawings

FIG. 1A illustrates an overall schematic diagram of an example detection device according to an embodiment of the disclosure;

FIG. 1B shows a schematic cross-sectional view of an example detection device according to an embodiment of the disclosure;

FIG. 2 shows a schematic exploded view of a partial structure of an example detection device according to an embodiment of the disclosure;

Fig. 3A-3B show schematic cross-sectional views of an example detection device in different states according to embodiments of the present disclosure;

FIG. 4 shows a flowchart of an example method for extracting nucleic acid according to an embodiment of the disclosure, an

FIG. 5 illustrates a flowchart of an example method 500 for preparing a methylation library according to an embodiment of the present disclosure.

Detailed Description

Various embodiments are now described with reference to the drawings, wherein like reference numerals are used to refer to like elements throughout. In the following description, for purposes of explanation, numerous specific details are set forth in order to provide a thorough understanding of one or more embodiments. However, it may be apparent that in some or all cases any of the embodiments described below may be practiced without resorting to the specific design details described below. In other instances, well-known structures and devices are shown in block diagram form in order to facilitate describing one or more embodiments. The following presents a simplified summary of one or more embodiments in order to provide a basic understanding of the embodiments. This summary is not an extensive overview of all contemplated embodiments, and is intended to neither identify key or critical elements of all embodiments nor delineate the scope of any or all embodiments.

References in the framework of the present description to "an embodiment" or "one embodiment" are intended to indicate that a particular configuration, structure, or characteristic described in connection with the embodiment is included in at least one embodiment. Thus, phrases such as "in an embodiment" or "in one embodiment" that may be present in one or more points of the present description do not necessarily refer to the same embodiment. Furthermore, the particular structures, or characteristics may be combined in any suitable manner in one or more embodiments.

As discussed above, NGS assays typically require the extraction of nucleic acids prior to the preparation of an NGS library. The nucleic acid extraction is, for example, a process of extracting DNA or RNA from a biological sample. Typical procedures for nucleic acid extraction may include sample collection and/or lysis. In this step, a biological sample (e.g., blood, tissue, or plant material) is collected and lysed to release the nucleic acids. A typical procedure for nucleic acid extraction may include removal of impurities. In this step, impurities such as cell debris and/or proteins are removed by centrifugation or filtration. A typical procedure for nucleic acid extraction may also include nucleic acid binding. In this step, the nucleic acid is bound to a solid support (e.g., a silica gel film or magnetic beads). A typical procedure for nucleic acid extraction may also include washing. In this step, the carrier is washed with a buffer to remove unbound impurities. A typical procedure for nucleic acid extraction may also include elution. In this step, the nucleic acid on the carrier is eluted with an elution buffer. Typical procedures for nucleic acid extraction may include precipitation and resuspension. In this step, the nucleic acid is precipitated and resuspended with an appropriate buffer.

NGS library preparation is then the process of preparing DNA samples into libraries that can be used for high throughput sequencing. A typical procedure for the preparation of an NGS library may include, for example, DNA fragmentation. In this step, the DNA sample is fragmented into smaller fragments. A typical procedure for NGS library preparation may include end repair. In this step, the fragmented DNA ends are repaired to have smooth ends. A typical procedure for NGS library preparation may include a tail addition. In this step, an A tail is added to the 3' -end of the DNA fragment. A typical procedure for NGS library preparation may involve linker ligation. In this step, a linker (short DNA sequence) is attached to the A-tail of the DNA fragment. A typical procedure for NGS library preparation may include PCR amplification. In this step, the adaptor-ligated DNA fragments are amplified using PCR. A typical procedure for NGS library preparation may include purification. In this step, the amplified library is purified to remove unreacted primers and dimers. A typical procedure for NGS library preparation may include quantification and quality control. In this step, the library is quantified and its quality assessed to ensure that it is suitable for sequencing.

It can be seen that the procedure for NGS library preparation involves a large number of steps, and that most of the steps in the procedure require separate instrumentation. Thus, multiple plate sites are required in NGS library preparation instruments. For example, in manual experiments, a number of instruments are required, such as a series of devices such as PCR instruments, thermostatted water baths, magnetomotive racks, pipette guns, centrifuges, etc. However, transferring the test tubes, well plates and liquids on individual devices is prone to losses, resulting in high costs and poor performance. In summary, in pipetting workstations, different modules require different plate positions, which results in problems of laborious operation, high costs, high losses and low performance.

In view of the foregoing, embodiments of the present disclosure provide a detection apparatus that combines vibration and magnetic bead manipulation functions. The detection device has a tray for realizing a vibration function. The tray is provided with a magnetic attraction component for providing a magnetic bead operation function. A carrier for receiving the sample container is provided on the magnetic assembly. In the step of executing vibration, the vibration function of the tray can be started to drive the magnetic attraction component in the tray and the sample container in the sample container carrier to vibrate. Meanwhile, in the step of controlling the magnetic beads in the sample container, the magnetic attraction component can be started to realize the magnetic bead control. In this way, the detection device can be used for example in the separation, extraction and/or purification of nucleic acids and/or proteins, and/or in multiple steps in library preparation, thereby reducing the occupation of plate sites, improving the flexibility of the instrument, while having an automated performance.

The structure of the detection apparatus according to the embodiment of the present disclosure will be described below with reference to fig. 1A to 2. Next, a state in which the detection apparatus according to the embodiment of the present disclosure is in use will be described with reference to fig. 3A to 3B. Finally, a procedure for preparing NGS libraries according to an embodiment of the present disclosure is described with reference to fig. 4 and 5. Fig. 1A shows an overall schematic diagram of an example detection device 100 according to an embodiment of the disclosure. As shown in fig. 1A, the inspection apparatus 100 includes a base 110 adapted to be placed on a countertop. The inspection apparatus 100 further includes a tray 120 elastically fixed to the base 110. Here, the tray 120 is fixed in a first direction D1 perpendicular to the slave operation table. The tray 120 can vibrate with respect to the base 110 under drive. In the embodiment shown in fig. 1A, the base 110 has a hollow rectangular parallelepiped-shaped body. At the upper edges of the four walls of the main body, a tray 120 is provided.

The detection apparatus 100 also includes a magnetic attraction assembly 130. The magnetic attraction assembly 130 is supported on a side of the tray 120 facing away from the base 110 in the first direction D1. The magnetic assembly 130 is capable of vibrating with the tray 120. Furthermore, the detection device 100 comprises a sample container carrier (not shown in detail) arranged on the side of the magnetic attraction assembly 130 facing away from the tray 120. The sample container carrier comprises a plurality of slots for receiving sample containers 160. In the embodiment shown in fig. 1, the sample container 160 is a deep well plate having 8x12 wells. The walls of the wells of the deep well plate are received in a plurality of wells of the sample container carrier. It should be appreciated that the sample container 160 may be any commercially available deep-well plate having a number of empty wells corresponding to the number of wells as a fitment for the detection device 100 and not belonging to the detection device 100.

Here, the magnetic attraction assembly 130 can control the magnetic force to the magnetic beads in the sample container. In some embodiments, the magnetic attraction assembly 130 may include a permanent magnet movable in a first direction D1. The magnet increases as it approaches the magnetic bead, and the magnetic force decreases as it moves away from the magnetic bead. In other embodiments, the magnetic attraction assembly 130 may also include an electromagnet positioned adjacent to the sample container carrier. When the electromagnet is electrified, the electromagnet is electrified and magnetized, so that the magnetic beads are attracted. Demagnetizing when the electromagnet is not energized, so that magnetic force to the magnetic beads is not generated, i.e. the magnetic force is eliminated. For example, the magnetic force of the magnetic attraction assembly 130 on the magnetic beads may be increased when pipetting to make the magnetic beads stationary relative to the sample container, thereby avoiding aspiration by the pipette. Accordingly, in operations requiring movement of the magnetic beads in the sample container, such as mixing, the magnetic force of the magnetic attraction assembly 130 on the magnetic beads may be reduced to enable movement of the magnetic beads relative to the sample container. In this way, the vibration and magnetic attraction functions in one board are realized.

Fig. 1B shows a schematic cross-sectional view of an example detection apparatus 100 according to an embodiment of the disclosure. As shown in fig. 1B, the base 110 of the inspection apparatus 100 includes a base bottom 111. The base bottom 111 includes a second plane A2 extending in the horizontal direction. The base 110 also includes a base wall 112. The base wall 112 extends from an edge of the base bottom 111 in the first direction D1 toward the tray 120, thereby forming a middle receiving cavity. The base 110 has an opening, and a tray 120 is provided at the opening.

To realize the vibration function, the tray 120 is connected to the second plane A2 of the base 110 via the elastic member 121. The elastic member 121 includes a guide rod 124. The guide bar 124 is fixed to the side of the tray 120 facing the base 110, i.e., the bottom of the tray 120, and extends toward the base bottom 111 and reaches near the second plane A2. One end of the guide rod 124 adjacent to the base bottom 111 is connected to a first end of the elastic body 125. The elastic body 125 may be, for example, a cylindrical spring. The other end of the elastic body 125 is connected to the second plane A2 of the base bottom 111. In addition, the peripheral wall of tray 120 is adjacent to base wall 112, but is not connected to base wall 112. Thus, the tray 120 can move relative to the base 110 by external force. The base wall 112 also provides some guiding action for the tray 120 during movement. The tray 120 also includes a vibration motor 122. The vibration motor 122 is disposed at the bottom of the tray 120 and configured to drive the tray 120 to vibrate. By providing the vibration motor 122 and the elastic member 121, a vibration function of the tray 120 is achieved.

A magnetic attraction assembly 130 is provided in the tray 120. The magnetic assembly 130 includes a support plate 133. The support plate 133 includes a first plane A1 parallel to a second plane A2. The magnet assembly 130 further includes a plurality of magnets 131. A plurality of magnets 131 are fixed on the first plane A1. The magnetic assembly 130 further includes a lifting device 132. The elevating device 132 is coupled to the support plate 133. The lifting device 132 is operable to drive the magnet support plate 133 to move in a first direction D1 perpendicular to the first plane A1.

In some embodiments, lifting device 132 may drive support plate 133 to move plurality of magnets 131 with it toward sample container carrier 140 if an increased magnetic force is desired, such that the magnetic force of plurality of magnets 131 on magnetic beads in a sample container in sample container carrier 140 increases with increasing distance of plurality of magnets 131 from sample container carrier 140. In contrast, the lifting device 132 may drive the support plate 133 to move the plurality of magnets 131 with it toward the tray 120 if a reduced magnetic force is desired, such that the magnetic force of the plurality of magnets 131 on the magnetic beads in the sample containers in the sample container carrier 140 decreases with a decrease in the distance of the plurality of magnets 131 from the sample container carrier 140.

In some embodiments, the lifting device 132 may include a motor 134 secured to the base 110. The motor 134 includes an output shaft. The lifting device 132 may also include a drive mechanism. The driving mechanism couples the output shaft of the motor to the support plate 133 and is capable of converting the rotational motion of the motor into the linear motion of the support plate 133. For example, the drive mechanism may be a geared screw mechanism. In such an embodiment, the gear can rotate with the output shaft and convert the rotation into linear motion of the screw, thereby enabling the screw to push the support plate 133 up and down. In the embodiment shown in fig. 1B, the driving mechanism is a rotating shaft 135. The first end of the rotation shaft 135 is coupled to the output shaft, and the second end thereof is screw-coupled to the magnet support plate 133 through the shaft hole of the tray 120. In such an embodiment, the shaft 135 can rotate with the output shaft of the motor 134. At this time, since the support plate 133 cannot rotate under the restriction of the tray peripheral wall, the support plate 133 will move in the first direction D1 by the screw connection.

In the embodiment shown in fig. 1B, the magnetic attraction assembly 130 further includes a spacer plate 137. The partition plate 137 is fixed to the tray 120. The partition plate 137 includes a plurality of through grooves 138 extending in the first direction D1. Each of the plurality of through slots 138 is respectively opposite to a corresponding magnet of the plurality of magnets such that the magnet is movable in the through slot along the first direction D1. A sample container carrier 140 is supported on the intermediate plate 137. The sample container carrier 140 comprises a plurality of slots 141 for receiving sample containers. The plurality of slots 141 are arranged in an array including a plurality of rows of slots and a plurality of columns of slots.

In the embodiment shown in fig. 1B, each row of slots corresponds to one through hole, so that after placing a sample container in a slot, the magnet can be moved in the through slot to the bottom of the sample container carrier 140 as close as possible to the sample container. In some embodiments, the support plate may push the plurality of magnets up against the bottom of the sample container carrier 140 under the drive of the lifting device. The position of the plurality of magnets against the bottom of the sample container carrier 140 may be referred to as the first position of the magnets. Accordingly, the support plate may lower the plurality of magnets to the bottom of the tray under the driving of the lifting device, and this position may be referred to as a second position of the magnets. In the illustrated embodiment, a buffer 136 is also provided on the support plate 133 in order to avoid a large impact on the bottom of the sample container carrier 140 during the magnet lifting.

The structure of the plurality of upper members such as the tray will be described in detail with reference to fig. 2. Fig. 2 shows a schematic exploded view of a portion 200 of an example detection device according to an embodiment of the disclosure. The detection device may correspond to the detection device in fig. 1A and 1B. As shown in fig. 2, portion 200 includes tray 120, a portion of the assembly of magnetic attraction assembly 130, and sample container 260. The tray 120 shown in fig. 2 includes a tray bottom 221. The tray bottom 221 extends in a direction parallel to the second plane A2. The tray 120 also includes a peripheral wall 222. The peripheral wall 222 extends from the edge of the tray bottom 221 in the first direction D1 to form a receiving chamber therebetween. Tray 120 also includes flange 223. The flange 223 extends outwardly from the top of the peripheral wall 222, i.e., the end adjacent the tray 120 in the first direction D1, in a direction parallel to the first plane. The tray 120 also includes a plurality of bumps 224. A plurality of bumps 224 extend from the edge of flange 223 in a first direction D1 away from the tray bottom 221.

The partial assembly of the magnetic assembly 130 includes a support plate 133, a plurality of magnets 131, a spacer plate 137, and a spacer plate spacer 239. In the embodiment shown in fig. 2, the support plate 133 is a chamfered rectangle corresponding to the peripheral wall 222 of the tray 120. The spacer 137 corresponds to the shape of the spacer pad 239 but is different in thickness. The partition plate 137 includes a plurality of through slots 138. Spacer plate pad 239 also includes a corresponding plurality of through slots. The partition plate 137 is rectangular parallelepiped. The plurality of through grooves 138 have a rectangular cross section, and the longitudinal direction of the through grooves, that is, the direction in which the length of the through grooves (third direction D3) is located corresponds to the lateral direction of the partition plate 137, that is, the direction in which the width of the partition plate 138 is located. The plurality of through grooves 138 are distributed in the longitudinal direction (second direction D2) of the partition plate 137. The magnet 131 is rectangular parallelepiped, its longitudinal direction extends in the third direction D2, its lateral direction extends in the second direction, and its thickness extends in the first direction D1. The cross section of the magnet corresponds to the cross section of the through slot so that the magnet can move in the through slot.

In the assembled state, the support plate 133 is placed on the tray bottom 221 of the tray 120. Spacer 137 and spacer washer 239 overlie flange 223 and are coupled to boss 224. In some embodiments, a temperature control device (TEC) is disposed between the bump 224 and the sidewall of the spacer 137. A space allowing the support plate 133 to move in the first direction D1 is formed between the partition plate 137 and the tray bottom 221 of the tray 120. The magnets 131 are arranged on the support plate 133 and extend into corresponding through slots 138 of the spacer plate 137. The guide of the magnet 131 by the through groove 138 of the partition plate 137 can be such that the magnet is accurately moved to a predetermined position.

The peripheral wall of the sample container 260 in fig. 2 shields the sample container carrier for carrying the sample container 260. The sample container carrier is fixed to the partition plate 137 and carries the sample container 260. The sample container 260 includes a plurality of deep holes 261. The walls of the plurality of deep holes 261 extend into corresponding slots of the sample container carrier.

Returning to fig. 1B, the inspection apparatus 100 further includes a temperature control device 150 also disposed around the side wall of the partition plate 137. The sample container carrier 140 comprises a metal bath block 142 on the surface of the spacer plate 137 facing away from the tray 120. The temperature control device 150 can be controlled to adjust the temperature of the partition plate 137 in contact therewith. Thereafter, since the partition plate 137 is in contact with the metal bath block 142, the temperature of the partition plate 137 affects the temperature of the metal bath block 142, thereby enabling the temperature control device 150 to control the temperature of the metal bath block 142 via the partition plate 137. In some embodiments, a temperature sensor may also be provided at the metal bath block 142. The temperature sensor may transmit the detected temperature of the metal bath block 142 to the control unit to cause the control unit to control the temperature control device 150 according to the detected temperature and the predetermined sum temperature. In some embodiments, a temperature protection switch may also be provided. The temperature protection switch may turn off the temperature control device 150 when the temperature of the metal bath block 142 exceeds a predetermined threshold.

In the embodiment shown in fig. 1B, the various components of the detection unit 100 are described. The vibration function of the sensing unit 100 can be achieved by driving the tray 120 to vibrate with respect to the base 110 using a vibration motor. The distance between the magnets and the sample container is controlled by lifting and lowering the plurality of magnets 131 by the support plate 133, so that the control of the magnetic beads can be realized. The temperature control function can be realized by controlling the temperature of the metal bath block 142 by the temperature control device 150. In this way, one detection device can be provided with the functions of PCR amplification, purification, sample extraction and/or lysis. The detection device can be used, for example, for the separation, extraction, and/or purification of nucleic acids and/or proteins, and/or library preparation, reducing plate occupancy, and increasing instrument flexibility. In addition, after the automatic pipetting device is set, the detection is controlled by a predetermined program of the controller, and automation can be realized.

The states of the detection device at different operations will be described below with reference to fig. 3A to 3B. Fig. 3A shows a cross-sectional view of an example detection device 100 in a state 300A in which magnetic beads are not attracted, according to an embodiment of the disclosure. The detection apparatus 100 in fig. 3A corresponds to the detection apparatus shown in fig. 1B, for example. The structure and the operation principle are the same, and for the sake of brevity, the description is omitted here. As shown in fig. 3A, a sample container 360 is placed on sample container carrier 140. Sample container 360 has a plurality of slots 361. In the embodiment shown in fig. 3A, sample container 360 is, for example, a deep well plate having a deep hole of 12x 8. That is, the plurality of grooves 361 are arranged in 12 columns 8 rows. Figure 3A shows a row of 12 slots. Correspondingly, the spacer 137 has 7 through slots, and the magnet assembly 130 has 7 magnets. The two outer channels on the spacer plate 137 are each adjacent to a row of channels on the sample container carrier 140. The central 5 through slots on the spacer plate 137 are respectively adjacent to two adjacent rows of slots on the sample container carrier 140.

As shown in fig. 3, a reaction solution and magnetic beads are placed in each well of the sample container 360. In state 300A, the support plate 133 of the detection device 100 is positioned at the bottom of the tray 120 such that the plurality of magnets 131 are positioned at the furthest distance from the magnetic beads in the sample container 360. In this position, the magnetic force of the magnet on the magnetic beads is minimal, enabling the magnetic beads to move freely in the grooves of the sample container 360. In this state 300A, for example, a vibration operation may be performed to mix the magnetic beads and the reaction liquid uniformly.

Fig. 3B shows a cross-sectional view of an example detection device 100 in a state 300B that attracts magnetic beads, according to an embodiment of the disclosure. As shown in fig. 3B, in state 300B, the plurality of magnets 131 rise from a position at the bottom of the tray 120 disk to abut the bottom of the sample container carrier 140, at a position closest to the distance of the magnetic beads in the sample container 360. In this position, the magnetic force of the magnet on the magnetic beads is maximized such that the magnetic beads are concentrated at the walls of the groove of the sample container 360 while being stationary with respect to the groove. In this state 300B, for example, a pipetting operation, such as extraction of supernatant or elution of eluted eluate, may be performed.

Fig. 4 shows a flowchart of an example method 400 for extracting nucleic acids, according to an embodiment of the disclosure. The method 400 may be performed manually, for example, using the detection device shown in fig. 1B, or may be performed by a controller (e.g., having a processor and memory) of the detection device executing a predetermined program. As shown in fig. 4, at 402, method 400 includes operating a magnetic attraction assembly of a detection device to attract a first set of magnetic beads added to a first mixed liquor that is subjected to a cleavage reaction such that the first set of magnetic beads is stationary relative to a first sample container. Here, the first mixed liquor is located in a first sample container which is placed in a sample container carrier.

In some embodiments, prior to this step, a lysis reaction may be performed in the first sample vessel. For example, the temperature control means of the detection device may be operated to maintain the first reaction liquid in the first sample container at the first temperature for a first predetermined time, thereby obtaining the first mixed liquid. Here, the first reaction liquid includes a sample and a lysate.

In some embodiments, a sample container containing a sample may be placed into a sample container carrier of a detection device and a lysis solution added to obtain a first reaction solution. Thereafter, a temperature control device of the detection apparatus, such as a thermoelectric cooler (TEC), may be operated to maintain the first reaction solution at 60 ℃ for one hour. Then, a first set of magnetic beads can be added, and a vibration motor is started after the magnetic beads are added, so that the tray drives the sample container carrier to vibrate together. Thereby, a sample and magnetic bead mixing operation is performed. After the sample is mixed with the magnetic beads, the magnetic attraction assembly may be operated to raise the magnet to a first position nearest the container to maximize attraction to the magnetic beads so that the magnetic beads collect on the walls of the sample container while stationary relative to the sample container.

At 404, method 400 includes operating a magnetic attraction assembly to reduce attraction to the first set of magnetic beads after the first mixed liquor is removed and ethanol is added. In some embodiments, the supernatant may be aspirated manually with a pipette or automatically with an automated pipetting device after the magnet is affixed to the wall of the sample container, and the aspirated supernatant discarded, leaving only the magnetic beads in the sample container. After that, ethanol is added to the sample container to perform a washing operation. In a washing operation, the magnet assembly is first operated to move the magnet to a second position furthest from the sample container to minimize attraction of the magnetic beads to enable movement of the magnetic beads relative to the sample container.

At 406, the method 400 includes operating the tray to shake to mix the first set of magnetic beads and ethanol in the first sample container. In some embodiments, the shaking motor may be activated to shake the sample container carrier with the tray for 5 minutes to mix the beads with the ethanol. Thereafter, the magnet may be operated to move to the first position to attract the magnetic beads. In the case where the magnetic beads are attracted, the ethanol may be sucked up and discarded to complete one washing operation. In some embodiments, multiple washing operations may be performed before elution is performed.

At 408, method 400 includes performing an elution operation after ethanol is removed and an eluent is added. At 410, method 400 includes operating a magnetic attraction assembly to attract a first set of magnetic beads such that the eluate is extracted to obtain nucleic acids. In some embodiments, the TEC may be operated for 10min at 30 ℃ after the eluent is added to the sample container. Thereafter, the magnet may be operated to move to the first position to attract the magnetic beads. After the magnetic beads are attracted, the eluate can be transferred to a new sample container for later use, and the magnetic beads discarded to obtain the extracted nucleic acids. Thereby, the flow of nucleic acid extraction is completed.

FIG. 5 illustrates a flowchart of an example method 500 for preparing a methylation library according to an embodiment of the present disclosure. The method 500 may be performed manually, for example, using the inspection apparatus shown in fig. 1B, or may be performed by detecting a controller (e.g., having a processor and memory) of the apparatus to execute a predetermined program. As shown in fig. 5, at 502, the method 500 includes operating a magnetic attraction assembly of a detection device to attract a second set of magnetic beads added to a second mixed liquor that is subjected to an amplification reaction, such that the second set of magnetic beads is stationary relative to a second sample container, the second mixed liquor being in a second sample container that is placed in a sample container carrier.

In some embodiments, the temperature control means of the detection device may be operated to maintain the second reaction solution at the third temperature for a third predetermined time after the enzyme deamination reaction reagent is added to the second reaction solution in the second sample container, thereby obtaining a third mixed solution. Here, the second reaction solution includes the extracted nucleic acid. Thereafter, the temperature control means of the detection apparatus may be operated to maintain the third mixed liquor at the fourth temperature for a fourth predetermined time. After the connection reagent is added to the third mixed solution, the temperature controlling means of the detecting apparatus may be operated to maintain the second reaction solution at the fifth temperature for a fifth predetermined time, thereby obtaining a fourth mixed solution. After the amplification reaction reagent is added to the fourth mixed solution, an amplification reaction may be performed using a temperature control apparatus to obtain a second mixed solution.

In some embodiments, the nucleic acid extraction step may be advanced. For example, a sample container containing 5mc/5hc of the oxidized biological sample may be placed onto the sample container carrier of the detection device. Thereafter, a third set of magnetic beads was added to the sample container using a pipette. After the magnetic beads are added, a vibration motor can be started to drive a tray to drive a sample container carrier to vibrate for 5min so as to uniformly mix the magnetic beads with a sample. After stopping the vibration motor, the stepper motor may be operated to raise the support plate through the spindle to raise the magnet to a second position against the bottom of the sample container carrier. Thereby, the magnetic beads are attracted to the walls of the sample carrier according to the magnetic field strength, stationary with respect to the sample carrier. Thereafter, the liquid in the sample may be withdrawn using a pipette and discarded. After only the magnetic beads remained in the sample container, 80% ethanol may be added to perform washing of the magnetic beads.

The washing step includes, for example, operating the stepper motor to lower the magnet to a second position at the bottom of the tray such that the magnet no longer attracts the magnetic beads. After the magnetic beads are released, the vibration motor may be started to mix the ethanol with the magnetic beads, and the mixing operation is performed for 5min. After the blending operation is completed, the vibration motor is stopped. Thereafter, the stepping motor is operated again to move the magnet to the first position, thereby attracting the magnetic beads. After the magnetic beads are attracted, the ethanol is sucked to complete one-time washing. Here, the washing operation may be repeated twice.

After the washing operation, an elution buffer may be added to the sample container. Thereafter, the temperature control device may be operated to heat the sample container at 25 ℃ for 5 minutes. Thereafter, the supernatant is aspirated into a new sample container, and the magnetic beads are discarded with the original sample container. Thereafter, a new sample container is placed on the sample container carrier of the detection device and an enzyme deamination reaction reagent (i.e. apodec reaction reagent) is added to the sample container. The temperature control device was operated to heat at 98 ℃ for 10min. After heating is completed, the temperature control device rapidly lowers the temperature of the sample container to 0 ℃ and maintains the temperature for 10min. Thereafter, a ligation reagent was added to the sample container, and the temperature control device was operated for 60min at 30℃to perform a ligation reaction.

After completion of the ligation reaction, PCR reagents are added to the sample container and ten cycles are performed according to a temperature control sequence, e.g., 98 ℃ 45s, 98 ℃ 15s,60 ℃ 30s,72 ℃ 30s, respectively. After cycling, the PCR reaction was completed to amplify the nucleic acid by maintaining at 72℃for 1min and finally at 4 ℃.

At 504, method 500 includes operating the magnetic attraction assembly to reduce attraction to the second set of magnetic beads after the second mixed liquor is removed and ethanol is added. At 506, the method 500 includes operating the tray to shake to mix the second set of magnetic beads and ethanol in the second sample container. At 508, method 500 includes performing an elution operation after the ethanol is removed and the eluent is added. Finally, at 510, the method 500 includes operating a magnetic attraction assembly to attract a second set of magnetic beads such that an eluate is extracted to obtain amplified nucleic acids. Here, after the amplification is completed, washing and elution with ethanol are performed at 504 to 510, and finally the supernatant is extracted to obtain an eluted product, i.e., amplified nucleic acid. The eluted product is transferred to a new sample vessel, thereby completing the methylated NGS library preparation procedure.

Throughout this specification, unless the context requires otherwise, the terms "comprise," "comprising," "includes," "including," and "having" will be understood to imply the inclusion of a stated step or element or group of steps or elements but not the exclusion of any other step or element or group of steps or elements. In particular embodiments, the terms "comprising," "including," "containing," and "having" are used synonymously.

As used herein, the term "or" is an inclusive "or" operator and is equivalent to the term "and/or" unless the context clearly indicates otherwise. The term "based on" is not exclusive and allows for being based on additional undescribed factors unless the context clearly indicates otherwise.

In the following disclosure, unless otherwise indicated, when absolute positional modifiers (such as the terms "front", "rear", "top", "bottom", "left", "right", etc.) or relative positional modifiers (such as the terms "above", "below", "higher", "lower", etc.) are referred to, or when directional modifiers (such as "horizontal", "vertical", etc.) are referred to, the orientations shown in the figures are referred to.

The indefinite articles "a" and "an" and the definite articles "the" and "the" as used herein in the specification and in the claims are to be understood to mean "at least one" unless explicitly indicated to the contrary. It should also be understood that, in any method claimed herein that includes more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited, unless clearly indicated to the contrary.

While preferred embodiments of the present invention have been shown and described herein, it should be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention.

Claims

1. A detection device (100), comprising:

Base (110);

a tray (120) elastically fixed on the base (110), and the tray (120) is configured to vibrate relative to the base (110) when driven;

a magnetic attraction component (130), supported on a side of the tray (120) facing away from the base (110) and configured to vibrate together with the tray (120); and

A sample container carrier (140) is arranged on a side of the magnetic attraction component (130) facing away from the tray (120), and the sample container carrier (140) includes a plurality of slots (141) for accommodating sample containers.

The magnetic attraction component (130) is configured to control the magnetic force on the magnetic beads in the sample container.

2. The detection device (100) according to claim 1, wherein the magnetic attraction component (130) further comprises:

A support plate (133) comprising a first plane (A1);

A plurality of magnets (131) are fixed on the first plane (A1); and

The lifting device (132) is coupled to the support plate (133), and the lifting device (132) is configured to drive the magnet support plate (133) to move in a first direction (D1) perpendicular to the first plane (A1).

3. The detection device (100) according to claim 2, wherein the lifting device (132) is configured to drive the support plate (133) to move the plurality of magnets (131) to a first position adjacent to the sample container carrier (140), and drive the first position to move to a second position spaced apart from the sample container carrier (140).

4. The detection device (100) according to claim 2, wherein the lifting device (132) comprises:

a motor (134) fixed to the base (110), wherein the motor (134) includes an output shaft; and

A rotating shaft (135) has a first end coupled to the output shaft, and a second end passing through the axial hole of the tray (120) and threadedly connected to the magnet support plate (133), and the rotating shaft (135) is configured to drive the support plate (133) to move along the first direction (D1) when rotating with the motor (134).

5. The detection device (100) according to claim 3, wherein the magnetic attraction component (130) further comprises:

A spacer plate (137) is fixed on the tray (120), and the spacer plate (137) includes a plurality of through slots (138) extending along the first direction (D1), wherein the plurality of magnets (131) are configured to move along the first direction (D1) in the plurality of through slots (138).

6. The detection device (100) according to claim 5, wherein the spacer plate (137) is a rectangular parallelepiped, the plurality of through grooves (138) have a rectangular cross-section, the longitudinal direction of the through grooves corresponds to the transverse direction of the spacer plate (137), and the plurality of through grooves (138) are distributed in the longitudinal direction of the spacer plate (137), wherein the magnet has a shape corresponding to the through grooves.

7. The detection device (100) according to claim 6, wherein the plurality of grooves (141) of the sample container carrier (140) includes a plurality of rows of grooves, and each row of the plurality of rows of grooves is adjacent to one of the plurality of through grooves (138).

8. The detection device (100) according to claim 1, wherein the tray (120) further comprises:

Plate bottom (221);

A peripheral wall (222) extending from the edge of the bottom (221) of the plate in a direction away from the base (110);

A flange (223) extending outward from the top of the peripheral wall (222) in a direction perpendicular to the peripheral wall (222); and

A plurality of protrusions (224) extend from the edge of the flange (223) in a direction away from the tray bottom (221).

9. The detection device (100) according to claim 8, wherein the base (110) comprises:

a base bottom (111); and

The base wall (112) extends from the edge of the base bottom (111) in a direction toward the tray (120) to a position adjacent to the peripheral wall (222).

10. The detection device (100) according to claim 9, wherein the tray (120) is connected to the base (110) via an elastic member (121), the elastic member (121) comprising:

a guide rod (124) fixed to a side of the tray (120) facing the base (110) and extending toward the base bottom (111); and

An elastic body (125) has one end connected to one end of the guide rod (124) adjacent to the base bottom (111), and the other end connected to the base bottom (111).

11. The detection device (100) according to claim 1, wherein the tray (120) comprises a vibration motor (122), and the vibration motor (122) is arranged at the bottom of the tray (120) and is configured to drive the tray (120) to vibrate.

12. The detection device (100) according to claim 1 further includes a temperature control device (150) arranged around the magnetic attraction component (130), and the sample container carrier (140) includes a metal bath block (142), wherein the temperature control device (150) is configured to transfer heat with the metal bath block (142) via the magnetic attraction component (130).

13. A method for extracting nucleic acid, comprising:

Operating the magnetic attraction component (130) of the detection device to attract a first group of magnetic beads added to a first mixed solution that has undergone a cleavage reaction, so that the first group of magnetic beads is stationary relative to a first sample container, and the first mixed solution is located in the first sample container placed in a sample container carrier (140);

In response to the first mixed solution being removed and ethanol being added, operating the magnetic attraction assembly (130) to reduce attraction to the first set of magnetic beads;

The operating tray (120) vibrates to mix the first group of magnetic beads and the ethanol in the first sample container;

In response to the ethanol being removed and the eluent being added, performing an elution operation; and

The magnetic attraction component (130) is operated to attract the first group of magnetic beads, so that the eluate is extracted to obtain nucleic acid.

14. The method according to claim 13, further comprising:

The temperature control device (150) of the detection device is operated to maintain the first reaction liquid in the first sample container at a first temperature for a first predetermined time, thereby obtaining the first mixed liquid, wherein the first reaction liquid includes a sample and a lysate.

15. The method of claim 14, wherein performing an elution operation comprises:

The temperature control device (150) is operated to maintain the eluent in the first sample container at a second temperature for a second predetermined time.

16. The method according to any one of claims 13 to 15, wherein the detection device is a detection device according to any one of claims 1 to 12.

17. A method for preparing a methylation library, comprising:

Operating the magnetic attraction component (130) of the detection device to attract a second group of magnetic beads added to a second mixed solution subjected to an amplification reaction, so that the second group of magnetic beads is stationary relative to a second sample container, and the second mixed solution is located in the second sample container placed in a sample container carrier (140);

In response to the second mixed solution being removed and ethanol being added, operating the magnetic attraction assembly (130) to reduce attraction to the second set of magnetic beads;

The operating tray (120) vibrates to mix the second group of magnetic beads and the ethanol in the second sample container;

The magnetic attraction component (130) is operated to attract the second group of magnetic beads so that the eluate is extracted to obtain the amplified nucleic acid.

18. The method according to claim 17, further comprising:

In response to the addition of an enzyme deamination reaction reagent to the second reaction liquid in the second sample container, operating the temperature control device (150) of the detection device to maintain the second reaction liquid at a third temperature for a third predetermined time, thereby obtaining a third mixed solution, wherein the second reaction liquid includes the extracted nucleic acid;

operating the temperature control device (150) of the detection device to maintain the third mixed liquid at a fourth temperature for a fourth predetermined time;

In response to the addition of a ligation reaction reagent into the third mixed solution, operating the temperature control device (150) of the detection device to maintain the second reaction solution at a fifth temperature for a fifth predetermined time, thereby obtaining a fourth mixed solution; and

In response to the addition of an amplification reaction reagent into the fourth mixed solution, an amplification reaction is performed using the temperature control device (150) to obtain a second mixed solution.

19. The method of claim 18, wherein performing the amplification reaction further comprises:

According to a predetermined temperature control sequence, the temperature control device (150) is operated to control the temperature of the fourth mixed solution and the amplification reaction reagent.

20. The method according to any one of claims 17 to 19, wherein the detection device is a detection device according to any one of claims 1 to 12.