Gene sequencing reaction equipment, MDA amplification method and device

Abstract

The invention discloses gene sequencing reaction equipment and an MDA amplification method and device.The gene sequencing reaction equipment comprises a dropping liquid reaction device, an external transfer device and a dropping liquid loading device, and the dropping liquid reaction device comprises a dropping liquid reaction area; the liquid dropping reaction area is used for accommodating a sequencing slide glass dropwise added with polymerase for gene sequencing reaction and providing a reaction environment with a preset temperature for the sequencing slide glass; the external transferring device comprises an external material transferring unit and an external driving unit which is in transmission connection with the external material transferring unit; the dropping liquid loading device is used for dropping polymerase on the sequencing slide glass, so that the polymerase is in contact with a primer on the sequencing slide glass, and the primer is positioned on an open reaction surface on one side of the sequencing slide glass. According to the gene sequencing reaction equipment disclosed by the embodiment of the invention, the activity of polymerase can be maintained, the problem of non-uniformity caused by liquid change of a reagent runner is solved, full-flow automatic operation of MDA of a plurality of sequencing slides is supported, and the overall sequencing efficiency is improved.

Description

Technical Field

[0001] The present invention relates to the technical field of gene sequencing, and particularly relates to a gene sequencing reaction device, an MDA amplification method using the gene sequencing reaction device, and an MDA amplification reaction device. Background Art

[0002] In the DNBSEQ technology, after the first-strand sequencing is completed, a multiple displacement amplification (hereinafter simply referred to as MDA) reaction is carried out. The phi29 DNA polymerase used in this reaction will continue to synthesize and extend the first-strand sequencing strand, and at the same time carry out strand displacement. The displaced complementary strand becomes a new template to achieve paired-end sequencing. Since the phi29 DNA polymerase has a certain sensitivity to the reaction temperature, its optimal reaction temperature is about 30°C - 42°C. When the temperature is too low, the reaction activity is weak and the reaction efficiency is low, and the paired-end (hereinafter simply referred to as PE) sequencing purpose cannot be achieved; when the temperature is too high, Phi29 will be inactivated and protein impurities will be produced. Therefore, in the MDA reaction process, ensuring that the phi29 DNA polymerase has a high reaction activity is a crucial step. Moreover, due to the high temperature sensitivity of Phi29, an immersion reaction is generally not used.

[0003] The MDA step in the related technology is completed by combining a glass cover plate with a flow channel, a sealing ring, and an MGIDL-T20A instrument and equipment, forming a sealed structure with a flow channel to complete a series of steps of MDA. Therefore, the maintenance of the instrument and equipment is relatively cumbersome, the degree of automation of the staff operation is low, and the progress and efficiency of the overall sequencing on the machine are affected. At present, the DNBSEQ-T20×2 platform supports simultaneous sequencing of 6 slides. In this way, two MGIDL-T20A instruments are required to complete the MDA operation, which will increase the manual operation time of the staff and greatly reduce the efficiency. It is estimated that it will take 4 - 5 hours to complete MDA for all 6 slides plus instrument maintenance. Summary of the Invention

[0004] In the first aspect of the present invention, a gene sequencing reaction device is proposed. After adding the polymerase to the sequencing slide in a droplet manner and then moving it into the droplet reaction device for reaction, the activity of the polymerase can be maintained, the unevenness problem caused by reagent flow channel liquid replacement is solved, the reagent consumption is reduced, and the sequencing cost is lowered. In addition, it also supports fully automated operation of the MDA process for multiple sequencing slides, improving the overall sequencing efficiency.

[0005] In the second aspect of the present invention, an MDA amplification method for a gene sequencing reaction device is proposed, which uses the aforementioned gene sequencing reaction device.

[0006] In the third aspect of the present invention, an MDA amplification reaction device is proposed.

[0007] According to the first aspect of the present invention, the gene sequencing reaction equipment comprises: a droplet reaction device, an external transfer device and a droplet loading device, wherein the droplet reaction device comprises a droplet reaction zone, wherein the droplet reaction zone is used to accommodate a sequencing carrier to which a polymerase for gene sequencing reaction is dropped and to provide a reaction environment with a preset temperature for the sequencing carrier; the external transfer device comprises an external material transfer unit and an external drive unit connected to the external material transfer unit in a transmission manner, wherein the external material transfer unit is used to obtain, carry and release the sequencing carrier, and the external drive unit is used to drive the external material transfer unit to insert the sequencing carrier into the droplet reaction zone or to withdraw the sequencing carrier from the droplet reaction zone; the droplet loading device is used to drop a polymerase on the sequencing carrier so that the polymerase contacts the primers on the sequencing carrier, and the primers are located on an open reaction surface on one side of the sequencing carrier.

[0008] According to the gene sequencing reaction equipment of the embodiment of the present invention, after adding the polymerase to the sequencing slide in a dripping manner, it is moved into a droplet reaction device for reaction, which can maintain the activity of the polymerase and solve the uneven problem caused by the liquid replacement of the reagent flow channel. At the same time, the reagent usage is reduced and the sequencing cost is reduced. In addition, it also supports the full-process automated operation of MDA for multiple sequencing slides, thereby improving the overall sequencing efficiency.

[0009] In addition, the gene sequencing reaction device according to the above embodiment of the present invention may also have the following additional technical features:

[0010] In some embodiments, the droplet loading device includes: a bracket, a droplet pipeline and a driving pump, one end of the droplet pipeline is arranged on the bracket, and is used to drop polymerase on the sequencing carrier; the driving pump is connected to the other end of the droplet pipeline.

[0011] In some embodiments, the support includes a crossbeam and a first positioning block, wherein the first positioning block is relatively fixedly connected to the crossbeam to position the one end of the dripping pipeline on the crossbeam and is configured to drip toward a bottom of the crossbeam.

[0012] In some embodiments, the bracket further includes a second positioning block, which is relatively fixedly connected to the cross beam and positions a portion of the middle section of the dripping pipeline on the cross beam to constrain the dripping pipeline to extend along the cross beam.

[0013] In some embodiments, the dripping pipeline includes multiple ones, one end of each of the multiple dripping pipelines is arranged side by side on the bracket for dripping polymerase side by side on the sequencing slide, and the other end of each of the multiple dripping pipelines is connected to the driving pump.

[0014] In some embodiments, the external driving unit is configured to drive the sequencing slide to be horizontally placed below the droplet loading device and drive the sequencing slide to move along an "S"-shaped path in the horizontal direction.

[0015] In some embodiments, the droplet reaction device includes: a main body and a positioning member. The main body has the droplet reaction area, and an opening for inserting and withdrawing the sequencing slide is formed on one side of the droplet reaction area; the positioning member is used to position the sequencing slide in the droplet reaction area or release the positioning of the sequencing slide.

[0016] In some embodiments, the opening of the main body is opposite to the space below the droplet loading device.

[0017] In some embodiments, the gene sequencing reaction device further includes an immersion reaction device and an internal transfer device. The immersion reaction device includes a plurality of first immersion reaction areas for containing chemical reagents for gene sequencing reactions, so as to realize gene sequencing reactions by immersing the sequencing slide with a DNA sample loading structure on its surface and loaded with a DNA sample into the first immersion reaction area; the internal transfer device includes an internal material transfer unit that moves along a predetermined trajectory, and the plurality of first immersion reaction areas are arranged in sequence along the predetermined trajectory. The internal material transfer unit is used to pick up, carry, and release the sequencing slide to insert the sequencing slide into the first immersion reaction area or withdraw it from the first immersion reaction area. Among them, the external driving unit is also used to insert the sequencing slide into the first immersion reaction area or withdraw it from the first immersion reaction area.

[0018] In some embodiments, the immersion reaction device further includes a loading storage area for placing the sequencing slide. The loading storage area is located near the first immersion reaction area, and the external driving unit is also used to withdraw the sequencing slide from the loading storage area.

[0019] In some embodiments, the immersion reaction device further includes an unloading storage area for placing the sequencing slide. The unloading storage area is located near the first immersion reaction area, and the external driving unit is also used to insert the sequencing slide into the unloading storage area or withdraw it from the unloading storage area.

[0020] In some embodiments, the immersion reaction device further includes at least one second immersion reaction area for containing chemical reagents for gene sequencing reactions, so as to realize gene sequencing reactions by immersing the sequencing slide with a DNA sample loading structure on its surface and loaded with a DNA sample into the second immersion reaction area. The external driving unit is also used to insert the sequencing slide into the second immersion reaction area or withdraw it from the second immersion reaction area.

[0021] The MDA amplification method according to the embodiment of the second aspect of the present invention uses the gene sequencing reaction device as described above. The MDA amplification method includes: using a droplet loading device to drop polymerase on the sequencing slide, so that the polymerase contacts the primer on the sequencing slide, and the primer is arranged on the open reaction surface of the sequencing slide; transferring the sequencing slide with the polymerase for gene sequencing reaction dropped thereon to the droplet reaction area, and controlling the reaction temperature of the droplet reaction area.

[0022] Another embodiment of the present invention provides an MDA amplification reaction device, including an enzyme reaction area, an immersion reaction area, and the above-mentioned external transfer device. The external transfer device is configured to move the sequencing slide to the enzyme reaction area or the immersion reaction area for reaction according to a preset reaction sequence; the enzyme reaction area includes the above-mentioned droplet loading device and the above-mentioned droplet reaction device. The droplet loading device is used to drop polymerase on the sequencing slide in real time so that the polymerase contacts the primer on the sequencing slide. The sequencing slide has an open reaction surface for fixing the primer; the droplet reaction area has a reaction chamber for accommodating the sequencing slide with the polymerase dropped thereon and providing a reaction environment with a preset temperature for the sequencing slide; the immersion reaction area includes an immersion tank storing reaction reagents, and the reaction reagents include strand displacement amplification reagents.

[0023] For the gene sequencing reaction device, MDA amplification method, and MDA amplification device according to the embodiments of the present invention, the MDA step is integrated into the sequencing biochemical platform. The bare slide replaces the closed flow channel type of other current platforms through the immersion type and the droplet type. It supports the full-process automated operation of MDA for multiple sequencing slides, improving the overall sequencing efficiency; the immersion type realizes the reuse of reagents, the droplet type solves the uneven problem caused by reagent flow channel liquid change, reduces the reagent consumption at the same time, and lowers the sequencing cost. Description of the Drawings

[0024] Figure 1 It is a schematic diagram of a gene sequencing reaction device according to an embodiment of the present invention.

[0025] Figure 2 It is a schematic diagram of a gene sequencing reaction device according to an embodiment of the present invention.

[0026] Figure 3 It is a schematic diagram of a gene sequencing reaction device according to an embodiment of the present invention.

[0027] Figure 4 It is a schematic flowchart of an MDA amplification method according to an embodiment of the present invention.

[0028] Reference numerals: Gene sequencing reaction device 100, droplet reaction device 10, main body 11, positioning member 12, opening 101, external transfer device 20, external material transfer unit 21, external drive unit 22, droplet loading device 30, bracket 31, cross beam 313, first positioning block 311, second positioning block 312, droplet pipeline 32, drive pump 33, immersion reaction device 40, first immersion reaction zone 41, second immersion reaction zone 42, feeding storage area 43, discharging storage area 44, internal transfer device 50, sequencing slide 200. Detailed implementation manners

[0029] The slides used in gene sequencing devices that support massively parallel sequencing (MPS, also known as next-generation sequencing technology) are mainly closed flow cell slides. By pumping liquid into the flow channels, rapid and efficient biochemical reactions can be achieved. However, there are drawbacks such as high reagent costs and complex fluids (especially when the slide size is large). The Dip immersion sequencing biochemical solution provided in the related technology supports biochemical reactions on ultra-large-sized sequencing slides (such as the DNBSEQ-T20×2 gene sequencer released at the AGBT conference in the United States in February 2023) by using sequencing slides that are not encapsulated into flow channels, effectively reducing the sequencing cost and achieving <$1 / Gb for the first time.

[0030] Paired End sequencing (abbreviated as PE sequencing), based on single-end sequencing (SE), obtains double-end sequence information by performing a second-round amplification on the template strand and sequencing the other end of the DNA insertion fragment, which plays an important role in genome assembly.

[0031] Multiple displacement amplification (MDA) technology is widely used in whole-genome amplification and is currently the whole-genome amplification method with the widest coverage of the entire genome and the smallest amplification bias at each locus. Currently, this method is also used in the field of high-throughput sequencing to achieve paired-end sequencing (abbreviated as PE sequencing) of DNBs based on the MDA principle for DNB complementary strand generation (Rongqin Ke, et.al., Patent., US20160237488 A1).

[0032] In the DNBSEQ technology, after the first-strand sequencing is completed, the MDA reaction will be carried out. The phi29 DNA polymerase used in this reaction will continue to synthesize and extend the first-strand sequencing strand, while strand displacement occurs. The displaced complementary strand then becomes a new template to achieve paired-end sequencing. Due to the certain sensitivity of phi29 DNA polymerase to the reaction temperature, its optimal reaction temperature is about 30°C - 42°C. When the temperature is too low, the reaction activity is weak and the reaction efficiency is low, unable to achieve the purpose of PE sequencing; when the temperature is too high, Phi29 will be inactivated and protein impurities will be produced. Therefore, in the process of the MDA reaction, ensuring that the phi29 DNA polymerase has a high reaction activity is a crucial step. When each sequencing platform conducts the MDA reaction, the Phi29 polymerase needs to be mixed with the reaction buffer before use and stored at 2 - 8°C. Then, through the fluid system, the reagents are pumped into the sealed slide through the flow channel, and then the platform adsorbing the slide is heated to make phi29 in a high activity to complete the strand displacement reaction.

[0033] The Dip immersion sequencing biochemical technology uses an open biochemical reaction scheme and also adopts the MDA technology for PE sequencing. Due to the temperature-sensitive characteristic of the aforementioned Phi29 DNA polymerase, when conducting the MDA reaction, the immersion method is not used for the reaction (this method is difficult to support the reaction of multiple slides, resulting in an increase in sequencing costs). Instead, a glass cover plate with a flow channel and a sealing ring are designed to form a sealed structure with a flow channel together with the sequencing slide, and a series of steps of MDA are completed with the help of a sample loader (such as MGIDL-T20A).

[0034] Taking DNBSEQ-T20×2 using the Dip immersion sequencing technology as an example, its MDA steps are completed by combining a glass cover plate with a flow channel, a sealing ring, and the MGIDL-T20A instrument and equipment to form a sealed structure with a flow channel to complete a series of steps of MDA. The sequencing slide of the DNBSEQ-T20×2 platform is the largest in area among the DNBSEQ sequencing platforms. The closed structure composed of a glass cover plate and a sealing ring uses the flow channel to pump liquid to achieve the purpose of liquid exchange and biochemical reaction. To a certain extent, there will be problems such as incomplete liquid exchange, insufficient reaction, and poor uniformity, ultimately resulting in poor or failed signal recovery of the second strand after MDA, affecting the final sequencing result. Therefore, the current MDA reagent has a large liquid exchange volume, increasing the reagent cost.

[0035] The present invention integrates the steps of operating MDA on the MGIDL-T20A instrument onto the DNBSEQ-T20×2 sequencer. Since most steps are carried out through immersion reactions, we named it "DIPMDA". Only need to prepare the MDA kit before loading the samples, and no further staff intervention is required afterwards. Also, the equipment maintenance is combined with the maintenance after the sequencer is unloaded, greatly improving the automation level of the MDA steps. It is estimated that it takes about 3 hours to complete MDA for all 6 slides, significantly improving the efficiency.

[0036] The immersion method of the present invention does not need to rely on a glass cover plate to form a sealed structure, nor does it need to pump liquid through a flow channel. Therefore, it can support the various requirements of an open reaction system, and to the greatest extent avoid the problem of poor uniformity caused by insufficient liquid exchange during flow channel liquid replacement. Moreover, the amount of MDA reagent used in the present invention is reduced by about 50% compared to the amount used in the prior art (here referring to the reagent with phi29 polymerase which has a higher cost), reducing the reagent cost.

[0037] Therefore, the present invention provides a gene sequencing reaction device and an MDA amplification method. The MDA step is integrated into the sequencing biochemical platform, and the bare slide is used to replace the closed flow channel type of the current DNBSEQ-T20×2 and other DNBSEQ platforms through immersion and dropping methods. It supports fully automated operation of the entire process of MDA for multiple sequencing slides, improving the overall sequencing efficiency; the immersion method realizes the reuse of reagents, and the dropping method solves the non-uniformity problem caused by liquid replacement in the reagent flow channel, while reducing the reagent consumption and the sequencing cost.

[0038] The embodiments of the present invention will be described in detail below. The examples of the embodiments are shown in the accompanying drawings, where the same or similar reference numerals denote the same or similar elements or elements with the same or similar functions throughout. The embodiments described below with reference to the accompanying drawings are exemplary and are intended to explain the present invention and should not be construed as limiting the present invention.

[0039] As Figures 1 to 3 , the gene sequencing reaction device 100 according to the first aspect embodiment of the present invention includes: a dropping reaction device 10, an external transfer device 20, and a dropping loading device 30.

[0040] Specifically, the droplet reaction device 10 includes a droplet reaction zone, which can provide a reaction environment for the sequencing slide 200, on which a polymerase for gene sequencing reaction can be dripped, and the droplet reaction zone can provide a reaction environment with a preset temperature for the sequencing slide 200, so that the sequencing slide 200 can be accommodated in the droplet reaction zone for reaction. The external transfer device 20 includes an external material transfer unit 21 and an external drive unit 22, the external drive unit 22 is connected to the external material transfer unit 21, the external material transfer unit 21 is used to obtain, carry and release the sequencing slide 200, and the external drive unit 22 is used to drive the external material transfer unit 21 to insert the sequencing slide 200 into the droplet reaction zone or extract it from the droplet reaction zone; the droplet loading device 30 is used to drip polymerase on the sequencing slide 200, so that the polymerase contacts the primer on the sequencing slide 200, and the primer is located on the open reaction surface on one side of the sequencing slide.

[0041] In the process of performing a droplet reaction using the gene sequencing equipment, the sequencing carrier 200 can be moved to a corresponding position by the external transfer device 20, and the droplet loading device 30 can be controlled to drop reaction reagents (such as polymerase) onto the sequencing carrier 200. After an appropriate amount of reaction reagents are dropped onto the sequencing carrier 200, the sequencing carrier 200 can be transferred to the droplet reaction area by the external transfer device 20. The droplet reaction device 10 can provide a preset temperature environment for the sequencing carrier 200 to facilitate a polymerization reaction. After the reaction is completed, the sequencing carrier 200 can be removed by the external transfer device 20 so that the sequencing carrier 200 can proceed to the next step.

[0042] According to the gene sequencing reaction equipment 100 of the embodiment of the present invention, after adding the polymerase to the sequencing carrier 200 in a dripping manner, it is moved into a droplet reaction device for reaction, which can maintain the activity of the polymerase and solve the uneven problem caused by the liquid replacement of the reagent flow channel. At the same time, the reagent usage is reduced and the sequencing cost is reduced. In addition, it also supports the full-process automated operation of multiple sequencing carriers 200MDA, thereby improving the overall sequencing efficiency.

[0043] The external material transfer unit 21 may be a clamping structure, a suction cup structure, etc. For example, the external material transfer unit 21 is a clamping structure, which is used to clamp the sequencing carrier 200, maintain the clamping state, and is driven by the external drive unit 22 to a predetermined position to achieve the carrying of the sequencing carrier 200. The clamping of the sequencing carrier 200 may also be released to release the sequencing carrier 200. The sequencing carrier 200 may have a reaction area and a non-reaction area, wherein the reaction area may be used for dripping reaction or immersion reaction, and the non-reaction area may be used for carrying the external material transfer unit 21 or the internal material transfer unit described below. The external drive unit 22 may be a structure such as a robotic arm.

[0044] likeFigure 2 and Figure 3 In some embodiments, the droplet loading device 30 includes: a bracket 31, a droplet pipeline 32, and a driving pump 33. The bracket 31 can provide support for the droplet pipeline 32. One end of the droplet pipeline 32 can be arranged on the bracket 31. The sequencing slide 200 can be moved to a position corresponding to one end of the droplet pipeline 32 below the bracket 31 through the external transfer device 20, so that it is convenient for the droplet pipeline 32 to drop polymerase on the sequencing slide 200. The driving pump 33 is connected to the other end of the droplet pipeline 32, so that the reagent can be driven to flow along the droplet pipeline 32 through the driving pump 33 to facilitate droplet dropping. The structure of the droplet loading device 30 is simple, which can achieve stable droplet dropping. Moreover, the provided bracket 31 can stably support the droplet pipeline 32 and facilitate the movement of the sequencing slide 200 to a preset position to receive the reaction reagent, so that the reagent can be laid on the sequencing slide 200, which can increase the droplet area and reduce the reagent dosage.

[0045] Among them, the bracket 31 includes a cross beam 313 and a first positioning block 311. The cross beam 313 can provide support for the first positioning block 311 and the droplet pipeline 32. The first positioning block 311 is fixedly connected to the cross beam 313 to facilitate stable droplet dropping. One end of the droplet pipeline 32 can be positioned on the cross beam 313 through the first positioning block 311. When one end of the droplet pipeline 32 is positioned on the cross beam 313, one end of the droplet pipeline 32 is configured to drop liquid downward toward the cross beam 313. Among them, one end of the droplet pipeline 32 can be installed on the first positioning block 311, or the first positioning block 311 can be fixed on the cross beam 313, and one end of the droplet pipeline 32 can be positioned between the first positioning block 311 and the cross beam 313. By providing the first positioning block 311 and the cross beam 313, the stability of the droplet pipeline 32 can be further improved to facilitate droplet dropping.

[0046] In addition, in order to position the droplet pipeline 32, the bracket 31 may further include a second positioning block 312. The second positioning block 312 is fixedly connected to the cross beam 313, and a part of the middle section of the droplet pipeline 32 is positioned on the cross beam 313 to restrict the droplet pipeline 32 from extending along the cross beam 313. Through the cooperation of the second positioning block 312 and the first positioning block 311, the droplet pipeline 32 can be arranged to move along the extension direction of the cross beam 313, avoiding the droplet pipeline 32 from being wound around other components or even falling onto the sequencing slide 200 and affecting the sequencing result. The stability and service life of the gene sequencing reaction device 100 can be improved.

[0047] In order to improve the efficiency of droplet dispensing, multiple droplet dispensing pipelines 32 can be provided in the present invention. One ends of the multiple droplet dispensing pipelines 32 are arranged side by side on the bracket 31 for dispensing polymerase side by side on the sequencing slide 200, and the other ends of the multiple droplet dispensing pipelines 32 are connected to the driving pump 33. Specifically, the sequencing slide 200 can be arranged to move along the first direction (refer to the left-right direction in the attached Figure 2 drawing), and droplet dispensing is performed using the droplet dispensing pipelines 32. One ends of the multiple droplet dispensing pipelines 32 can be arranged in a form of being side by side along the second direction (refer to the front-back direction in the attached Figure 2 drawing). In this way, relative to one end of the droplet dispensing pipeline 32, when the sequencing slide 200 moves from one end along the first direction to the other end, one ends of the multiple droplet dispensing pipelines 32 arranged side by side can achieve a relatively large range of dispensing and laying of the reagent, thereby improving the dispensing efficiency and uniformity of the reagent and reducing the dosage of the reagent.

[0048] Among them, the external driving unit 22 is configured to drive the sequencing slide 200 to be horizontally placed under the droplet dispensing device 30 and drive the sequencing slide 200 to move in an "S"-shaped path in the horizontal direction. Specifically, the external driving unit 22 can drive the sequencing slide 200 to be located under the droplet dispensing device 30. Among them, the external driving unit 22 drives the sequencing slide 200 to move along the first direction. When the sequencing slide 200 moves from one end in the first direction to the other end relative to the droplet dispensing device 30, the laying of the reagent on a partial range of the sequencing slide 200 can be completed; subsequently, the external driving unit 22 drives the sequencing slide 200 to move along the second direction, and at this time the droplet dispensing device 30 can stop dispensing; subsequently, the external driving unit 22 drives the sequencing slide 200 to move along the first direction and controls the droplet dispensing device 30 to dispense. When the sequencing slide 200 moves from the other end in the first direction to the one end relative to the droplet dispensing device 30, the laying of the reagent on another part of the area of the sequencing slide 200 can be completed. By reciprocally moving the sequencing slide 200 in the "S"-shaped path described above and cooperating with the droplet dispensing device 30, the dispensing of the reagent on the sequencing slide 200 can be completed. By dispensing the reagent in this way, the efficiency of droplet dispensing can be improved, the dosage of the reagent can be reduced, and a relatively large range of laying of the reagent on the sequencing slide 200 can be achieved, facilitating a stable reaction.

[0049] Such as Figure 2 and Figure 3, in some embodiments of the present invention, the droplet reaction device 10 includes: a main body 11 and a positioning member 12. The main body 11 has a droplet reaction area, and an opening 101 for inserting and withdrawing the sequencing slide 200 is provided on one side of the droplet reaction area; the positioning member 12 is used to position the sequencing slide 200 in the droplet reaction area or release the positioning of the sequencing slide 200. After the polymerase is added to the sequencing slide 200, the external driving unit 22 can drive the sequencing slide 200 to insert the sequencing slide 200 into the droplet reaction area from the opening 101, and then use the positioning member 12 to position the sequencing slide 200. The positioning member 12 can be a positioning clamp, a positioning suction cup or a positioning magnetic structure, etc. For example, the suction cup can be attached to the sequencing slide 200, and the sequencing slide 200 can be positioned by evacuating the suction cup. After the sequencing slide 200 is positioned, the stable reaction of the polymerase can be achieved by using the temperature environment in the droplet reaction area. After the sequencing slide 200 completes the reaction in the droplet reaction area, the sequencing slide 200 can be taken out of the droplet reaction area by the external driving unit 22. Subsequently, the sequencing slide 200 can be moved to other processes of other devices. The structure of the droplet reaction device 10 is simple, which is convenient to insert the sequencing slide 200 into the droplet reaction area and also convenient to withdraw the sequencing slide 200 from the droplet reaction area. When the sequencing slide 200 is located in the droplet reaction area, the stable reaction of the polymerase can be achieved, improving the sequencing efficiency.

[0050] As Figure 2 and Figure 3 , the opening 101 of the main body 11 is opposite to the lower space of the droplet loading device 30. Thus, the sequencing slide 200 can be quickly placed into the droplet reaction area after the droplet is added, so as to improve the efficiency of gene sequencing and at the same time improve the stability of the polymerase.

[0051] As Figure 2 and Figure 3 , in some embodiments of the present invention, the gene sequencing reaction device 100 further includes an immersion reaction device 40 and an internal transfer device 50. The immersion reaction device 40 includes a plurality of first immersion reaction areas 41, and the first immersion reaction areas 41 are used to hold chemical reagents for gene sequencing reactions, so as to realize gene sequencing reactions by immersing the sequencing slide 200 with a DNA sample loading structure on the surface and loaded with a DNA sample in the first immersion reaction areas 41; the internal transfer device 50 includes an internal material transfer unit that moves along a predetermined trajectory, and a plurality of first immersion reaction areas 41 are arranged in sequence along the predetermined trajectory. The internal material transfer unit is used to obtain, carry and release the sequencing slide 200 to insert the sequencing slide 200 into the first immersion reaction area 41 or withdraw the sequencing slide 200 from the first immersion reaction area 41. Among them, the external driving unit 22 is also used to insert the sequencing slide 200 into the first immersion reaction area 41 or withdraw the sequencing slide 200 from the first immersion reaction area 41.

[0052] Combined with the foregoing, the external drive unit 22 can be used to extract the sequencing slide 200 from a first immersion reaction zone 41 and move it to a predetermined position to complete liquid dropping and reaction. Additionally, the sequencing slide 200 that needs to undergo an immersion reaction can be inserted into a first immersion reaction zone 41 to perform the immersion reaction. Further, when the sequencing slide 200 is moved to an immersion reaction zone by the external drive unit 22, or after manually moving the sequencing slide 200 to an immersion reaction zone, it can be moved between multiple immersion reaction zones by the internal transfer device 50 to achieve an orderly immersion reaction, improving the intelligence of the gene sequencing reaction device 100, facilitating the sequential and automated operation of the sequencing reaction, etc., reducing the reagent consumption, and increasing the sequencing efficiency and reducing the sequencing time.

[0053] Among them, the immersion reaction device 40 further includes a loading storage area 43 for placing the sequencing slide 200. The loading storage area 43 is located near the first immersion reaction zone 41, and the external drive unit 22 is further used to extract the sequencing slide 200 from the loading storage area 43. By using the loading storage area 43, the sequencing slide 200 can be temporarily stored and processed, etc. The external transfer device 20 can be used to transfer the sequencing slide 200 from the loading storage area 43 to the first immersion reaction zone 41, so as to facilitate the sequential movement of the sequencing slide 200 between different first immersion reaction zones 41 by the internal transfer device 50.

[0054] In addition, the immersion reaction device 40 further includes an unloading storage area 44 for placing the sequencing slide 200. The unloading storage area 44 is located near the first immersion reaction zone 41, and the external drive unit 22 is further used to insert the sequencing slide 200 into the unloading storage area 44 or extract it from the unloading storage area 44. By using the unloading storage area 44, the sequencing slide 200 can be temporarily stored. The external transfer device 20 can be used to transfer the sequencing slide 200 from the unloading storage area 44 to the dropping reaction zone, or transfer the sequencing slide 200 from the first immersion reaction zone 41 to the unloading storage area 44.

[0055] In some embodiments of the present invention, the immersion reaction device 40 further includes at least one second immersion reaction zone 42. The second immersion reaction zone 42 is used to hold chemical reagents for gene sequencing reactions, so as to achieve gene sequencing reactions by immersing the sequencing slide 200 with a DNA sample loading structure on its surface and loaded with a DNA sample into the second immersion reaction zone 42. The external drive unit 22 is further used to insert the sequencing slide 200 into the second immersion reaction zone 42 or extract it from the second immersion reaction zone 42. Among them, through the cooperation of the first immersion reaction zone 41, the second immersion reaction zone 42, and the dropping reaction zone, it is possible to achieve the orderly and stable operation of the gene sequencing reaction according to the requirements of different reagents, different reaction temperatures, and different reaction durations during the gene sequencing reaction process.

[0056] In addition, the internal transfer device 50 may include an internal material transfer unit and an internal drive unit, and the internal material transfer unit may be used to obtain, carry and release the sequencing carrier 200, and the internal drive unit is connected to the internal material transfer unit through transmission to move the sequencing carrier 200. The second immersion reaction area 42 in the present invention may include multiple, and therefore, multiple internal transfer devices 50 may be provided to move the sequencing carrier 200 more quickly, so as to shorten the cycle of the sequencing reaction.

[0057] like Figure 4 According to the MDA amplification method of the second aspect of the embodiment of the present invention, the MDA amplification method uses the aforementioned gene sequencing reaction equipment 100, and the MDA amplification method includes: using a drop loading device 30 to drip polymerase on the sequencing slide 200; transferring the sequencing slide 200 dripped with the polymerase for gene sequencing reaction to the drip reaction area, and controlling the reaction temperature of the drip reaction area. According to the gene sequencing reaction equipment 100 and the MDA amplification method of the embodiment of the present invention, the MDA step is integrated into the sequencing biochemical platform, and the closed flow channel type of other current platforms is replaced by the exposed slide through the immersion and dripping methods. The MDA full-process automated operation of multiple sequencing slides 200 is supported, which improves the overall sequencing efficiency; the immersion method realizes the reuse of reagents, and the dripping method solves the uneven problem caused by the replacement of reagent flow channels, while reducing the amount of reagents and the sequencing cost.

[0058] Another embodiment of the present invention provides an MDA amplification reaction device, including an enzyme reaction area, an immersion reaction area, and the above-mentioned external transfer device 20, wherein the external transfer device 20 is configured to move the sequencing carrier to the enzyme reaction area or the immersion reaction area for reaction according to a preset reaction sequence. The specific structure of the external reaction device 20 is similar to the external transfer device in the above-mentioned gene sequencing reaction equipment, and will not be repeated here.

[0059] The enzyme reaction area includes the above-mentioned drop loading device 30 and the above-mentioned drop reaction device 10. The drop loading device 30 is used to drip polymerase in real time when the sequencing carrier 200 is moved to the drop loading device 30 so that the polymerase contacts the primer on the sequencing carrier 200. The sequencing carrier 200 has an open reaction surface for fixing the primer. The drop reaction device 10 has a reaction chamber that accommodates the sequencing carrier to which the polymerase is dripped and provides a reaction environment with a preset temperature for the sequencing carrier 200. The specific structures of the drop loading device 30 and the drop reaction device 10 are respectively similar to the drop loading device and the drop reaction device in the above-mentioned gene sequencing reaction equipment, and are not described in detail here.

[0060] The immersion reaction zone includes an immersion tank storing reaction reagents, and the reaction reagents include strand displacement amplification reagents. The specific structure of the immersion reaction zone is similar to that of the above-mentioned immersion reaction device 40, and will not be elaborated here.

[0061] The gene sequencing reaction process of a specific embodiment of the present invention will be described below with reference to the accompanying drawings.

[0062] Since the MDA reagent containing phi29 DNA polymerase will cause enzyme inactivation and generate impurities when heated in the reagent tank for a long time, the present invention designs the reaction mode of phi29 DNA polymerase in the form of dripping liquid on the surface of the bare slide using eight pipelines, and then heats the platform for adsorbing the slide to provide the optimal reaction conditions for phi29 DNA polymerase. Therefore, the present invention combines the slide immersion and dripping methods to complete all the processes of MDA, and the process is shown in Table 1.

[0063] Table 1

[0064] Step Reagent Name Reaction Temperature (°C) Reaction Time (s) Reaction Method 1 DIPSEQ Regeneration Buffer 57 10 Immersion 2 DIPSEQ Regeneration Reagent 58 60 Immersion 3 DIPSEQ Regeneration Buffer 40 10 Immersion 4 DIPSEQ Sequencing Buffer 40 10 Immersion 5 DIPSEQ Sequencing Buffer 40 5 Immersion 6 DIPSEQ Sequencing Buffer 55 5 Immersion 7 MDA Primer 1 Working Solution 50 300 Immersion 8 1×phi29 buffer Room Temperature Indeterminate Immersion 9 MDA Reagent + phi29 DNA Polymerase 40 300 Dropwise 10 MDA Reagent 42 1500 Immersion 11 Elution Reagent 2 Room Temperature Indeterminate Immersion 12 DIPSEQ Regeneration Buffer 40 10 Immersion 13 DIPSEQ Sequencing Buffer 40 10 Immersion 14 DIPSEQ Sequencing Buffer 40 5 Immersion 15 Blocking Reagent 55 300 Immersion 16 Elution Reagent 2 55 10 Immersion 17 Elution Reagent 2 40 10 Immersion 18 Sequencing Primer 3 Working Solution 50 300 Immersion 19 Elution Reagent 2 Room Temperature Indeterminate Immersion

[0065] Among them, the immersion step is the same as the biochemical part of the DNBSEQ-T20×2 sequencer (reference can be made to the PCT international application with the international application number PTC / CN2017 / 104587), and the reaction is carried out by mechanically clamping the slide and vertically immersing it in the reagent tank. The dripping step is that the manipulator clamps the slide, the slide is placed horizontally, eight dripping pipelines are arranged in a row at equal intervals, and the MDA reagent containing phi29 DNA polymerase is spread on the slide along the "S" path, and then the mechanical clamp sends the slide to the adsorption platform. After the slide is stably adsorbed, it is heated to make phi29 DNA polymerase combine with the DNA strand and carry out strand displacement reaction.

[0066] The following will Figure 3 describe the working process of the gene sequencing reaction device according to the embodiment of the present invention in combination with the attached

[0067] 1. Insert the sequencing slide into the loading storage area. There can be multiple loading storage areas, and multiple sequencing slides can be placed in multiple loading storage areas respectively, and they can be placed manually or automatically.

[0068] 2. Transfer the sequencing slide in the loading storage area to a first immersion reaction zone through an external transfer device, and carry out the reaction process according to step 1 in Table 1.

[0069] 3. Transfer the sequencing slide through the internal transfer device in the corresponding multiple first immersion reaction zones in sequence according to Steps 1 to 7 in Table 1 to implement Steps 1 to 7 in Table 1. The reagent corresponding to Step 8 is stored in the discharge storage area 44, and the external drive unit (22) inserts the sequencing slide 200 after the reaction in Step 7 into the discharge storage area 44 to complete Step 8.

[0070] 4. Transfer the sequencing slide in the discharge storage area 44 corresponding to Step 8 to the corresponding position of the droplet loading device through the external transfer device, use the droplet loading device to add liquid, and then put the sequencing slide with polymerase dropped thereon into the droplet reaction zone to complete Step 9.

[0071] 5. After the droplet reaction is completed, transfer it to the second immersion reaction zone corresponding to Step 10. The second immersion reaction zone may include multiple ones, and the multiple second immersion reaction zones can react in parallel or in series. For example, the second immersion reaction zone includes two, and the two second immersion reaction zones are in series. The sequencing slide after Step 9 is completed can be moved to one second immersion reaction zone to run for a period of time, and then transferred to another second immersion reaction zone to run for a period of time to complete Step 10; for another example, the second immersion reaction zone includes two, and the two second immersion reaction zones are in parallel. The sequencing slide after Step 9 is completed can be moved to one second immersion reaction zone to run, and at the same time, another sequencing slide is moved to another second immersion reaction zone to run.

[0072] 6. Transfer the sequencing slide after Step 10 is completed to the loading storage area for temporary storage through the external transfer device to perform Step 11; and then transfer the sequencing slide to the first immersion reaction zone corresponding to Step 12 through the external transfer device, and then use the internal transfer device to transfer the sequencing slide in sequence in the corresponding multiple first immersion reaction zones according to Steps 12 to 18 in Table 1 to implement Steps 12 to 18 in Table 1.

[0073] 7. Transfer the sequencing slide after Step 18 is completed to the discharge storage area through the external transfer device to perform Step 19.

[0074] The present invention combines the Dip immersion sequencing biochemical technology and the droplet method to complete the entire process of MDA, realizing the automation of the MDA step in the DNBSEQ-T20×2 sequencing platform. The MDA step is shortened from 4-5 hours in the original operation mode to 3 hours, improving the efficiency of the entire sequencing process. Most of the steps in the MDA process of the present invention use immersion liquid exchange. After the slide is immersed in the reagent, the surface of the slide is in full contact with the reagent, avoiding the drawbacks of insufficient liquid exchange in the flow channel type liquid exchange and the problem of the sequence of liquid exchange in different regions of the slide. This invention brings obvious advantages to the liquid exchange on the surface of large-sized slides. Before the most important droplet reaction step, the present invention uses 1×phi29 buffer immersion. This reagent is one component of the droplet reagents in 8 pipelines, which can form a liquid film of a similar component on the surface of the slide in advance, avoiding the problem of insufficient reagent exchange caused by droplet liquid exchange. The dosage of the phi29 DNA polymerase reagent with the highest cost in the present invention is reduced from 10 ml in the set package to 5 ml, a 50% reduction, greatly reducing the reagent cost.

[0075] The MDA amplification method according to the embodiment of the present invention can be applied to DNA nanosphere loading and fixation, realizing the automation of the entire DNBSEQ-T20×2 sequencing process. This method can be extended to other steps of open platform sequencing and applied to reaction systems of high-value components or temperature-sensitive components, forming a complement with the immersion scheme; this method can be extended to other applications beyond sequencing, such as fluorescence probe detection, construction of spatio-temporal omics capture slides, etc.

[0076] In the description of the present invention, it should be understood that the orientation or positional relationship indicated by the terms "center", "longitudinal", "transverse", "length", "width", "thickness", "upper", "lower", "front", "rear", "left", "right", "vertical", "horizontal", "top", "bottom", "inner", "outer", "clockwise", "counterclockwise", "axial", "radial", "circumferential", etc. is based on the orientation or positional relationship shown in the drawings, and is only for the convenience of describing the present invention and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore should not be construed as a limitation to the present invention.

[0077] In addition, the terms "first" and "second" are only used for descriptive purposes and cannot be understood as indicating or implying relative importance or implicitly specifying the quantity of the indicated technical features. Thus, the features defined with "first" and "second" may explicitly or implicitly include at least one of such features. In the description of the present invention, the meaning of "plurality" is at least two, such as two, three, etc., unless otherwise specifically defined.

[0078] In the present invention, unless otherwise clearly defined or limited, terms such as "installed", "connected", "joined", "fixed", etc. shall be understood in a broad sense. For example, it may be a fixed connection, a detachable connection, or an integral one; it may be a mechanical connection or an electrical connection; it may be directly connected or indirectly connected through an intermediate medium, and it may be the internal communication between two components or the interaction relationship between two components, unless otherwise clearly defined. For those of ordinary skill in the art, the specific meanings of the above terms in the present invention can be understood according to specific circumstances.

[0079] In the present invention, unless otherwise clearly defined or limited, the first feature being "on" or "under" the second feature may be that the first and second features are in direct contact, or the first and second features are indirectly in contact through an intermediate medium. Moreover, the first feature being "above", "over" and "on top of" the second feature may be that the first feature is directly above or obliquely above the second feature, or merely indicates that the first feature is at a higher level than the second feature in terms of horizontal height. The first feature being "under", "beneath" and "underneath" the second feature may be that the first feature is directly below or obliquely below the second feature, or merely indicates that the first feature is at a lower level than the second feature in terms of horizontal height.

[0080] In the description of this specification, the description with reference to terms such as "one embodiment", "some embodiments", "examples", "specific examples", or "some examples", etc. means that the specific features, structures, materials, or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials, or characteristics described can be combined in a suitable manner in any one or more embodiments or examples. In addition, without contradiction, those skilled in the art can combine and combine the different embodiments or examples described in this specification and the features of different embodiments or examples.

[0081] Although the embodiments of the present invention have been shown and described above, it can be understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those of ordinary skill in the art can make changes, modifications, substitutions, and variations to the above embodiments within the scope of the present invention.

Claims

1. A gene sequencing reaction device (100), characterized in that, include: A droplet reaction device (10), the droplet reaction device (10) comprising a droplet reaction zone, the droplet reaction zone being used to accommodate a sequencing carrier (200) to which a polymerase for a gene sequencing reaction is dropped and to provide a reaction environment with a preset temperature for the sequencing carrier (200); An external transfer device (20), the external transfer device (20) comprising an external material transfer unit (21) and an external drive unit (22) drivingly connected to the external material transfer unit (21), the external material transfer unit (21) being used to obtain, carry and release the sequencing carrier (200), and the external drive unit (22) being used to drive the external material transfer unit (21) to insert the sequencing carrier (200) into the droplet reaction area or to extract it from the droplet reaction area; A drop loading device (30) is used to drop a polymerase on the sequencing slide (200) so that the polymerase contacts the primer on the sequencing slide (200), wherein the primer is located on an open reaction surface on one side of the sequencing slide.

2. The gene sequencing reaction device (100) according to claim 1, characterized in that, The drop loading device (30) comprises: Bracket (31); A dripping pipeline (32), one end of which is disposed on the support (31) and is used for dripping polymerase onto the sequencing slide (200); A driving pump (33) is connected to the other end of the dripping pipeline (32).

3. The gene sequencing reaction device (100) according to claim 2, characterized in that, The support (31) comprises: beam (313); A first positioning block (311), the first positioning block (311) is relatively fixedly connected to the crossbeam (313) so as to position the one end of the dripping pipeline (32) on the crossbeam (313) and be configured to drip liquid toward the bottom of the crossbeam (313).

4. The gene sequencing reaction device (100) according to claim 3, characterized in that, The support (31) further comprises: A second positioning block (312), the second positioning block (312) is relatively fixedly connected to the cross beam (313), and positions a portion of the middle section of the dripping pipeline (32) on the cross beam (313) to constrain the dripping pipeline (32) to extend along the cross beam (313).

5. The gene sequencing reaction device (100) according to claim 2, wherein, The dripping pipeline (32) includes a plurality of dripping pipelines (32), one end of which is arranged side by side on the support (31) for dripping polymerase side by side on the sequencing slide (200), and the other end of which is connected to the driving pump (33).

6. The gene sequencing reaction device (100) according to any one of claims 1-5, characterized in that, The external driving unit (22) is configured to drive the sequencing carrier (200) to be placed horizontally below the droplet loading device (30), and to drive the sequencing carrier (200) to move in an "S"-shaped path in the horizontal direction.

7. The gene sequencing reaction device (100) according to any one of claims 1-5, characterized in that, The droplet reaction device (10) comprises: A main body (11), wherein the main body (11) has the droplet reaction area, and an opening (101) for inserting and removing the sequencing slide (200) is provided on one side of the droplet reaction area; A positioning member (12), wherein the positioning member (12) is used to position the sequencing carrier (200) in the droplet reaction area or release the positioning of the sequencing carrier (200).

8. The gene sequencing reaction device (100) according to claim 7, wherein, The opening (101) of the main body (11) faces the space below the liquid dripping loading device (30).

9. The gene sequencing reaction device (100) according to claim 1, wherein, The gene sequencing reaction device (100) further comprises: An immersion reaction device (40), which includes a plurality of first immersion reaction zones (41) for containing chemical reagents for gene sequencing reactions, so as to realize gene sequencing reactions by immersing a sequencing slide (200) with a DNA sample loading structure on its surface and loaded with a DNA sample into the first immersion reaction zone (41); An internal transfer device (50), which includes an internal material transfer unit moving along a predetermined trajectory, and the plurality of first immersion reaction zones (41) are arranged in sequence along the predetermined trajectory. The internal material transfer unit is used to acquire, carry and release the sequencing slide (200) to insert the sequencing slide (200) into the first immersion reaction zone (41) or withdraw it from the first immersion reaction zone (41); Wherein, the external driving unit (22) is further used to insert the sequencing slide (200) into the first immersion reaction zone (41) or withdraw it from the first immersion reaction zone (41).

10. The gene sequencing reaction device (100) according to claim 9, characterized in that, The immersion reaction device (40) further includes a loading storage area (43) for placing the sequencing slide (200). The loading storage area (43) is located near the first immersion reaction zone (41), and the external driving unit (22) is further used to withdraw the sequencing slide (200) from the loading storage area (43); And / or, the immersion reaction device (40) further includes a discharging storage area (44) for placing the sequencing slide (200). The discharging storage area (44) is located near the first immersion reaction zone (41), and the external driving unit (22) is further used to insert the sequencing slide (200) into the discharging storage area (44) or withdraw it from the discharging storage area (44); And / or, the immersion reaction device (40) further includes at least one second immersion reaction zone (42) for containing chemical reagents for gene sequencing reactions, so as to realize gene sequencing reactions by immersing a sequencing slide (200) with a DNA sample loading structure on its surface and loaded with a DNA sample into the second immersion reaction zone (42). The external driving unit (22) is further used to insert the sequencing slide (200) into the second immersion reaction zone (42) or withdraw it from the second immersion reaction zone (42).

11. A MDA amplification method using the gene sequencing reaction device according to any one of claims 1-10, characterized in that, Comprising: Using a liquid dripping loading device to drip polymerase onto the sequencing slide, so that the polymerase contacts the primer on the sequencing slide, and the primer is arranged on the open reaction surface of the sequencing slide; Transferring the sequencing slide with polymerase for gene sequencing reaction to a liquid dripping reaction zone and controlling the reaction temperature of the liquid dripping reaction zone.

12. An MDA amplification reaction device, characterized in that, Comprising an enzyme reaction zone, an immersion reaction zone, and the external transfer device (20) in the gene sequencing reaction device according to any one of claims 1-8. The external transfer device (20) is configured to move the sequencing slide to the enzyme reaction area or the immersion reaction area for reaction according to a preset reaction sequence; The enzyme reaction area includes a droplet loading device (30) and a droplet reaction device (10) in the gene sequencing reaction device according to any one of claims 1-8. The droplet loading device (30) is used to drop polymerase onto the sequencing slide (200) in real time so that the polymerase contacts the primer on the sequencing slide (200). The sequencing slide has an open reaction surface for fixing the primer. The droplet reaction device (10) has a reaction chamber for accommodating the sequencing slide (200) with the dropped polymerase and providing a controllable reaction temperature for the sequencing slide (200); The immersion reaction area includes an immersion tank storing reaction reagents, and the reaction reagents include strand displacement amplification reagents.

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