A system and method for directly extracting and enriching trace microorganisms from complex matrices

Abstract

The application discloses a system and method directly used for extracting and enriching trace microorganisms from a complex matrix, the system comprises a hardware connection module, a business logic scheduling module, a data processing module, an interface display module and a data storage module connected in sequence; and the interface display module is further connected with a software configuration module and a sample management module respectively. The system can realize the functions of trace microorganism extraction, enrichment and equipment control, a user can enrich trace microorganisms to obtain high-throughput samples, and high-throughput sample processing and data analysis are realized through an artificial intelligence technology.

Description

Technical Field

[0001] This invention relates to the field of microbial detection technology, and more specifically to a system and method for directly extracting and enriching trace microorganisms from complex matrices. Background Technology

[0002] Infectious diseases are common in clinical practice. Globally, 31.5 million people suffer from bloodstream infections and 5 million from meningitis each year. Half of all women and 1 in 20 men worldwide suffer from urinary tract infections. In addition, chest and abdominal infections are also very common. Pathogen diagnosis is a crucial step in their treatment. The conventional method for pathogen detection is microbial culture, but this method is not the ideal gold standard because it is time-consuming (1-5 days), results are often unavailable, the positive rate is low (detection rate less than 15%), the results are incomplete or not sensitive enough, and it can be misleading. Furthermore, it is relatively labor-intensive, thus failing to meet clinical needs.

[0003] Infectious diseases, especially bloodstream infections, often require a 1-5 day diagnostic time between sample collection and infection results due to the low pathogen concentration in samples (1-10 CFU / mL). Consequently, 19.4 million people worldwide experience severe bloodstream infections annually, with an average mortality rate of 16-40%. It is estimated that 3 million cases of neonatal sepsis occur globally each year, resulting in 1.2 million child deaths annually, a mortality rate of 11-19%. Furthermore, over 75,000 women die annually from postpartum sepsis. In US hospitals, sepsis is not only the most expensive disease to treat but also a leading cause of death. The US sees up to 3.1 million cases of sepsis annually, costing $24 billion, with a mortality rate between 20% and 50%. In Europe, the probability of death from sepsis is even higher, approximately 10-15% higher than in the US. Recent studies indicate that patients with severe or shock-type bloodstream infections have a 7.6% increased risk of death per hour without antibiotic treatment. Survival rates are significantly reduced within the first six hours of recognizing low blood pressure without the use of antibiotics. Therefore, shortening the diagnostic time can greatly improve patient survival. Rapid early diagnosis is thus the most reliable hope for improving the survival rate of patients with infectious diseases and limiting survivor disability, and highly sensitive products that can provide diagnostic information rapidly within a few hours, with small sample sizes, are urgently needed in clinical practice.

[0004] Antimicrobial susceptibility testing is a method used to detect the resistance and sensitivity of bacteria or other microorganisms to antibiotics or other drugs. It is an important means to guide rational drug use in clinical practice, reduce drug abuse, and improve treatment efficacy. Traditional antimicrobial susceptibility testing mainly includes disk diffusion, dilution, and automated antimicrobial susceptibility testing systems. To meet the needs of rapid clinical diagnosis, antimicrobial susceptibility testing needs to develop towards faster and simpler methods. By adopting new technologies and methods, the time cycle of antimicrobial susceptibility testing can be shortened, providing clinicians with more timely medication guidance. However, antimicrobial susceptibility test results are often affected by various factors such as culture conditions and microbial species. Infected samples often need to be cultured before antimicrobial susceptibility testing, and the sample enrichment and isolation culture time is relatively long, making it difficult to quickly select appropriate antimicrobial drugs and reasonable antimicrobial dosages.

[0005] MALDI-TOF MS is a novel soft ionization biomolecular mass spectrometry technique developed in recent years. Clinical microbiology laboratories use it to identify large numbers of bacteria and fungi after culture. The identification results are automatically interpreted, analyzed, and reported, with automatic sample unloading. The detection cost is low; the instrument only requires sample plates and a dedicated mass spectrometry matrix, without any additional reagents, and the technical requirements for staff are not high. Due to its high efficiency, accuracy, and low cost, it has become a popular method for microbial identification.

[0006] Pathogen culture remains the gold standard for detecting infectious samples, but it has several drawbacks. Firstly, the diagnostic time is long, typically 1-5 days from patient sampling to obtaining infection results. Further time is required for purified pathogens to undergo drug susceptibility testing. During this period, treatment usually involves broad-spectrum, non-targeted antibiotics, which may be effective in treating the disease but can lead to multidrug resistance in many microorganisms. Secondly, the variability of pathogens in a sample results in significant differences in their culture requirements, meaning the dominant bacteria cultured may not be the original state found in the sample. Thirdly, the sample pretreatment process for drug susceptibility testing or MALDI-TOF MS detection is overly complex. Pathogen culture requires substantial investment of manpower, resources, time, and money, resulting in high costs and demanding skilled operators, hindering its widespread adoption. Finally, bacteria cultured over extended periods may be contaminated, leading to inaccurate results for clinicians.

[0007] Currently, MALDI-TOF MS mass spectrometry cannot be used for direct detection of clinical specimens and mixed cultures. The analyte must be a single, pure microorganism. Its high accuracy and reproducibility in identifying microorganisms are based on the detection of microbial ribosomal proteins (relative molecular mass 2000–20000), a class of highly abundant proteins immobilized and expressed intracellularly. Therefore, theoretically, different culture conditions should not lead to significant differences in MALDI-TOF MS identification results. However, to achieve more ideal identification results, laboratories currently need to culture the bacteria twice and then pick single-clone colonies for MALDI-TOF MS identification, which is time-consuming and labor-intensive. Furthermore, MALDI-TOF MS identification typically requires a bacterial load of 10⁵–10⁷ colony-forming units (CFU). Therefore, if direct detection is performed, samples from early stages of bloodstream infections or meningitis infections may not be enriched to a sufficient bacterial load or may require an excessive amount of sample. Moreover, if the sample contains two or more mixed infections, the detection results will usually fail or contain errors.

[0008] None of the above methods have been able to achieve satisfactory automated control of the experiment. Summary of the Invention

[0009] To address the aforementioned technical problems, this invention provides a system for directly extracting and enriching trace microorganisms from complex matrices. The system comprises a hardware connection module, a business logic scheduling module, a data processing module, an interface display module, and a data storage module connected in sequence.

[0010] The interface display module is also connected to the software configuration module and the sample management module.

[0011] According to an embodiment of the present invention, the interface display module is used to provide user graphical interface interaction.

[0012] According to an embodiment of the present invention, the interface display module includes a main interface control module, a hardware parameter module, a user login module, and an engineer parameter module; the main interface control module, hardware parameter module, user login module, and engineer parameter module are arranged in parallel. The user login module is used for users to log in to the system; the hardware parameter module is used for setting system parameters; the main interface control module is used for system monitoring; and the engineer parameter module is used for engineers to maintain the system.

[0013] According to an embodiment of the present invention, the data processing module is connected to the main interface control module of the interface display module.

[0014] According to an embodiment of the present invention, the hardware parameter module is also directly connected to the hardware connection module.

[0015] According to an embodiment of the present invention, the hardware connection module is connected to the business logic scheduling module and is used to control the workstation hardware to execute relevant instructions for extraction operations (such as sample separation, solvent injection, etc.). The hardware connection module specifically includes a robotic arm module, a mixing module, a centrifuge module, a sample position module, and a reagent position module. The hardware includes a robotic arm, a mixing assembly, a centrifuge assembly 6, a sample position assembly 17, and a reagent position assembly 14, etc., with each module responsible for executing the action instructions of its respective hardware. Specifically, the robotic arm module controls the robotic arm; the mixing module controls the mixing assembly; the centrifuge module controls the centrifuge assembly 6; the sample position module controls the sample position assembly 17; and the reagent position module controls the reagent position assembly 14.

[0016] According to an embodiment of the present invention, the user login module, data storage module, software configuration module, sample management module, and data processing module are all parallel components.

[0017] According to an embodiment of the present invention, the engineer parameter module is stored in the data storage module, and it is parallel to the software configuration module and the sample management module.

[0018] According to an embodiment of the present invention, the hardware parameter module is stored in the data storage module; the hardware parameters in the hardware parameter module are transmitted to the hardware connection module through the software configuration module to realize the execution of the action; the hardware parameter module and the sample management module are parallel.

[0019] In this invention, the main interface control module issues instructions through the business logic scheduling module. These instructions are then processed by the data processing module, which reads relevant parameters from the data storage module. Finally, the software configuration module transmits these parameters to the hardware connection module to execute the instruction. The main interface control module and the sample management module are parallel components.

[0020] According to an embodiment of the present invention, the workstation includes:

[0021] It includes a main frame 1, a robotic arm, a mixing assembly, a centrifuge assembly 6, a sample position assembly 17, a workbench assembly 15, and a reagent position assembly 14;

[0022] The robotic arm is located on the upper part of the main frame, and the robotic arm is controlled by the control system.

[0023] The mixing component, sample position component 17, workbench component 15 and reagent position component 14 are arranged side by side in the middle of the main frame and located below the robotic arm.

[0024] The centrifuge assembly is located at the lower part of the main frame.

[0025] According to an embodiment of the present invention, the robotic arm includes a pipetting robotic arm 4 and a tube transfer and cap opening / closing robotic arm 5, which are placed side by side. The pipetting robotic arm is used to add or remove liquid into a reagent tube, for example, a sample tube or a centrifuge tube. The tube transfer and cap opening / closing robotic arm is used to move the reagent tube or to open or close the cap of the reagent tube.

[0026] According to an embodiment of the present invention, the mixing assembly includes an inverting mixing assembly 8 and a heating assembly 9.

[0027] Preferably, the mixing component further includes an oscillation component 10.

[0028] Preferably, the inverted mixing component 8, the heating component 9, and the oscillation component 10 are arranged side by side in sequence.

[0029] According to an embodiment of the present invention, a platform assembly 11 is further provided in the middle of the main frame, and the platform assembly 11 is placed side by side with the reagent position assembly 14. The platform assembly 11 is used for balancing when centrifuge tubes enter the centrifuge.

[0030] The reagent position assembly 14 is used to place an auxiliary agent, for example, the auxiliary agent includes at least one of lysis buffer, microbial sedimentation agent and washing liquid.

[0031] According to an embodiment of the present invention, the middle portion of the main frame is further provided with at least one set of pipette tip assemblies 12, which are placed side by side with the dispensing platform assembly 11; the pipette tip assemblies are used to hold consumable pipette tips. Preferably, the middle portion of the main frame contains at least two sets of pipette tip assemblies, including a 200μL pipette tip assembly and a 5mL pipette tip assembly 13. Preferably, the at least two sets of pipette tip assemblies are respectively used to hold 200μL consumable pipette tips and 5mL consumable pipette tips.

[0032] According to an embodiment of the present invention, the workstation further includes a waste liquid and waste material disposal assembly 16, which is located in the middle of the main frame 1. Preferably, the waste liquid and waste material disposal assembly 16 is placed side by side with the workbench assembly 15.

[0033] According to an embodiment of the present invention, the workstation further includes a waste liquid and waste material collection tank 7 located at the lower part of the main frame. The waste liquid and waste material collection tank 7 is placed side by side with the centrifuge assembly 6 for collecting waste liquid or waste material.

[0034] According to an embodiment of the present invention, an air filter assembly 2 is further provided on the top of the main frame, the air filter assembly being used to filter air.

[0035] According to an embodiment of the present invention, an operation screen 3 is further provided on the outside of the main frame 1. The operation screen 3 is used to control the robotic arm for subsequent experimental operations. Preferably, the operation screen 3 is located on the outside side of the main frame 1, for example, on the right or left side.

[0036] According to an embodiment of the present invention, a barcode scanner assembly 18 is further provided in the middle of the main frame, and the barcode scanner assembly is placed side by side with the mixing assembly. Preferably, the barcode scanner assembly is placed side by side with the inverted mixing assembly 8.

[0037] According to an embodiment of the present invention, the method for extracting and enriching trace microorganisms using the workstation includes:

[0038] (1) A robotic arm is used to move the sample tube containing the blood sample to the mixing assembly to mix the sample. After mixing, the robotic arm is used to move the mixed sample tube and centrifuge tube to the workbench assembly 5 respectively.

[0039] (2) A robotic arm is used to transfer the sample in the sample tube to a centrifuge tube to obtain a centrifuge tube containing blood.

[0040] (3) Use a robotic arm to transfer the auxiliary agent from the reagent position assembly and add it to the centrifuge tube containing the blood sample. Then use the robotic arm to move the centrifuge tube to the mixing assembly to mix the sample evenly.

[0041] (4) Use a robotic arm to move the centrifuge tubes into the centrifuge assembly for centrifugation;

[0042] (5) After centrifugation, a robotic arm is used to move the centrifuge tube to the workbench assembly to remove the supernatant, thus completing the extraction and enrichment of trace microorganisms.

[0043] According to an embodiment of the present invention, in step (1), the robotic arm includes a pipetting robotic arm and a tube transfer and cap opening / closing robotic arm; the pipetting robotic arm is used to add or remove liquid into the centrifuge tube. The tube transfer and cap opening / closing robotic arm is used to move the centrifuge tube, or to open or close the cap of the centrifuge tube.

[0044] According to an embodiment of the present invention, in step (1), the mixing component includes an inverting mixing component 8 and a heating component 9.

[0045] Preferably, the mixing component further includes an oscillation component 10.

[0046] According to an embodiment of the present invention, step (1) further includes the process of collecting waste pipette tips; after the robotic arm transfers blood samples from the reagent position assembly through the pipette tips and adds them to the centrifuge tube, the waste pipette tips are placed in the waste liquid and waste material collection bucket; the waste liquid and waste material collection bucket is set in the waste liquid and waste material position assembly.

[0047] According to an embodiment of the present invention, in step (3), the robotic arm removes the adjuvant from the reagent position assembly via the consumable nozzle. Preferably, the nozzle is placed in the suction tip assembly 12.

[0048] According to an embodiment of the present invention, step (3) further includes the process of collecting waste pipette tips; after the robotic arm removes the auxiliary agent from the reagent position assembly through the pipette tip and adds it to the centrifuge tube containing the blood sample, the waste pipette tip is placed in the waste liquid and waste material collection bucket; the waste liquid and waste material collection bucket is set in the waste liquid and waste material position assembly.

[0049] According to an embodiment of the present invention, steps (1) and (2) are specifically as follows:

[0050] A robotic arm is used to move the sample tube containing the blood sample to the mixing assembly to mix the sample. After mixing, the robotic arm is used to move the centrifuge tube and the sample tube containing the blood sample to the workbench assembly. The robotic arm opens the caps of the centrifuge tube and the sample tube. Then, the robotic arm takes the consumable pipette tip from the pipette tip assembly 12 and moves the blood sample from the sample tube to the centrifuge tube through the pipette tip to obtain a centrifuge tube containing the blood sample.

[0051] According to an embodiment of the present invention, in step (1), the centrifuge tube and sample tube may be scanned first; the scanning operation is implemented by the barcode scanner assembly 18.

[0052] According to an embodiment of the present invention, in step (1), before the operation is performed, the workstation can be evacuated and ventilated to achieve aseptic operation; the evacuation and ventilation operation is achieved through the air filter assembly 2.

[0053] According to an embodiment of the present invention, in step (4), the centrifuge tube containing the sample can also be weight balanced, and the balancing operation is achieved by the balancing platform assembly 11.

[0054] According to an embodiment of the present invention, in step (5), the robotic arm removes the supernatant from the centrifuge tube through a consumable nozzle.

[0055] According to an embodiment of the present invention, the robotic arm is controlled via an operation screen, which is connected to an external control system.

[0056] According to an embodiment of the present invention, the business logic scheduling module is used to provide execution logic scheduling related to hardware control, and includes a program execution unit, a time control unit, a state management unit, an exception handling unit, a motion control unit, etc., all of which are devices known in the art. The units in the business logic scheduling module cooperate with each other to provide strong support for the correct and efficient operation of the entire enrichment process.

[0057] According to an embodiment of the present invention, the program execution unit is used to execute a fully automated program for microbial enrichment operations according to a preset procedure.

[0058] For example, the program execution unit can parse the experimental parameters input by the user (such as centrifugation speed, mixing time, heating temperature, etc.) and generate an executable instruction sequence.

[0059] For example, the program execution unit is used to control the hardware connection module to perform operations such as sample transfer, reagent addition, centrifugation, and mixing in sequence.

[0060] For example, the program execution unit can adjust experimental parameters (such as sample volume and oscillation time) and achieve flexible adaptation by calling pre-stored parameter templates.

[0061] According to an embodiment of the present invention, the time control unit precisely manages the time nodes and synchronization of each step, ensuring the efficiency and accuracy of the experimental process.

[0062] Preferably, the time control unit can set and monitor the duration of each operation step (such as centrifugation time, mixing time, etc.).

[0063] Preferably, the time control unit coordinates the parallel operation of multiple hardware connection modules (e.g., synchronously preheating the heating component while the centrifuge assembly is running).

[0064] Preferably, the time control unit provides a countdown function, which automatically triggers an alarm or switches to an abnormal handling process when the time expires.

[0065] According to an embodiment of the present invention, the status management unit monitors the system hardware and software status in real time to ensure a stable operating environment.

[0066] Preferably, the status management unit is used to detect the real-time status of the hardware connection module (such as whether the centrifuge assembly is ready, whether the positions of the pipetting robot arm 4 and the tube transfer and cap opening / closing robot arm 5 are normal, and whether the reagent balance is sufficient).

[0067] Preferably, the status management unit is used to record the current stage of the experimental process (e.g., "adding liquid" or "oscillating").

[0068] Preferably, the status management unit dynamically adjusts operating parameters based on sensor data feedback (such as temperature and position) to avoid process interruption.

[0069] According to an embodiment of the present invention, the anomaly handling unit automatically identifies and responds to faults or deviations during workstation operation, ensuring system security and reliability.

[0070] Preferably, the anomaly handling unit detects common anomalies (such as pipetting + opening / closing robotic arm, pipetting robotic arm assembly jamming, centrifuge assembly malfunction, etc.) and triggers preset emergency measures (such as pausing the process, alarm prompts).

[0071] Preferably, the anomaly handling unit supports error logging and fault location, facilitating remote diagnosis or on-site maintenance by engineers.

[0072] Preferably, the anomaly handling unit is used to provide an anomaly prompt interface (such as a pop-up alarm or sound reminder) and recommend solutions.

[0073] According to an embodiment of the present invention, the motion control unit is used to precisely control the path planning and action execution of the hardware.

[0074] Preferably, the motion control unit plans the movement path of the pipetting robot arm 4 or the tube transfer and cap opening / closing robot arm 5 according to the experimental procedure (such as picking up test tubes from the workbench and transferring them to the centrifuge assembly, etc.).

[0075] Preferably, the motion control unit calibrates the aspiration and dispensing accuracy of the pipetting robot arm (e.g., ensuring that the 200μL pipette tip accurately aspirates the sample).

[0076] Preferably, the motion control unit supports multi-axis linkage control (such as simultaneously operating the pipetting robot arm and the tube transfer and cap opening / closing robot arm 5) to improve operating efficiency.

[0077] According to an embodiment of the present invention, the data processing module interacts directly with the data storage module, including reading and writing parameters such as nozzle parameters, robotic arm parameters, centrifuge parameters, step parameters, access control parameters, and engineer maintenance parameters.

[0078] According to an embodiment of the present invention, the data storage module is used to store data received from the data processing module, and it uses the Android local file JSON format to store the data, thereby improving the read and write efficiency of the program.

[0079] According to an embodiment of the present invention, the software configuration module is used to provide data interaction support between the hardware connection module and the data storage module (hardware parameter data obtained from the data storage module), parse the hardware parameter data obtained from the data storage module, and drive the hardware connection module to perform precise hardware operations based on the parameter data. For example, it can effectively interact with the lower-level machine system through Ethernet communication, serial communication, etc., to achieve effective control of the instrument and ensure the consistency of parameters and operational reliability during the extraction process.

[0080] For example, the interface display module directly implements TCP / IP communication via Ethernet;

[0081] The robotic arm, centrifuge, and other components in the workstation communicate via serial port.

[0082] According to an embodiment of the present invention, the system further includes a sample management module. The sample management module is connected to the main interface control module.

[0083] According to an embodiment of the present invention, the sample management module is used to place reagents / samples at a designated location on the workstation.

[0084] The system works as follows:

[0085] (1) First, power on the workstation, then log in to the system through the user login module on the interface display module. Then, set the engineer parameters in the engineer parameter module, and then set the system parameters in the hardware parameter module (such as read / write gun head parameters, robotic arm parameters, centrifuge parameters, step parameters (including heating parameters, oscillation parameters, shaking parameters), permission management parameters, engineer maintenance parameters, etc.) (this function is set according to the test to be performed). The above parameters will be directly stored in the data storage module for later system retrieval and use.

[0086] (2) Then place the sample in the designated sample location on the workstation, then set the sample management module on the interface display module, and then place the corresponding reagents / consumables in the designated location on the workbench.

[0087] (3) Start the workstation test process through the main interface control module. The system first operates in the main interface control module. The business logic scheduling module issues the sample scanning command. The command reads the sample scanning related parameters in the data storage module through the data processing module. Then the sample scanning parameters are transmitted to the hardware connection module through the software configuration module to execute the sample scanning.

[0088] (4) Then the business logic scheduling module issues a mixing instruction. The instruction reads the mixing parameters in the data storage module through the data processing module. Then the mixing parameters are transmitted to the hardware connection module through the software configuration module to execute the sample mixing action.

[0089] (5) Next, the business logic scheduling module issues an instruction to place the mixed sample and centrifuge tube consumables onto the workbench. The instruction reads the robotic arm parameters in the data storage module through the data processing module. Then, the robotic arm parameters are transmitted to the hardware connection module through the software configuration module to execute the transfer action.

[0090] (6) Then the business logic scheduling module issues a sample packaging instruction. The instruction reads the relevant packaging parameters in the data storage module through the data processing module. Then the packaging parameters are transmitted to the hardware connection module through the software configuration module to execute the packaging action.

[0091] (7) Then the business logic scheduling module issues a reagent dispensing instruction. The instruction reads the relevant parameters of the reagent dispensing from the data storage module through the data processing module. Then the reagent dispensing parameters are transmitted to the hardware connection module through the software configuration module to execute the reagent dispensing action.

[0092] (8) Then the business logic scheduling module issues an oscillation command. The command reads the oscillation-related parameters in the data storage module through the data processing module. Then the oscillation parameters are transmitted to the hardware connection module through the software configuration module to execute the transfer oscillation action.

[0093] (9) Then the business logic scheduling module issues a heating instruction. The instruction reads the heating-related parameters in the data storage module through the data processing module. Then the heating parameters are transmitted to the hardware connection module through the software configuration module to execute the transfer heating action.

[0094] (10) Then the heated sample needs to be centrifuged in the centrifuge assembly. The business logic scheduling module issues a centrifugation command. The command reads the centrifugation-related parameters in the data storage module through the data processing module. Then the centrifugation parameters are transmitted to the hardware connection module through the software configuration module to execute the transfer centrifugation action.

[0095] (11) The centrifuged sample needs to be taken out and the supernatant needs to be taken out. The business logic scheduling module issues the supernatant taking instruction. The instruction reads the supernatant taking parameters from the data storage module through the data processing module. Then the supernatant taking parameters are transmitted to the hardware connection module through the software configuration module to execute the transfer and supernatant taking action.

[0096] (12) After the final test is completed, the sample is returned to the sample position on the workbench. The business logic scheduling module issues a sample return instruction. The instruction reads the sample return-related parameters from the data storage module through the data processing module. Then, the sample return clearing parameters are transmitted to the hardware connection module through the software configuration module to execute the sample return action.

[0097] The present invention also provides a method for directly extracting and enriching trace microorganisms from complex matrices using the above-described system, the method comprising:

[0098] (S1) Log in to the system and set system parameters through the interface display module, and store the system parameters in the data storage module;

[0099] (S2) By setting the sample management module on the interface display module, reagents / consumables can be placed in a designated location on the workstation;

[0100] (S3) Operate on the main interface control module, and issue corresponding instructions to the workstation through the business logic scheduling module. The instructions read relevant parameters in the data storage module through the data processing module, and transmit the relevant parameters to the hardware connection module through the software configuration module to execute the instructions.

[0101] (S4) After the relevant instructions are completed, the extraction and enrichment of microorganisms are completed.

[0102] According to an embodiment of the present invention, in step (S3), the instructions include at least one of the following: sample scanning instruction, mixing instruction, transfer instruction, sample dispensing instruction, reagent dispensing instruction, heating instruction, centrifugation instruction, and supernatant collection instruction.

[0103] According to an embodiment of the present invention, in step (S3), the relevant parameters include at least one of the following: sample scanning parameters, mixing parameters, robotic arm parameters, dispensing parameters, reagent dispensing parameters, shaking parameters, heating parameters, centrifugation parameters, and supernatant collection parameters.

[0104] The beneficial effects of this invention are:

[0105] 1. The enrichment system of this invention can use conventional consumables and pre-packaged reagent plates or strips in conjunction with an automated processing workbench for separation. No manual operation or complex personnel training is required, saving time, effort, and manpower.

[0106] 2. The system of the present invention can realize the extraction, enrichment and equipment control functions of microorganisms with extremely low abundance. Users can enrich microorganisms with extremely low abundance to obtain high-throughput samples, and realize high-throughput sample processing and data analysis through artificial intelligence technology.

[0107] 3. The workstation of this invention is used to extract and enrich trace microorganisms from complex matrices. Its workflow includes sample identification, cap opening and closing, consumable handling, sample addition, mixing (including shaking, heating, etc.), centrifugation, and liquid aspiration and disposal, achieving a fully automated separation process. The cap opening and closing mechanism is more compact, has a lower failure rate, and is more stable than other similar products. The pipetting robotic arm adopts a more stable piston rod-type liquid aspiration / discharge mode, resulting in higher precision and stronger stability. The centrifuge assembly integrates high speed, positioning, and cooling, achieving high-speed centrifugation while rapidly reducing the temperature rise during operation through its cooling function. Compared to similar products, it offers higher rotation speed, more accurate positioning, and more precise temperature control. The workstation of this application also features an air filtration system, which maintains a sterile experimental environment throughout the instrument's operation, making the experiment more accurate and effectively preventing contamination.

[0108] 4. The workstation of this invention can enrich microorganisms at extremely low abundance, significantly improving the accuracy and reliability of detection. Combined with artificial intelligence technology, it realizes high-throughput sample processing and data analysis, and is suitable for trace microbial detection in multiple fields, with broad application prospects.

[0109] 5. The method for extracting and enriching trace microorganisms of the present invention can effectively remove most of the human cells, proteins, sugars, salts and other impurities from uncultured or cultured clinical samples such as blood, cerebrospinal fluid, urine, pleural effusion and peritoneal fluid, and obtain relatively pure live pathogens.

[0110] 6. In the method of this invention, pathogen products isolated from uncultured samples can be subjected to drug susceptibility testing or MALDI-TOF MS detection after simple culture (only one step of liquid culture) and bacterial collection. Cultured sample products, such as blood, cerebrospinal fluid, pleural effusion, and ascites, can be directly used for drug susceptibility testing or MALDI-TOF MS detection after simple concentration. Compared to pathogen diagnosis and identification of clinical infection samples, the method of this invention shortens the culture time and enables faster acquisition of drug susceptibility results and mass spectrometry microbial identification results, providing clinicians with more timely diagnostic and treatment references.

[0111] 7. The workstation of this invention uses conventional consumables and pre-packaged reagents to isolate microorganisms from blood samples. It requires no manual operation, no complex personnel training or practice, saving time, effort, and manpower. Attached Figure Description

[0112] Figure 1 This is a schematic diagram of the enrichment system of the present invention.

[0113] Figure 2 This is the front view of the workstation of this invention.

[0114] Figure 3 This is a top view of section A of the workstation of the present invention.

[0115] 1. Main unit frame; 2. Air filter assembly; 3. Operation screen; 4. Pipetting robotic arm; 5. Tube transfer and cap opening / closing robotic arm; 6. Centrifuge assembly; 7. Waste liquid and waste material collection container; 8. Inverting and mixing assembly; 9. Heating assembly; 10. Shaking assembly; 11. Preparation platform assembly; 12. 200μL pipette tip assembly; 13. 5mL pipette tip assembly; 14. Reagent assembly; 15. Workbench assembly; 16. Waste liquid and waste material disposal assembly; 17. Sample assembly; 18. Barcode scanner assembly.

[0116] Figure 4 This is the process of separating and enriching microorganisms at the workstation in Example 2. Detailed Implementation

[0117] The technical solution of the present invention will be further described in detail below with reference to specific embodiments. It should be understood that the following embodiments are merely illustrative and explanatory of the present invention, and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are covered within the scope of protection intended by the present invention.

[0118] Unless otherwise stated, the raw materials and reagents used in the following examples are commercially available products or can be prepared by known methods.

[0119] Example 1

[0120] A system for directly extracting and enriching trace microorganisms from complex matrices, the system comprising a hardware connection module, a business logic scheduling module, a data processing module, an interface display module, and a data storage module connected in sequence;

[0121] The interface display module is also connected to the software configuration module and the sample management module respectively;

[0122] The interface display module includes a main interface control module, a hardware parameter module, a user login module, and an engineer parameter module; the main interface control module, hardware parameter module, user login module, and engineer parameter module are arranged side by side.

[0123] The data processing module is connected to the main interface control module of the interface display module.

[0124] The hardware parameter module is also directly connected to the hardware connection module.

[0125] The hardware connection module is connected to the business logic scheduling module and is used to control the workstation hardware to execute relevant instructions for extraction operations (such as sample separation, solvent injection, etc.). The hardware connection module specifically includes a robotic arm module, a mixing module, a centrifuge module, a sample position module, and a reagent position module. The hardware includes a robotic arm, a mixing assembly, a centrifuge assembly 6, a sample position assembly 17, and a reagent position assembly 14, etc., with each module responsible for executing the action instructions of its respective hardware. Specifically, the robotic arm module controls the robotic arm; the mixing module controls the mixing assembly; the centrifuge module controls the centrifuge assembly 6; the sample position module controls the sample position assembly 17; and the reagent position module controls the reagent position assembly 14.

[0126] The workstation includes a main frame 1, a pipetting robotic arm 4, a tube transfer and cap opening / closing robotic arm 5, an inverting and mixing assembly 8, a heating assembly 9, a shaking assembly 10, a centrifuge assembly 6, a sample position assembly 17, a workbench assembly 15, and a reagent position assembly 14.

[0127] The robotic arms (including the pipetting robotic arm 4, the tube transfer and cap opening / closing robotic arm 5) are located on the upper part of the main frame, and the robotic arms are controlled by the control system.

[0128] The main frame is also equipped with a platform assembly 11 and two sets of suction head assemblies 12 in the middle.

[0129] The inverted mixing component 8, heating component 9, oscillation component 10, platform component 11, and two sets of suction head components 12 are arranged side by side in the middle of the main frame and located below the robotic arm.

[0130] The sample position component 17 and the heating component 9 are placed side by side;

[0131] The workstation also includes a waste liquid and waste material disposal assembly 16, which is located in the middle of the main frame 1; the vibration assembly 10, the worktable assembly 15, and the waste liquid and waste material disposal assembly 16 are placed side by side in sequence.

[0132] The platform assembly 11 and the reagent placement assembly 14 are placed side by side; the platform assembly 11 is used for balancing when centrifuge tubes enter the centrifuge. The reagent placement assembly 14 is used to place reagents or additives, such as lysis buffer, microbial sedimentation aid, and washing solution.

[0133] The centrifuge assembly is located at the lower part of the main frame.

[0134] The pipetting robotic arm is used to add or remove liquid into reagent tubes (sample tubes or centrifuge tubes), and the tube transfer and cap opening / closing robotic arm is used to move reagent tubes or to open or close the caps of reagent tubes.

[0135] Preferably, the middle part of the main frame contains two sets of pipette tip assemblies 12, including a 200μL pipette tip assembly and a 5mL pipette tip assembly 13. Preferably, the two sets of pipette tip assemblies are respectively used to hold 200μL pipette tips and 5mL pipette tips.

[0136] The workstation also includes a waste liquid and waste material collection tank 7, located at the lower part of the main frame. The waste liquid and waste material collection tank 7 is placed side by side with the centrifuge assembly 6 and is used to collect waste liquid or waste material.

[0137] The top of the main frame is also provided with an air filter assembly 2, which is used to filter air.

[0138] An operation screen 3 is also provided on the outside of the main frame 1. The operation screen 3 is used to control the robotic arm for subsequent experimental operations. Preferably, the operation screen 3 is located on the outside side of the main frame 1, such as the right or left side.

[0139] The main frame also includes a barcode scanner assembly 18, which is placed side-by-side with the mixing assembly. Preferably, the barcode scanner assembly is placed side-by-side with the inverted mixing assembly 8.

[0140] The working principle of this invention's workstation is as follows:

[0141] (1) Place the sample tubes and centrifuge tubes in the corresponding positions of the sample position assembly 17, the reagent kit in the corresponding position of the reagent position assembly 14, the 200μL pipette tip consumable in the corresponding position of the 200μL pipette tip position assembly 12, and the 5mL pipette tip consumable in the corresponding position of the 5mL pipette tip position assembly 13. Then, start the test procedure by running the instrument through the operation screen 3.

[0142] (2) First, the sample tube and centrifuge tube consumables in the sample position assembly 17 are transferred to the barcode scanner assembly 18 by the tube transfer and cap opening / closing robotic arm 5 for barcode scanning. Then, the sample tube is transferred to the inverting and mixing assembly 8 for sample blood mixing. After mixing, the sample tube and centrifuge tube are transferred to the corresponding position of the workbench assembly 15 and the caps of the sample tube and centrifuge tube are opened. At the same time, the pipetting robotic arm 4 is moved to the 5mL pipette tip position assembly 13 to insert the 5mL pipette tip. Then, it is moved to the workbench assembly 15 to dispense the sample from the sample tube into the centrifuge tube. Finally, the pipetting robotic arm 4 runs to the waste liquid and waste material position assembly 16 to discard the used 5mL pipette tip.

[0143] (3) The tube transfer and cap opening / closing robotic arm 5 closes the cap on the sample tube on the workbench assembly 15 and picks it up and puts it back in the corresponding position on the sample position assembly 17. Then the pipetting robotic arm 4 moves to the 200μL pipette tip position assembly 12 to insert a 200μL pipette tip, then moves to the reagent position assembly 14 to draw the corresponding reagent, then moves to the workbench assembly 15 to add the corresponding auxiliary agent into the centrifuge tube, and finally the pipetting robotic arm 4 moves to the waste liquid and waste material position assembly 16 to discard the used 200μL pipette tip.

[0144] (4) Then the pipetting robot arm 4 moves to the 5mL pipette tip assembly 13 to insert the 5mL pipette tip, then moves to the reagent assembly 14 to pick up the corresponding auxiliary agent, then moves to the worktable assembly 15 to add the corresponding auxiliary agent into the corresponding centrifuge tube, and finally the pipetting robot arm 4 moves to the waste liquid and waste material assembly 16 to discard the used 5mL pipette tip.

[0145] (5) Then the tube transfer and cap opening / closing robot arm 5 moves to the worktable assembly 15 to close the cap of the centrifuge tube and pick it up and transfer it to the corresponding position of the oscillation assembly 10 for oscillation. After the oscillation is completed, the tube transfer and cap opening / closing robot arm 5 puts the centrifuge tube into the centrifuge assembly 6 in sequence and automatically calculates whether balancing is needed based on the number of centrifuge tubes put in.

[0146] (6) If balancing is required, the tube transfer and cover opening / closing robotic arm 5 will move to the balancing platform assembly 11 for balancing. The balancing centrifuge tubes will be placed in the corresponding position of the centrifuge assembly 6, and then the centrifuge assembly 6 will start running to perform centrifugation.

[0147] (7) After centrifugation, the transfer and cap opening / closing robotic arm 5 picks up the centrifuge tube and places it on the workbench assembly 15 to open the cap. Then, the pipetting robotic arm 4 inserts the 5ml pipette tip from the 5ml pipette tip assembly and moves it to the opened centrifuge tube to collect the supernatant. After that, it moves to the waste liquid / waste material disposal assembly 16 to discard the waste liquid / waste pipette tip. Finally, the transfer and cap opening / closing robotic arm 5 closes the cap on the centrifuge tube on the workbench assembly 15.

[0148] (8) Repeat steps (5)-(7) 6 times. After the shaking is complete, the centrifuge tube is transferred to the heating component 9 by the tube transfer and cap opening / closing robotic arm 5 for heating. After the heating is complete, the centrifuge tube is returned to the corresponding position of the sample position component 17.

[0149] Example 2

[0150] The extraction and enrichment process of microorganisms using the system of Example 1 is as follows:

[0151] (1) First, power on the workstation, then log in to the system through the user login module on the interface display module, then set the engineer parameters in the engineer parameter module, and then set the system parameters in the hardware parameter module. The above parameters will be directly stored in the data storage module for later system retrieval.

[0152] (2) Then place the sample in the designated sample location on the workstation, then set the sample management module on the interface display module, and then place the corresponding reagents / consumables in the designated location on the workbench.

[0153] (3) Start the workstation test process through the main interface control module. The system first operates in the main interface control module. The business logic scheduling module issues the sample scanning command. The command reads the sample scanning related parameters in the data storage module through the data processing module. Then the sample scanning parameters are transmitted to the hardware connection module through the software configuration module to execute the sample scanning.

[0154] (4) Then the business logic scheduling module issues a mixing instruction. The instruction reads the mixing parameters in the data storage module through the data processing module. Then the mixing parameters are transmitted to the hardware connection module through the software configuration module to execute the sample mixing action.

[0155] (5) Next, the business logic scheduling module issues an instruction to place the mixed sample and centrifuge tube consumables onto the workbench. The instruction reads the robotic arm parameters in the data storage module through the data processing module. Then, the robotic arm parameters are transmitted to the hardware connection module through the software configuration module to execute the transfer action.

[0156] (6) Then the business logic scheduling module issues a sample packaging instruction. The instruction reads the relevant packaging parameters in the data storage module through the data processing module. Then the packaging parameters are transmitted to the hardware connection module through the software configuration module to execute the packaging action.

[0157] (7) Then the business logic scheduling module issues a reagent dispensing instruction. The instruction reads the relevant parameters of the reagent dispensing from the data storage module through the data processing module. Then the reagent dispensing parameters are transmitted to the hardware connection module through the software configuration module to execute the reagent dispensing action.

[0158] (8) Then the business logic scheduling module issues an oscillation command. The command reads the oscillation-related parameters in the data storage module through the data processing module. Then the oscillation parameters are transmitted to the hardware connection module through the software configuration module to execute the transfer oscillation action.

[0159] (9) Then the business logic scheduling module issues a heating instruction. The instruction reads the heating-related parameters in the data storage module through the data processing module. Then the heating parameters are transmitted to the hardware connection module through the software configuration module to execute the transfer heating action.

[0160] (10) Then the heated sample needs to be centrifuged in the centrifuge assembly. The business logic scheduling module issues a centrifugation command. The command reads the centrifugation-related parameters in the data storage module through the data processing module. Then the centrifugation parameters are transmitted to the hardware connection module through the software configuration module to execute the transfer centrifugation action.

[0161] (11) The centrifuged sample needs to be taken out and the supernatant needs to be taken out. The business logic scheduling module issues the supernatant taking instruction. The instruction reads the supernatant taking parameters from the data storage module through the data processing module. Then the supernatant taking parameters are transmitted to the hardware connection module through the software configuration module to execute the transfer and supernatant taking action.

[0162] (12) After the final test is completed, the sample is returned to the sample position on the workbench. The business logic scheduling module issues a sample return instruction. The instruction reads the sample return-related parameters from the data storage module through the data processing module. Then, the sample return clearing parameters are transmitted to the hardware connection module through the software configuration module to execute the sample return action.

[0163] The operating principle of the workstation is as follows:

[0164] S1. Scanning and identification: Under the action of the tube transfer and cap opening / closing robotic arm 5, the sample tube and centrifuge tube are transferred to the barcode scanner assembly 18 for scanning and identification / recording.

[0165] S2. Sample mixing: The sample tube is transferred to the mixing assembly by the tube transfer and cap opening / closing robotic arm 5 for inverting and mixing, and then transferred to the workbench assembly 15 together with the centrifuge tube.

[0166] S3. Blood Sample Dispensing and Reagent Addition: First Step of Lysis: The sample tube and centrifuge tube are opened using the pipetting and opening / closing robotic arm 5. The blood sample is quantitatively dispensed into the corresponding centrifuge tube by inserting a 5ml pipette tip from the 5mL pipette tip assembly 13 using the pipetting robotic arm 4. The discarded pipette tip is then sent to the waste liquid and waste material assembly 16 to complete the discarding of the pipette tip. Next, the pipetting robotic arm 4 inserts the corresponding consumable pipette tip to quantitatively add lysis buffer A and microbial sedimentation agent to the centrifuge tube that has just been dispensed with blood sample. The discarded pipette tip is then sent to the waste liquid and waste material assembly 16. The sample tube and centrifuge tube are closed using the pipetting and opening / closing robotic arm 5, and the sample tube is returned to its original position.

[0167] S4. The centrifuge tubes are transferred to the shaking assembly 10 for 1 minute of shaking by the tube transfer and cap opening / closing robotic arm 5, and then transferred to the centrifuge assembly 6 for 2-3 minutes of centrifugation (the balancing is automatically performed during this process).

[0168] S5. Discarding supernatant: After centrifugation, the centrifuge tubes are transferred from the centrifuge 6 to the workbench 15 via the tube transfer and cap opening / closing robotic arm 5 and the caps are opened. Then, the supernatant is quantitatively collected by inserting a 5ml pipette tip into the centrifuge tube using the pipette robotic arm. The supernatant is then discarded at the waste liquid outlet of the waste liquid and waste material disposal assembly, and the waste pipette tip is discarded at the waste material outlet of the waste liquid and waste material disposal assembly.

[0169] S6. Blood sample lysis for the 2nd-4th time: Use a pipette tip to draw a fixed amount of B, C, or D lysis buffer using a pipette arm and add it to the centrifuge tube from which the supernatant has just been collected. Then, use the pipette and capping arm to close the cap of the centrifuge tube and transfer it to the shaking assembly for 1 minute of shaking. Then, transfer it to the centrifuge for 2-3 minutes of centrifugation (the balance is automatically performed during this process). Repeat the above operation 3 times (adding lysis buffer B, C, and D in sequence).

[0170] S7. Washing: Using the tube transfer and cap opening / closing robotic arm, remove the centrifuge tubes after the fourth lysis from the centrifuge and transfer them to the workbench assembly 15, then open the caps. Next, using the pipetting robotic arm, insert a 5ml pipette tip to draw washing solution A, B, or C and add it to the centrifuge tubes. The tube transfer and cap opening / closing robotic arm 5 closes the caps on the centrifuge tubes and transfers them to the shaking assembly for 1 minute of shaking. Then, transfer them to the centrifuge for 2 minutes of centrifugation (automatic balancing occurs during this process). After centrifugation, perform the supernatant disposal step S5. Repeat the above operation three times (adding washing solutions AB and C in sequence).

[0171] S8. Collection: The product after the last S7 wash is transferred to the designated location of the sample position by a tube transfer and cap opening / closing robotic arm.

[0172] The above is a summary of the entire process of separating and enriching microorganisms using the workstation, which saves approximately 25% of the time compared to manual operation. Depending on the sample size, it can enrich the bacterial cells to a concentration 5-10 times higher than the original concentration.

[0173] The embodiments of the present invention have been described above by way of example. However, the scope of protection of the present invention is not limited to the above embodiments. Any modifications, equivalent substitutions, improvements, etc., made by those skilled in the art within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A system for directly extracting and enriching trace microorganisms from complex matrices, characterized in that, The system comprises a hardware connection module, a business logic scheduling module, a data processing module, an interface display module, and a data storage module connected in sequence. The interface display module is also connected to the software configuration module and the sample management module.

2. The system according to claim 1, characterized in that, The interface display module is used to provide users with a graphical interface interaction; Preferably, the interface display module includes a main interface control module, a hardware parameter module, a user login module, and an engineer parameter module; the main interface control module, hardware parameter module, user login module, and engineer parameter module are arranged in parallel.

3. The system according to claim 1, characterized in that, The interface display module includes a main interface control module, a hardware parameter module, a user login module, and an engineer parameter module; the main interface control module, hardware parameter module, user login module, and engineer parameter module are arranged side by side; The user login module is used for users to log in to the system; the hardware parameter module is used for setting system parameters; the main interface control module is used for system monitoring; and the engineer parameter module is used for engineers to maintain the system.

4. The system according to claim 1, characterized in that, The data processing module is connected to the main interface control module of the interface display module; Preferably, the hardware parameter module is also directly connected to the hardware connection module.

5. The system according to claim 1, characterized in that, The hardware connection module is connected to the business logic scheduling module and is used to control the hardware of the workstation to execute relevant instructions for extraction operations. The hardware connection module specifically includes a robotic arm module, a mixing module, a centrifuge module, a sample position module and a reagent position module. The hardware includes a robotic arm, a mixing component, a centrifuge component (6), a sample position component (17) and a reagent position component (14), and each module is responsible for the execution logic of the action instructions of its respective hardware.

6. The system according to claim 1, characterized in that, The workstation includes: It includes a main frame (1), a robotic arm, a mixing assembly, a centrifuge assembly (6), a sample position assembly (17), a workbench assembly (15), and a reagent position assembly (14); The robotic arm is located on the upper part of the main frame, and the robotic arm is controlled by the control system. The mixing component, sample position component, worktable component, and reagent position component are arranged side by side in the middle of the main frame and located below the robotic arm. The centrifuge assembly is located at the lower part of the main frame.

7. The system according to claim 6, characterized in that, The robotic arm includes a pipetting robotic arm (4) and a tube transfer and cap opening / closing robotic arm (5), which are placed side by side.

8. The system according to claim 6, characterized in that, The method for extracting and enriching trace microorganisms using the workstation described above includes: (1) A robotic arm is used to move the sample tube containing the blood sample to the mixing assembly to mix the sample. After mixing, the robotic arm is used to move the mixed sample tube and centrifuge tube to the workbench assembly 5 respectively. (2) A robotic arm is used to transfer the sample in the sample tube to a centrifuge tube to obtain a centrifuge tube containing blood. (3) Use a robotic arm to transfer the auxiliary agent from the reagent position assembly and add it to the centrifuge tube containing the blood sample. Then use the robotic arm to move the centrifuge tube to the mixing assembly to mix the sample evenly. (4) Use a robotic arm to move the centrifuge tubes into the centrifuge assembly for centrifugation; (5) After centrifugation, a robotic arm is used to move the centrifuge tube to the workbench assembly to remove the supernatant, thus completing the extraction and enrichment of trace microorganisms.

9. The system according to claim 1, characterized in that, The business logic scheduling module is used to provide execution logic scheduling related to hardware control. It includes a program execution unit, a time control unit, a state management unit, an exception handling unit, and a motion control unit. The above units are devices known in the art.

10. A method for directly extracting and enriching trace microorganisms from a complex matrix using the system described in any one of claims 1-9, the method comprising: (S1) Log in to the system and set system parameters through the interface display module, and store the system parameters in the data storage module; (S2) By setting the sample management module on the interface display module, reagents / consumables can be placed in a designated location on the workstation; (S3) Operate on the main interface control module, and issue corresponding instructions to the workstation through the business logic scheduling module. The instructions read relevant parameters in the data storage module through the data processing module, and transmit the relevant parameters to the hardware connection module through the software configuration module to execute the instructions. (S4) After the relevant instructions are completed, the extraction and enrichment of microorganisms are completed.

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