A cell indicating virus multimodal rapid detection integrated system

Abstract

The application discloses a cell-indicating virus multi-modal rapid detection integrated system, which comprises an indicating cell module configured with multiple standardized indicating cell lines adapted to different types of viruses; and a inoculation culture module comprising an integrated culture cabin, a culture well plate arranged in the integrated culture cabin and a closed micro-fluidic channel automatic sample adding system; the closed micro-fluidic channel automatic sample adding system is provided with multiple-channel independent pipelines which are physically isolated from each other and are used for respectively adding a test sample and each reagent, and is used for quantitatively inoculating the test sample into the indicating cells in the culture well plate. The cell-indicating virus multi-modal rapid detection integrated system is characterized in that: firstly, multi-dimensional three-index integrated synchronous detection is adopted, and the three indexes of CPE, hemadsorption and hemagglutination are integrated and in-situ continuously detected, so that multi-dimensional cross verification is performed from cell morphology and virus biological characteristics, and the probability of missed detection and false positive is greatly reduced.

Description

Technical Field

[0001] This invention relates to the field of virus detection, specifically to a cell-indicating multimodal rapid detection integrated system for viruses. Background Technology

[0002] Currently, in the field of biomedicine, the core method for in vitro detection of exogenous viruses in cell bank test samples is to use indicator cells inoculated with the test samples as the test object. The method combines methods such as cytopathic effect observation, blood adsorption, and blood agglutination reaction. However, each detection method is still an independent operation mode, and there is no dedicated integrated operating system. Sample processing, cell culture, and various detection and analysis steps all need to be carried out manually. After cell culture is completed, samples need to be manually transferred to different detection devices for subsequent operations. The results of different detection methods need to be manually analyzed and summarized separately. The overall detection process is fragmented, has poor connectivity, and has a long detection cycle. It is also difficult to achieve both high efficiency and high specificity in detection.

[0003] Conventional techniques suffer from the following problems: First, their operation modes are independent. Although multiple methods can be combined for detection, the various methods lack integration and still require step-by-step operation, failing to leverage the synergistic advantages of multi-dimensional detection and limiting detection efficiency. Second, the detection process is fragmented. Sample processing, culture, and detection are carried out manually in each step, requiring samples to be transferred to different devices multiple times. This is not only cumbersome and costly in terms of manpower and time, but also unsuitable for the rapid detection needs of large-scale cell banks. Sample transfer carries a high risk. During manual sample transfer after cell culture, sample contamination and loss are easily caused, directly affecting the authenticity and reliability of the test results. Furthermore, the level of integration is low. There is no dedicated integrated operating equipment for each step, making it impossible to achieve seamless integration of rapid initial screening and accurate verification, and failing to meet the high standards and high efficiency requirements of cell bank quality control for virus detection. Summary of the Invention

[0004] (a) Technical problems to be solved To address the shortcomings of existing technologies, this invention provides an integrated system for rapid multimodal detection of cell-indicating viruses, thereby resolving the aforementioned problems.

[0005] (II) Technical Solution To achieve the above objectives, at least one embodiment of this disclosure provides an integrated system for rapid multimodal detection of cell-indicator viruses, comprising: The indicator cell module is equipped with a variety of standardized indicator cell lines adapted to different types of viruses; The inoculation and culture module includes an integrated culture chamber, a culture plate disposed within the integrated culture chamber, and a closed-loop microfluidic channel automatic sample dispensing system. The closed-loop microfluidic channel automatic sample dispensing system is equipped with physically isolated multi-channel independent tubing for separately dispensing test samples and reagents, used to quantitatively inoculate the test samples into indicator cells within the culture plate. The inoculation and culture module is also configured to maintain a controllable culture environment within the integrated culture chamber and complete the culture according to a preset culture cycle. The multimodal detection module includes a cytopathic effect observation unit, a hemoadsorption test unit, and a hemagglutination reaction unit integrated into the integrated culture chamber. It is configured to sequentially and continuously perform detection on the same cultured sample after culture, without manual transfer. Specifically, the cytopathic effect observation unit images the indicator cells after culture and identifies the cytopathic effect; the hemoadsorption test unit is configured to: add a red blood cell suspension, incubate, and then image and interpret the red blood cell adsorption state; the hemagglutination reaction unit is configured to: add a hemagglutination reaction reagent, complete hemagglutination incubation, and then image and interpret the results. An integrated control and result analysis module is connected to both the inoculation and culture module and the multimodal detection module. It stores dedicated programs for each indicator cell line, used to regulate culture environment parameters, detection procedures, and reagent addition according to these programs. The module also performs comprehensive interpretation of three types of detection results: cytopathic effects, hemoadsorption, and hemagglutination. The three types of results are quantified into scores according to preset grading rules. A score reaching the preset positive score is recorded as a single positive result, while a score not reaching the preset positive score is recorded as a single negative result. When all three test results are single negative, the test sample is determined to be negative for exogenous virus. When any single test result is positive, the built-in interference database is retrieved to identify interference features to distinguish false positives, and a virus detection conclusion is output based on the identification results.

[0006] For example, in a cell-indicating virus multimodal rapid detection integrated system provided in at least one embodiment of this disclosure, the closed microfluidic channel automatic sampling system is further provided with a detachable disposable sampling tip, and is configured to automatically discard the currently used disposable sampling tip and replace it with a new disposable sterile sampling tip after each reagent is dispensed.

[0007] For example, in a cell-indicating virus multimodal rapid detection integrated system provided in at least one embodiment of this disclosure, the closed microfluidic channel automatic sample dispensing system is further configured to automatically perform a cleaning procedure before the same tube in the multi-channel independent tubing is reused. The cleaning procedure includes sequentially performing forward and reverse rinsing of the inner wall of the tube with 75% ethanol, forward and reverse rinsing three times with sterile PBS buffer, and sterile air purging to remove residual liquid.

[0008] For example, in a cell-indicating virus multimodal rapid detection integrated system provided in at least one embodiment of this disclosure, the inoculation and culture module is configured to maintain the integrated culture chamber in a slightly negative pressure sealed environment.

[0009] For example, in a multimodal rapid detection integrated system for cell-indicating viruses provided in at least one embodiment of this disclosure, the parameters of the adjustable culture environment include temperature, carbon dioxide concentration, and humidity; the preset culture period is 5 to 7 days.

[0010] For example, in a multimodal rapid detection integrated system for cell-indicator viruses provided in at least one embodiment of this disclosure, the various standardized indicator cell lines are selected from at least two of BHK-21 hamster kidney cells, Vero African green monkey kidney cells, HeLa human cervical cancer cells, MRC-5 human diploid cells, CHO-K1 Chinese hamster ovary cells, MEF mouse embryonic fibroblasts, NIH / 3T3 mouse fibroblasts, MDBK bovine kidney cells, and BT bovine nasal turbinate bone cells.

[0011] For example, in a cell-indicator-based multimodal rapid detection integrated system for viruses provided in at least one embodiment of this disclosure, the various standardized indicator cell lines are configured to screen for corresponding exogenous viruses; wherein, BHK-21 hamster kidney cells are used to screen for various arboviruses; Vero African green monkey kidney cells are used to screen for herpesviruses, poxviruses, arboviruses, primate parvoviruses, and human susceptible enveloped viruses; HeLa human cervical cancer cells are used to screen for human adenoviruses, human papillomaviruses, and human respiratory susceptible viruses; and MRC-5 human diploid cells are used to screen for human cytomegaloviruses, human adenoviruses, and respiratory syncytial virus. Human herpesvirus; CHO-K1 Chinese hamster ovary cells are used to screen for mouse parvovirus, mousepoxvirus, hamster retrovirus, and hamster susceptible exogenous viruses; MEF mouse embryonic fibroblasts are used to screen for mouse parvovirus, mouse leukemia virus, and mouse herpesvirus; NIH / 3T3 mouse fibroblasts are used to screen for mouse parvovirus, mouse polyomavirus, and mouse retrovirus; MDBK bovine kidney cells are used to screen for bovine viral diarrhea virus, bovine coronavirus, bovine parvovirus, and vaccinia virus; BT bovine nasal turbinate bone cells are used to screen for bovine viral diarrhea virus, bovine parainfluenza virus, and vaccinia virus.

[0012] For example, in a multimodal rapid detection integrated system for cell-indicating viruses provided in at least one embodiment of this disclosure, the dedicated program includes the culture medium formulation, serum concentration, plated cell density, and incubation time for the corresponding indicator cell line.

[0013] For example, in a cell-indicator virus multimodal rapid detection integrated system provided in at least one embodiment of this disclosure, the integrated control and result analysis module is configured to: when only one test result is positive, the score of that positive result does not reach the preset strong positive score, and the other two test results are negative, retrieve the built-in interference database to identify interference features including cell aging, serum impurities, non-specific adsorption of red blood cells, and non-specific agglutination; when confirmed as a false positive, correct the judgment to negative for exogenous virus; when interference cannot be completely eliminated, mark suspected positive and prompt for retesting and verification; when the score of any positive result reaches the preset strong positive score, or two or more test results are positive, after excluding conventional non-specific interference, determine that the exogenous virus is positive.

[0014] According to another aspect of the present invention, a method for detecting exogenous viruses is also provided, which is executed using a cell-indicator virus multimodal rapid detection integrated system, including the following steps: The test sample is simultaneously and quantitatively inoculated into various standardized indicator cells in the culture plate through the multi-channel independent tubing of the closed microfluidic channel automatic sample addition system; culture is completed according to a preset culture cycle in the adjustable culture environment of the integrated culture chamber; multimodal detection is performed sequentially and continuously on the same cultured sample after culture without manual transfer: the cultured indicator cells are imaged to identify cytopathic effects; red blood cell suspension is added, and the red blood cell adsorption state is interpreted after incubation; hemagglutination reaction reagent is added, and hemagglutination incubation and imaging interpretation are completed; the three types of detection results—cytopathic effect, hemagglutination, and hemagglutination—are quantified into scores according to preset grading rules, wherein: 0 points are awarded for no cytopathic effect, 1 point for mild local cytopathic effect, and 2 points for large-area typical cytopathic effect; 0 points are awarded for negative hemagglutination detection, and 0 points for weak hemagglutination detection. A positive result is scored as 1 point, and a strong positive result as 2 points; a negative result for hemagglutination is scored as 0 points, a weak positive result as 1 point, and a strong positive result as 2 points; a score of 1 or 2 points is recorded as a single positive result, and a score of 0 points is recorded as a single negative result; the scores of the three test results are weighted and summed according to their respective preset weights for cytopathic effect, hemagglutination and hemagglutination, to obtain a comprehensive reference score, which is calculated as: cytopathic effect score × 0.5 + hemagglutination score × 0.3 + hemagglutination score × 0.2. The comprehensive reference score is used to quantify the severity of the positive result and as a reference for reporting and tracing the source; when all three test results are single negative, the test sample is determined to be negative for exogenous virus; when any single test result is positive, the built-in interference database is retrieved to identify interference features to identify false positives, and a virus detection conclusion is output based on the identification results; after outputting the virus detection conclusion, all original images, the scores of each test item, the comprehensive reference score, the judgment conclusion and the corresponding indicator cell line are bound together by the dedicated program to form a traceability dataset.

[0015] (III) Beneficial Effects Compared with the prior art, the present invention provides an integrated system for rapid detection of cell-indicating viruses in multiple modalities, which has the following advantages: Firstly, the system integrates and simultaneously detects three indicators across multiple dimensions for more accurate collaborative verification: It integrates CPE, hemagglutination, and hemagglutination into a single, continuous in-situ detection system, enabling multi-dimensional cross-verification based on cell morphology and viral biological characteristics, significantly reducing the probability of missed detections and false positives. Secondly, it offers flexible adaptation of cell culture parameters, supporting independent customization of culture medium types, serum concentrations, and cell density for BHK-21, Vero, HeLa, MRC-5, CHO-K1, MEF, NIH / 3T3, MDBK, and BT cells. Dedicated culture programs can be accessed with a single click, adapting to different cell growth characteristics. Thirdly, the system is fully integrated, significantly improving detection efficiency. It integrates sample inoculation, cell culture, multiple detections, and result analysis into a single system, with seamless integration and automatic linkage between modules. This eliminates the need for manual sample transfer and step-by-step operations, simplifying the process. The detection cycle can be customized as needed, perfectly adapting to different scales. This system meets the rapid detection needs of cell banks; it eliminates the risk of contamination and loss by eliminating sample transfer throughout the entire process. All culture and detection operations are completed within the same integrated culture chamber, featuring a dual-isolation and anti-contamination structure with multiple tubing and disposable pipette tips, fundamentally preventing cross-contamination and cell loss, and ensuring accurate and stable test results. Automated and standardized operation ensures excellent repeatability; cell resuscitation, plating, incubation, and reagent dispensing are all quantified and solidified, and cell-specific parameters can be pre-stored and locked, minimizing human error and ensuring parallelism meets pharmacopoeia quality control standards. Autonomous intelligent integrated interpretation reduces subjective bias, abandoning the manual item-by-item summary and interpretation mode. The system automatically quantifies and scores, automatically identifies false positives, and uses unified judgment standards, resulting in objective and traceable conclusions. It has a wide range of compatibility and strong compliance, carrying indicator cells covering multiple species including human, mouse, hamster, and bovine, and can be matched with various cell banks and exogenous virus screening of biological raw materials. Attached Figure Description

[0016] To more clearly illustrate the technical solutions of the embodiments of this disclosure, the accompanying drawings of the embodiments will be briefly described below. Obviously, the drawings described below only relate to some embodiments of this disclosure and are not intended to limit this disclosure.

[0017] Figure 1 This is a schematic diagram of the system architecture of a cell-indicator virus multimodal rapid detection integrated system according to the present invention. Detailed Implementation

[0018] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. 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.

[0019] It should be understood that in the various embodiments of the present invention, the sequence number of each process does not imply the order of execution. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of the present invention.

[0020] It should be understood that in this invention, "comprising" and "having," and any variations thereof, are intended to cover non-exclusive inclusion, for example, a process, method, system, product, or device that includes a series of steps or units is not necessarily limited to those steps or units that are explicitly listed, but may include other steps or units that are not explicitly listed or that are inherent to such process, method, product, or device.

[0021] The applicant's research revealed the following problems with conventional techniques: First, the operational modes are independent. Although multiple methods can be combined for detection, the various methods lack integration and still require step-by-step operation, failing to leverage the synergistic advantages of multi-dimensional detection and limiting detection efficiency. Second, the detection process is fragmented. Sample processing, culture, and detection are carried out manually in each step, requiring multiple transfers of samples to different devices. This is not only cumbersome and costly in terms of manpower and time, but also unsuitable for the rapid detection needs of large-scale cell banks. Furthermore, sample transfer carries a high risk; manual transfer after cell culture can easily lead to sample contamination and loss, directly affecting the authenticity and reliability of the test results. Finally, the level of integration is low. There is no dedicated integrated operating equipment for each step, making it impossible to achieve seamless integration of rapid initial screening and accurate verification, and failing to meet the high standards and efficiency requirements of cell bank quality control for virus detection.

[0022] Based on the above ideas, at least one embodiment of this disclosure provides an integrated system for rapid detection of multimodal cell indicator viruses. The following describes an integrated system for rapid detection of multimodal cell indicator viruses through several specific embodiments.

[0023] This embodiment provides an integrated system for rapid multimodal detection of cell-indicator viruses, including an indicator cell module, an inoculation and culture module, a multimodal detection module, and an integrated control and result analysis module. The system can be used for screening exogenous viruses in various cell bank samples. Through multimodal integration of indicator cell culture combined with cytopathic effect observation, hemoadsorption assay, and hemagglutination reaction, it achieves simultaneous and quantitative inoculation of samples, as well as continuous, automated detection and comprehensive interpretation of cultured samples without manual transfer.

[0024] Indicator Cell Module: The indicator cell module is equipped with a variety of standardized indicator cell lines adapted to different types of viruses. These standardized indicator cell lines are obtained through standardized resuscitation, passage, plating, and culture procedures to ensure that the morphology, viability, and viral sensitivity of each batch of indicator cells are consistent, thereby guaranteeing the repeatability and comparability of test results.

[0025] In one specific implementation, multiple standardized indicator cell lines are selected from at least two of the following cell lines: BHK-21 hamster kidney cells, Vero African green monkey kidney cells, HeLa human cervical cancer cells, MRC-5 human diploid cells, CHO-K1 Chinese hamster ovary cells, MEF mouse embryonic fibroblasts, NIH / 3T3 mouse fibroblasts, MDBK bovine kidney cells, and BT bovine nasal turbinate bone cells, to meet the detection requirements of multiple viruses in test samples from a multi-species cell bank of humans, monkeys, hamsters, mice, and cattle.

[0026] Different indicator cell lines have different susceptibility profiles to viruses, thus adapting to different types of viruses. In one specific embodiment, the correspondence between each standardized indicator cell line and the exogenous virus it screens for is as follows: BHK-21 hamster kidney cells are used to screen for various arboviruses; Vero African green monkey kidney cells are used to screen for herpesviruses, poxviruses, arboviruses, primate parvoviruses, and human susceptible enveloped viruses; HeLa human cervical cancer cells are used to screen for human adenoviruses, human papillomaviruses, and human respiratory susceptible viruses; MRC-5 human diploid cells are used to screen for human cytomegaloviruses, human adenoviruses, respiratory syncytial virus, and human herpesviruses; CHO-K1... Chinese hamster ovary cells are used to screen for mouse parvovirus, mousepox virus, hamster retrovirus, and hamster-susceptible exogenous viruses; MEF mouse embryonic fibroblasts are used to screen for mouse parvovirus, mouse leukemia virus, and mouse herpesvirus; NIH / 3T3 mouse fibroblasts are used to screen for mouse parvovirus, mouse polyomavirus, and mouse retrovirus; MDBK bovine kidney cells are used to screen for bovine viral diarrhea virus, bovine coronavirus, bovine parvovirus, and vaccinia virus; and BT bovine nasal turbinate bone cells are used to screen for bovine viral diarrhea virus, bovine parainfluenza virus, and vaccinia virus. Based on this, the above indicator cell lines can be flexibly combined according to the type of exogenous virus to be screened and the detection requirements to form customized screening combinations suitable for large-scale detection needs.

[0027] In one specific implementation, cell resuscitation is performed under uniform resuscitation water bath conditions, namely a constant temperature water bath at 37°C for 90 seconds; after resuscitation, cells are centrifuged at 1000 rpm for 5 minutes to remove the cryoprotectant DMSO. Cells are then seeded into culture plates at the pre-set cell density for the corresponding cell line.

[0028] After plating, the indicator cell monolayer can be evaluated for its suitability. Evaluation criteria include: ① Cell adhesion coverage ≥ 90%; ② Dead cells and vacuolated / lysed cells < 5%; ③ Uniform cell distribution, with no localized stacking or large sparse areas. Evaluation thresholds can be customized in the background. Wells that fail the evaluation will not be used for testing or will need to be re-plated.

[0029] In one embodiment, the culture plate also includes blank control wells or positive control wells for monitoring the effectiveness of the detection system.

[0030] Inoculation and Culture Module: The inoculation and culture module includes an integrated culture chamber and culture plates disposed within the integrated culture chamber. In one specific embodiment, the culture plates are 6-well culture plates, with a uniform plate volume of 2.5 mL per well. The accompanying culture medium is automatically supplied synchronously according to the preset protocol for each cell line. In other embodiments, culture plates of other specifications can be used according to the detection throughput requirements, and the plate volume can be adjusted accordingly. The wells carrying different standardized indicator cell lines can be arranged according to cell line zones. The integrated culture chamber is a sealed chamber that maintains a controllable culture environment. The environmental parameters include temperature, carbon dioxide concentration, and humidity. In one specific embodiment, the culture environment is a temperature of 37°C, a carbon dioxide concentration of 5% (volume fraction), and a humidity of 95%. In one embodiment, the inoculation and culture module is configured to maintain the integrated culture chamber in a slightly negative pressure sealed environment, in conjunction with a closed microfluidic channel automatic sample dispensing system, to block cross-contamination caused by aerosols and reagent residues, thereby preventing the escape of aerosols from the chamber and cross-contamination.

[0031] The closed-loop microfluidic channel automated sample dispensing system adopts a non-open dispensing method and is equipped with multi-channel independent tubing that is physically isolated from each other for dispensing the test sample and each reagent separately.

[0032] In one specific implementation, the multi-channel independent tubing includes a dedicated tubing for the test sample, as well as dedicated tubing for each detection reagent, such as red blood cell suspension, washing solution, sterile PBS buffer, and hemagglutination reaction reagent. Each tubing is physically completely separated and not interconnected.

[0033] The test samples and reagents are all added through their respective dedicated tubing: the closed-loop microfluidic channel automated sample addition system quantitatively inoculates the test samples into the indicator cells in the culture plate; the red blood cell suspension, washing solution and other detection reagents, as well as the hemagglutination reaction reagents, are all added to the culture plates in the integrated culture chamber through the corresponding dedicated tubing of the closed-loop microfluidic channel automated sample addition system, realizing closed-loop reagent delivery from inoculation to detection and avoiding contamination introduced by open operation.

[0034] The closed-loop microfluidic channel automated sample dispensing system is configured to synchronously and quantitatively seed test samples into multiple standardized indicator cells within a culture plate. Under the scheduling of the integrated control and result analysis module, the sample dispensing system quantitatively distributes test samples at a preset volume (e.g., 100–200 μL per well, depending on the plate specifications and detection requirements) to each well containing different indicator cell lines in the same dispensing process. This ensures that each indicator cell line is seeded synchronously, avoiding inconsistencies in the culture start point caused by seeding time differences, and supporting parallel screening of multiple indicator cell lines.

[0035] In one embodiment, the closed-loop microfluidic channel automated sampling system is further equipped with a detachable disposable sampling tip, and is configured to automatically discard the currently used disposable sampling tip and replace it with a new disposable sterile sampling tip after each reagent is dispensed, so as to avoid cross-contamination of reagents through the pipette tip.

[0036] In one implementation, before reusing the same tubing in a multi-channel independent tubing system, an automatic cleaning procedure is performed. This procedure includes sequentially flushing the tubing walls with 75% ethanol (both forward and reverse), flushing three times with sterile PBS buffer (both forward and reverse), and purging with sterile air to remove residual liquid, thus removing reagent residue from the tubing walls. Combined with a slightly negative pressure sealed environment and disposable pipette tips, this forms a dual-isolation, cross-contamination-free anti-pollution system integrating closed channels, independent tubing, and tip replacement with tubing cleaning.

[0037] After inoculation, the inoculation culture module completes the culture in the adjustable culture environment of the integrated culture chamber according to the preset culture cycle; In one specific implementation, the preset culture period is 5-7 days, during which indicator cells can be imaged and observed at preset time points. The integrated culture chamber directly interfaces with the multimodal detection module, and multimodal detection can be carried out directly after culture without the need for manual sample transfer.

[0038] Multimodal Detection Module: The multimodal detection module includes a cytopathic effect observation unit, a hemoadsorption assay unit, and a hemagglutination reaction unit, sequentially integrated into the integrated culture chamber. It is configured to sequentially and continuously perform tests on the same culture sample after culture, without manual transfer, verifying the presence of the virus from three dimensions: morphology, biological characteristics, and antigenicity. After culture, the culture plate does not need to be removed from the integrated culture chamber, and no manual plate or sample transfer is required; the three detection units sequentially perform tests on the same culture sample according to a preset time sequence.

[0039] Cytopathic Effect Observation Unit: This unit images and identifies cytopathic effects in cultured indicator cells. In one specific embodiment, the cytopathic effect observation unit is equipped with a high-magnification, high-resolution imaging system that performs fully automated morphological imaging and intelligent analysis of cultured indicator cells, and automatically identifies typical pathological effects such as virus-induced cell shrinkage, fusion, and detachment through a built-in image recognition algorithm.

[0040] Blood adsorption assay unit: Red blood cell suspension is added, and imaging is performed after incubation to determine the adsorption status of red blood cells. The red blood cell suspension is added to the cultured indicator cell monolayer via a dedicated tubing of a closed microfluidic automated sample dispensing system. After incubation, imaging is performed to determine whether red blood cells are adsorbed onto the surface of the indicator cells and their distribution range. The appropriate red blood cell type can be selected based on the virus to be screened, such as guinea pig red blood cells, human type O red blood cells, or chicken red blood cells, at a concentration of, for example, 0.5%–1% (volume fraction).

[0041] Hemagglutination reaction unit: This unit dispenses hemagglutination reaction reagents, completing hemagglutination incubation and imaging interpretation. The hemagglutination reaction reagents are dispensed via a dedicated tubing within a closed microfluidic automated dispensing system. In one specific embodiment, the hemagglutination reaction reagents include a red blood cell suspension for the hemagglutination reaction and a sterile buffer solution, such as sterile PBS buffer, for dilution. The red blood cell suspension can be selected based on the type of red blood cells to be screened, such as chicken red blood cells, guinea pig red blood cells, or human type O red blood cells, with a concentration, for example, 0.5%–1% (volume fraction). After incubation, imaging is performed to determine whether red blood cell agglutination has occurred and the degree of agglutination.

[0042] Integrated Control and Result Analysis Module: This module connects to both the inoculation and culture module and the multimodal detection module; the connection can be, for example, a communication connection or an electrical connection. The integrated control and result analysis module can automatically retrieve raw data and results from the three main detection units and precisely control the working sequence and operating parameters of each module, including adjusting culture environment parameters, detection procedures, and reagent additions according to the specific programs for each indicator cell line, achieving automated and standardized operation of the entire culture-detection process.

[0043] The integrated control and results analysis module stores dedicated programs for each indicator cell line. These programs include specific culture parameters for each cell line, such as culture medium formulation, serum concentration, cell density, and incubation time. In one specific implementation, each cell line can be configured with different basal culture medium types, such as DMEM, MEM, or RPMI-1640. The serum concentration is adjustable from 0% to 20%, with the default configuration adding 10% fetal bovine serum to the basal medium. The adjustable cell density range is 1×10⁻⁶. 4 ~5×10 5 cells / mL. In this application, the specific culture parameters of the corresponding indicator cell line bound to the traceability dataset are the culture-related parameters stored in the specific program of the indicator cell line.

[0044] The integrated control and result analysis module has a built-in interference database, which includes known interference features and their interpretation rules. Interference features include cell aging, serum impurities, non-specific adsorption of red blood cells, and non-specific agglutination. During false positive identification, the image features and reaction patterns of the current test result are compared with the interference features included in the interference database to determine whether the positive result is caused by the aforementioned interference factors.

[0045] The integrated control and results analysis module performs comprehensive interpretation of three types of test results: cytopathic effects, blood adsorption, and blood coagulation. Specifically, this includes: Step 1, Grading and Quantification: The three types of test results are quantified into scores according to preset grading rules. In one specific implementation, cytopathic effect: no lesion, 1 point; mild local lesion, 2 points; large area typical lesion, 0 points; hemoadsorption test: negative, 1 point; weak positive, 2 points; hemagglutination test: negative, 0 points; weak positive, 1 point; strong positive, 2 points. The preset positive score is 1 point. A score of 1 or 2 points (i.e., reaching the preset positive score) is recorded as a single positive, and a score of 0 points (i.e., not reaching the preset positive score) is recorded as a single negative; the preset strong positive score is 2 points.

[0046] Step 2, Weighted Calculation: The scores of the three test results are weighted and summed according to their respective preset weights for cytopathic effect, hemoadsorption, and hemagglutination to obtain a comprehensive reference score. In one specific implementation, the comprehensive reference score = cytopathic effect test score × 0.5 + hemoadsorption test score × 0.3 + hemagglutination test score × 0.2. The comprehensive reference score is used to quantify the severity of a positive result and as a reference for report traceability. This application does not set a threshold for determining positive or negative results for the comprehensive reference score, and the comprehensive reference score is not used as the basis for determining positive or negative results. The determination of positive or negative results is based solely on the score of each test item and the judgment rules.

[0047] Step 3, initial screening and determination: When all three test results are negative, the test sample is determined to be negative for exogenous virus; when any single test result is positive, proceed to step 4 for false positive identification.

[0048] Step 4, False Positive Identification: When only one test result is positive, the score for that positive result does not reach the preset strong positive score, and the other two test results are both negative, the built-in interference database is retrieved to identify interference features including cell aging, serum impurities, non-specific adsorption of red blood cells, and non-specific agglutination. If confirmed as a false positive, the conclusion is corrected to negative for exogenous virus. If interference cannot be completely eliminated, a suspected positive is marked and a retest is requested. When the score for any single positive result reaches the preset strong positive score, or two or more test results are positive, after excluding routine non-specific interference, the result is determined to be positive for exogenous virus.

[0049] Step 5, Archiving and Reporting: After outputting the virus detection results, bind all original images, scores for each detection item, comprehensive reference scores, judgment conclusions, and the corresponding indicator cell line's dedicated program (including culture-related parameters, i.e., dedicated culture parameters) to form a complete source tracing dataset and generate a detection report; the report may include the detection results, item scores, comprehensive reference scores, judgment conclusions, and source tracing information for each indicator cell line.

[0050] V. Post-testing processing In one implementation, the system is configured to perform high-temperature sterilization on the integrated culture chamber, its waste liquid, and pipelines after the detection is completed, in order to inactivate any pathogenic microorganisms that may be present and ensure biosafety.

[0051] Example 2: Method for detecting exogenous viruses This embodiment provides a method for detecting exogenous viruses, which is executed using the cell-indicator virus multimodal rapid detection integrated system of Embodiment 1, and includes the following steps: S1. Inoculation: The test sample is simultaneously and quantitatively inoculated into various standardized indicator cells within the culture plate via the dedicated tubing of the closed microfluidic channel automated sample addition system; detection reagents such as red blood cell suspension, washing buffer, and hemagglutination reaction reagent are also added via their respective physically isolated dedicated tubing. In one embodiment, blank control wells or positive control wells are simultaneously set up during inoculation.

[0052] S2. Cultivation: The cultivation is completed in the adjustable cultivation environment of the integrated cultivation chamber according to the preset cultivation cycle; no manual transfer of samples is required after cultivation.

[0053] S3. Multimodal Detection: Multimodal detection is performed sequentially and continuously on the same cultured sample after culture without manual transfer. The morphology of the indicator cells after culture is automatically captured and intelligently analyzed by a high-magnification, high-definition imaging system to identify typical pathological effects such as cell shrinkage, fusion, and detachment. Red blood cell suspension is added through a dedicated tubing, and the adsorption state of red blood cells is determined by imaging after incubation. Hemagglutination reagent is added through a dedicated tubing to complete hemagglutination incubation and imaging interpretation.

[0054] S4. Grading, Quantification and Weighting: The three types of test results, namely cytopathic effect, blood adsorption and blood coagulation, are quantified into scores according to the preset grading rules, and a comprehensive reference score is calculated. The grading rules, the identification of single positive and single negative results, the preset positive score, the preset strong positive score and the weighting calculation method are the same as steps one and two of Example 1.

[0055] S5. Judgment and identification: When all three test results are negative, the test sample is determined to be negative for exogenous virus; when any single test result is positive, the built-in interference database is retrieved to identify interference features to identify false positives, and the virus detection conclusion is output according to the identification results; the identification rules are the same as steps three and four of Example 1.

[0056] S6. Archiving and Reporting: After outputting the virus detection results, the system binds all original images, scores for each detection item, comprehensive reference scores, judgment conclusions, and corresponding indicator cell lines to form a source tracing dataset and generates a detection report.

[0057] It should be noted that the system composition, parameters and interpretation rules not detailed in this embodiment are all based on Embodiment 1.

[0058] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A cell-indicator-based multimodal rapid detection integrated system for viruses, characterized in that, include: The indicator cell module is equipped with a variety of standardized indicator cell lines adapted to different types of viruses; The inoculation and culture module includes an integrated culture chamber, a culture plate disposed within the integrated culture chamber, and a closed-loop microfluidic channel automatic sample dispensing system. The closed-loop microfluidic channel automatic sample dispensing system is equipped with physically isolated multi-channel independent tubing for separately dispensing test samples and reagents, used to quantitatively inoculate the test samples into indicator cells within the culture plate. The inoculation and culture module is also configured to maintain a controllable culture environment within the integrated culture chamber and complete the culture according to a preset culture cycle. The multimodal detection module includes a cytopathic effect observation unit, a hemoadsorption test unit, and a hemagglutination reaction unit integrated into the integrated culture chamber. It is configured to sequentially and continuously perform detection on the same cultured sample after culture, without manual transfer. Specifically, the cytopathic effect observation unit images the indicator cells after culture and identifies the cytopathic effect; the hemoadsorption test unit is configured to: add a red blood cell suspension, incubate, and then image and interpret the red blood cell adsorption state; the hemagglutination reaction unit is configured to: add a hemagglutination reaction reagent, complete hemagglutination incubation, and then image and interpret the results. An integrated control and result analysis module is connected to both the inoculation and culture module and the multimodal detection module. It stores dedicated programs for each indicator cell line, used to regulate culture environment parameters, detection procedures, and reagent addition according to these programs. The module also performs comprehensive interpretation of three types of detection results: cytopathic effects, hemoadsorption, and hemagglutination. The three types of results are quantified into scores according to preset grading rules. A score reaching the preset positive score is recorded as a single positive result, while a score not reaching the preset positive score is recorded as a single negative result. When all three test results are single negative, the test sample is determined to be negative for exogenous virus. When any single test result is positive, the built-in interference database is retrieved to identify interference features to distinguish false positives, and a virus detection conclusion is output based on the identification results.

2. The system according to claim 1, characterized in that, The closed-loop microfluidic channel automated sampling system is also equipped with detachable disposable sampling tips, and is configured to automatically discard the currently used disposable sampling tip and replace it with a new disposable sterile sampling tip after each reagent is dispensed.

3. The system according to claim 1, characterized in that, The closed microfluidic channel automatic sample dispensing system is also configured to automatically perform a cleaning procedure before the same tubing in the multi-channel independent tubing is reused. The cleaning procedure includes sequentially performing forward and reverse flushing of the tubing wall with 75% ethanol, forward and reverse flushing with sterile PBS buffer three times, and sterile air purging to remove residual liquid.

4. The system according to claim 1, characterized in that, The inoculation and culture module is configured to maintain the integrated culture chamber in a slightly negative pressure sealed environment.

5. The system according to claim 1, characterized in that, The parameters of the adjustable culture environment include temperature, carbon dioxide concentration, and humidity; the preset culture period is 5 to 7 days.

6. The system according to claim 1, characterized in that, The various standardized indicator cell lines are selected from at least two of the following: BHK-21 hamster kidney cells, Vero African green monkey kidney cells, HeLa human cervical cancer cells, MRC-5 human diploid cells, CHO-K1 Chinese hamster ovary cells, MEF mouse embryonic fibroblasts, NIH / 3T3 mouse fibroblasts, MDBK bovine kidney cells, and BT bovine nasal turbinate bone cells.

7. The system according to claim 6, characterized in that, The various standardized indicator cell lines are configured to screen for corresponding exogenous viruses; among them, BHK-21 hamster kidney cells are used to screen for various arboviruses; Vero African green monkey kidney cells are used to screen for herpesviruses, poxviruses, arboviruses, primate parvoviruses, and human susceptible enveloped viruses; HeLa human cervical cancer cells are used to screen for human adenoviruses, human papillomaviruses, and human respiratory susceptible viruses; MRC-5 human diploid cells are used to screen for human cytomegaloviruses, human adenoviruses, respiratory syncytial virus, and human herpesviruses; and CHO-K1 Chinese hamster kidney cells are used to screen for these viruses. Mouse ovarian cells are used to screen for mouse parvovirus, mousepox virus, hamster retrovirus, and hamster-susceptible exogenous viruses; MEF mouse embryonic fibroblasts are used to screen for mouse parvovirus, mouse leukemia virus, and mouse herpesvirus; NIH / 3T3 mouse fibroblasts are used to screen for mouse parvovirus, mouse polyomavirus, and mouse retrovirus; MDBK bovine kidney cells are used to screen for bovine viral diarrhea virus, bovine coronavirus, bovine parvovirus, and vaccinia virus; BT bovine nasal turbinate bone cells are used to screen for bovine viral diarrhea virus, bovine parainfluenza virus, and vaccinia virus.

8. The system according to claim 1, characterized in that, The proprietary program includes the culture medium formulation, serum concentration, plated cell density, and incubation duration for the corresponding indicator cell line.

9. The system according to claim 1, characterized in that, The integrated control and result analysis module is configured to: when only one test result is positive, the score of that positive result does not reach the preset strong positive score, and the other two test results are negative, retrieve the built-in interference database to identify interference features including cell aging, serum impurities, non-specific adsorption of red blood cells, and non-specific agglutination; if a false positive is confirmed, correct the judgment to negative for exogenous virus; if interference cannot be completely eliminated, mark it as a suspected positive and prompt for retesting and verification; when the score of any positive result reaches the preset strong positive score, or two or more test results are positive, after excluding conventional non-specific interference, determine that the exogenous virus is positive.

10. A method for detecting exogenous viruses, performed using the cell-indicator virus multimodal rapid detection integrated system as described in any one of claims 1 to 9, characterized in that, The process includes the following steps: The test sample is simultaneously and quantitatively inoculated into various standardized indicator cells within the culture plate via the multi-channel independent tubing of the closed microfluidic automatic sample addition system; culture is completed according to a preset culture cycle in the adjustable culture environment of the integrated culture chamber; multimodal detection is performed sequentially and continuously on the same cultured sample without manual transfer after culture: the indicator cells are imaged to identify cytopathic effects; a red blood cell suspension is added, and the red blood cell adsorption state is interpreted after incubation; a hemagglutination reaction reagent is added, and hemagglutination incubation and imaging interpretation are completed; the results of the three types of detection—cytopathic effect, hemagglutination, and hemagglutination—are quantified into scores according to preset grading rules, where: 0 points for no cytopathic effect, 1 point for mild localized cytopathic effect, and 2 points for large-area typical cytopathic effect; 0 points for negative hemagglutination test, 1 point for weak positive hemagglutination test, and 2 points for strong positive hemagglutination test; 0 points for negative hemagglutination test, 1 point for weak positive hemagglutination test, and 2 points for strong positive hemagglutination test. A positive result is scored as follows: 1 point for a positive result and 2 points for a strong positive result. A score of 1 or 2 points is considered a single positive result, and a score of 0 points is considered a single negative result. The scores of the three test results are weighted and summed according to their preset weights for cytopathic effect, hemoadsorption, and hemagglutination to obtain a comprehensive reference score. The comprehensive reference score is calculated as follows: cytopathic effect test score × 0.5 + hemoadsorption test score × 0.3 + hemagglutination test score × 0.

2. This comprehensive reference score is used to quantify the severity of the positive result and serves as a reference for reporting and tracing the source. When all three test results are single negative, the test sample is determined to be negative for exogenous virus. When any single test result is positive, the built-in interference database is retrieved to identify interference features to identify false positives, and a virus detection conclusion is output based on the identification results. After outputting the virus detection conclusion, all original images, scores of each test item, comprehensive reference score, judgment conclusion, and the corresponding indicator cell line are bound together by the dedicated program to form a traceability dataset.

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