A method for preparing a reaction mixture and a testing apparatus

Abstract

The application provides a reaction mixture preparation method and a testing device. The method adds a first premix and a second premix to a mixing well in a certain order to form a reaction mixture, then mixes the reaction mixture in the mixing well, and then distributes the mixed reaction mixture into multiple reaction wells. This pre-mixing, mixing and distribution mode can minimize the filling error and ensure that the composition ratio in each reaction well is completely consistent, thereby making the experimental results more reliable. Specifically, when adding two premixes, the second premix with a lower density is added to the mixing well first, and then the first premix with a higher density is added to the mixing well. When the high-density liquid flows into the low-density liquid, the high-density first premix will penetrate downward to achieve initial mixing. On this basis, the mixing operation further fully mixes the reaction mixture. The method can improve the uniformity between the reaction wells.

Description

Technical Field

[0001] This application relates to the field of in vitro diagnostic equipment technology, and in particular to a method for preparing a reaction mixture and a testing device. Background Technology

[0002] Nucleic acid testing is widely used in clinical diagnosis, agricultural monitoring, and food safety, especially during large-scale infectious disease outbreaks, where it serves as the gold standard for disease diagnosis. The most commonly used nucleic acid testing method is PCR (polymerase chain reaction). The general principle of PCR is the in vitro replication of complementary daughter DNA strands, catalyzed by DNA polymerase using parent DNA as a template and specific primers as extension start points. This process is typically completed in a multi-well plate of a nucleic acid testing machine.

[0003] To improve detection sensitivity, exponential amplification of the parent DNA is necessary. The amplification reagent plays a crucial role in this amplification. In nucleic acid detection systems, the consistency of the amplification reagent ratio across the wells of a multi-well plate is extremely important. Even minute differences in concentration will be amplified dramatically during exponential amplification, leading to inconsistent amplification results across wells and ultimately severely impacting the accuracy of the detection results.

[0004] Existing methods have shortcomings in achieving consistent ratios between reaction wells: the amplification reagent consists of two components, a reaction solution and an enzyme solution. The viscosity and density of these components differ, making them prone to uneven distribution during mixing and dispensing. This leads to inconsistent ratios of the amplification reagent between reaction wells, affecting the accuracy and reliability of the detection results. Summary of the Invention

[0005] To address the existing technical problems, this application provides a method for preparing a reaction mixture and a testing device, which can prepare a reaction mixture for each reaction well that is well mixed and in the same proportion, making the experimental results more reliable and more repeatable.

[0006] In a first aspect, a method for preparing a reaction mixture is provided, comprising controlling a pipette to perform the following steps: A first premixed liquid and a second premixed liquid are added to a mixing well to form a reaction mixture, wherein the density of the first premixed liquid is higher than that of the second premixed liquid, and the first premixed liquid is added after the second premixed liquid. The reaction mixture in the mixing hole is mixed. The thoroughly mixed reaction mixture is distributed into multiple reaction wells; The mixing operation includes at least one of the following two mixing operations: The first mixing operation: the pipette needle performs multiple cycles of aspirating at a first depth range and discharging at a second depth range in the reaction mixture, where the depth of each aspiration may be the same or different, and the depth of each discharge may be the same or different; the first depth range is [D1,D2], and the second depth range is [D3,D4], where 0<D1<D2<D3<D4<H, and D3≥K×D2, with 1.5≤K≤3, where the liquid surface depth is 0, and H is the total depth of the reaction mixture; The second mixing operation: the pipette needle travels axially along the mixing well inside the reaction mixture, and performs multiple cycles of follow-up aspiration-discharge operations during the traveling process; wherein the depth of the axial travel does not exceed the depth of the reaction mixture.

[0007] In a second aspect, a testing apparatus is provided, comprising a reagent preparation mechanism and a controller, wherein the reagent preparation mechanism comprises a pipette, the pipette comprises a pipette needle at the distal end and a driving device, the driving device is connected to the controller, the controller controls the driving device to drive the pipette needle to move, and the controller implements the reaction mixture preparation method described in the above embodiments.

[0008] According to the reaction mixture preparation method provided in the above embodiments, the first premix and the second premix are added in a certain order to one mixing well to form a reaction mixture, then the reaction mixture in the mixing well is subjected to a mixing operation, and the fully mixed reaction mixture is dispensed into a plurality of reaction wells. This working mode of premixing-mixing-dispensing can minimize the dispensing error to the greatest extent, ensure that the component ratio in each reaction well is completely consistent, thus making the experimental results more reliable and having better repeatability. Specifically, when adding the two premixes, the different densities of the two different premixes are fully considered: after the second premix with lower density is added to the mixing well, the first premix with higher density is added to the mixing well. When the high-density liquid flows into the low-density liquid, the high-density first premix will penetrate downward, and this process itself will generate a certain degree of convection and agitation to achieve preliminary mixing. On this basis, the reaction mixture is further mixed through the first mixing operation and / or the second mixing operation, so that the two premixes with different densities in the premixing process are fully mixed. By adopting this method, the reaction mixture required by each reaction well that is fully mixed and has a consistent ratio can be prepared, and this method can improve the uniformity between reaction wells.

[0009] The testing apparatus provided in the above embodiments belongs to the same inventive concept as the corresponding embodiments of the reaction mixture preparation method, and thus has the same technical effects as the corresponding embodiments of the reaction mixture preparation method, which will not be repeated here. Brief Description of the Drawings

[0010] Figure 1This is a flowchart of a method for preparing a reaction mixture in one embodiment.

[0011] Figure 2 This is a schematic diagram illustrating the operating range of pipetting in a mixing well in one embodiment.

[0012] Figure 3 This is a flowchart of a method for preparing a reaction mixture in another embodiment. Detailed Implementation

[0013] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0014] To make the objectives, technical solutions, and advantages of this application clearer, the application will be further described in detail below with reference to the accompanying drawings. The described embodiments should not be regarded as limitations on this application. All other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0015] In the following description, the phrase "some embodiments" refers to a subset of all possible embodiments. It should be noted that "some embodiments" can be the same subset or different subsets of all possible embodiments, and can be combined with each other without conflict.

[0016] In the following description, the terms "first, second, and third" are used merely to distinguish similar objects and do not represent a specific ordering of objects. It is understood that "first, second, and third" may be interchanged in a specific order or sequence where permitted, so that the embodiments of this application described herein can be implemented in an order other than that illustrated or described herein.

[0017] Nucleic acid testing is widely used in clinical diagnosis, agricultural monitoring, and food safety, especially during large-scale infectious disease outbreaks, where it serves as the gold standard for disease diagnosis. The most commonly used nucleic acid testing method is PCR (polymerase chain reaction). The general principle of PCR is the in vitro replication of complementary daughter DNA strands, catalyzed by DNA polymerase using parent DNA as a template and specific primers as extension start points. This process is typically completed in a multi-well plate of a nucleic acid testing machine.

[0018] To improve detection sensitivity, exponential amplification of the parent DNA is necessary. The amplification reagent plays a crucial role in this amplification. In nucleic acid detection systems, the consistency of the amplification reagent ratio across the wells of a multi-well plate is extremely important. Even minute differences in concentration will be amplified dramatically during exponential amplification, leading to inconsistent amplification results across wells and ultimately severely impacting the accuracy of the detection results.

[0019] The existing solutions have deficiencies in achieving the consistency of the ratio between reaction wells: the amplification reagent is divided into two reagent components, namely the reaction solution and the enzyme solution. There are differences in viscosity and density between different components such as the reaction solution and the enzyme solution, which are very prone to uneven distribution during the mixing and dispensing process, further leading to inconsistent ratios of the amplification reagent between reaction wells, thus affecting the accuracy and reliability of detection results.

[0020] To address the above problems, the present application provides a testing device, which includes a reagent preparation mechanism and a controller. The reagent preparation mechanism includes a pipettor, the pipettor comprises a pipette tip located at the distal end and a driving device, the driving device is connected to the controller, and the controller controls the driving device to drive the pipette tip to move. The controller can implement the reaction mixture preparation method of the present application.

[0021] In one embodiment, the driving device includes a movement drive assembly for driving the pipette tip to move two-dimensionally or three-dimensionally in space, and a pipetting drive assembly for driving the pipette tip to aspirate or dispense liquid.

[0022] Specifically, the controller controls the pipettor to perform the following steps: respectively adding the first premix solution and the second premix solution into one mixing well to form a reaction mixture, wherein the density of the first premix solution is higher than the density of the second premix solution, and the first premix solution is added after the second premix solution; Performing a mixing operation on the reaction mixture in the mixing well; Dispensing the mixed reaction mixture into a plurality of reaction wells; Wherein, the mixing operation includes at least one of the following two mixing operations: The first mixing operation: the pipette tip performs multiple cycles of aspirating liquid at a first depth range and dispensing liquid at a second depth range in the reaction mixture, the depth of each aspiration is the same or different, and the depth of each dispensing is the same or different; the first depth range is [D1,D2], the second depth range is [D3,D4], where 0<D1<D2<D3<D4<H, and D3≥K×D2, where 1.5≤K≤3, wherein the liquid surface depth is 0, and H is the total depth of the reaction mixture; The second mixing operation: the pipette tip travels along the axial direction of the mixing well, and performs multiple cycles of follow-up aspiration and dispensing operations during the traveling process; wherein the depth of the axial travel does not exceed the depth of the reaction mixture.

[0023] This allows the testing equipment to first add the first and second premixed solutions into a mixing well to form a reaction mixture, then homogenize the reaction mixture in the mixing well, and finally distribute the homogenized reaction mixture into multiple reaction wells. This premix-homogenization-dispensing operation mode minimizes dosing errors and ensures that the component ratios in each reaction well are completely consistent, thus making the experimental results more reliable and reproducible. When adding the two premixed solutions, the different densities of the two different premixed solutions are fully considered. The second premixed solution, which has a lower density, is added to the mixing well first, followed by the first premixed solution, which has a higher density. When the high-density liquid flows into the low-density liquid, the high-density first premixed solution will penetrate downwards, and this process itself will generate a certain amount of convection and agitation to achieve preliminary mixing. On this basis, the reaction mixture is further homogenized through the first and / or second homogenization operations, so that the two premixed solutions of different densities are fully mixed during the premixing process. This testing equipment is capable of preparing the reaction mixture required for each reaction well to be thoroughly mixed and in the same proportion.

[0024] This application provides a method for preparing a reaction mixture, such as... Figure 1 As shown, this method controls the pipette to perform the following steps: Step 102: The first premixed liquid and the second premixed liquid are added to a mixing well to form a reaction mixture, wherein the density of the first premixed liquid is higher than that of the second premixed liquid, and the first premixed liquid is added after the second premixed liquid.

[0025] The density of the first premixed liquid is higher than that of the second premixed liquid. Therefore, for the same volume, the first premixed liquid weighs more than the second premixed liquid. If the first premixed liquid is added to the mixing hole first, and then the second premixed liquid is added to the mixing hole, the first premixed liquid will remain at the bottom of the mixing hole.

[0026] In this embodiment, a second premixed liquid with a lower density is first added to the mixing hole, and then a first premixed liquid with a higher density is added to the mixing hole. When the high-density liquid flows into the low-density liquid, the high-density first premixed liquid will penetrate downwards. This process itself will generate a certain amount of convection and agitation to achieve preliminary mixing.

[0027] Specifically, within a PCR apparatus, the amplification reagents consist of two components: the reaction solution and the enzyme solution. The reaction solution includes buffer, dNTPs, MgCl2, etc. The buffer maintains a stable pH in the reaction system and provides important ions (Na+, K+) to make the PCR reaction more stable. dNTPs are essential raw materials in the PCR reaction, providing the mononucleotides required for DNA chain synthesis. MgCl2 is used to enhance polymerase activity and increase the product yield. The main component of the enzyme solution is DNA polymerase, responsible for catalyzing the synthesis of new DNA chains.

[0028] Wherein the density of the enzyme solution is higher than that of the reaction solution, the enzyme solution is taken as the first premix solution, and the reaction solution is taken as the second premix solution. During addition, a certain amount of the reaction solution is first added into the mixing well according to a preset premixing ratio, and then a certain amount of the enzyme solution is added into the mixing well. Since the density of the enzyme solution is higher than that of the reaction solution, the high-density enzyme solution flows into the low-density reaction solution after being added. This process itself generates a certain convection and agitation to achieve preliminary mixing.

[0029] To ensure the uniformity of detection, for example, to ensure that the preparation of amplification reagents in each reaction well of a multi-well plate in the same batch of experiments is consistent, it is necessary to dispense the mixed reaction mixture into the reaction wells of the multi-well plate. Therefore, the mixing well usually has a relatively large volume to accommodate the premix solutions.

[0030] Optionally, the mixing well comes from a premix container, which may be a large-capacity multi-well plate or a bottle-shaped container.

[0031] In one embodiment, the integrated nucleic acid detection apparatus has a nucleic acid extraction function. To accommodate large-volume nucleic acid extraction reagents, a large-capacity multi-well plate is required. Therefore, the large-capacity multi-well plate for nucleic acid extraction can be directly used for mixing the reaction mixture, so that no additional type of consumable is needed.

[0032] In one embodiment, the mixing well generally adopts a conical bottom to reduce the dead volume of the mixing well, thereby reducing the loss of the reaction mixture.

[0033] Step 104: performing a mixing operation on the reaction mixture in the mixing well; wherein the mixing operation comprises at least one of at least two mixing operations: The first mixing operation: the pipette needle performs multiple cycles of aspirating the liquid in the reaction mixture at a first depth range and discharging the liquid at a second depth range, wherein the depth of each aspiration may be the same or different, and the depth of each discharge may be the same or different; the first depth range is [D1, D2], and the second depth range is [D3, D4], where 0<D1<D2<D3<D4<H, and D3≥K×D2, where 1.5≤K≤3, the liquid surface depth is 0, and H is the total depth of the reaction mixture.

[0034] The second mixing operation: the pipette needle travels along the axial direction of the mixing well, and during the traveling process, performs multiple cycles of following aspiration and liquid discharge; wherein the depth of the axial traveling does not exceed the depth of the reaction mixture.

[0035] Wherein the pipette needle is the end effector of the pipettor.

[0036] Optionally, the pipette needle can be directly used for liquid aspiration, liquid discharge and mixing operations. After use, the pipette needle needs to be cleaned to avoid reagent contamination.

[0037] Optionally, the pipette needle can be fitted with a tip to cooperate to complete liquid aspiration, liquid dispensing and mixing operations, and the tip can be unloaded after use.

[0038] Wherein, the first depth range and the second depth range in the first mixing operation refer to the depth ranges located below the liquid surface. The first depth range is [D1, D2], and the second depth range is [D3, D4], where 0<D1<D2<D3<D4<H, and D3≥K×D2. In this way, the first depth range is located in the upper layer of the reaction mixture, and the second depth range is located in the lower layer of the reaction mixture.

[0039] Figure 2 For example, first control the pipette needle to detect the liquid surface of the reaction mixture in the mixing well, which is taken as the reference datum with a depth of 0. Based on the preset specification parameters of the mixing well or the movement of the pipette needle, the total depth H of the reaction mixture is obtained. The first depth range [D1, D2] and the second depth range [D3, D4] are determined. Wherein, D1 is greater than 0, so that the pipette needle is located below the liquid surface to avoid sucking in air. D3≥K×D2, which can ensure that the liquid dispensing layer is always located below the liquid aspiration layer.

[0040] It can be understood that the specific values of D1, D2, D3 and D4 can be obtained by conversion according to the preset functional relationship with the total depth H of the reaction mixture, or can be preset according to the user's experimental experience, which is not specifically limited herein.

[0041] The first mixing operation is specifically to perform multiple cycles of liquid aspiration in the first depth range and liquid dispensing in the second depth range of the reaction mixture, that is, to perform multiple cycles of liquid aspiration in the upper layer and liquid dispensing in the deep layer of the reaction mixture. Wherein, one cycle operation is liquid aspiration in the upper layer - liquid dispensing in the deep layer. After completing one cycle operation, the pipette needle is controlled to move upward to the first depth range, and the cycle operation of liquid aspiration in the upper layer - liquid dispensing in the deep layer is performed again. In this way, the mixing operation is cycled for multiple times to realize the exchange of upper and lower layer liquids in the mixing well.

[0042] In one embodiment, the depth of each upper layer liquid aspiration is the same, and the depth of each deep layer liquid dispensing is the same. That is, the pipette needle of the pipettor is controlled to first aspirate liquid at a fixed position in the shallow layer of the reaction mixture, and then the pipettor is controlled to move down to dispense liquid at a fixed position in the deep layer of the reaction mixture. In this way, after completing one mixing operation, the pipettor is controlled to move up to make the pipette needle aspirate liquid at the fixed position in the shallow layer of the reaction mixture, and then the pipettor is controlled to move down to make the pipette needle dispense liquid at the fixed position in the deep layer of the reaction mixture. This is repeated many times to fully exchange the upper layer reaction mixture and the lower layer reaction mixture, so as to achieve mixing.

[0043] In one embodiment, the depth of liquid aspiration from the upper layer varies each time, and the depth of liquid ejection from the deeper layer also varies each time. The pipette is controlled to first aspirate liquid at a first position in the shallow layer of the reaction mixture, and then the pipette is controlled to move downwards to eject liquid at a second position in the deeper layer of the reaction mixture. After completing one mixing operation, the pipette is controlled to move upwards to a third position in the shallow layer of the reaction mixture, and then the pipette is controlled to move downwards to eject liquid at a fourth position in the deeper layer of the reaction mixture. The depths of the first and third positions are different (e.g., the depth of the first position is lower than the depth of the third position), and the depths of the second and fourth positions are different (e.g., the depth of the second position is lower than the depth of the fourth position). This process is repeated multiple times. Through the continuously changing liquid flow path, the liquid in each layer within the mixing orifice is sufficiently agitated and exchanged, thereby achieving a more uniform mixture.

[0044] It is understandable that other methods can be used to achieve thorough mixing, such as taking the same amount of liquid from the upper layer each time and taking different amounts of liquid from the deeper layer each time; or taking different amounts of liquid from the upper layer each time and taking the same amount of liquid from the deeper layer each time, etc., which are not limited here.

[0045] In the second mixing operation, the axis of the mixing orifice is the height direction of the mixing orifice. The pipette is controlled to move along the axis of the mixing orifice, and multiple cycles of aspiration and dispensing are performed during this movement. That is, one cycle of aspiration and dispensing constitutes a single complete mixing operation. In the second mixing operation, multiple cycles of aspiration and dispensing are performed as the pipette moves along the axis of the mixing orifice.

[0046] Follow-up aspiration refers to the simultaneous and continuous aspiration operation during the movement of the pipette; that is, the pipette aspirates liquid while moving. Thus, in a single aspiration, the pipette can pick up liquid at different heights in the reaction mixture, and after aspiration, it ejects liquid to exchange and mix the liquids at different heights. This achieves vertical mixing of the liquids, improving mixing efficiency.

[0047] In this embodiment, the direction in which the pipette travels axially through the mixing hole is not limited. It can travel from top to bottom along the axial direction of the mixing hole. Alternatively, it can travel from bottom to top along the axial direction of the mixing hole. It can also travel first from top to bottom and then from bottom to top along the axial direction of the mixing hole. Finally, it can travel first from bottom to top and then from top to bottom along the axial direction of the mixing hole.

[0048] The pipette begins its movement from the top layer of the reaction mixture, dynamically aspirating liquid as it moves. When the pipette reaches position B, a single aspiration is completed, and then the liquid is immediately expelled at position B. This process exchanges and mixes liquids at different heights in the reaction mixture. Alternatively, the pipette can begin its movement from position B and move downwards, dynamically expelling liquid as it moves. This releases the mixed liquid back to different depths, further improving mixing efficiency.

[0049] The preparation of amplification reagents requires following specific ratios, which vary depending on the detection target and the type of reagent. Before nucleic acid amplification, the reaction solution and enzyme solution need to be mixed together in a certain ratio; the uniformity of mixing directly affects the amplification effect. Since the density and viscosity of the enzyme solution differ significantly from those of the reaction solution, it is crucial to find a way to better mix these two liquids.

[0050] In this embodiment, the mixing operation includes at least one of the two mixing operations described above.

[0051] In one embodiment, the mixing operation of the reaction mixture in the mixing hole can be a first mixing operation. Thus, by drawing in and releasing liquid at different depths, diffusion is promoted using the difference in liquid density, achieving mixing.

[0052] In one embodiment, the mixing operation of the reaction mixture in the mixing hole can be a second mixing operation. This allows for the exchange and mixing of liquids at different heights within the reaction mixture.

[0053] In one embodiment, the mixing operation of the reaction mixture in the mixing well can be performed by first performing a first mixing operation on the reaction mixture in the mixing well, and then performing a second mixing operation. This method first uses the first mixing operation to preliminarily resuspend and mix precipitates or components of different densities, and then uses the second mixing operation to further improve the mixing effect through fine mixing.

[0054] In one embodiment, the mixing operation of the reaction mixture in the mixing hole can be performed by first performing a second mixing operation on the reaction mixture in the mixing hole, and then performing a second mixing operation on the reaction mixture. This method improves the mixing effect through two dynamic axial mixing operations.

[0055] Step 106: Distribute the well-mixed reaction mixture into multiple reaction wells.

[0056] The first two steps involve mixing and thoroughly integrating the first and second premixed solutions in a specific ratio. When applied to a nucleic acid detection integrated machine, this process allows for the precise mixing and homogenization of the reaction solution and enzyme solution, resulting in a fully homogenized reaction mixture.

[0057] Furthermore, the well-mixed reaction mixture is distributed to multiple reaction wells. This premix-mix-dispensing operation mode can minimize dispensing errors and ensure that the component ratio in each reaction well is completely consistent, thereby making the experimental results more reliable and reproducible. In addition, since this operation mode can mix the reaction mixture required for multiple reaction wells at once, without having to mix them one by one, it improves the preparation efficiency of amplification reagents.

[0058] The above-described method for preparing the reaction mixture involves adding a first premix and a second premix into a mixing well to form the reaction mixture. The reaction mixture in the mixing well is then homogenized, and finally, the homogenized reaction mixture is distributed to multiple reaction wells. This premix-homogenization-dispensing process minimizes filling errors and ensures that the component ratios in each reaction well are completely consistent, resulting in more reliable and reproducible experimental results. When adding the two premixes, their different densities are carefully considered. The lower-density second premix is ​​added to the mixing well first, followed by the higher-density first premix. When the higher-density liquid flows into the lower-density liquid, the higher-density first premix penetrates downwards, generating convection and agitation to achieve initial mixing. Based on this, the reaction mixture is further homogenized through the first and / or second homogenization operations, ensuring thorough mixing of the two premixes with different densities during the premixing process. Using this method, the reaction mixture required for each reaction well can be prepared with sufficient mixing and consistent proportions, which can improve the uniformity between reaction wells.

[0059] In one embodiment, the mixing operation includes a first mixing operation and a second mixing operation. The mixing operation of the reaction mixture in the mixing well includes: The reaction mixture in the mixing well is first mixed in the first way, and then the second mixing operation is performed.

[0060] In other words, the pipette first performs a first mixing operation on the reaction mixture in the mixing well, so that the upper and lower liquid layers are exchanged, which initially breaks the liquid stratification caused by density difference. Then, the pipette is controlled to travel along the axis of the mixing well. During the travel, multiple cycles of aspiration and dispensing are performed, thereby further improving the mixing effect by dynamically sampling and redistributing the liquid at various vertical levels.

[0061] In one embodiment, during the first mixing operation, it is prevented that after a certain amount of liquid is aspirated during the liquid aspiration process, the tip of the pipette needle is exposed from the liquid surface due to the drop of the liquid level, which leads to the aspiration of an air column. Therefore, the height ΔH of the liquid level drop caused by a single aspiration can be determined according to the aspiration volume and the cross-sectional area of the mixing well, and then D1 is set to be greater than ΔH. In this way, during the upper-layer column aspiration in the first mixing operation, the pipette needle always aspirates liquid below the liquid surface without drawing in an air column.

[0062] In one embodiment, during the first mixing operation, the single aspiration volume does not exceed 1 / 4 to 1 / 3 of the total liquid volume. This volume can not only generate a sufficiently large fluid to effectively mix the liquid, but also ensure that only part of the liquid is moved in one cycle, forcing the upper-layer and lower-layer liquids to exchange through the pipette needle. Efficient mixing is achieved through multiple cycles.

[0063] In one embodiment, during the second mixing operation, the pipette needle travels within the third depth range [D0, D4] of the reaction mixture; 0 < D0, and the tip of the pipette needle is kept below the liquid surface during the traveling process.

[0064] Specifically, during the second mixing operation, the pipette needle travels within the third depth range [D0, D4] of the reaction mixture. Wherein, 0 < D0, D4 < H, and the tip of the pipette needle is kept below the liquid surface during the traveling process. In this way, during the process of the pipette needle traveling along the axial direction of the mixing well, the tip is always below the liquid surface, which effectively avoids aspirating air or touching the bottom, thus ensuring the mixing quality and operation reliability.

[0065] In one embodiment, the pipette needle travels along the axial direction of the mixing well, and performs multiple repeated aspiration-dispensing cycle operations during the traveling process, including the following modes: The first mode: the pipette needle moves downward starting from the depth D0 along the axial direction of the mixing well, performs multiple repeated aspiration-dispensing cycle operations during the moving process, and stops when moving to the depth D4 or when the number of repeated aspiration-dispensing cycles reaches a preset number.

[0066] This mode is a top-down unidirectional traveling mode, in which multiple repeated aspiration-dispensing cycle operations are performed during the top-down unidirectional traveling process. This top-down path can effectively bring the upper-layer liquid to the lower layer to achieve preliminary vertical mixing.

[0067] The second mode: the pipette needle moves upward starting from the depth D4 along the axial direction of the mixing well, performs multiple repeated aspiration-dispensing cycle operations during the moving process, and stops when moving to the depth D0 or when the number of repeated aspiration-dispensing cycles reaches a preset number.

[0068] This method involves a bottom-up, unidirectional flow, during which multiple cycles of aspiration and dispensing are performed. The pipette starts working directly from the location of the precipitate, quickly disrupting it.

[0069] The third method: Move downwards along the axis of the mixing hole starting from depth D0. When it reaches depth D4, it starts moving upwards. During the movement, perform multiple cycles of suction and discharge. Stop when it reaches depth D0 or when the number of suction and discharge cycles reaches the preset number.

[0070] This method first travels from top to bottom, then from bottom to top, performing multiple cycles of suction and discharge during the unidirectional travel. By covering a complete path from shallow to deep and then from deep to shallow, this method ensures that the liquid is cyclically stirred in the axial direction, improving the mixing effect and further enhancing the uniformity of the mixed liquid.

[0071] The fourth method involves moving upwards along the axis of the mixing hole from depth D4, then moving downwards when it reaches depth D0. During this movement, multiple cycles of suction and discharge are performed. The process stops when the hole reaches depth D4 or when the number of suction and discharge cycles reaches a preset number.

[0072] This method involves first moving the pipette from bottom to top, then from top to bottom, performing multiple cycles of aspiration and ejection during the process. As the pipette rises from the bottom, it draws the liquid upwards, and as it descends from the top, it pushes the liquid downwards. This creates a vertical convection cycle that rapidly breaks up liquid stratification, improving mixing and further enhancing the uniformity of the mixed liquid.

[0073] In one embodiment, the aspiration-expelling operation includes one of the following: The first method involves aspirating liquid during movement, stopping the movement when the aspirated amount reaches a preset level, and then spitting out the liquid at the current location.

[0074] The aspiration-expelling method involves moving the pipette upwards or downwards from point A and continuously aspirating liquid. When the aspirated volume reaches the target, the pipette is stopped, and then the liquid is expelled back while the pipette is at rest.

[0075] Because the pipette needle remains stationary during dispensing, the ejected liquid stream impacts a specific depth. This helps create a local mixing vortex at that location, achieving a localized mixing effect and continuous mixing in different areas to achieve uniformity. Furthermore, stationary dispensing is gentler and more stable, as the liquid is not violently agitated by rapid needle movement, greatly reducing the risk of bubble formation.

[0076] The second method involves aspirating liquid during movement, and continuing to move and spitting out liquid when the aspirated amount reaches the preset level.

[0077] In one embodiment, the aspiration-expelling method specifically involves moving the pipette upwards or downwards from point A and continuously aspirating liquid. When the aspirated volume reaches the target, the aspiration stops, and then the pipette continues to move upwards or downwards while continuously expelling liquid during the movement.

[0078] Since both suction and discharge are completed during continuous movement without interruption, more suction-discharge mixing cycles can be performed per unit time, improving mixing efficiency. Furthermore, the number of times the robotic arm starts and stops during mixing is reduced, contributing to increased overall operating speed and equipment lifespan.

[0079] In one embodiment, during the second mixing operation, when the pipette moves downward, the downward speed of the pipette matches the rate at which the liquid level drops.

[0080] In this way, during the liquid aspiration process, the pipette can descend with the liquid level, thereby maintaining the relative depth of the needle tip below the liquid level (or within a safe range) and preventing the needle tip from protruding from the liquid surface.

[0081] In one embodiment, when the pipette reaches depth D4, the dispensing is accelerated. High-speed dispensing at the bottom (D4) of the premixing container generates a strong bottom-up vortex, achieving thorough vertical mixing. Furthermore, the rapid dispensing creates a momentary localized high-pressure zone in front of the pipette tip, forcing the liquid within this zone to accelerate its diffusion outwards and upwards. The bottom structure further enhances the mixing effect by backflushing the liquid. Thus, the dispensed liquid can be efficiently mixed with the surrounding liquid in a very short time, significantly improving the mixing efficiency per unit time.

[0082] In one embodiment, when the pipette moves to depth D4, it remains at that position to perform at least one aspiration-expelling cycle. The bottom is the most difficult part to mix during the entire mixing process. When the pipette moves to the bottom, performing at least one aspiration-expelling cycle at the bottom continuously impacts the bottom, improving mixing efficiency.

[0083] In one embodiment, adding a first premixed liquid and a second premixed liquid to a mixing well to form a reaction mixture includes: The first addition amount of the first premixed liquid and the second addition amount of the second premixed liquid are determined based on the number of reaction wells to be allocated, the preset premixing ratio of the first premixed liquid and the second premixed liquid, and the transfer loss amount redundant according to the preset premixing ratio; wherein, the number of reaction wells to be allocated is determined based on the sample amount to be tested and the preset quality control sample amount, and / or, the transfer loss amount is determined based on the preset redundant number of reaction wells and the preset premixing ratio. Take the second amount of the second premixed liquid into the mixing hole, and then take the first amount of the first premixed liquid into the mixing hole.

[0084] In this embodiment, the preset premixing ratio refers to the volume ratio of the first premixed solution and the second premixed solution required for a single reaction well. Therefore, the first addition amount of the first premixed solution and the second addition amount of the second premixed solution both take into account the actual demand and redundant transfer losses. This ensures that the total amount of premixed solution prepared meets the needs of all reaction wells (including samples and quality control), compensates for inevitable losses during operation, and maintains a consistent proportion of each component. In this embodiment, reaction mixtures with different proportions can be flexibly mixed according to the testing process ratio requirements.

[0085] In existing technologies, the method of preparing and mixing according to a preset premixing ratio requires the system to calculate the specific volume under this fixed ratio when the total volume to be prepared changes. This is effective in simple scenarios, but lacks error tolerance.

[0086] Furthermore, in one embodiment, the determination of the first amount of the first premixed liquid added further considers the amount of the first premixed liquid required in each reaction well, and the determination of the second amount of the second premixed liquid added further considers the amount of the second premixed liquid required in each reaction well. This embodiment ensures that the required volume of reaction mixture is prepared.

[0087] In view of this, in another embodiment, adding the first premixed liquid and the second premixed liquid to a mixing well to form a reaction mixture includes: The second addition amount of the second premixed liquid is determined based on the sum of the number of reaction holes to be assigned and the number of preset redundant reaction holes, as well as the amount of the second premixed liquid required in each reaction hole. The number of times the second premixed liquid is drawn is determined based on the second addition volume and the single absorption volume. The second premixed liquid is drawn according to the number of times and the single absorption volume and added to the mixing well. The first addition amount of the first premixed liquid is determined based on the sum of the number of reaction holes to be assigned and the number of preset redundant reaction holes, as well as the amount of the first premixed liquid required in each reaction hole. The number of times the first premixed liquid is drawn is determined based on the first added liquid volume and the single aspiration volume. The first premixed liquid is drawn according to the number of aspirations and the single aspiration volume and added to the mixing hole that already contains the second premixed liquid.

[0088] Understandably, regardless of whether the pipette directly aspirates liquid or a disposable tip is used, the volume of liquid that can be aspirated in a single operation is limited. If the maximum volume that either the pipette or the disposable tip can aspirate in one operation is taken as the single aspiration volume, then the number of times the pipette aspirates the first premixed liquid can be determined based on the first added volume and the single aspiration volume. Similarly, the number of times the pipette aspirates the second premixed liquid can be determined based on the second added volume and the single aspiration volume. This reduces the number of aspirations and improves the liquid transfer efficiency of the pipette. If a disposable tip is used, reducing the number of aspirations also avoids waste of consumables.

[0089] It is understandable that when the pipette is used N times, the amount of liquid drawn in the Nth time is determined based on the actual amount of liquid remaining to be transferred, thereby avoiding abnormal preparation of the reaction mixture.

[0090] In this embodiment, with the goal of filling a certain number of reaction wells, uncertainties such as losses and errors are actively incorporated into the calculation model to independently calculate the exact total demand for each component, thereby enabling more flexible response to detection requirements of different energies.

[0091] In one embodiment, the first premix and the second premix satisfy at least one of the following requirements: The first premixed solution and the second premixed solution are compatible liquids; The first addition amount of the first premixed solution is less than the second addition amount of the second premixed solution; The viscosity of the first premix is ​​higher than that of the second premix.

[0092] The first premixed liquid and the second premixed liquid are compatible liquids. The two liquids will not undergo adverse chemical reactions such as precipitation, denaturation, or gas generation, and can form a homogeneous and stable mixture.

[0093] The first premixed liquid requires less than the second premixed liquid. The viscosity of the first premixed liquid is higher than that of the second premixed liquid, and the density of the first premixed liquid is higher than that of the second premixed liquid. Utilizing the above characteristics, the small volume of the high-viscosity first premixed liquid is added to the large volume of the low-viscosity second premixed liquid, which is beneficial for the rapid diffusion and dilution of the high-viscosity liquid.

[0094] In one embodiment, the pipette uses the same disposable tip to perform the following operations: aspirating and dispensing the first premix, mixing, and dispensing the reaction mixture.

[0095] Because the first premixed solution has a high density and viscosity, residue of the first premixed solution may remain on the inner wall of the disposable pipette tip. If the disposable pipette tip is replaced before mixing, the ratio of the two reagent components may be abnormal, affecting the subsequent reaction results. Therefore, the disposable pipette tip is not replaced here; the residual enzyme solution is carried into the reaction solution through subsequent mixing.

[0096] In one embodiment, the pipette uses different disposable tips to aspirate and dispense the first premixed solution and the second premixed solution.

[0097] By using different disposable pipette tips to aspirate and expel the first and second premixed solutions separately—that is, after using one tip to aspirate and expel the premixed solution, a new disposable tip is used to aspirate and expel the second premixed solution—residue on the inner wall of the pipette tip used for aspirating and expelling the first premixed solution can be prevented from affecting the amount of the second premixed solution aspirated and expelled, thus avoiding abnormal ratios of the two reagent components and impacting subsequent reaction results.

[0098] In one embodiment, the homogenized reaction mixture is distributed into multiple reaction wells, including: The well-mixed reaction mixture is distributed to multiple reaction wells using a single suction and multiple discharge method.

[0099] "One aspiration, multiple dispensing" refers to a single aspiration followed by multiple dispensings to achieve multiple dispensings from a single aspiration operation. This process distributes a uniformly mixed reaction solution to multiple reaction wells through a single aspiration operation, thereby further improving the consistency of distribution to each well.

[0100] In one embodiment, the method for preparing the reaction mixture is as follows: Figure 3 As shown, it includes the following steps: Step 302: The first premixed liquid and the second premixed liquid are added to a mixing well to form a reaction mixture, wherein the density of the first premixed liquid is higher than that of the second premixed liquid, and the first premixed liquid is added after the second premixed liquid.

[0101] Specifically, the first addition amount of the first premixed solution and the second addition amount of the second premixed solution can be determined based on the number of reaction wells to be allocated, the preset premixing ratio of the first and second premixed solutions, and the redundant transfer loss amount according to the preset premixing ratio. The number of reaction wells to be allocated is determined based on the sample size to be tested and the preset quality control sample size, and / or the transfer loss amount is determined based on the preset redundant reaction well number and the preset premixing ratio. The second addition amount of the second premixed solution is drawn into the mixing well, and then the first addition amount of the first premixed solution is drawn into the mixing well. In this embodiment, both the first addition amount of the first premixed solution and the second addition amount of the second premixed solution take into account the actual demand and the redundant transfer loss amount. This ensures that the total amount of premixed solution prepared can meet the needs of all reaction wells (including samples and quality control) and compensate for the inevitable losses during the operation process, while maintaining a consistent ratio of each component. In this embodiment, reaction mixtures with different ratios can be flexibly mixed according to the testing process ratio requirements.

[0102] The second addition amount of the second premixed liquid can be determined based on the sum of the number of reaction wells to be assigned and the number of preset redundant reaction wells, as well as the amount of the second premixed liquid required in each reaction well. The number of times the second premixed liquid is aspirated is determined based on the second addition amount and the single aspiration volume, and the second premixed liquid is aspirated according to the number of aspirations and the single aspiration volume and added to the mixing well. The first addition amount of the first premixed liquid is determined based on the sum of the number of reaction wells to be assigned and the number of preset redundant reaction wells, as well as the amount of the first premixed liquid required in each reaction well. The number of times the first premixed liquid is aspirated is determined based on the first addition amount and the single aspiration volume, and the first premixed liquid is aspirated according to the number of aspirations and the single aspiration volume and added to the mixing well already containing the second premixed liquid. In this embodiment, with the goal of filling a certain number of reaction wells, uncertainties such as loss and error are actively incorporated into the calculation model to independently calculate the exact total required amount of each component, thereby enabling greater flexibility in meeting the detection requirements of different energy levels.

[0103] Understandably, regardless of whether the pipette directly aspirates liquid or a disposable tip is used, the volume of liquid that can be aspirated in a single operation is limited. If the maximum volume that either the pipette or the disposable tip can aspirate in one operation is taken as the single aspiration volume, then the number of times the pipette aspirates the first premixed liquid can be determined based on the first added volume and the single aspiration volume. Similarly, the number of times the pipette aspirates the second premixed liquid can be determined based on the second added volume and the single aspiration volume. This reduces the number of aspirations and improves the liquid transfer efficiency of the pipette. If a disposable tip is used, reducing the number of aspirations also avoids waste of consumables.

[0104] It can be understood that when the pipette needle has performed N aspiration operations, the liquid aspiration volume of the Nth time is determined according to the actual remaining volume of the liquid to be transferred, so as to avoid abnormal preparation of the reaction mixture.

[0105] Step 304: performing a first mixing operation on the reaction mixture in the mixing well, and then performing a second mixing operation thereon.

[0106] Wherein, the first mixing operation: the pipette needle performs multiple cycles of aspirating liquid at a first depth range and discharging liquid at a second depth range for the reaction mixture, the depth of each aspiration may be the same or different, and the depth of each discharge may be the same or different; the first depth range is [D1, D2], the second depth range is [D3, D4], where 0<D1<D2<D3<D4<H, and D3≥K×D2, where 1.5≤K≤3, wherein the liquid surface depth is 0, and H is the total depth of the reaction mixture; The second mixing operation: the pipette needle travels along the axial direction of the mixing well, and during the traveling process, performs multiple cycles of follow-up aspiration-discharge operations; wherein the depth of the axial travel does not exceed the depth of the reaction mixture.

[0107] Wherein, in the first mixing operation, D1 is greater than the liquid level drop height caused by a single aspiration, and / or the single aspiration volume does not exceed 1 / 4 to 1 / 3 of the total liquid volume. In this way, it is possible to prevent an air column from being aspirated when aspirating liquid at the upper layer.

[0108] In the second mixing operation, the pipette needle travels in the third depth range [D0, D4] of the reaction mixture; 0<D0, and the tip of the pipette needle is kept below the liquid surface during the traveling process. In this way, during the process that the pipette needle travels along the axial direction of the mixing well, the tip is always kept below the liquid surface, which effectively avoids aspirating air or touching the bottom, thereby ensuring the mixing quality and operation reliability.

[0109] The pipette first performs the first mixing operation on the reaction mixture in the mixing well, so that the upper and lower layers of liquid are interchanged, and the liquid stratification caused by the density difference is initially broken, then the pipette is controlled to travel along the axial direction of the mixing well, and multiple cycles of follow-up aspiration-discharge operations are performed during the traveling process, so that dynamic sampling and redistribution are performed at each vertical level of the liquid, further improving the mixing effect.

[0110] In one embodiment, the follow-up aspiration-discharge operation includes one of the following: Aspirating liquid during the moving process, stopping moving when the aspiration volume reaches a preset volume, and discharging the liquid at the current position; Aspirating liquid during the moving process, and when the aspiration volume reaches a preset volume, continuing to move and discharging the liquid during the moving process.

[0111] In one embodiment, during the second mixing operation, the downward movement speed of the pipette matches the rate at which the liquid level drops. Thus, during aspiration, the pipette descends along with the liquid level, maintaining a constant (or within a safe range) relative depth of the needle tip below the liquid surface, preventing the needle tip from protruding above the liquid.

[0112] In one embodiment, the ejection of liquid is accelerated when the pipette reaches depth D4. This allows for efficient mixing of the ejected liquid with the surrounding liquid in a very short time, significantly improving the mixing efficiency per unit time.

[0113] In one embodiment, during the second mixing operation, when the pipette reaches depth D4, it remains at that position and performs at least one aspiration-expulsion cycle. The bottom is the most difficult area to mix during the entire mixing process. When the pipette reaches the bottom, performing at least one aspiration-expulsion cycle at the bottom continuously impacts the bottom, improving mixing efficiency.

[0114] Step 306: The mixed reaction solution is distributed into multiple reaction wells using a one-suction-multiple-discharge method.

[0115] "One aspiration, multiple dispensing" refers to the process of aspirating once and dispensing multiple times to achieve multiple dispensings from a single aspiration operation. By dispensing a well-mixed reaction solution into multiple reaction wells through a single aspiration operation, the efficiency of amplification reagent preparation and the consistency of dispensing to each reaction well are further improved.

[0116] This method involves adding a first premix and a second premix to a mixing well to form a reaction mixture, then homogenizing the reaction mixture in the mixing well, and finally distributing the homogenized reaction mixture to multiple reaction wells. This premix-homogenization-dispensing operation mode minimizes dosing errors and ensures that the component ratios in each reaction well are completely consistent, thus making the experimental results more reliable and reproducible. When adding the two premixes, their different densities are fully considered. The lower-density second premix is ​​added to the mixing well first, followed by the higher-density first premix. When the higher-density liquid flows into the lower-density liquid, the higher-density first premix penetrates downwards, generating convection and agitation to achieve initial mixing. Based on this, the reaction mixture is further homogenized through the first and / or second homogenization operations, ensuring thorough mixing of the two premixes of different densities during the premixing process. Using this method, the reaction mixture required for each reaction well can be prepared with sufficient mixing and consistent proportions, which can improve the uniformity between reaction wells.

[0117] Taking the preparation of amplification reagents for a nucleic acid detection all-in-one machine as an example, the preparation method of the reaction mixture is explained.

[0118] In the amplification reagent preparation stage, liquids of different densities (reaction solution and enzyme solution) need to be transferred to a premixing container for mixing, and the mixed premix is ​​then transferred to the reaction wells of a multi-well plate. To ensure that the two liquids are thoroughly mixed and that the reaction solution distributed to the reaction wells is homogeneous, the following steps can be taken: Step 1: Calculate the volume of reaction solution and enzyme solution to be added separately.

[0119] Step 2: Based on the calculated amount of liquid to be added, first add the reaction solution to the mixing well of the premix container, and then add the enzyme solution to the mixing well that already contains the reaction solution.

[0120] The reasons are as follows: First, the enzyme solution has a higher density than the reaction solution, so dispensing the enzyme solution first will cause it to remain at the bottom of the liquid. Dispensing the enzyme solution into the reaction solution effectively eliminates this phenomenon. Second, the enzyme solution has a high viscosity, making it difficult to dispense accurately when the liquid volume is low and the premixing container is empty. This can lead to enzyme solution sticking to the tip, inner wall, or end of the pipette tip. In such cases, subsequent mixing is necessary to completely dispense the enzyme solution from the pipette tip.

[0121] Because the enzyme solution has high viscosity, enzyme residue may remain on the inner wall of the pipette tip. If the pipette tip is replaced before mixing, the ratio of the two reagent components may be abnormal, affecting the subsequent reaction results. Therefore, the pipette tip is not replaced here; the residual enzyme solution is carried into the reaction solution through subsequent mixing.

[0122] It should be noted that the air column should not be aspirated after the liquid is aspirated to prevent air from mixing into the reagent and causing bubbles during the liquid expulsion.

[0123] Step 3: Control the pipette to perform the first mixing operation on the reaction mixture in the mixing well.

[0124] Specifically, the pipette performs multiple cycles of aspirating liquid at the first depth range and dispensing liquid at the second depth range in the reaction mixture, i.e., aspirating liquid at the upper layer and dispensing liquid at the lower layer.

[0125] For example, a pipette is used to aspirate liquid from the upper layer of the reaction mixture, taking one-quarter of the total liquid volume. Then, it is moved to the bottom of the mixing well to expel liquid, and then returned to the upper layer to aspirate liquid. This process of aspirating liquid from the upper layer and expelling liquid from the bottom is repeated 8 times to ensure that the liquid in the upper layer is fully exchanged with the liquid in the lower layer.

[0126] Since the density of the enzyme solution is higher than that of the reaction solution, the enzyme solution will settle after being discharged in this step. At this time, the enzyme concentration in the lower layer of the reaction mixture is higher than that in the upper layer. The upper layer of low-concentration reaction mixture is sucked up and moved to the bottom of the premixing container. After the pump speed is increased, it is discharged. The conical bottom of the premixing container is used to backflush the enzyme solution to each layer of the reaction mixture. This process is repeated multiple times to ensure sufficient liquid exchange.

[0127] The volume of liquid aspirated or expelled should be adjusted according to the total amount of amplification reagents prepared; for example, it can be one-quarter of the total liquid volume in the premixed container.

[0128] Step 4: Control the pipette to perform a second mixing operation on the reaction mixture in the mixing well.

[0129] Specifically, the pipette travels axially through the mixing hole in the reaction mixture and performs multiple cycles of aspiration and dispensing during the travel; wherein the depth of axial travel does not exceed the depth of the reaction mixture.

[0130] After the upper and lower liquid layers are exchanged, the pipette's Z-axis moves to the surface of the reaction mixture to aspirate more liquid. After aspiration, the Z-axis remains stationary and the liquid is ejected. Then, the pipette's Z-axis descends to a certain height to continue aspiration and ejection. This cycle is repeated until the bottom of the container is reached, ensuring that all layers of the reaction mixture are thoroughly mixed. For example, assuming a total of 9 ml of liquid, divided into 8 cycles, after aspirating 1 ml of liquid and ejecting it, the pipette's Z-axis moves down to aspirate another 1 ml and eject it. This cycle is repeated 8 times to thoroughly mix all layers of liquid.

[0131] In the second mixing operation, to ensure that each liquid layer undergoes aspiration and expulsion, a follow-up aspiration mode is adopted during aspiration, meaning the Z-axis follows the liquid level as it descends, while the Z-axis remains stationary during expulsion. Therefore, as the number of aspiration and expulsion cycles increases, the Z-axis height continuously decreases, achieving the effect of ensuring that each liquid layer undergoes aspiration and expulsion. The amount of aspirated / expelled liquid in this step can also be adjusted according to the total amount of amplification reagent prepared.

[0132] A well-mixed reaction mixture can be prepared by performing two different mixing operations.

[0133] Step 5: Distribute the well-mixed reaction solution into multiple reaction wells of the multi-well plate using a one-suction-multiple-discharge method.

[0134] The premix-mixing-dispensing operation mode of this method minimizes dosing errors and ensures that the composition ratio of the reaction mixture in each reaction well is completely consistent, thus making the experimental results more reliable and reproducible. When adding the two premixed solutions, the different densities of the two solutions are fully considered. The lower-density reaction solution is added to the mixing well first, followed by the higher-density enzyme solution. When the high-density liquid flows into the low-density liquid, the high-density enzyme solution will penetrate downwards, and this process itself generates some convection and agitation, achieving preliminary mixing. Based on this, the reaction mixture is further homogenized through the first and second homogenization operations, ensuring that the two premixed solutions of different densities are thoroughly mixed during the premixing process. Using this method, the required reaction mixture for each reaction well can be prepared with sufficient homogenization and consistent proportions. This method improves the uniformity between reaction wells, making the experimental results more reliable and reproducible.

[0135] It should be noted that, in this document, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Unless otherwise specified, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes that element.

[0136] Through the above description of the embodiments, those skilled in the art can clearly understand that the methods of the above embodiments can be implemented by means of software plus necessary general-purpose hardware platforms. Of course, they can also be implemented by hardware, but in many cases the former is a better implementation method. Based on this understanding, the technical solution of the present invention, in essence, or the part that contributes to the prior art, can be embodied in the form of a software product. This computer software product is stored in a storage medium (such as ROM / RAM, magnetic disk, optical disk) and includes several instructions to cause a terminal (which may be a mobile phone, computer, server, or network device, etc.) to execute the methods described in the various embodiments of the present invention.

[0137] The above are merely specific embodiments of this application, but the scope of protection of this application is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in this application should be included within the scope of protection of this application. Therefore, the scope of protection of this application should be determined by the scope of the claims.

Claims

1. A method for preparing a reaction mixture, comprising controlling a pipette to perform the following steps: The first premix and the second premix were added to a mixing well to form a reaction mixture, wherein, The density of the first premix is higher than the density of the second premix, and the first premix is added after the second premix; Performing a mixing operation on the reaction mixture in the mixing well; Dispensing the mixed reaction mixture into a plurality of reaction wells; Wherein, the mixing operation comprises at least one of the following two mixing operations: The first mixing operation: the pipette tip performs multiple cycles of aspirating the reaction mixture at a first depth range and discharging at a second depth range, wherein the depth of each aspiration may be the same or different, and the depth of each discharge may be the same or different; the first depth range is [D1, D2], the second depth range is [D3, D4], where 0<D1<D2<D3<D4<H, and D3≥K×D2, where 1.5≤K≤3, the liquid surface depth is 0, and H is the total depth of the reaction mixture; The second mixing operation: the pipette tip travels axially along the mixing well inside the reaction mixture, and during the traveling process, performs multiple cycles of follow-up aspiration-discharge operations; wherein the depth of the axial traveling does not exceed the depth of the reaction mixture.

2. The method for preparing the reaction mixture according to claim 1, characterized in that, The mixing operation comprises the first mixing operation and the second mixing operation; Said performing a mixing operation on the reaction mixture in the mixing well comprises: Performing the first mixing operation on the reaction mixture in the mixing well first, and then performing the second mixing operation.

3. The method for preparing the reaction mixture according to claim 1 or 2, characterized in that, D1 is greater than the liquid level drop height caused by a single aspiration, and / or the single aspiration volume does not exceed 1 / 4 to 1 / 3 of the total liquid volume.

4. The method for preparing the reaction mixture according to claim 1 or 2, characterized in that, In the second mixing operation, the pipette tip travels within the third depth range [D0, D4] of the reaction mixture; 0<D0, and the tip of the pipette tip is kept below the liquid surface during the traveling process.

5. The method for preparing the reaction mixture according to claim 4, characterized in that, Said traveling axially along the mixing well and performing multiple cycles of follow-up aspiration-discharge operations during the traveling process comprises one of the following: Moving downward axially along the mixing well starting from the depth D0, performing multiple cycles of follow-up aspiration-discharge operations during the movement, and stopping when moving to the depth D4 or when the number of cycles of the follow-up aspiration-discharge operations reaches a preset number; Moving upward axially along the mixing well starting from the depth D4, performing multiple cycles of follow-up aspiration-discharge operations during the movement, and stopping when moving to the depth D0 or when the number of cycles of the follow-up aspiration-discharge operations reaches a preset number; First moving downward axially along the mixing well starting from the depth D0, starting to move upward after moving to the depth D4, performing multiple cycles of follow-up aspiration-discharge operations during the movement, and stopping when moving to the depth D0 or when the number of cycles of the follow-up aspiration-discharge operations reaches a preset number; Moving upward axially along the mixing well starting from the depth D4, moving downward when moving to the depth D0, performing multiple cycles of follow-up aspiration-discharge operations during the movement, and stopping when moving to the depth D4 or when the number of cycles of the follow-up aspiration-discharge operations reaches a preset number.

6. The method for preparing the reaction mixture according to claim 5, characterized in that, The follow-up aspiration-discharge operation comprises one of the following: The device aspirates liquid during movement, stops moving when the aspirated amount reaches a preset level, and then expels the liquid at the current location. The liquid is aspirated during movement, and when the aspirated amount reaches the preset amount, the movement continues and the liquid is expelled during the movement.

7. The method for preparing the reaction mixture according to claim 4, characterized in that, In the second mixing operation, when the pipette moves downward, the downward speed of the pipette matches the rate at which the liquid level drops.

8. The method for preparing the reaction mixture according to claim 4, characterized in that, When the pipette moves to depth D4, the discharge is accelerated, or when the pipette moves to depth D4, it remains at that position and performs at least one aspiration-discharge cycle.

9. The method for preparing the reaction mixture according to claim 1, characterized in that, The step of adding the first premixed liquid and the second premixed liquid into a mixing well to form a reaction mixture includes: The first addition amount of the first premixed liquid and the second addition amount of the second premixed liquid are determined based on the number of reaction wells to be allocated, the preset premixing ratio of the first premixed liquid and the second premixed liquid, and the transfer loss amount redundant according to the preset premixing ratio; wherein, the number of reaction wells to be allocated is determined based on the sample amount to be tested and the preset quality control sample amount, and / or, the transfer loss amount is determined based on the preset redundant reaction wells and the preset premixing ratio; The second amount of the second premixed liquid is drawn into the mixing hole, and then the first amount of the first premixed liquid is drawn into the mixing hole.

10. The method for preparing the reaction mixture according to claim 1, characterized in that, The step of adding the first premixed liquid and the second premixed liquid into a mixing well to form a reaction mixture includes: The second addition amount of the second premixed liquid is determined based on the sum of the number of reaction holes to be assigned and the number of preset redundant reaction holes, as well as the amount of second premixed liquid required in each reaction hole. The number of times the second premixed liquid is drawn is determined based on the second added liquid and the single drawing volume. The second premixed liquid is drawn according to the number of times and the single drawing volume and added to the mixing hole. The first addition amount of the first premixed liquid is determined based on the sum of the number of reaction holes to be assigned and the number of preset redundant reaction holes, as well as the amount of first premixed liquid required in each reaction hole. The number of times the first premixed liquid is drawn is determined based on the first added liquid volume and the single aspiration volume. The first premixed liquid is drawn according to the number of times and the single aspiration volume and added to the mixing hole that already contains the second premixed liquid.

11. The method for preparing the reaction mixture according to claim 9 or 10, characterized in that, The first premix and the second premix satisfy at least one of the following requirements: The first premixed liquid and the second premixed liquid are compatible liquids; The required amount of the first premixed liquid in the reaction well is less than the required amount of the second premixed liquid; The viscosity of the first premixed liquid is higher than that of the second premixed liquid.

12. The method for preparing the reaction mixture according to claim 1, 9, or 10, characterized in that, The pipette uses the same disposable tip to perform the following operations: aspirate and dispense the first premixed solution, mix and dispense the reaction mixture; and / or, the pipette uses different disposable tips to aspirate and dispense the first premixed solution and the second premixed solution.

13. The method for preparing a reaction mixture according to claim 1, wherein distributing the homogenized reaction mixture into a plurality of reaction wells comprises: The well-mixed reaction mixture is distributed into multiple reaction wells using a one-intake-multiple-exhaust method.

14. A testing device comprising a reagent preparation mechanism and a controller, the reagent preparation mechanism comprising a pipette, the pipette comprising a pipette needle at its end and a driving device, the driving device being connected to the controller, the controller controlling the driving device to drive the pipette needle to move, the controller implementing the reaction mixture preparation method as described in any one of claims 1 to 13.

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