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CN-121992148-A - Automatic liquid mixing method for detecting virus titer based on plaque method

CN121992148ACN 121992148 ACN121992148 ACN 121992148ACN-121992148-A

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 move liquid, divide liquid and mix, greatly improves the liquid mixing efficiency, particularly greatly reduces the manual workload and saves time and cost for detection tasks of batch liquid preparation and liquid mixing, and utilizes the execution module to strictly acquire liquid according to a preset sequence and inject the liquid into a mixing tube, so that host cell liquid is injected into the same mixing tube first, then a virus test sample is injected, then liquid agar is injected, the steps in the whole liquid mixing flow are linked smoothly and high in efficiency, only one virus invades one host cell as much as possible is ensured, and the accuracy of a virus titer detection result based on the plaque method is improved.

Inventors

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Assignees

  • 杭州纽创生物检测有限公司

Dates

Publication Date
20260508
Application Date
20251231

Claims (11)

  1. 1. An automatic liquid mixing method for detecting virus titer based on plaque method is characterized by comprising the following steps, Step one, an execution module acquires a clean suction head and controls the suction head to execute an operation A1, wherein the operation A1 comprises the steps of sucking host cell liquid with the volume of X 1 *K 1 *V 1 through the suction head at one time, injecting the host cell liquid with the volume of V 1 into K 1 mixing pipes respectively, and repeating the operation A1 for N 1 times; Step two, the execution module replaces a clean suction head and controls the suction head to execute operation A2, wherein the operation A2 comprises the steps of sucking a virus test sample with the volume of X 2 *K 2 *V 2 through the suction head once, injecting a virus test sample with the volume of V 2 into K 2 mixing pipes which already contain host cell liquid, and repeating the operation A2 for N 2 times; Step three, a clean suction head is replaced by an execution module, and the suction head is controlled to execute an operation A3, wherein the operation A3 comprises the steps of sucking liquid agar with the volume of X 3 *V 3 through the suction head at one time, injecting liquid agar with the volume of V 3 into a mixing tube which already contains the host cell liquid and the virus test sample, and repeating the operation A3 for N 3 times, wherein N 3 ＝K 1 *N 1 ＝K 2 *N 2 is carried out to obtain N 3 parts of mixed liquid, and each part of mixed liquid consists of the host cell liquid with the volume of V 1 , the virus test sample with the volume of V 2 and the liquid agar with the volume of V 3 ; And X 1 is 1-2, X 2 is 1-2 and X 3 is 1-2, and K1, K2, N1, N2, N3 are integers of 1 or more.
  2. 2. The automated mixing method of claim 1, wherein at least one of the following conditions is satisfied, (1) X 1 is not more than 1.1, (2) X 2 is not more than 1.1, and (3) X 3 is not more than 1.1.
  3. 3. The automated mixing method of claim 1, wherein at least one of the following is performed, (1) In the first step, the operation of sucking up the host cell liquid with the volume X 1 *K 1 *V 1 by the suction head comprises an operation S1-1 and an operation S1-2 which are sequentially executed, wherein the sucking up volume of the suction head in the operation S1-1 is larger than X 1 *K 1 *V 1 , and the sucking up volume exceeding X 1 *K 1 *V 1 in the operation S1-2 is extruded, so that the volume of the host cell liquid remained in the suction head is equal to X 1 *K 1 *V 1 ; (2) In the second step, the operation of sucking the virus test sample with the volume X 2 *K 2 *V 2 by the suction head comprises an operation S2-1 and an operation S2-2 which are sequentially executed, wherein the sucking volume of the suction head in the operation S2-1 is larger than X 2 *K 2 *V 2 , and the sucking volume exceeding X 2 *K 2 *V 2 is extruded in the operation S2-2, so that the volume of the virus test sample remained in the suction head is equal to X 2 *K 2 *V 2 ; (3) In the third step, the operation of sucking the virus test sample with the volume X 3 *V 3 by the suction head comprises an operation S3-1 and an operation S3-2 which are sequentially executed, the sucking volume of the suction head in the operation S3-1 is larger than X 3 *V 3 , and the sucking volume exceeding X 3 *V 3 in the operation S3-2 is extruded, so that the volume of the liquid agar remained in the suction head is equal to X 3 *V 3 .
  4. 4. The automated mixing method of claim 3, wherein at least one of the following is performed, (1) In the first step, after the execution module executes the operation S1-2, the execution module also executes the operation P1, wherein the operation P1 is to suck quantitative air into the bottom of the suction head; (2) In the second step, after the execution module executes the operation S2-2, the execution module also executes the operation P2, wherein the operation P2 is to suck quantitative air into the bottom of the suction head; (3) In the third step, after the execution module executes the operation S3-2, the execution module also executes the operation P3, where the operation P3 is to suck quantitative air into the bottom of the suction head.
  5. 5. The automated mixing method of claim 3, wherein at least one of the following is performed, (1) In the first step, after the execution module executes the operation S1-2, the execution module further executes the operation Q1, where the operation Q1 refers to that the execution module controls the axial movement of the suction head, the residence time is T1, and then the suction head moves axially; (2) In the second step, after the execution module executes the operation S2-2, the execution module also executes the operation Q2, wherein the operation Q2 refers to that the execution module controls the axial movement of the suction head, the retention time is T2, and then the suction head moves axially; (3) In the third step, after the execution module executes the operation S3-2, the execution module further executes the operation Q3, where the operation Q3 refers to that the execution module controls the axial movement of the suction head, the residence time is T3, and then the axial movement is performed.
  6. 6. The automatic liquid mixing method according to claim 5, wherein the residence time T1 in the operation Q1 is 0.2 to 2s, and/or the residence time T2 in the operation Q2 is 0.2 to 2s, and/or the residence time T3 in the operation Q3 is 0.2 to 2s.
  7. 7. The automated mixing method of claim 5, wherein in operation Q1, the axial movement of the tip is first axially downward, then axially upward, or first axially upward, then axially downward; And/or, in the operation Q2, the axial movement process of the suction head is firstly axially downward movement, then axially upward movement or firstly axially upward movement and then axially downward movement; And/or, in the operation Q3, the axial movement process of the suction head is firstly axially downward movement, then axially upward movement or firstly axially upward movement and then axially downward movement.
  8. 8. The automated mixing method of claim 1, wherein at least one of the following is performed, (1) In the operation A1, when the suction head sucks the host cell liquid each time, the suction head moves downwards to a certain height of which the tail end is lower than the liquid level after the suction head sucks the host cell liquid for the first time; (2) In the operation A2, when the suction head sucks the virus test sample each time, the suction head moves downwards to a certain height of the tail end of the suction head lower than the liquid level after the suction head sucks the virus test sample for the first time; (3) In the operation A3, each time the suction head sucks the liquid agar, the suction head moves down to a position where the tail end of the suction head is lower than the liquid level of the liquid agar sucked by the suction head for the previous time by a certain height.
  9. 9. The automatic mixing method according to any one of claims 1 to 8, wherein in the third step, the execution module further performs an operation A3-1, the operation A3-1 refers to sucking out at least part of the mixed liquid in each mixing tube and re-injecting the sucked out mixed liquid into the original mixing tube.
  10. 10. The automatic liquid mixing method as claimed in claim 9, wherein the operation A3-1 is performed by moving the tip downward to a position where its end is lower than the initial liquid level of the mixed liquid, sucking at least part of the mixed liquid, then moving the tip upward to a position not lower than the initial liquid level of the mixed liquid, and then squeezing the mixed liquid in the tip back into the original mixing tube, and/or wherein at least half of the mixed liquid in each mixing tube is sucked out and injected again into the original mixing tube in the operation A3-1.
  11. 11. The automatic liquid mixing method according to claim 1, wherein the temperature of the mixed liquid is 38-42 ℃ and the temperature of the liquid agar is 45-60 ℃.

Description

Automatic liquid mixing method for detecting virus titer based on plaque method Technical Field The invention relates to the technical field of virus detection, in particular to an automatic liquid mixing method for detecting virus titer based on a plaque method. Background In the processing technology of biological agents, virus removal has become an indispensable step to meet the virus safety requirements. In the production process of the biological agent, virus inactivation and/or virus removal procedures are required, and a common virus removal method is to remove viruses through interception of a filter membrane, and the virus removal capacity of the filter membrane directly determines the virus safety of the biological agent. The membrane separation technology uses a filter membrane as a separation medium, when a certain driving force (such as pressure difference, concentration difference and the like) exists on two sides of the filter membrane, raw material components selectively permeate the filter membrane, and viruses are physically trapped due to the fact that the size of the raw material components is larger than the aperture of the filter membrane. Plaque method based on virus infectivity detection of virus titer is classical virus titer detection technique, the basic principle is that after host cells are cultured to a monolayer, properly diluted virus is inoculated and cultured, virus is adsorbed and invaded into host cells, and enzyme in the host cells is utilized to synthesize self-components, so that the host cells are cracked and dead, and plaque is formed, and one plaque represents one living virus particle. For example, the American society PDA (Parenteral Drug Association) refers to the biological agent's Virus Filtration test method and the manual file "Virus Filtration," TECHNICAL REPORT No.41 (recycled 2008), abbreviated as TR41, issued for Virus removal verification. The TR41 document specifies that the model virus for virus challenge of the virus removal membrane/filter is phage PR772 or PP7 (PR 772 phage is a large virus, PP7 phage is a small virus, optionally) and the material stream is immunoglobulin IVIG (or bovine serum albumin BSA). Titer detection of both PR772 phage and PP7 phage was achieved using the plaque method described previously. More specifically, the minivirus-entrapment filter-test protocol, which uses PP7 phage as a nominally challenge virus model, is well described in annex III of TR41, with human immunoglobulin (IVIG) for protein transmittance assessment. In order to calculate the viral retention, the titers of PP7 phage in the challenge and filtrate, respectively, were measured and the Log Reduction (LRV) of the viral titers was calculated using the corresponding formula. The method for detecting the titer of PP7 phage is well described in TR41, and the method uses a double-layer plate counting method to count the samples, wherein the detection system is obtained by mixing 1mL of a filtrate sample containing PP7 phage with a specific concentration, 2mL of host cells (Pseudomonas aeruginosa) and 9mL of warm agar, or by mixing about 0.1mL of a filtrate sample containing PP7 phage, 1mL of host bacteria and 4.5mL of agar solution. At present, the operations of liquid preparation and liquid mixing of host cell liquid, virus test samples and liquid agar are completely carried out manually, and the detection quantity is limited by manual work and has extremely low efficiency. Because the mixing process of the detection system is often strictly according to the relevant regulations in the document such as TR41, the mixing process is sequentially carried out according to the steps. Specifically, 1mL of a filtrate sample containing PP7 phage, 2mL of host cell liquid, and 9mL of warm agar were sequentially aspirated by a pipette, poured into a mixing tube, mixed well, poured into a plate, and then cultured and counted. In order to avoid cross-mixing of different samples, which leads to inaccurate detection results, different tips are required to be used for sucking different samples, and therefore, each time a sample is sucked, the previously used tip is discarded, a clean tip is obtained, and then the next sample is sucked. That is, if 10 mixes are to be performed, 30 clean tips are taken, 30 samples are aspirated, and 30 used tips are discarded. The whole liquid mixing process is complex in flow, easy to make mistakes, long in time consumption and excessive in number of suction heads required to be used. In addition, for the method for detecting virus titer in the ultralow virus titer system as disclosed in the prior patent CN115181816B, since the titer of PP7 phage in the filtrate is lower than the detection limit, in order to ensure the accuracy of the detection result, the method is adopted in which a filtrate with a sufficiently large detection volume must be accumulated for a certain volume of filtrate, i.e. the detected filtrate volume must be sufficiently larg