Automatic liquid mixing method for virus titer detection based on plaque method

Abstract

The invention discloses an automatic liquid mixing method for virus titer detection based on a plaque method, which utilizes an execution module to automatically transfer liquid, separate the liquid and mix the liquid, greatly improves the liquid mixing efficiency, and can greatly reduce the manual workload and save time and cost especially for the detection task of batch liquid preparation and liquid mixing. Liquid is obtained strictly according to a preset sequence by means of the execution module and injected into the mixing tube, so that the host cell liquid, the virus test sample and the liquid agar are injected into the same mixing tube firstly, the steps in the whole liquid mixing process are smoothly linked and are high in efficiency, and it is guaranteed as much as possible that only one virus invades one host cell; and the accuracy of the virus titer detection result based on the plaque method is improved.

Description

Technical Field

[0001] This invention relates to the field of virus detection technology, and in particular to an automated liquid mixing method for virus titer detection based on plaque assay. Background Technology

[0002] In the processing of biopharmaceuticals, virus removal has become an essential step to meet viral safety requirements. The production of biopharmaceuticals requires virus inactivation and / or virus removal processes. A common method for virus removal is membrane filtration, and the virus removal capacity of the membrane directly determines the viral safety of the biopharmaceutical. Membrane separation technology uses a membrane as the separation medium. When a driving force exists across the membrane (such as pressure difference or concentration difference), the raw material components selectively permeate through the membrane, while viruses, due to their larger size than the membrane pores, are physically retained.

[0003] Plaque assay, which detects viral titers based on viral infectivity, is a classic viral titer detection technique. Its basic principle is as follows: after culturing host cells to a monolayer, an appropriately diluted virus is inoculated and cultured. The virus adsorbs and invades the host cells, uses enzymes within the host cells to synthesize its own components, causing the host cells to lyse and die, forming plaques. Each plaque represents a live viral particle.

[0004] For example, the Parenteral Drug Association (PDA) issued a guidance document, "Virus Filtration, Technical Report No. 41 (Revised 2008)," or TR41, for virus filtration testing methods and virus removal validation for biologics. TR41 specifies that the model virus used for virus removal membrane / filter challenges is bacteriophage PR772 or PP7 (PR772 is a large virus, PP7 is a small virus, and the choice can be made as needed), and the material stream is immunoglobulin IVIG (or bovine serum albumin BSA). The titer detection of both PR772 and PP7 bacteriophages is achieved using the aforementioned plaque assay.

[0005] More specifically, Appendix III of TR41 clearly describes the small virus-retention filter-test protocol, which uses PP7 phage as the nominal challenge virus model, and human immunoglobulin (IVIG) is used to assess protein penetration. To calculate the virus rejection rate, the titers of PP7 phage in the challenge solution and filtrate need to be detected separately, and the logarithmic decrease in viral titer (LRV) is calculated using the corresponding formula. TR41 clearly describes the method for detecting the PP7 phage titer, which uses a double-layer plate counting method to culture and count the samples. The detection system is prepared by mixing 1 mL of filtrate sample containing a specific concentration of PP7 phage, 2 mL of host cells (Pseudomonas aeruginosa), and 9 mL of warm agar; or by mixing approximately 0.1 mL of filtrate sample containing PP7 phage, 1 mL of host cells, and 4.5 mL of agar solution.

[0006] Currently, the preparation and mixing of host cell fluid, virus test samples, and liquid agar are entirely manual, resulting in limited testing capacity and extremely low efficiency. This is because the mixing process for the detection system often strictly follows the relevant regulations in documents such as TR41, proceeding step-by-step. Specifically, it requires using a pipette to sequentially aspirate 1 mL of a filtrate sample containing PP7 phage at a specific concentration, 2 mL of host cell fluid, and 9 mL of warm agar, injecting them into a mixing tube, mixing thoroughly, pouring the mixture into a plate, and then incubating and counting. To avoid cross-contamination of different samples and inaccurate results, different pipette tips are needed for different samples. Therefore, before aspirating each sample, previously used pipette tips must be discarded, and clean tips obtained before aspirating the next sample. In other words, to mix the samples 10 times, 30 clean pipette tips must be obtained, 30 samples must be aspirated, and 30 used pipette tips must be discarded. The entire mixing process is cumbersome, error-prone, time-consuming, and requires an excessive number of pipette tips.

[0007] Furthermore, regarding the method for detecting virus titers in ultra-low virus titer systems disclosed in existing patents such as CN115181816B, since the titer of PP7 phage in the filtrate is below the detection limit, to ensure accurate detection results, the approach is to accumulate a sufficiently large volume of filtrate for testing for a given volume of filtrate; that is, the amount of filtrate tested must be sufficiently large. For example, if the filtrate volume is 100 ml and contains 20 PP7 phages, if only 1 ml is tested, the probability of detecting 0 PP7 phages is relatively high, and the detection result is not statistically significant (the detection result must meet the 95% confidence interval). Therefore, at least 30 ml of filtrate, or even more, must be tested, which requires mixing the solution 30 times. Considering that virus challenge tests are often performed in batches using multiple virus-removing membranes / filters, the workload is extremely large. Moreover, manual sampling is prone to sampling volume deviations, which can accumulate and reduce the reliability of the detection results.

[0008] Therefore, there is a need for an automated mixing method for virus titer detection based on plaque assay, which can improve detection efficiency, save pipette tips, and provide accurate and reliable detection results. Summary of the Invention

[0009] To address the shortcomings of existing technologies, the present invention aims to provide an automated liquid mixing method for virus titer detection based on plaque assay, which solves the problems of cumbersome, error-prone, time-consuming, and excessive number of pipette tips required by existing manual liquid mixing processes.

[0010] To achieve the above objectives, the present invention adopts the following technical solution:

[0011] An automated liquid mixing method for virus titer detection based on plaque assay includes the following steps:

[0012] Step 1: The execution module obtains a clean pipette tip and controls the pipette tip to perform operation A1. Operation A1 includes aspirating a volume of host cell fluid of X1*K1*V1 through the pipette tip once, and injecting a volume of V1 of the host cell fluid into each of the K1 mixing tubes, and repeating operation A1 N1 times.

[0013] Step 2: The execution module replaces the clean pipette tip and controls the pipette tip to perform operation A2. Operation A2 includes aspirating a virus test sample with a volume of X2*K2*V2 through the pipette tip, and injecting a virus test sample with a volume of V2 into K2 mixing tubes that already contain the host cell fluid. The operation A2 is repeated N2 times.

[0014] Step 3: The execution module replaces the clean pipette tip and controls the pipette tip to perform operation A3. Operation A3 includes drawing a volume of liquid agar of X3*V3 through the pipette tip once, and injecting a volume of liquid agar of V3 into a mixing tube that already contains the host cell fluid and the virus test sample. Operation A3 is repeated N3 times, where N3 = K1*N1 = K2*N2, to obtain N3 portions of the mixture. Each portion of the mixture consists of a volume of host cell fluid of V1, a volume of virus test sample of V2, and a volume of liquid agar of V3.

[0015] And X1 is 1 to 2, X2 is 1 to 2 and X3 is 1 to 2, and K1, K2, N1, N2 and N3 are all integers greater than or equal to 1.

[0016] The automated liquid mixing method of the present invention is applicable to virus titer detection methods based on plaque assay. It utilizes an execution module to automatically perform liquid transfer, separation and mixing operations, which greatly improves the efficiency of liquid mixing. Especially for detection tasks that require batch preparation and mixing, it can greatly reduce manual workload and save time and costs.

[0017] Each time the execution module uses a pipette tip to pick up a different liquid, the tip needs to be changed to ensure the purity of each liquid and avoid cross-contamination. Each removal and reloading of the pipette tip takes time. Therefore, to reduce the number of tip changes, the execution module can use the same pipette tip to transfer a certain multiple of a predetermined amount of the same liquid at once, or use the same pipette tip to transfer a certain multiple of a predetermined amount of the same liquid multiple times consecutively, or use the same pipette tip to transfer a predetermined amount of the same liquid multiple times consecutively until the previous liquid is completely transferred, then change the pipette tip to transfer the next liquid. This operation, where the number of tip changes depends on the type of liquid to be mixed, is suitable for batch virus challenge testing of virus-free membranes / filters. It requires mixing fewer types of liquids, simplifies the entire mixing process, eliminates the need for frequent tip changes, ensures high operational continuity, greatly improves efficiency, provides accurate test results, and saves pipette tips. As for step three, on the one hand, the amount of agar required for a single test is already quite large, and on the other hand, agar solidifies rapidly at room temperature. Both of these factors mean that when automatically mixing the solutions, the amount of agar taken in a single test should only be the amount required for that single measurement, and should not be double or triple the amount of agar required for the test.

[0018] Meanwhile, the automated liquid mixing method of the present invention relies on the execution module to strictly obtain liquids and inject them into the mixing tube in a predetermined order. This ensures that the host cell fluid is injected first, followed by the virus test sample and then liquid agar in the same mixing tube. In this order, the host cell fluid is injected into the mixing tube and evenly dispersed in the mixing tube before the virus test sample is added. Since the virus test sample is added last, the host cell fluid is in large excess relative to the gradually injected virus. Moreover, during the injection of the virus test sample, based on the release force of the pipette tip, the virus test sample forms a vortex in the host cell fluid, allowing the virus test sample to quickly and evenly diffuse throughout the liquid environment and fully mix with the host cell fluid. This allows the virus test sample to randomly collide and adsorb with the host cells, which helps to ensure that only one virus invades a host cell, thus improving the accuracy of virus titer detection results based on plaque assay. More importantly, both the host cell solution and the virus test sample are at room temperature, while agar is in a warm liquid state. Injecting the host cell solution first, followed by the virus test sample, results in only one heating process for the virus test sample. If the order is reversed, the virus test sample is heated first, then rapidly cooled by a larger volume of host cell solution, and then heated again. This can negatively impact the viral activity in the test sample, potentially leading to distorted test results. It should be noted that manual operation is performed at room temperature, therefore this issue does not exist.

[0019] The benefits of injecting liquid agar last include: to maintain the activity of the host cell fluid and virus test sample, the temperature of the mixing tube is lower than the storage temperature of the liquid agar. If the liquid agar is added too early, it may cause local solidification. Therefore, injecting the liquid agar last allows it to disperse quickly and evenly in the mixture, shortening the residence time of the liquid agar in the relatively cooler mixing tube. This ensures that the agar remains in a liquid state, making it easier to pour the mixture into plates and spread it evenly. After the liquid agar solidifies, the plates can be inverted for incubation. The high transparency of the agar also facilitates subsequent plaque counting.

[0020] It should be noted that if there are multiple types of virus test samples, step two should be performed for each type of virus test sample. When the virus types in multiple virus test samples are the same (e.g., all are PP7 bacteriophage), then K1*N1 is the total number of tests for all virus test samples (e.g., if a single virus test sample requires 10mL of sample, and 1mL is taken at a time, then the number of tests is 10; if the number of virus test samples is 3, then the total number of tests for all virus test samples is 30). In this case, K1*N1 = 30. The statement that K1*N1 = K2*N2 means that the sum of the number of tests for all virus test samples in step two should also be 30, not that the number of tests for a single virus test sample satisfies K2*N2 = 30. If the virus types in the multiple virus test samples are different, since they correspond to different host cell fluids, K1*N1=K2*N2 still refers to multiple virus test samples with the same type of virus. If K1*N1=30 in step one, then the sum of the number of tests for all virus test samples in step two should also be 30.

[0021] It should be noted that in step one, operation A1 includes drawing a volume of host cell fluid of X1*K1*V1 through the pipette tip in one go, and injecting a volume of V1 of host cell fluid into each of the K1 mixing tubes. Since X1 is 1-2, a certain amount of host cell fluid may remain in the pipette tip after operation A1. This operation aims to ensure that the host cell fluid squeezed into each mixing tube is relatively accurate, and to avoid some air being squeezed out when K1>1, leaving a small amount of host cell fluid remaining in the pipette tip during the last squeeze. Of course, in order to obtain higher pipetting efficiency, X1 can also be 1, but it should not be too large to avoid material waste and possible contamination (the same applies below). If there is little host cell fluid remaining in the pipette tip (i.e., X1 is small), then the remaining host cell fluid can be left in the pipette tip, and operation A1 can be repeated. However, if there is relatively more host cell fluid remaining in the pipette tip (i.e., X1 is large), then before performing the next operation A1, the remaining host cell fluid in the pipette tip can be squeezed back into the original container, and then operation A1 can be repeated. This can improve the accuracy of pipetting and separation, and also prevent excessive accumulation of host cell fluid in the pipette tip.

[0022] In step two, operation A2 includes drawing a volume of virus test sample of X2*K2*V2 using the pipette tip, and injecting a volume of V2 of virus test sample into each of the K2 mixing tubes already containing host cell fluid. Since X2 is 1-2, a certain amount of virus test sample may remain in the pipette tip after operation A2. If the amount of virus test sample remaining in the pipette tip is small, it can be left in the pipette tip, and operation A2 can be repeated. However, if the amount of virus test sample remaining in the pipette tip is relatively large, the remaining virus test sample in the pipette tip should be squeezed back into the original container before performing the next operation A2, and then operation A2 can be repeated.

[0023] In step three, operation A3 includes drawing a volume of liquid agar (X3*V3) through the pipette tip and injecting a volume of virus test sample (V3) into a mixed tube already containing host cell fluid and virus test sample. Since X3 is 1-2, a certain amount of liquid agar may remain in the pipette tip after operation A3. If the amount of liquid agar remaining in the pipette tip is small, it can be left in the pipette tip, and operation A3 can be repeated. Although the agar will solidify, it will melt again when the pipette tip is re-immersed in warm agar. However, if there is a relatively large amount of liquid agar remaining in the pipette tip, it should be squeezed back into the original container before performing the next operation A3, and then operation A3 can be repeated.

[0024] Preferably, the automated mixing method satisfies at least one of the following conditions:

[0025] (1) X1 is not greater than 1.1; (2) X2 is not greater than 1.1; (3) X3 is not greater than 1.1.

[0026] Since X1 is no greater than 1.1, after performing operation A1 in step one, there is relatively little host cell fluid remaining in the pipette tip. Therefore, the remaining host cell fluid can be retained in the pipette tip, and operation A1 can be repeated, resulting in higher operational efficiency. Of course, it is also possible to squeeze the remaining host cell fluid in the pipette tip back into the original container before repeating operation A1. This is not a limitation and will be applied hereinafter.

[0027] Similarly, since X2 is not greater than 1.1, in step two, after completing operation A2 once, there is less virus test sample remaining in the pipette tip. Therefore, the remaining virus test sample can be left in the pipette tip, and operation A2 can be repeated.

[0028] Similarly, since X3 is not greater than 1.1, in step three, after performing operation A3 once, there is less liquid agar remaining in the pipette tip. Therefore, the remaining liquid agar can be left in the pipette tip, and operation A3 can be repeated.

[0029] Preferably, the automated mixing method includes at least one of the following operations:

[0030] (1) In step one, the operation of the pipette tip to draw host cell fluid with a volume of X1*K1*V1 includes operation S1-1 and operation S1-2 performed sequentially. In operation S1-1, the volume drawn by the pipette tip is greater than X1*K1*V1. In operation S1-2, the volume drawn by the pipette tip that exceeds X1*K1*V1 is squeezed out, so that the volume of host cell fluid retained in the pipette tip is equal to X1*K1*V1.

[0031] (2) In step two, the operation of the pipette tip to draw a virus test sample with a volume of X2*K2*V2 includes operations S2-1 and S2-2 executed sequentially. In operation S2-1, the volume drawn by the pipette tip is greater than X2*K2*V2. In operation S2-2, the volume of the drawn sample exceeding X2*K2*V2 is squeezed out, so that the volume of the virus test sample retained in the pipette tip is equal to X2*K2*V2.

[0032] (3) In step three, the operation of the pipette tip to draw a virus test sample with a volume of X3*V3 includes operations S3-1 and S3-2 executed sequentially. In operation S3-1, the volume of the pipette tip drawn is greater than X3*V3. In operation S3-2, the volume of the pipette tip drawn is squeezed out beyond X3*V3, so that the volume of liquid agar retained in the pipette tip is equal to X3*V3.

[0033] When aspirating various materials, a small amount of gas may inevitably be drawn in. Due to surface tension, viscosity, and other factors, the gas remains at the bottom of the pipette tip. This results in the final amount of liquid aspirated being lower than the preset amount. The purpose of the above three operations is to ensure that the volume of liquid retained in the pipette tip is as close as possible to or equal to the preset amount, thereby improving the accuracy of the liquid volume injected into the mixing tube and the accuracy of the proportions of each liquid in the mixture, ultimately improving the detection accuracy.

[0034] Preferably, the automated mixing method includes at least one of the following operations:

[0035] (1) In step one, after the execution module performs operation S1-2, the execution module also performs operation P1, which is to draw a certain amount of air into the bottom of the suction head;

[0036] (2) In step two, after the execution module performs operation S2-2, the execution module also performs operation P2, which is to draw a certain amount of air into the bottom of the suction head;

[0037] (3) In step three, after the execution module performs operation S3-2, the execution module also performs operation P3, which is to draw a certain amount of air into the bottom of the suction head.

[0038] The purpose of the above three operations is to draw up any dripping liquid that may be protruding from the bottom of the pipette tip. Based on the principle of equal volume replacement, the dripping liquid is also part of the predetermined volume. If the dripping liquid falls off during the process of the execution module moving the pipette tip, it may not only cause contamination, but may also cause the volume of liquid injected into the mixing tube to be lower than the predetermined volume. This operation can further ensure that the volume of liquid inside the pipette tip is closer to or equal to the preset volume, prevent random dripping, and improve detection accuracy.

[0039] It should be noted that:

[0040] In step one, when K1 equals 1, in operation A1, the volume of gas extruded into the mixing tube must be greater than or equal to the sum of the volume of gas drawn back (the volume of a fixed amount of air drawn into the bottom of the pipette tip in operation P1) and V1 in order to expel all the liquid in the pipette tip into the mixing tube.

[0041] In step one, when K1 is greater than or equal to 2, in operation A1, the volume of gas extruded into the first mixing tube is equal to the sum of the volume of gas drawn back (the volume of a fixed amount of air drawn into the bottom of the suction head in operation P1) and V1. Only in this way can the gas drawn back be accurately extruded and liquid of volume V1 be squeezed into the mixing tube. Then, liquid of volume V1 can be directly extruded each time.

[0042] In step two, when K2 equals 1, in operation A2, the volume of gas extruded into the mixing tube must be greater than or equal to the sum of the volume of gas drawn back (operation P2 is the volume of a fixed amount of air drawn into the bottom of the pipette tip) and V2 in order to squeeze all the liquid in the pipette tip into the mixing tube.

[0043] In step two, when K2 is greater than or equal to 2, in operation A2, the volume of gas extruded into the first mixing tube is equal to the sum of the volume of gas drawn back (operation P2 is the volume of a fixed amount of air drawn into the bottom of the suction head) and V2. Only in this way can the gas drawn back be accurately extruded and liquid of volume V2 be squeezed into the mixing tube. Then, liquid of volume V2 can be directly extruded each time.

[0044] In step three, during operation A3, the volume extruded into the mixing tube must be greater than or equal to the sum of the volume of gas drawn back (operation P3 is the volume of a fixed amount of air drawn into the bottom of the pipette tip) and V3 in order to extrude all the liquid in the pipette tip into the mixing tube.

[0045] Preferably, the automated mixing method includes at least one of the following operations:

[0046] (1) In step one, after the execution module performs operation S1-2, the execution module also performs operation Q1. Operation Q1 refers to the execution module controlling the suction head to move axially upward, the dwell time being T1, and then moving axially.

[0047] (2) In step two, after the execution module performs operation S2-2, the execution module also performs operation Q2. Operation Q2 refers to the execution module controlling the suction head to move axially upward, the dwell time being T2, and then moving axially.

[0048] (3) In step three, after the execution module performs operation S3-2, the execution module also performs operation Q3. Operation Q3 refers to the execution module controlling the suction head to move axially upward, the dwell time being T3, and then moving axially.

[0049] The purpose of the above three operations is to control the pipette tip to move axially upwards (actually a reset action after the pipette tip probes down to collect liquid) and remain there after the liquid in the pipette tip reaches the set amount of the execution module. This extends the time the pipette tip stays within the projection range of the opening of the original container, providing time for the liquid adhering to the outer wall of the pipette tip to collect downwards to the bottom of the pipette tip. Then, the pipette tip is moved axially (not limited to upwards or downwards). The liquid adhering to the outer wall of the pipette tip, having collected at the bottom of the pipette tip, separates from the pipette tip under inertia, thereby removing the liquid adhering to the outer wall of the pipette tip and allowing it to fall back into the original container. This avoids random dripping when the execution module moves the pipette tip, which could cause potential contamination and safety risks, ensuring the cleanliness of the entire operating environment, reducing the possibility of cross-contamination, and thus improving detection accuracy.

[0050] Preferably, in operation Q1, the dwell time T1 is 0.2-2s; and / or, in operation Q2, the dwell time T2 is 0.2-2s; and / or, in operation Q3, the dwell time T3 is 0.2-2s.

[0051] This operation allows the liquid adhering to the outer wall of the pipette tip to drip back into the original container naturally, instead of dripping onto the work surface or containers along the movement path as the execution module moves. This avoids contaminating the working environment and cross-contamination, reducing waste. It also improves the accuracy, repeatability, and aseptic safety of pipetting. If the residence time is less than 0.2 seconds, it is too short, and the liquid on the outer wall of the pipette tip has not yet moved to the bottom and fallen off. This is especially true for high-viscosity products such as warm agar. If the residence time is greater than 2 seconds, it is too long, which prolongs the overall operation time, reduces mixing efficiency, and may also cause warm agar to solidify upon cooling.

[0052] Preferably, in operation Q1, the axial movement process of the suction head is either axially downward first and then axially upward, or axially upward first and then axially downward.

[0053] And / or, in operation Q2, the axial movement process of the suction head is either first moving axially downward and then moving axially upward, or first moving axially upward and then moving axially downward.

[0054] And / or, in operation Q3, the axial movement process of the suction head is either first moving axially downward and then moving axially upward, or first moving axially upward and then moving axially downward.

[0055] This operation is equivalent to axially shaking the suction tip within the projection range of the opening of the original container, which can quickly and completely drip the liquid adhering to the outer wall of the suction tip back into the original container, improving operational efficiency. At the same time, it avoids liquid from falling indiscriminately and contaminating the working environment or other containers.

[0056] Preferably, the automated mixing method includes at least one of the following operations:

[0057] (1) In operation A1, each time the pipette tip draws host cell fluid, the pipette tip moves axially downward until its end is a certain height below the liquid level after the previous draw of host cell fluid by the pipette tip.

[0058] (2) In operation A2, each time the pipette tip draws a virus test sample, the pipette tip moves axially downward until its end is a certain height below the liquid level after the previous virus test sample was drawn by the pipette tip.

[0059] (3) In operation A3, each time the pipette tip draws liquid agar, the pipette tip moves axially downward until its end is a certain height below the liquid surface after the previous draw of liquid agar.

[0060] After each material is drawn, the liquid level drops, and the pipette tip needs to be lowered to an even lower position for the next liquid drawing operation. This ensures that the bottom opening of the pipette tip is always below the liquid surface, reducing the probability of air being drawn in while the tip is drawing in liquid. This allows the amount of liquid drawn by the tip to be closer to the set amount of liquid, improving the accuracy of the drawing. In turn, this ensures that the liquid injected into the mixing tube is as close as possible to or equal to the preset amount, thus improving the detection accuracy. For materials with low liquid levels, designing the depth of the pipette tip to be closer to the bottom of the container is certainly a possible solution. However, this means that initially, the tip may be too deep below the liquid surface, causing more liquid to adhere to the outer wall of the tip and forming more droplets, which will also affect the final test results.

[0061] Preferably, in step three, the execution module also performs operation A3-1, which refers to drawing out at least a portion of the mixture from each mixing tube and re-injecting the drawn-out mixture into the original mixing tube.

[0062] This setup improves the uniformity of the mixture, allowing the virus test sample to randomly collide and adsorb with the host cell, ensuring that only one virus invades a host cell as much as possible, thus improving the accuracy of virus titer detection results based on plaque assay.

[0063] It is important to note that operation A3-1 can be performed in two ways: first, using a single pipette tip to sequentially pipette the required agar into all the mixing tubes; then, using a new pipette tip to perform operation A3-1 on the mixture in each tube (after the pipette tip used to aspirate the agar has finished dispensing the agar into the last mixing tube, operation A3-1 can be performed directly using that same pipette tip); or, after pipetting the required agar into a single mixing tube with a single pipette tip, immediately perform operation A3-1 on the mixture in that tube with the same tip, then discard the pipette tip and repeat the above operation with a new pipette tip. The number of times operation A3 is performed, N3, refers to the number of times the execution module completes the pipetting of the required agar into all the mixing tubes, and is not limited by whether a new pipette tip needs to be replaced repeatedly.

[0064] Preferably, the specific operation of operation A3-1 is to move the pipette tip down to a position where its end is below the initial liquid level of the mixture, draw up at least a portion of the mixture, then move the end of the pipette tip up to a position not below the initial liquid level of the mixture, and then squeeze the mixture in the pipette tip back into the original mixing tube.

[0065] This operation allows the pipette tip to draw up the hotter, denser mixture closer to the bottom of the mixing tube, and mix it again with the upper layer of mixture, which helps to improve the overall temperature uniformity and concentration uniformity of the mixture.

[0066] Preferably, in operation A3-1, at least half of the mixture in each mixing tube is drawn out and re-injected into the original mixing tube; this operation can greatly improve the overall temperature uniformity and concentration uniformity of the mixture.

[0067] Preferably, the temperature of the mixture is 38-42°C, and the temperature of the liquid agar is 45-60°C.

[0068] Within the aforementioned temperature range, both the host cells and viruses in the mixture maintain good activity to meet the requirements of the plaque assay. The incubation temperature of the mixture is 38-42℃, and the temperature of the liquid agar is 45-60℃. Even if the temperature drops when the liquid agar is injected into the mixing tube, it will not fall below the solidification temperature of the agar, allowing the agar to remain liquid for a certain period of time. This ensures that the agar is quickly and evenly dispersed in the mixture. When the mixture is then poured into a plate, the agar will not solidify locally or form a skin. The mixture can be evenly spread in the plate, and the liquid agar solidifies quickly. After inverting the plate for incubation, the high transparency of the agar facilitates subsequent plaque counting.

[0069] In summary, compared with the prior art, the present invention has at least the following beneficial effects:

[0070] The automated liquid mixing method of this invention is applicable to virus titer detection methods based on plaque assays. It utilizes an execution module to automatically perform liquid transfer, dispensing, and mixing operations, significantly improving mixing efficiency. Especially for detection tasks requiring batch preparation and mixing, it greatly reduces manual workload, saving time and costs. Since the execution module needs to change pipette tips each time it aspirates different liquids to ensure the purity of each liquid and avoid cross-contamination, and each tip removal and reloading takes time, the execution module can use the same pipette tip to reduce the frequency of tip changes. This method involves transferring a predetermined amount of the same liquid at once, or repeatedly transferring a predetermined amount of the same liquid using the same pipette tip, or repeatedly transferring a predetermined amount of the same liquid using the same pipette tip until the previous liquid has been transferred, then changing the pipette tip and transferring the next liquid. The number of times the pipette tip needs to be changed depends on the type of liquid to be mixed. This method is suitable for batch virus challenge testing of virus-free membranes / filters. It requires mixing fewer types of liquids, simplifies the entire mixing process, eliminates the need for frequent pipette tip changes, ensures high operational continuity, greatly improves efficiency, provides accurate test results, and saves pipette tips.

[0071] The automated liquid mixing method of this invention relies on the execution module to strictly follow a predetermined sequence to obtain liquids and inject them into the mixing tube. This ensures that the host cell fluid is injected first, followed by the virus test sample, and then liquid agar in the same mixing tube. The steps in the entire liquid mixing process are smoothly connected and highly efficient. In the above process, the host cell fluid is injected into the mixing tube and evenly dispersed in the mixing tube. The virus test sample is added later, so the host cell fluid is in large excess relative to the gradually injected virus. Moreover, during the injection of the virus test sample, based on the release force of the pipette tip, the virus test sample forms a vortex in the host cell fluid, which allows the virus test sample to quickly and evenly diffuse throughout the liquid environment and fully mix with the host cell fluid. This allows the virus test sample to randomly collide and adsorb with the host cells, ensuring that only one virus invades a host cell as much as possible, thus improving the accuracy of virus titer detection results based on plaque assay. Attached Figure Description

[0072] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0073] Figure 1 This is a flowchart of an automated liquid mixing method for virus titer detection based on plaque assay, according to Embodiment 1 of the present invention. Detailed Implementation

[0074] The technical solution of the present invention will now be clearly and completely described with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0075] In the description of this invention, it should be noted that the terms "center," "upper," "lower," "left," "right," "vertical," "horizontal," "inner," and "outer," etc., indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings. They are used only for the convenience of describing the invention and for simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, they should not be construed as limitations on the invention. Furthermore, the terms "first," "second," and "third" are used for descriptive purposes only and should not be construed as indicating or implying relative importance.

[0076] In the description of this invention, it should be noted that, unless otherwise explicitly specified and limited, the terms "installation," "connection," and "linking" should be interpreted broadly. For example, they can refer to a fixed connection, a detachable connection, or an integral connection; they can refer to a mechanical connection or an electrical connection; they can refer to a direct connection or an indirect connection through an intermediate medium; and they can refer to the internal connection of two components. Those skilled in the art can understand the specific meaning of the above terms in this invention based on the specific circumstances.

[0077] Example 1

[0078] This invention provides an automated liquid mixing method for virus titer detection based on plaque assay, comprising the following steps:

[0079] Step 1: The execution module obtains a clean pipette tip and controls the pipette tip to perform operation A1. Operation A1 includes aspirating a volume of host cell fluid of X1*K1*V1 through the pipette tip once, and injecting a volume of host cell fluid of V1 into each of the K1 mixing tubes, and repeating operation A1 N1 times.

[0080] Step 2: The execution module replaces the clean pipette tip and controls the pipette tip to perform operation A2. Operation A2 includes aspirating a virus test sample with a volume of X2*K2*V2 through the pipette tip, and injecting a virus test sample with a volume of V2 into each of the K2 mixing tubes that already contain host cell fluid. Operation A2 is repeated N2 times.

[0081] Step 3: The execution module replaces the clean pipette tip and controls the pipette tip to perform operation A3. Operation A3 includes drawing a volume of liquid agar of X3*V3 through the pipette tip once, and injecting a volume of liquid agar of V3 into a mixing tube already containing host cell fluid and virus test sample. Operation A3 is repeated N3 times, where N3 = K1*N1 = K2*N2, resulting in N3 portions of the mixture. Each portion of the mixture consists of a volume of host cell fluid of V1, a volume of virus test sample of V2, and a volume of liquid agar of V3. In this embodiment, X1 is 1, X2 is 1, and X3 is 1, and K1, K2, N1, N2, and N3 are all integers greater than or equal to 1.

[0082] In step one above, the execution module uses a pipette tip to draw a fixed amount of host cell fluid, and then sequentially injects the predetermined amount of host cell fluid into one or more mixing tubes, thereby completing the pipetting and dispensing operation of the host cell fluid. V1 is the predetermined amount of host cell fluid required for each mixing tube, and K1 and N1 are natural numbers. Taking 10 mixing tubes as an example, if K1 is 1, the execution module draws a predetermined amount V1 of host cell fluid each time and injects it into one mixing tube, and then repeats operation A1 10 times to complete the pipetting and dispensing of the 10 mixing tubes; if K1 is 2, the execution module draws twice the predetermined amount V1 of host cell fluid each time and injects it into two mixing tubes respectively, and then repeats operation A1 5 times to complete the pipetting and dispensing of the host cell fluid in the 10 mixing tubes; and so on, ensuring that K1*N1=10.

[0083] In step two above, the execution module uses a pipette tip to draw a fixed amount of virus test sample, and then sequentially injects the predetermined amount of virus test sample into one or more mixing tubes already containing host cell fluid, thereby completing the pipetting and dispensing operation of the virus test sample. V2 is the predetermined amount of virus test sample required for each mixing tube, and K2 and N2 are natural numbers. Taking 10 mixing tubes and one type of virus test sample as an example, if K2 is 1, the execution module draws a predetermined amount V2 of virus test sample each time and injects it into a mixing tube already containing host cell fluid, and then repeats operation A2 10 times to complete the pipetting and dispensing of 10 mixing tubes; if K2 is 2, the execution module draws twice the predetermined amount V2 of virus test sample each time and injects it into two mixing tubes already containing host cell fluid, and then repeats operation A2 5 times to complete the pipetting and dispensing of virus test sample for 10 mixing tubes; and so on, ensuring that K2*N2=10.

[0084] If there are multiple types of virus test samples, then perform step two for each type of virus test sample.

[0085] In step three above, the execution module uses a pipette tip to draw a fixed amount of liquid agar, and then, carrying the liquid agar, sequentially injects a predetermined amount of liquid agar into multiple mixing tubes already containing host cell fluid and virus test samples. This completes the liquid agar pipetting and separation operation, and also achieves the mixing of liquid agar with virus test samples and host cell fluid to obtain the final mixture. V3 is the predetermined amount of liquid agar required for each mixing tube. The execution module draws only V3 volume of liquid agar each time, quickly transfers it to the mixing tube, and injects it to avoid the liquid agar solidifying due to prolonged time in the pipette tip. N3 is a natural number, N3 = K1 * N1 = K2 * N2, resulting in N3 portions of mixture. Taking 10 mixing tubes as an example, the execution module draws a predetermined amount V3 of liquid agar each time and injects it into a mixing tube already containing host cell fluid and virus test samples. This process is repeated 10 times to complete the pipetting, separation, and mixing of 10 mixing tubes.

[0086] It should be noted that the execution module controls the pipette tip to draw a fixed amount of liquid each time. The fixed amount is not necessarily equal to the predetermined amount of liquid, and may be a multiple of the predetermined amount of liquid.

[0087] When pipetting and separating host cell fluid or virus test samples, the same pipette tip can transfer a certain multiple of the predetermined amount of liquid at once, and then inject the predetermined amount in multiple times. By using the precise operation of the execution module, the amount of liquid injected into the mixing tube each time is as close as possible to or equal to the preset amount, eliminating the error of manual operation. At the same time, it can also reduce the error of residual liquid inside the pipette tip due to liquid tension when injecting liquid, thus improving the accuracy of pipetting and separating.

[0088] The execution module automatically performs pipetting, dispensing, and mixing operations, significantly improving mixing efficiency. This is especially beneficial for batch testing tasks requiring liquid preparation and mixing, greatly reducing manual workload and saving time and costs. Since the execution module requires changing pipette tips each time a different liquid is drawn to ensure purity and prevent cross-contamination, and each tip removal and reloading takes time, the module can reduce tip changes by using the same tip to transfer a predetermined amount of the same liquid at once, or repeatedly transferring a predetermined amount of the same liquid using the same tip, or repeatedly transferring a predetermined amount of the same liquid using the same tip until the previous liquid is completely transferred, then changing the tip before transferring the next liquid. This method, where the number of tip changes depends on the type of liquid being mixed, is suitable for batch virus challenge testing of virus-free membranes / filters, where fewer types of liquids need to be mixed. The simplified mixing process eliminates the need for frequent tip changes, ensures high operational continuity, significantly improves efficiency, provides accurate test results, and saves on pipette tips.

[0089] The execution module strictly follows the steps sequentially, first injecting host cell fluid, then the virus test sample, and finally liquid agar into the same mixing tube. This sequence ensures the host cell fluid is evenly dispersed within the mixing tube before the virus test sample is added. Because the virus test sample is added last, the host cell fluid is significantly excessive compared to the gradually injected virus. Furthermore, during the injection of the virus test sample, the release force of the pipette tip creates a vortex in the host cell fluid, allowing the virus test sample to rapidly and evenly diffuse throughout the liquid environment and mix thoroughly with the host cell fluid. This ensures that the virus test sample randomly collides and adsorbs with the host cells, minimizing the possibility of only one virus invading a single host cell, thus improving the accuracy of virus titer detection results based on plaque assays.

[0090] The benefits of injecting liquid agar last include: to maintain the activity of the host cell fluid and virus test sample, the temperature of the mixing tube is lower than the storage temperature of the liquid agar. If the liquid agar is added too early, it may cause local solidification. Therefore, injecting the liquid agar last allows it to disperse quickly and evenly in the mixture, shortening the residence time of the liquid agar in the relatively cooler mixing tube. This ensures that the agar remains in a liquid state, making it easier to pour the mixture into plates and invert them for culture after the liquid agar has solidified. The high transparency of the agar also facilitates subsequent plaque counting.

[0091] In this embodiment, the temperature of the mixture is 38-42℃, and the temperature of the liquid agar is 45-60℃. Within this temperature range, both the host cells and viruses in the mixture maintain good activity to meet the requirements of the plaque assay. Even if the temperature decreases after the liquid agar is injected into the mixing tube, it will not fall below the solidification temperature of the agar, allowing the agar to remain liquid for a certain period of time. This ensures that the agar disperses quickly and evenly in the mixture, and when the mixture is poured into a plate, the agar will not locally solidify or form a skin. This prevents interference with the even spreading of the mixture, allowing it to spread evenly in the plate. The liquid agar solidifies quickly, and the plate is then inverted for incubation. Due to the high transparency of the agar, subsequent plaque counting is convenient.

[0092] The execution module in this embodiment can be a conventional, commercially available robotic arm. Under the control of a conventional control system, the robotic arm automatically acquires a clean pipette tip, automatically draws up the corresponding sample through the tip, and automatically injects the sample from the tip into the appropriate mixing tube. The loading and unloading method between the robotic arm's execution end and the pipette tip is existing technology. The execution end of the robotic arm can be equipped with a pipetting air pump, which is connected to the pipette tip. The air pump controls the liquid intake and discharge from the pipette tip through air pressure changes, offering advantages such as high precision, speed, and ease of operation. The execution module can internally set the suction volume of the pipette tip, i.e., the set volume, including a predetermined volume, a multiple of the predetermined volume, an excess exceeding the predetermined volume or a multiple of the predetermined volume, a certain extrusion volume, and other settable amounts. The pipette tip is also a conventional, commercially available tip, and the mixing tube can be a conventional, commercially available test tube, centrifuge tube, etc.

[0093] Specifically, such as Figure 1 As shown, in step one, the operation of the pipette tip to draw host cell fluid with a volume of K1*V1 includes operations S1-1 and S1-2 executed sequentially. In operation S1-1, the execution module controls the pipette tip to draw a volume greater than K1*V1. In operation S1-2, the execution module squeezes out the draw volume exceeding K1*V1, so that the volume of host cell fluid retained in the pipette tip is equal to K1*V1.

[0094] This is because, after aspiration, a small amount of air may occasionally remain at the bottom of the pipette tip, leading to inaccurate volume of the aspirated host cell fluid. The actual volume of the host cell fluid is smaller than the aspirated volume. Therefore, under the control of the execution module, the pipette tip first obtains a precise aspiration volume greater than K1*V1. This aspiration volume includes a small amount of air at the bottom of the pipette tip and excess host cell fluid. Then, under the control of the control module, an aspiration volume exceeding K1*V1 is squeezed out. This squeezed-out volume includes excess host cell fluid and air, ensuring that the volume of host cell fluid retained in the pipette tip equals K1*V1, and that there is no air at the bottom of the pipette tip. The amount of host cell fluid retained in the pipette tip is consistent with the set amount set by the execution module, improving the accuracy of the volume of host cell fluid injected into the mixing tube. During this process, at least part of the bottom of the pipette tip remains in the original container containing the host cell fluid to reduce aerosol contamination in the working environment. For the host cell fluid, this operation ensures that the amount of host cells in each mixing tube is basically consistent, which is the basis for the repeatability and accuracy of virus titer detection based on plaque assay.

[0095] More preferably, in operation A1, each time the pipette tip draws host cell fluid from the container, the tip moves axially downwards until its end is a certain height below the liquid surface after the previous draw. This operation ensures that the bottom opening of the pipette tip is always below the liquid surface of the host cell fluid, reducing the probability of air being drawn in when the pipette tip draws in host cell fluid. This ensures that the amount of host cell fluid drawn by the pipette tip matches the set amount of the execution module, thereby improving the accuracy of the aspiration.

[0096] After performing operation S1-2, there may be droplets protruding from the tip opening at the bottom of the pipette tip. Based on the principle of equal volume replacement, these droplets are also part of the set volume. If these droplets fall off during the movement of the pipette tip by the execution module, it may not only cause contamination but also result in the volume of host cell fluid injected into the mixing tube being lower than the predetermined amount. Therefore, further, in step one, after the execution module performs operation S1-2, it also performs operation P1. Operation P1 is to draw a fixed amount of air into the bottom of the pipette tip, thereby drawing any possible droplets into the pipette tip. After operations S1-1 and S1-2, the volume of host cell fluid inside the pipette tip is equal to K1*V1. At this point, drawing in the amount of air set by the execution module will not affect the accuracy of the volume of host cell fluid injected into the mixing tube.

[0097] When the pipette tip is immersed in the host cell fluid, due to reasons such as adhesion to the outer wall and surface tension adsorption, the host cell fluid may be adsorbed on the outer wall of the pipette tip to form droplets. Since the execution module moves the pipette tip at a relatively fast speed, the droplets may fall randomly during the movement, contaminating the working environment or causing cross-contamination. Therefore, in step one, after the execution module performs operations S1-2, the execution module also performs operation Q1. Operation Q1 refers to the execution module controlling the pipette tip to move axially upward, with a dwell time of T1, and then moving axially again.

[0098] Once the host cell fluid in the pipette tip reaches the set volume of the execution module, controlling the pipette tip to move axially and remain stationary extends the time the pipette tip stays within the projected range of the original container's opening. This provides time for any dripping fluid adhering to the outer wall of the pipette tip to collect downwards. Then, the pipette tip moves axially, and the fluid adhering to the outer wall separates from the pipette tip due to inertia, thus removing the fluid and allowing it to fall back into the original container. This prevents random dripping during the movement of the pipette tip by the execution module, avoiding potential contamination and safety risks. It ensures the cleanliness of the entire operating environment, reduces the possibility of cross-contamination, and thereby improves detection accuracy.

[0099] More specifically, in operation Q1, the residence time T1 is 0.2-2s, which balances operational efficiency and pipetting accuracy. If the residence time is less than 0.2s, it is too short, and the liquid on the outer wall of the pipette tip has not yet moved to the bottom and fallen off. If the residence time is greater than 1s, it is too long, which prolongs the overall operation time and reduces pipetting efficiency.

[0100] To improve the efficiency of liquid dripping from the outer wall of the pipette tip, in operation Q1, the axial movement of the pipette tip is either axially downward first and then axially upward, or axially upward first and then axially downward. This is equivalent to shaking the pipette tip within the projection range of the opening of the original container, which can quickly and completely drip the liquid adhering to the outer wall of the pipette tip back into the original container, avoiding cross-contamination and improving operational efficiency.

[0101] In this embodiment, operation P1 and operation Q1 can be executed selectively, or both can be executed. Preferably, as follows: Figure 1 As shown, in step one of the automated mixing method of this embodiment, after the execution module obtains a clean pipette tip and controls the pipette tip to perform operations S1-1 and S1-2, operation P1 is then performed, followed by operation Q1. Then, host cell fluid of volume V1 is injected into each of the K1 mixing tubes, and the above operations are repeated N1 times. The execution order of operations P1 and Q1 can also be reversed.

[0102] After injecting host cell fluid from V1 into all the mixing tubes, the execution module begins to execute step two, performing pipetting and separation operations on at least one viral test sample.

[0103] In step two of this embodiment, the execution module replaces the clean pipette tip and controls the pipette tip to perform operation A2. Operation A2 includes operations S2-1 and S2-2 executed sequentially. In operation S2-1, the suction volume of the pipette tip is greater than K2*V2. In operation S2-2, the suction volume exceeding K2*V2 is squeezed out, so that the volume of the virus test sample retained in the pipette tip is equal to K2*V2.

[0104] For the same reasons as in step one above, this operation ensures that there is no air at the bottom of the pipette tip, and the volume of the virus test sample retained in the pipette tip remains consistent with the set amount in the execution module, improving the accuracy of the volume of virus test sample injected into the mixing tube. During this process, at least part of the bottom of the pipette tip remains in the original container containing the virus test sample, reducing aerosol contamination in the working environment. For the virus test sample, this operation ensures that the virus content in each mixing tube is essentially consistent, which is the basis for the repeatability and accuracy of virus titer detection based on the plaque method.

[0105] Similarly, in operation A2, each time the pipette tip draws a virus test sample from the container, the tip moves axially downwards until its end is a certain height below the liquid level after the previous virus test sample was drawn. This operation ensures that the bottom opening of the pipette tip is always below the liquid level of the virus test sample, reducing the probability of air being drawn in when the pipette tip draws in the virus test sample. This ensures that the amount of virus test sample drawn by the pipette tip meets the set amount of the execution module, thereby improving the accuracy of the drawing.

[0106] After performing operation S2-2, there may be droplets protruding from the tip opening at the bottom of the pipette tip. Based on the principle of equal volume replacement, these droplets are also part of the set volume. If these droplets fall off during the movement of the pipette tip by the execution module, it may not only cause contamination but also result in the volume of the virus test sample injected into the mixing tube being lower than the predetermined amount. Therefore, after performing operation S2-2, the execution module also performs operation P2, which involves drawing a fixed amount of air into the bottom of the pipette tip to draw any potential droplets into the pipette tip. After operations S2-1 and S2-2, the volume of host cell fluid inside the pipette tip is equal to K2*V2. At this point, drawing in the air volume set by the execution module will not affect the accuracy of the volume of the virus test sample injected into the mixing tube.

[0107] When the pipette tip is immersed in the virus test sample, due to adhesion to the outer wall and surface tension adsorption, virus test sample may adhere to the outer wall of the pipette tip, forming droplets. Since the execution module moves the pipette tip at a relatively high speed, these droplets may randomly fall during the movement, contaminating the working environment or causing cross-contamination. Especially when multiple virus test samples are generally arranged in a matrix, with containers containing other virus test samples surrounding one sample, droplets are more likely to fall off when the pipette tip first begins to move. Therefore, it is necessary to ensure that the droplets fall back into the original container of the current virus test sample, i.e., return to the original container. Based on this, after executing operation S2-2, the execution module also executes operation Q2. Operation Q2 refers to the execution module controlling the pipette tip to move axially upwards for a dwell time of T2, and then moving axially again. This operation can extend the time the pipette tip stays within the projection range of the opening of the original container, thus providing time for the drips attached to the outer wall of the pipette tip to collect downwards. Then, the pipette tip moves axially, and the liquid attached to the outer wall of the pipette tip separates from the pipette tip due to inertia, thereby removing the liquid attached to the outer wall of the pipette tip and allowing it to fall back into the original container. This avoids random dripping when the execution module moves the pipette tip, which could cause potential contamination and safety risks, ensuring the cleanliness of the entire operating environment, reducing the possibility of cross-contamination, and thus improving detection accuracy.

[0108] As a preferred option, in operation Q2, the residence time T2 is 0.2-2s, which balances operational efficiency and pipetting accuracy.

[0109] More specifically, in operation Q2, the axial movement of the pipette tip is either axially downward first, then axially upward, or axially upward first, then axially downward. This is equivalent to shaking the pipette tip within the projection range of the original container's opening, which quickly and completely removes liquid adhering to the outer wall of the pipette tip back into the original container, preventing contamination of other virus test samples. Preferably, during the above operation, at least part of the bottom of the pipette tip remains inside the container, preventing drips from splashing outwards or causing aerosol contamination.

[0110] In this embodiment, operation P2 and operation Q2 can be executed selectively, or both can be executed. Preferably, as follows: Figure 1 As shown, in step two of the automated mixing method of this embodiment, after the execution module obtains a clean pipette tip and controls the pipette tip to perform operations S2-1 and S2-2, operation P2 is then performed, followed by operation Q2. Then, a volume of V2 of virus test sample is injected into each of the K2 mixing tubes already containing host cell fluid, and operation A2 is repeated N2 times. If there are multiple types of virus test samples, step two is performed the corresponding number of times until all mixing tubes have been injected with a predetermined amount of virus test sample. The execution order of operations P2 and Q2 can also be reversed.

[0111] Then the execution module begins to execute step three, which involves pipetting and separating the liquid agar.

[0112] In step three, the execution module replaces the clean pipette tip and controls the pipette tip to perform operation A3. Operation A3 includes operations S3-1 and S3-2 executed sequentially. In operation S3-1, the pipette tip's suction volume is greater than V3. In operation S3-2, the suction volume exceeding V3 is squeezed out, so that the volume of liquid agar retained in the pipette tip is equal to V3.

[0113] For the same reasons as steps one and two, after this operation, there is no air at the bottom of the pipette tip, and the volume of liquid agar retained in the pipette tip is consistent with the set amount set by the execution module, which improves the accuracy of the volume of liquid agar injected into the mixing tube. As a result, the ratio of host cell fluid, virus test sample and liquid agar in the resulting mixture is very accurate and meets the requirements, ultimately improving the detection accuracy.

[0114] In operation A3, each time the pipette tip draws liquid agar from the container, the tip moves axially downwards until its end is below the liquid surface after the previous draw. This operation ensures that the bottom opening of the pipette tip is always below the liquid agar surface, reducing the probability of air being drawn in while the tip is drawing in liquid agar. This ensures that the amount of liquid agar drawn by the tip matches the set amount of the execution module, thereby improving the accuracy of the agar draw.

[0115] In operation S3-2, the viscosity of liquid agar is relatively high, and there may be droplets protruding from the bottom of the pipette tip. Based on the principle of equal volume replacement, these droplets are also part of the set volume. Liquid agar exposed in the bottom opening of the pipette tip is prone to cooling and forming a skin during movement, which can clog the pipette tip. Therefore, in step three, after the execution module performs operation S3-2, it also performs operation P3. Operation P3 is to draw a fixed amount of air into the bottom of the pipette tip so that all liquid agar is located inside the pipette tip. After operations S3-1 and S3-2, the volume of liquid agar inside the pipette tip is equal to V3. At this time, drawing in the amount of air set by the execution module will not affect the accuracy of the volume of liquid agar injected into the mixing tube, and can also ensure that the liquid agar is smoothly injected into the mixing tube and mixed evenly with the host cell fluid and virus test sample to form a mixture.

[0116] Due to the high viscosity of liquid agar, when the pipette tip enters the liquid agar for aspiration, liquid agar may adhere to the outer wall of the tip, forming droplets. These droplets may condense or randomly fall during the transfer process, contaminating the working environment or clogging the pipette tip. Therefore, in step three, after the execution module performs operation S3-2, it also performs operation Q3. Operation Q3 refers to the execution module controlling the pipette tip to move axially upwards for a residence time of T3, and then moving axially again, so that the droplets on the outer wall of the pipette tip can fall back into the agar bottle. Preferably, the residence time T3 in operation Q3 is 0.2-2s. More preferably, considering the cooling rate of liquid agar, the residence time T3 in operation Q3 is 0.2-1.5s to shorten the liquid agar transfer time and reduce the probability of condensation and skin formation.

[0117] To improve the efficiency of liquid agar dripping from the outer wall of the pipette tip, in operation Q3, the axial movement of the pipette tip is either axially downward first, then axially upward, or axially upward first, then axially downward. This is equivalent to shaking the pipette tip within the projection range of the original container's opening, which can quickly and completely drip the liquid adhering to the outer wall of the pipette tip back into the original container, reducing the total time the pipette tip is placed within the projection range of the original container's opening and improving operational efficiency.

[0118] In this embodiment, operation P3 and operation Q3 can be executed selectively, or both can be executed. Preferably, as follows: Figure 1As shown, in step one of the automated mixing method of this embodiment, after the execution module obtains a clean pipette tip and controls the pipette tip to perform operations S3-1 and S3-2, operation P3 is then performed, followed by operation Q3. Then, a volume of V3 of liquid agar is injected into a mixing tube already containing host cell fluid and virus test sample, and operation A3 is repeated N3 times, where N3 = K1 * N1 = K2 * N2, resulting in N3 portions of the mixture. Each portion of the mixture consists of a volume of V1 of host cell fluid, a volume of V2 of virus test sample, and a volume of V3 of liquid agar. The execution order of operations P3 and Q3 can also be reversed.

[0119] Better, such as Figure 1 As shown, in step three, after injecting liquid agar into all mixing tubes to obtain N3 portions of the mixture, the execution module also performs operation A3-1. Operation A3-1 refers to aspirating at least a portion of the mixture from each mixing tube and re-injecting the aspirated mixture back into the original mixing tube. Specifically, operation A3-1 involves lowering the pipette tip until its end is below the initial liquid level of the mixture, aspirating at least a portion of the mixture, then raising the end of the pipette tip to a position not lower than the initial liquid level of the mixture, and then squeezing the mixture in the pipette tip back into the original mixing tube. This operation allows the pipette tip to aspirate the hotter and denser mixture closer to the bottom of the mixing tube, and then squeezing it back into the mixing tube to mix with the upper mixture again. This helps improve the overall temperature and concentration uniformity of the mixture, allowing the virus test sample to randomly collide and adsorb with the host cell, ensuring that only one virus invades a host cell as much as possible, and improving the accuracy of virus titer detection results based on plaque assay. Of course, as mentioned above, you can also directly proceed to operation A3-1 after obtaining one sample of the mixture, performing agar transfer and operation A3-1 N3 times in total.

[0120] It is important to note that a new pipette tip must be used each time operation A3-1 is performed to avoid mixing of liquids in different mixing tubes.

[0121] In other embodiments, operation A3-1 may involve drawing out at least half of the mixture from each mixing tube and re-injecting it into the original mixing tube; this operation can also greatly improve the overall temperature uniformity and concentration uniformity of the mixture.

[0122] The automated liquid mixing method provided in this invention can be used not only for titer detection of PR772 bacteriophage and PP7 bacteriophage mentioned in the background art, but also for titer determination of PhiX174 bacteriophage, MS2 bacteriophage, and all other viruses whose titer can be detected by plaque assay.

[0123] Example 2

[0124] The only difference between Example 2 and Example 1 is that X1 is 2, X2 is 2, and X3 is 2. Accordingly,

[0125] In step one, after operation A1 is completed, there is a relatively large amount of host cell fluid remaining in the pipette tip. Therefore, before performing the next operation A1, squeeze the remaining host cell fluid in the pipette tip back into the original container, and then repeat operation A1.

[0126] In step two, after operation A2 is completed, there is a relatively large amount of virus test sample remaining in the pipette tip. Therefore, before performing the next operation A2, squeeze the remaining virus test sample in the pipette tip back into the original container, and then repeat operation A2.

[0127] In step three, after operation A3 is completed, there is a relatively large amount of liquid agar remaining in the pipette tip. Therefore, before performing the next operation A3, squeeze the remaining liquid agar in the pipette tip back into the original container, and then repeat operation A3.

[0128] Example 3

[0129] The only difference between Example 3 and Example 1 is that X1 is 1.1, X2 is 1.1, and X3 is 1.1. Accordingly,

[0130] In step one, after performing operation A1 once, there is a small amount of host cell fluid remaining in the pipette tip. Therefore, the remaining host cell fluid can be left in the pipette tip, and operation A1 can be repeated.

[0131] In step two, if there is a small amount of virus test sample remaining in the pipette tip after completing operation A2 once, the remaining virus test sample can be left in the pipette tip and operation A2 can be repeated.

[0132] In step three, after completing operation A3 once, there is a small amount of liquid agar remaining in the pipette tip. Therefore, the remaining liquid agar can be left in the pipette tip, and operation A3 can be repeated.

[0133] Example 4

[0134] The only difference between Example 4 and Example 1 is that X1 is 1.1, X2 is 1.1, and X3 is 1.1. Accordingly,

[0135] In step one, after performing operation A1 once, there is less host cell fluid remaining in the pipette tip. However, before performing the next operation A1, the remaining host cell fluid in the pipette tip is squeezed back into the original container, and then operation A1 is repeated.

[0136] In step two, after performing operation A2 once, there is less virus test sample remaining in the pipette tip. However, before performing the next operation A2, the remaining virus test sample in the pipette tip is squeezed back into the original container, and then operation A2 is repeated.

[0137] In step three, after performing operation A3 once, there is less liquid agar remaining in the pipette tip. However, before performing the next operation A3, the remaining liquid agar in the pipette tip is squeezed back into the original container, and then operation A3 is repeated.

[0138] The above embodiments are merely preferred embodiments of the present invention and should not be construed as limiting the scope of protection of the present invention. Any non-substantial changes and substitutions made by those skilled in the art based on the present invention shall fall within the scope of protection claimed by the present invention.

Claims

1. An automated liquid mixing method for virus titer detection based on plaque assay, characterized in that, Includes the following steps, Step 1: The execution module obtains a clean pipette tip and controls the pipette tip to perform operation A1. Operation A1 includes aspirating a volume of host cell fluid of X1*K1*V1 through the pipette tip once, and injecting a volume of V1 of the host cell fluid into each of the K1 mixing tubes, and repeating operation A1 N1 times. Step 2: The execution module replaces the clean pipette tip and controls the pipette tip to perform operation A2. Operation A2 includes aspirating a virus test sample with a volume of X2*K2*V2 through the pipette tip, and injecting a virus test sample with a volume of V2 into K2 mixing tubes that already contain the host cell fluid. The operation A2 is repeated N2 times. Step 3: The execution module replaces the clean pipette tip and controls the pipette tip to perform operation A3. Operation A3 includes drawing a volume of liquid agar of X3*V3 through the pipette tip once, and injecting a volume of liquid agar of V3 into a mixing tube that already contains the host cell fluid and the virus test sample. Operation A3 is repeated N3 times, where N3 = K1*N1 = K2*N2, to obtain N3 portions of the mixture. Each portion of the mixture consists of a volume of host cell fluid of V1, a volume of virus test sample of V2, and a volume of liquid agar of V3. And X1 is 1 to 2, X2 is 1 to 2 and X3 is 1 to 2, and K1, K2, N1, N2 and N3 are all integers greater than or equal to 1.

2. The automatic mixing method as described in claim 1, characterized in that, At least one of the following conditions must be met: (1) X1 is not greater than 1.1; (2) X2 is not greater than 1.1; (3) X3 is not greater than 1.

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3. The automatic mixing method as described in claim 1, characterized in that, At least one of the following operations, (1) In step one, the operation of the pipette tip to draw host cell fluid with a volume of X1*K1*V1 includes operation S1-1 and operation S1-2 performed sequentially. In operation S1-1, the volume drawn by the pipette tip is greater than X1*K1*V1. In operation S1-2, the volume drawn by the pipette tip that exceeds X1*K1*V1 is squeezed out, so that the volume of host cell fluid retained in the pipette tip is equal to X1*K1*V1. (2) In step two, the operation of the pipette tip to draw a virus test sample with a volume of X2*K2*V2 includes operations S2-1 and S2-2 executed sequentially. In operation S2-1, the volume drawn by the pipette tip is greater than X2*K2*V2. In operation S2-2, the volume of the drawn sample exceeding X2*K2*V2 is squeezed out, so that the volume of the virus test sample retained in the pipette tip is equal to X2*K2*V2. (3) In step three, the operation of the pipette tip to draw a virus test sample with a volume of X3*V3 includes operations S3-1 and S3-2 executed sequentially. In operation S3-1, the volume of the pipette tip drawn is greater than X3*V3. In operation S3-2, the volume of the pipette tip drawn is squeezed out beyond X3*V3, so that the volume of liquid agar retained in the pipette tip is equal to X3*V3.

4. The automatic mixing method as described in claim 3, characterized in that, At least one of the following operations, (1) In step one, after the execution module performs operation S1-2, the execution module also performs operation P1, which is to draw a certain amount of air into the bottom of the suction head; (2) In step two, after the execution module performs operation S2-2, the execution module also performs operation P2, which is to draw a certain amount of air into the bottom of the suction head; (3) In step three, after the execution module performs operation S3-2, the execution module also performs operation P3, which is to draw a certain amount of air into the bottom of the suction head.

5. The automatic mixing method as described in claim 3, characterized in that, At least one of the following operations, (1) In step one, after the execution module performs operation S1-2, the execution module also performs operation Q1. Operation Q1 refers to the execution module controlling the suction head to move axially upward, the dwell time being T1, and then moving axially. (2) In step two, after the execution module performs operation S2-2, the execution module also performs operation Q2. Operation Q2 refers to the execution module controlling the suction head to move axially upward, the dwell time being T2, and then moving axially. (3) In step three, after the execution module performs operation S3-2, the execution module also performs operation Q3. Operation Q3 refers to the execution module controlling the suction head to move axially upward, the dwell time being T3, and then moving axially.

6. The automatic mixing method as described in claim 5, characterized in that, In operation Q1, the dwell time T1 is 0.2-2s; and / or, in operation Q2, the dwell time T2 is 0.2-2s; and / or, in operation Q3, the dwell time T3 is 0.2-2s.

7. The automatic mixing method as described in claim 5, characterized in that, In operation Q1, the axial movement of the suction head is either axially downward first and then axially upward, or axially upward first and then axially downward. And / or, in operation Q2, the axial movement process of the suction head is either first moving axially downward and then moving axially upward, or first moving axially upward and then moving axially downward. And / or, in operation Q3, the axial movement of the suction head is either axially downward first, then axially upward, or axially upward first, then axially downward.

8. The automatic mixing method as described in claim 1, characterized in that, At least one of the following operations, (1) In operation A1, each time the pipette tip draws host cell fluid, the pipette tip moves axially downward until its end is a certain height below the liquid level after the previous draw of host cell fluid by the pipette tip. (2) In operation A2, each time the pipette tip draws a virus test sample, the pipette tip moves axially downward until its end is a certain height below the liquid level after the previous virus test sample was drawn by the pipette tip. (3) In operation A3, each time the pipette tip draws liquid agar, the pipette tip moves axially downward until its end is a certain height below the liquid surface after the previous draw of liquid agar.

9. The automated mixing method according to any one of claims 1-8, characterized in that, In step three, the execution module also performs operation A3-1, which refers to drawing out at least a portion of the mixture from each mixing tube and re-injecting the drawn-out mixture into the original mixing tube.

10. The automatic mixing method as described in claim 9, characterized in that, The specific operation of operation A3-1 is as follows: the pipette tip is lowered to a position where its end is below the initial liquid level of the mixture, at least a portion of the mixture is drawn up, and then the end of the pipette tip is raised to a position not lower than the initial liquid level of the mixture, and the mixture in the pipette tip is squeezed back into the original mixing tube; and / or, in operation A3-1, at least half of the mixture in each mixing tube is drawn out and re-injected into the original mixing tube.

11. The automatic mixing method as described in claim 1, characterized in that, The temperature of the mixture is 38-42℃, and the temperature of the liquid agar is 45-60℃.

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