Integrated method for bacterium enrichment and sample preparation based on Coulter counting

Abstract

The invention provides an integrated method for bacterium enrichment and sample preparation based on Coulter counting, which can greatly shorten the detection time and improve the detection accuracy and is low in cost. The integrated method comprises the following specific steps in sequence: 1, designing a culture bottle; 2, configuring a transport case; 3, performing differential centrifugal operation; 4, preparing bacterial liquid of a microorganism pretreatment system; 5, growth situation judgment and sample application: a user only needs to operate according to equipment prompts, such as reagent adding and sample putting, the equipment can give a drug sensitivity detection result within three hours, and an accurate basis is provided for subsequent treatment.

Description

Technical Field

[0001] This application relates to the field of medicine, specifically to the field of precise drug sensitivity testing for sepsis, and particularly to an integrated method for bacterial enrichment and sample preparation based on Coulter counting. Background Art

[0002] Precise drug sensitivity testing for sepsis belongs to the cross-field of infectious diseases and clinical pharmacy. It mainly involves determining the sensitivity of the pathogenic bacteria causing sepsis to antibacterial drugs quickly and accurately through precise drug sensitivity testing techniques, so as to provide precise medication guidance for clinicians. This requires microbiological techniques to detect pathogenic bacteria, pharmaceutical knowledge to understand the antibacterial mechanism and pharmacokinetics and pharmacodynamics characteristics of drugs, as well as clinical diagnosis and treatment knowledge to comprehensively judge the patient's condition and apply appropriate drug treatment regimens.

[0003] In the prior art, medical staff first use a suitable puncture needle and vacuum blood collection tube to draw several milliliters to dozens of milliliters of blood samples from the patient's arm vein, label them with patient information tags after collection according to aseptic operation. After sample collection, it is quickly placed in a transport box with appropriate temperature and buffer device and delivered to the laboratory within 1 - 2 hours. In the laboratory biosafety cabinet, the blood is inoculated into a blood culture bottle, and at the same time, it is centrifuged at a speed of 3000 - 5000 revolutions per minute for 10 - 15 minutes to separate blood cells and plasma. The inoculated blood culture bottle is placed in an automated blood culture instrument that can provide a temperature of 35 - 37 °C, appropriate humidity and a 5% carbon dioxide gas environment. The instrument monitors the growth of bacteria by detecting the production of carbon dioxide or changes in bacterial metabolites. This culture process takes 1 - 7 days depending on the type and quantity of bacteria. When the instrument indicates that bacteria have grown, in the biosafety cabinet, bacteria are taken out from the culture bottle with a sterile inoculation loop, dropper or cotton swab and evenly spread on the surface of the agar plate medium for drug sensitivity testing.

[0004] There are many problems in the existing blood sample detection schemes.

[0005] In the sample collection and transportation link, after sample collection, it does not directly enter a suitable culture environment. Even if it is quickly placed in a transport box, it still takes 1 - 2 hours to be delivered to the laboratory. During this period, bacteria cannot be in the best growth environment because the traditional collection and transportation methods lack an integrated culture start-up design. This may cause the decline of bacterial activity, affect the quantity and type of bacteria in subsequent cultures, reduce the detection accuracy, and easily result in false negative results, thus delaying treatment.

[0006] In the sample processing stage, complex pre-treatment, including centrifugation and stratification, is required in the laboratory in a biosafety cabinet. This is because the traditional sample processing process is cumbersome and has not been effectively integrated with the collection and transportation stages, which increases the risk of sample contamination and interferes with subsequent testing. In the bacterial growth monitoring stage, the traditional carbon dioxide culture and monitoring method is used, relying on carbon dioxide incubators and automated blood culture instruments to monitor bacterial growth by detecting carbon dioxide production or changes in bacterial metabolites. Because bacteria need to grow for a certain period of time and metabolites accumulate to a certain degree before they can be detected, the culture time is long and the equipment dependence is high. This not only has a long detection cycle and makes it difficult to provide timely clinical treatment guidance for acutely infected patients, but also increases laboratory resource consumption and operating costs, raising the technical threshold. In the drug sensitivity test preparation stage, because it is based on original manual operations and lacks automation and integrated design, the bacteria extraction operation is cumbersome, manual-dependent, and requires transfer culture, which is time-consuming. This not only reduces detection efficiency and increases the risk of contamination, but also easily reduces bacterial activity, leading to deviations in drug sensitivity results, affecting the formulation of treatment plans, and may even lead to antibiotic abuse or poor treatment effects.

[0007] Chinese patent document CN116042773A discloses a method for rapid bacterial species identification and drug sensitivity detection, which can target a certain type of RNA bacteria, and achieve online ion concentration extreme difference enrichment based on complementary DNA-modified nanogold probes and microfluidic chips, so as to be used for ultra-low concentration pathogen identification and drug sensitivity testing. It can cope with the monitoring of various complex samples, complete bacterial species identification within 30 minutes, and quickly detect the sensitivity of bacteria to antibiotics in 2.5 hours.

[0008] Chinese patent document CN119061136A discloses a primer combination and kit for detecting sepsis resistance genes. The primer combination is designed for 6 regions of 12 sepsis resistance genes, specifically identifying multiple independent regions on each resistance gene, improving the sensitivity and specificity of resistance gene detection, and ensuring the accuracy of monitoring results. This technical solution can stably, efficiently and specifically monitor more than ten sepsis resistance genes.

[0009] However, there is no bacterial detection solution that integrates bacterial enrichment and sample preparation in the prior art. Summary of the invention

[0010] In order to solve the technical problems of long time and high cost in the prior art, especially for bacterial detection of sepsis, the present invention provides a low-cost integrated method for bacterial enrichment and sample preparation based on Coulter counting, which can significantly reduce detection time and improve detection accuracy.

[0011] According to one aspect of the present invention, a method for integrated bacterial enrichment and sample preparation based on Coulter counting is provided, and the specific steps are as follows: 1. Culture bottle design: The culture bottle is produced by an aseptic process, and precisely proportioned nutrients such as peptone, yeast extract, and inorganic salts are pre-added into the bottle to provide carbon source, nitrogen source, vitamins, and minerals for the growth of bacteria. 2. Transport box configuration: The transport box is equipped with a precise temperature control system and an efficient buffer device. High-precision temperature sensors and heating / cooling elements are built into the transport box, and precise temperature control is achieved through a microprocessor; the temperature setting range is adjusted according to the optimal growth temperature of the target bacteria. 3. Differential centrifugation operation: Transfer the bacterial liquid in the culture bottle after 20 hours of cultivation to a sterile centrifuge tube, and set appropriate centrifugation parameters according to the characteristics of the target bacteria; first centrifuge at a lower speed for 5 - 10 minutes to precipitate larger cell debris and impurities to the bottom of the centrifuge tube, while the bacteria remain suspended in the supernatant; carefully aspirate the supernatant and transfer it to a new centrifuge tube, then centrifuge at a higher speed for 10 - 15 minutes, at this time the bacteria will precipitate to the bottom of the centrifuge tube; discard the supernatant, add an appropriate amount of sterile normal saline or buffer solution, and gently pipette to resuspend the bacterial precipitate to obtain an enriched bacterial suspension. 4. Preparation of bacterial liquid in the microbial pretreatment system: Connect the enriched bacterial suspension to the microbial pretreatment system using the Coulter principle; when the bacterial suspension passes through the small hole, each bacterium will change the resistance inside and outside the small hole, generating an electrical pulse signal; the signal processing module inside the system amplifies, discriminates, and counts these electrical pulses, and at the same time automatically performs different operations according to the counting results. 5. Judgment of growth trend and sample application: The user performs differential centrifugation on the sample, then counts using the Coulter principle, and compares with the standard to judge whether the sample is positive or not; the system compares the counting result with the pre-set positive judgment standard; if the counting result is lower than this standard, the system automatically determines that the sample is negative, stops the subsequent operations, and gives a clear prompt; if the counting result is higher than this standard, the system determines that the sample is positive, and at this time the precise quantitative pipetting device and mixing module inside the system are activated, and an appropriate amount of sterile diluent is automatically added according to the required bacterial liquid concentration for the drug sensitivity test to prepare for the next drug sensitivity test; the user then uses the microbroth dilution method to perform drug sensitivity detection; the user only needs to operate according to the device prompts, such as adding reagents, putting in samples, etc., and the device will give the drug sensitivity test result within three hours, providing an accurate basis for subsequent treatment.

[0012] According to the present invention, it is possible to achieve: Quick start of cultivation: The sample can be directly collected into the culture bottle and placed in a transport box with appropriate temperature and buffer device to start cultivation immediately, greatly saving the transport time and the centrifugation step of sample pretreatment, and reducing the risk of loss and deterioration of the sample during transport and processing.

[0013] Innovative Growth Monitoring and Red Blood Cell Processing: Abandon the traditional method of monitoring bacterial growth using carbon dioxide, and adopt a unique design that does not require this monitoring means. At the same time, the culture bottle contains components such as hemolysin, which can directly break down blood red blood cells, simplify the sample processing process, and avoid errors and increased costs caused by additional operations.

[0014] Efficient and Accurate Bacterial Counting and Determination: All bacteria in the enrichment bottle are concentrated by differential centrifugation in 20 hours, and then the Coulter counting method is used to accurately count the number of bacteria. Using a more accurate value as the positive determination standard, the determination process is clear and the result is accurate, ensuring the consistency and comparability of the detection results.

[0015] Rapid Antibiotic Sensitivity Testing: The enriched positive bacteria are directly diluted to an appropriate concentration, and the microbroth dilution method for rapid antibiotic sensitivity testing technology is used to complete the entire process from sample collection to antibiotic sensitivity results within two days, greatly shortening the detection time and improving the experimental efficiency.

[0016] According to a further optimized solution based on the technical concept of the present invention, the temperature of the transport box is stabilized at 37 ± 0.5 °C.

[0017] According to a further optimized solution based on the technical concept of the present invention, the buffer device uses multi-layer seismic materials such as sponge, foam or plastic to tightly wrap the culture bottle and reduce vibration transmission.

[0018] According to a further optimized solution based on the technical concept of the present invention, an appropriate amount of buffer substance, such as phosphate buffer solution, is added to the culture bottle to stabilize the pH value in the bottle; in addition, an appropriate amount of hemolysin is added to the bottle.

[0019] According to a further optimized solution based on the technical concept of the present invention, the lower rotation speed is 500 - 1000 revolutions per minute and the higher rotation speed is 5000 - 8000 revolutions per minute.

[0020] According to a further optimized solution based on the technical concept of the present invention, the positive determination standard is 600.

[0021] According to a further optimized solution based on the technical concept of the present invention, as an alternative: Alternative Solution to Change the Composition of the Culture Bottle Technical Principle: On the basis of the original culture bottle components, a new combination of nutrients and a red blood cell lysing agent are introduced. The traditional culture bottle nutrient components are mainly common peptone, yeast extract, etc. The new solution adds an optimized ratio of specific amino acids and vitamins to enhance the culture effect on various pathogenic bacteria. At the same time, a new type of biological enzyme is selected as the red blood cell lysing agent, which can specifically recognize a specific glycoprotein structure on the red blood cell membrane, destroy the cell membrane through an enzymatic reaction, and has less impact on bacteria.

[0022] Operation method: When producing culture bottles, add the optimized nutrients and the new biological enzyme in precise proportions. When collecting samples, directly inject the blood into the culture bottle and gently shake to start the culture. During transportation, the culture bottle maintains suitable conditions inside the transport box. After arriving at the laboratory, subsequent detection steps can be directly carried out without additional red blood cell treatment operations.

[0023] In this alternative, the optimized nutrient components can increase the bacterial growth rate and activity, reduce the time required for detection, and improve the positive detection rate. The new red blood cell lysing agent precisely lyses red blood cells, reduces interference with bacteria, and improves detection accuracy.

[0024] Explanation of professional terms counted in the present invention Coulter counting method: This is a cell or particle counting technique based on the principle of electrical impedance. When an electrolyte solution containing suspended particles (such as bacteria) passes through a narrow small hole, each particle passing through the small hole will instantaneously change the resistance inside and outside the small hole, thereby generating an electrical pulse signal. By counting these electrical pulse signals, the number of particles passing through the small hole can be accurately counted, thereby achieving the precise determination of the number of bacteria.

[0025] Drug susceptibility test: That is, the drug sensitivity test, whose main purpose is to determine the inhibitory effect of antibacterial drugs on bacteria in vitro. It determines the sensitivity of bacteria to various drugs by detecting the effects of different antibacterial drugs on bacterial growth, thereby providing a scientific basis for clinicians to select appropriate antibacterial drugs for treatment. For example, in the present invention, the drug susceptibility test can help doctors understand which antibiotic is most effective against the enriched positive bacteria to achieve precise treatment.

[0026] Minimum inhibitory concentration (MIC): It refers to the lowest drug concentration that can inhibit the growth of bacteria in the culture medium after culturing bacteria in vitro for 18 - 24 hours. It is an important indicator to measure the antibacterial activity of antibacterial drugs. In the microbroth dilution method, the minimum inhibitory concentration is determined by observing the growth of bacteria in broth media with different concentrations of antibiotics. Once the MIC of a certain bacterium to a specific antibiotic is determined, doctors can select appropriate drug doses based on this to ensure the treatment effect while reducing the adverse reactions and the generation of drug resistance of the drugs.

[0027] Microbroth dilution method: It is a commonly used drug susceptibility test method. It dissolves different concentrations of antibacterial drugs in broth media, and then inoculates the same number of bacteria to be tested respectively. After culturing for a certain period of time, observe the growth of bacteria in drugs with different concentrations. Take the lowest drug concentration that can inhibit the growth of bacteria as the minimum inhibitory concentration (MIC). This method is relatively simple to operate and can more accurately reflect the sensitivity of bacteria to different antibacterial drugs, and is widely used in clinical microbiology laboratories.

[0028] The following briefly describes the implementation principle.

[0029] 1. Principle of sample transportation and culture initiation: The culture bottle is designed to directly collect samples. Various nutrients, buffers, and specific components (such as hemolysin) suitable for microbial growth are pre-added inside it. These components ensure that bacteria have sufficient nutrients and a stable acid-base environment after collection, and hemolysin can lyse red blood cells in the subsequent process. When the sample is collected into the culture bottle, since the transport box is equipped with a suitable temperature and buffer device, it can effectively isolate adverse factors such as external temperature changes and vibrations, ensuring that the activity of microorganisms in the sample is not affected during transportation. This enables the culture to start immediately after the sample is collected. The principle is to simulate a stable culture environment in the laboratory, allowing microorganisms to be in the best growth state during transportation. The temperature control system maintains the suitable temperature for bacterial growth, and the buffer device prevents damage to the sample caused by vibrations during transportation.

[0030] 2. Principle of integrating bacterial growth monitoring and sample preparation based on Coulter counting: This invention breaks through the tradition and adopts an innovative method. It uses Coulter counting to directly collect bacteria in the bottle within 20 hours to monitor the growth of bacteria and prepare for subsequent drug sensitivity tests. During the 20-hour process of bacterial culture, bacteria continuously grow and reproduce in the nutrient environment of the culture bottle, and the quantity keeps changing. When the culture time reaches the critical node of 20 hours, the Coulter counting method is directly used to collect and count all bacteria in the bottle. Since the growth of bacteria is closely related to their growth state, if the bacteria grow well, their quantity will reach a corresponding scale within 20 hours. The result of the bacterial quantity obtained through Coulter counting can not only intuitively reflect the growth trend of bacteria during this period, but more importantly, it provides a sample that meets the requirements for the subsequent experimental process. These bacteria whose quantity has been determined through counting can be directly used for subsequent drug sensitivity tests. It omits the cumbersome steps such as additional sample collection and processing in the traditional process after monitoring the growth of bacteria, making the entire experimental process closely connected, greatly improving the experimental efficiency, reducing the errors that may be introduced due to multiple sample transfers and processing, and laying a solid foundation for quickly and accurately conducting drug sensitivity tests.

[0031] 3. Principle of red blood cell lysis: Components such as hemolysin in the culture bottle can specifically damage the cell membrane structure of blood red blood cells, releasing their contents without affecting the activity and growth of target bacteria. Its mechanism of action is based on the interaction between hemolysin and specific components on the red blood cell membrane to achieve directional lysis of red blood cells.

[0032] 4. Bacterial enrichment and counting principle: Differential centrifugation utilizes the difference in the sedimentation rates of particles at different rotational speeds to separate and enrich bacteria from other impurities. Coulter counting is based on the principle of electrical impedance. When bacteria pass through a small pore, they change the resistance inside and outside the pore, generating electrical pulse signals. By counting these signals, the accurate determination of the number of bacteria can be achieved.

[0033] 5. Antibiotic susceptibility testing principle: The microbroth dilution method determines the minimum inhibitory concentration (MIC) by culturing different concentrations of antibiotics with bacteria in a broth medium. By optimizing the detection process, using rapid-response reagents, or utilizing advanced detection equipment, the determination process of MIC is accelerated, thus enabling rapid antibiotic susceptibility testing.

[0034] In the present invention, during the integration process, the interaction scheme for user intervention can be as follows: Interaction between the user and sample collection and transportation: The user only needs to collect the sample into a specially designed culture bottle, ensuring that the collection process complies with aseptic operation specifications. Then, place the culture bottle into a transportation box equipped with an appropriate temperature and buffer device and start the transportation. During the entire transportation process, the user does not need to perform additional operations, and the transportation box will automatically maintain the appropriate culture environment for the sample.

[0035] Interaction between the user and bacterial culture and monitoring: During transportation, the culture proceeds simultaneously. The user does not need to monitor the carbon dioxide concentration as in the traditional method, reducing the operational complexity. When the sample is transported to the laboratory, the user can directly proceed to the next step without performing the centrifugation step of sample pretreatment.

[0036] Interaction for bacterial counting and determination: After culturing for 20 hours, the user performs differential centrifugation on the sample and then counts using the Coulter principle, and compares with the standard to determine whether the sample is positive or negative. After adding the bacterial suspension dilution step, the user needs to first gradient-dilute the enriched bacterial suspension to determine the optimal ratio and then perform counting and determination.

[0037] Interaction between the user and antibiotic susceptibility testing: If the sample is determined to be positive, the user then uses the microbroth dilution method for antibiotic susceptibility testing. The user only needs to operate according to the device prompts, such as adding reagents and placing the sample, etc. The device will give the antibiotic susceptibility testing result within three hours, providing an accurate basis for subsequent treatment.

[0038] According to the technical solution of the present invention, it has the following outstanding technical effects: Significantly shorten the detection time: Traditional drug susceptibility testing usually takes several days to obtain results. However, through optimized reaction conditions, innovative signal transduction mechanisms, and rapid detection and analysis methods, this solution can determine the minimum inhibitory concentration (MIC) in a short time. With an integrated design starting from sample collection, it simplifies transportation and pretreatment steps. The rapid drug susceptibility testing technology is integrated, using advanced sensing mechanisms and rapid reaction systems, significantly shortening the detection time, improving accuracy and stability, and accelerating the culture and detection processes. From sample processing to finally obtaining drug susceptibility results, the entire process can be completed within two days, greatly shortening the detection cycle and winning precious time for clinical treatment.

[0039] Simplify the operation process: Abandon complex intermediate steps and cumbersome operation processes. Traditional methods may involve multiple sample transfers, complex monitoring means, and a large number of manual operation links. This invention integrates multiple technologies, making the operation more concise and efficient.

[0040] Improve the detection accuracy: It can more precisely capture the subtle changes during the interaction between bacteria and antibiotics. By combining Coulter counting with the statistical analysis of the number of bacteria at specific time points to monitor growth, without the need for additional carbon dioxide monitoring equipment, it is more simple and efficient. Compared with traditional methods, it can more accurately determine the minimum inhibitory concentration, providing a more reliable basis for clinical medication and avoiding treatment failures or antibiotic abuse caused by inaccurate drug susceptibility results.

[0041] Reduce costs: Although some advanced technologies and equipment are introduced, overall, due to the shortening of the detection time and the simplification of the operation process, the input of human, material, and time costs is reduced. The consumption of culture media required for long-term sample culture is reduced, and the additional costs caused by repeated detections are lowered. Brief Description of the Drawings

[0042] Figure 1 It is a flowchart of the integration of sample collection, transportation, and culture in the integrated method for bacterial enrichment and sample preparation based on Coulter counting according to an embodiment of the present invention; Figure 2 It is a graph showing the growth of bacteria within 72 hours in the integrated method for bacterial enrichment and sample preparation based on Coulter counting according to an embodiment of the present invention. Detailed Embodiments

[0043] To make the objectives, technical solutions, and advantages of the present invention clearer and more understandable, the present invention will be described in detail below with reference to the accompanying drawings and specific embodiments. It should be noted that the embodiments in this application are exemplary, and the present invention is not limited to this specific implementation. Example 1

[0044] Objective: To verify that the enrichment experiment of the sterile culture bottle in the spesis precise medication method can meet the drug sensitivity conditions and the time for normal drug sensitivity tests.

[0045] Materials and methods: 1. Bacterial strain preparation: Number the Escherichia coli ATCC25922 as 1-1, 1-2, 1-3, the Staphylococcus aureus ATCC25923 as 2-1, 2-2, 2-3, and the Pseudomonas aeruginosa ATCC27853 as 3-1, 3-2, 3-3. Transfer and inoculate for standby and incubate at 37 °C for 18 hours.

[0046] 2. Reagent preparation: Prepare the bacterial strains, grind them on the wall of the AST (antibiotic susceptibility test) broth, mix well, cover the bottle cap, measure the turbidity with a turbidimeter (BD Company PhoenixSpec Nephelomter), the turbidity is 0.5 McFarland units, dilute the bacterial liquid and add 5 - 30 bacterial strains into the sterile culture bottle of this patent for standby.

[0047] After reaching the test time, inoculate the bacteria to be tested at the inoculation concentration of the CLSI (Clinical and Laboratory Standards Institute) broth dilution method for drug sensitivity test.

[0048] Instrument preparation: Blood culture instrument, high-speed centrifuge, microorganism pretreatment system.

[0049] Experimental method Place three bottles of each of the three prepared bacterial liquids in the incubator of this patent for 2 hours to simulate the transportation state, set the temperature at 37 °C, then transfer them to the automatic blood culture instrument for cultivation. Take out three bottles of bacterial liquid in sequence at 15h, 20h, 24h, and 72h. First, centrifuge at a speed of 800 revolutions per minute for 8 minutes, take the supernatant, and then centrifuge at a speed of 6000 revolutions per minute for 12 minutes. Discard the supernatant, add 5 ml of sterile normal saline, gently pipette to resuspend the bacterial precipitate, obtain the enriched bacterial suspension, mix well and measure the bacterial liquid with the microorganism pretreatment system.

[0050] The test results are shown in Table 1:

[0051] Conclusion: The sterile culture bottle of the Spesis precise medication method can detect the number of bacteria that meet the drug sensitivity test conditions at 20 hours.

[0052] Example 2 Objective: To verify the detection consistency of the method of the present invention for Gram-positive bacteria (Staphylococcus aureus) and Gram-negative bacteria (Escherichia coli) and the accuracy of drug sensitivity results.

[0053] Materials and methods Bacterial strain preparation: Escherichia coli ATCC25922 (Gram-negative bacterium), numbered 1-1, 1-2, 1-3.

[0054] Staphylococcus aureus ATCC25923 (Gram-positive bacterium), numbered 2-1, 2-2, 2-3.

[0055] The strains were incubated at 37 °C for 18 hours, and the turbidity was adjusted to 0.5 McFarland units.

[0056] Reagents and instruments: Antibiotics: Vancomycin (for Gram-positive bacteria), Ceftazidime (for Gram-negative bacteria).

[0057] Culture bottles (containing hemolysin and optimized nutritional components).

[0058] Instrument preparation High-speed centrifuge, microbial pretreatment system, transport box (37 ± 0.5 °C), blood culture instrument, high-speed centrifuge.

[0059] Experimental method Sample treatment: The bacterial suspension was injected into the culture bottle under aseptic operation and immediately placed in the transport box to simulate a 2-hour transportation.

[0060] After transportation, the blood culture instrument was used to culture for 20 hours, and the bacterial suspension was transferred to a centrifuge tube for differential centrifugation: Centrifuge at a low speed of 800 rpm for 8 minutes, and take the supernatant. Then centrifuge at a high speed of 6000 rpm for 12 minutes, discard the supernatant, and resuspend with 5 mL of sterile normal saline.

[0061] Coulter counting and determination: Use the microbial pretreatment system to count the bacteria to determine whether it is a positive sample. If it is a positive sample, a bacterial suspension with a concentration of 5×10 5 cfu / ml is prepared for the drug sensitivity test.

[0062] Drug sensitivity detection: Use drug sensitivity plates coated with different concentrations of vancomycin and ceftazidime antibiotics respectively, together with positive and negative control wells, as the experimental drug sensitivity plates in this experiment. Add 500 μL of the bacterial suspension 5×10 5 CFU / mL (the recommended inoculation concentration by CLSI), and gently shake and mix well.

[0063] Cover the microplate with a sealing film, and use a drug sensitivity instrument to test its MIC.

[0064] Test results

[0065] Conclusion Bacterial count repeatability: The CV value of Gram-negative bacteria (Escherichia coli) is 1.7%, and the CV value of Gram-positive bacteria (Staphylococcus aureus) is 7.6%, both meeting the CLSI requirements for repeatability (CV < 10%). MIC coincidence rate: The MIC values of 6 test samples are exactly the same as the CLSI standard, with a coincidence rate of 100% and an error rate of 0%. Example 3 Experimental purpose: Systematically verify the optimal combination of differential centrifugation parameters (low speed 500 - 1000 rpm, high speed 5000 - 8000 rpm), and clarify the influence of rotation speed and time on the enrichment efficiency of the target bacteria.

[0066] Materials and Methods 1. Bacterial strain culture and sample preparation Bacterial strain: Staphylococcus aureus ATCC25923, cultured at 37°C for 18 hours, and then the turbidity of the bacterial solution was adjusted to 0.5 McFarland units using a BD turbidimeter. At this time, the corresponding concentration was 1.5×10 8 CFU / mL.

[0067] Simulated clinical sample: Take 10 μL of the above bacterial solution and inoculate it into 990 μL of sterile broth medium to prepare a sample with an initial bacterial concentration of 1.5×10 6 CFU / mL. Inject this sample into the culture bottle, and the culture bottle is added with the nutritional components of the patented formula to provide a good growth environment for the bacteria.

[0068] Synchronous culture: Place the culture bottle in a transport box at 37 ± 0.5°C and culture for 20 hours to allow the bacteria to enter the logarithmic growth phase. After the culture is completed, use the Coulter counting method to determine the total number of bacteria in the cultured bacterial solution.

[0069] 2. Group design and centrifugation operation

[0070] 3. Detection indicators Bacterial recovery rate (%): Use the Coulter counting method to measure the total number of bacteria in the cultured bacterial solution / the number of bacteria in the resuspended solution after centrifugation × 100%.

[0071] Enrichment time (minutes): Low-speed time + high-speed time Test results

[0072] Data analysis and optimization Recovery rate comparison The recovery rates of Group A and Group B are both significantly higher than those of the control group. This indicates that the differential centrifugation parameters can more effectively enrich the target bacteria and reduce bacterial loss. Enrichment Time and Efficiency Group B completed enrichment within 15 minutes and had the highest recovery rate. This shows the advantage of high rotational speed (8000 revolutions per minute) in achieving efficient enrichment in a short time, which is suitable for the detection of emergency samples with high time requirements. Although the enrichment time of Group A was 25 minutes, the recovery rate was also maintained at a high level, indicating that its parameter settings can also well meet the bacterial enrichment requirements.

[0073] Conclusion Effectiveness of Differential Centrifugation Parameters The high recovery rates of the differential centrifugation groups (Group A and Group B) verified the effectiveness of the differential centrifugation parameters. By the method of "preliminary concentration at low rotational speed + precise enrichment at high rotational speed", the recovery rate of the target bacteria can be significantly improved, ensuring sufficient bacterial quantity for subsequent detection such as drug sensitivity tests. Rationality of Parameter Range The parameter ranges of low rotational speed (500 - 1000 revolutions per minute) and high rotational speed (5000 - 8000 revolutions per minute) are set reasonably. Different parameter combinations can be selected according to actual needs (such as time urgency, sample characteristics, etc.) to achieve the best enrichment effect. Support for Patent Technology The results of this experiment provided strong experimental support for the differential centrifugation link of "preparation of bacterial liquid in the microbial pretreatment system" in the patent, proving that this technology can improve the enrichment efficiency of the target bacteria, and thus enhance the accuracy and reliability of the entire detection process.

[0074] Example 4 Objective: To verify the differences in detection time, bacterial count, and drug sensitivity results between the method of the present invention (directly incubating in a transport box for 2 hours and then transferring to a blood culture instrument for a total incubation time of 20 hours) and the traditional method, and to evaluate its superiority.

[0075] II. Materials and Methods (I) Bacteria Strains and Reagents Bacteria strains: Escherichia coli ATCC25922 (Gram-negative bacterium), Staphylococcus aureus ATCC25923 (Gram-positive bacterium), 3 strains each, incubated at 37°C for 18 hours, and the turbidity was adjusted to 0.5 McFarland units.

[0076] Reagents: Special culture bottles of the present invention (containing hemolysin and optimized nutritional components), traditional blood culture bottles, sterile normal saline, antibiotic drug sensitivity plates (containing vancomycin, ceftazidime).

[0077] Instruments: Transport box (37 ± 0.5°C), blood culture instrument, high-speed centrifuge, microbial pretreatment system (Coulter counting), turbidimeter, rapid drug sensitivity instrument.

[0078] Experimental method: In the group of the present invention (experimental group): The sample is injected into a dedicated culture flask → incubated in a transport box at 37°C for 2 hours → transferred to a blood culture instrument and continuously cultured for 20 hours → differential centrifugation (low speed 800 rpm × 8 min to take the supernatant, high speed 6000 rpm × 12 min, the precipitate is resuspended with normal saline) → Coulter counting → drug sensitivity test (micro broth dilution method, bacterial concentration 5×10 5 CFU / mL).

[0079] Traditional method group (control group): Traditional intravenous blood collection → transported in a transport box for 2 hours (without constant temperature culture) → centrifuged in the laboratory (3000 rpm × 15 min) → inoculated into the original blood culture flask → cultured in a blood culture instrument until positive (average 48 hours, with CO2 monitoring alarm as the positive standard) → the bacteria are taken and spread on an agar plate → subcultured for 18 hours → prepare a bacterial suspension → drug sensitivity test (same as the experimental group).

[0080] Detection indexes Total detection time: The time from sample collection to obtaining the drug sensitivity result.

[0081] Bacterial count: The concentration of enriched bacteria (CFU / mL) is measured by the Coulter counting method.

[0082] Drug sensitivity result: Minimum inhibitory concentration (MIC), coincidence rate compared with the CLSI standard.

[0083] Test results (I) Comparison of total detection time

[0084] Bacterial count and positive determination

[0085] Drug sensitivity result (comparison of MIC values)

[0086] IV. Conclusion The detection time is significantly shortened: The total time-consuming of the group of the present invention is 23 hours, which is shorter than that of the traditional method group (66 - 68 hours). This is mainly due to the synchronous culture in the transport box (avoiding bacterial stagnation during traditional transportation) and the efficient enrichment of differential centrifugation and Coulter counting, eliminating the cumbersome steps such as centrifugation stratification and subculturing in the traditional method.

[0087] The bacterial count is consistent with the positive determination: Positive samples (the number of bacteria is much higher than the determination standard of 600) are obtained in both groups after culture, indicating that the method of the present invention is equivalent to the traditional method in terms of bacterial enrichment efficiency, but does not rely on the monitoring of CO2 metabolites, and can be directly determined by regular counting at 20 hours, simplifying the monitoring process.

[0088] The drug sensitivity results are highly accurate: the MIC values are completely consistent with the CLSI standards and traditional methods, with a coincidence rate of 100%, verifying the reliability of the method of the present invention in the accuracy of drug sensitivity detection, and avoiding the risk of decreased bacterial activity or contamination that may be caused by subculture in traditional methods.

[0089] The above are only the preferred embodiments of the present invention. Of course, the present invention can also have many other embodiments. Without departing from the spirit and essence of the present invention, those skilled in the art can make various corresponding changes and deformations according to the present invention. However, these corresponding changes and deformations should all fall within the protection scope of the appended claims of the present invention.

Claims

1. An integrated method for bacterial enrichment and sample preparation based on Coulter counting, characterized in that, The specific steps are as follows: (1)Culture bottle design: The culture bottle is produced by aseptic technology, and nutrient components are pre-added in the bottle to provide carbon source, nitrogen source, vitamins and minerals for bacterial growth; (2)Transport box configuration: The transport box is equipped with a temperature control system and a buffer device, with a temperature sensor and heating / cooling elements built-in, and the temperature is controlled through a microprocessor; the temperature setting range is adjusted according to the optimal growth temperature of the target bacteria; (3)Differential centrifugation operation: Transfer the bacterial liquid in the cultured culture bottle to a sterile centrifuge tube, and set appropriate centrifugation parameters according to the characteristics of the target bacteria; centrifuge at a lower speed to precipitate larger cell debris and impurities to the bottom of the centrifuge tube, aspirate the supernatant and transfer it to a new centrifuge tube, centrifuge at a higher speed, discard the supernatant, and add an appropriate amount of sterile physiological saline or buffer to obtain an enriched bacterial suspension; (4)Preparation of bacterial liquid in the microbial pretreatment system: Transfer the enriched bacterial suspension into the microbial pretreatment system using the Coulter principle; when the bacterial suspension passes through the small hole, each bacterium generates an electrical pulse signal; amplify, discriminate and count these electrical pulses, and automatically perform different operations according to the counting results; (5)Judgment of growth trend and sample application: The user performs differential centrifugation on the sample, then counts using the Coulter principle, and compares with the standard to judge whether the sample is positive or not; the system compares the counting result with the pre-set positive judgment standard; if the counting result is lower than this standard, the system automatically determines that the sample is negative and stops subsequent operations; if the counting result is higher than this standard, the system determines that the sample is positive, and automatically adds an appropriate amount of sterile diluent according to the required bacterial liquid concentration for the drug sensitivity test to prepare for the next drug sensitivity test.

2. The method according to claim 1, characterized in that, The temperature of the transport box is stabilized at 37 ± 0.5 °C.

3. The method according to claim 2, wherein The buffer device uses multi-layer shock-absorbing materials such as sponge, foam or plastic to tightly wrap the culture bottle and reduce vibration transmission.

4. The method according to claim 3, characterized in that, An appropriate amount of buffer substance is added to the culture bottle to stabilize the pH value in the bottle.

5. The method according to claim 4, wherein The appropriate buffer substance added to the culture bottle is phosphate buffer solution.

6. The method according to claim 4, wherein In addition, an appropriate amount of hemolysin is added to the bottle.

7. The method according to claim 1, characterized in that, The lower speed is 500 - 1000 revolutions per minute.

8. The method according to claim 1, characterized in that, The higher speed is 5000 - 8000 revolutions per minute.

9. The method according to claim 1, wherein The positive judgment standard is 600.

10. The method according to claim 1, characterized in that When producing the culture bottle, the optimized nutrients and new biological enzymes are added in precise proportions. When collecting the sample, directly inject the blood into the culture bottle, gently shake it to start the culture. During transportation, the culture bottle maintains suitable conditions in the transport box.

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