A kit for detecting a blood stream infection pathogen

Abstract

The application provides a kit for detecting blood stream infection pathogens, and belongs to the technical field of immunoassay. The kit comprises an optimized sample processing solution and a coating solution. The sample processing solution takes PBS buffer as a base and comprises Triton X-100, saponin, polyethylene glycol 6000, PC300, EDTA-Na2, BSA and sodium citrate. The coating solution takes carbonate buffer as a base and comprises trehalose, SDS, methanol, BSA, PC300 and proline. The kit provided by the application can realize high-sensitivity, high-specificity and rapid detection of blood stream infection pathogens, and has the advantages of storage stability, wide application range, strong compatibility and suitability for rapid detection of common bacteria, fungi and other pathogens in clinical blood stream infection.

Description

Technical Field

[0001] This invention belongs to the field of immunoassay technology, specifically relating to a kit for detecting bloodstream pathogens. Background Technology

[0002] Bloodstream infections (BSIs) are common and serious infectious diseases in clinical practice. Pathogens mainly include Gram-positive bacteria (such as Staphylococcus aureus), Gram-negative bacteria (such as Escherichia coli and Klebsiella pneumoniae), and fungi such as Candida. They have a rapid onset and progression; if pathogens are not detected promptly and accurately, clinical treatment can be delayed, leading to serious complications such as sepsis and septic shock, and even endangering the patient's life. Currently, immunoassay methods for detecting bloodstream infection pathogens are widely used in clinical emergency screening due to their simplicity and speed. These methods rely on the effective release and purification of pathogen antigens in the sample, as well as the specific binding efficiency of antigens and antibodies.

[0003] Existing immunoassay kits face key technical bottlenecks: First, sample processing solutions are ineffective. Blood samples contain a large number of interfering substances such as red blood cells, white blood cells, and plasma proteins. Furthermore, pathogens are often encapsulated within blood cells or bound to plasma proteins. Conventional processing solutions can only lyse red blood cells, failing to fully release pathogen antigens and effectively remove interfering proteins. This results in low detection sensitivity and a high likelihood of false negatives, especially for low-load pathogens (e.g., ≤10). The detection results for CFU / mL and residual pathogens after antibiotic treatment are extremely poor. Secondly, the coating solution formulation is unreasonable; conventional coating solutions often use a single buffer system, which makes the coated antibodies prone to conformational changes and reduced activity. Furthermore, the coating stability is poor, leading to antibody shedding and decreased antigen-antibody binding efficiency, further affecting detection accuracy and the storage stability of the kit. Thirdly, existing kits lack anti-interference adaptation designs and cannot handle common clinical samples such as hemolysis, lipemia, and jaundice, which severely interfere with immune binding reactions, resulting in distorted test results. Fourthly, the kits have poor adaptability, failing to meet the diverse needs of rapid emergency testing, low-cost testing at the grassroots level, and accurate detection of difficult infections, and lack compatibility with existing testing equipment.

[0004] Therefore, developing an immunoassay kit that can effectively solve the above-mentioned technical bottlenecks, achieve high sensitivity, high specificity, rapid detection of bloodstream pathogens, and is stable in storage, has a wide range of applications, and strong compatibility has become one of the key issues that urgently need to be addressed in this field. Summary of the Invention

[0005] To address the problems existing in the prior art, the present invention aims to provide a reagent kit for detecting bloodstream infection pathogens and its preparation method. By optimizing the formulation of the sample processing solution and coating solution, the kit achieves high sensitivity, high specificity, and rapid detection of bloodstream infection pathogens, while also being stable in storage, widely applicable, and highly compatible.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a kit for detecting bloodstream infection pathogens, comprising a sample processing solution and a coating solution; The sample processing solution, based on PBS buffer, contains, by weight and volume percentage: 0.18%–0.22% Triton X-100, 0.09%–0.11% saponins, 0.35%–0.45% polyethylene glycol 6000, 0.02%–0.04% PC300, 0.05%–0.07% EDTA-Na2, 0.01%–0.02% BSA, and 0.005%–0.01% sodium citrate. The coating solution is based on carbonate buffer and contains, by mass-volume percentage, 0.12%~0.14% trehalose, 0.095%~0.105% SDS, 0.03%~0.05% methanol, 0.02%~0.03% BSA, 0.01%~0.015% PC300 and 0.003%~0.005% proline.

[0007] Preferably, the concentration of the PBS buffer is 0.01 mol / L and the pH value is 7.2~7.4.

[0008] Preferably, the concentration of the carbonate buffer solution is 0.05 mol / L, and the pH value is 9.5~9.8.

[0009] Preferably, it also includes a detection plate, monoclonal antibody, enzyme-labeled secondary antibody, washing solution, colorimetric solution and stop solution.

[0010] Preferably, the monoclonal antibody includes any one or more of the following: Escherichia coli monoclonal antibody, Klebsiella pneumoniae monoclonal antibody, Acinetobacter baumannii monoclonal antibody, Pseudomonas aeruginosa monoclonal antibody, Staphylococcus aureus monoclonal antibody, and Candida albicans monoclonal antibody.

[0011] Preferably, the enzyme-labeled secondary antibody is horseradish peroxidase-labeled goat anti-mouse IgG antibody.

[0012] Preferably, the washing solution is based on PBS buffer and comprises, by mass-volume percentage, 0.05% Tween-20, 0.01% PC300, and 0.005% EDTA-Na2.

[0013] Preferably, the colorimetric solution is a TMB colorimetric solution.

[0014] Preferably, the terminating solution is a 2 mol / L H2SO4 solution.

[0015] The present invention also provides a method for preparing the above-mentioned kit, wherein the method for preparing the sample processing solution includes: adding Triton X-100, saponin, PEG 6000, PC300, EDTA-Na2, BSA, and sodium citrate sequentially to PBS buffer, stirring magnetically for 30-40 min until completely dissolved, adjusting the pH, and aseptically filtering; the method for preparing the coating solution includes: adding trehalose, SDS, methanol, BSA, PC300, and proline sequentially to carbonate buffer, stirring magnetically for 20-30 min until completely dissolved, adjusting the pH, and aseptically filtering.

[0016] Compared with the prior art, the beneficial effects of the technical solution of the present invention are as follows: This invention addresses the problems of existing immunoassay kits, such as poor sample processing solution lysis, incomplete interference removal, inability to handle hemolyzed / lipemic / jaundiced samples, poor antibody activity maintenance and inadequate stability in the coating solution, poor kit adaptability and compatibility, and poor synergy of supporting components, leading to low detection sensitivity, low specificity, short storage period, and limited applicability. Based on the principle of double-antibody sandwich immunoassay, this invention optimizes the formulation of the sample processing solution and coating solution to achieve efficient release and purification of pathogen antigens and efficient antigen-antibody binding, resulting in high-sensitivity and high-specificity detection. It also improves the kit's anti-interference ability, adaptability, and compatibility, making it particularly suitable for low-viral-load infections, residual pathogen detection after antibiotic treatment, and use in primary healthcare institutions. It can effectively handle abnormal blood samples such as hemolysis, lipemia, and jaundice, meeting the needs for rapid and accurate detection in various scenarios, including clinical emergency, primary care, and complex infections. Detailed Implementation

[0017] This invention provides a kit for detecting bloodstream pathogens, including a sample processing solution and a coating solution.

[0018] The sample processing solution of this invention uses PBS buffer as a base and, by weight / volume percentage (w / v), contains 0.18%~0.22% Triton X-100, 0.09%~0.11% saponins, 0.35%~0.45% polyethylene glycol 6000, 0.02%~0.04% PC300, 0.05%~0.07% EDTA-Na2, 0.01%~0.02% BSA, and 0.005%~0.01% sodium citrate; preferably, it contains 0.2% Triton X-100, 0.1% saponins, 0.4% polyethylene glycol 6000, 0.03% PC300, 0.06% EDTA-Na2, 0.015% BSA, and 0.008% sodium citrate. The concentration of the PBS buffer is 0.01 mol / L, and the pH value is 7.2~7.4.

[0019] The preparation method of the sample processing solution of the present invention includes: adding Triton X-100, saponins, PEG 6000, PC300, EDTA-Na2, BSA, and sodium citrate sequentially to PBS buffer, stirring magnetically for 30-40 min until completely dissolved, adjusting the pH, and aseptically filtering; the aseptic filtration is preferably performed using a 0.22 μm filter membrane. The prepared sample processing solution of the present invention is sealed and stored at 4℃ for later use.

[0020] In the sample processing solution described in this invention, Triton X-100 is a nonionic surfactant that gently lyses the membranes of erythrocytes and pathogens, avoiding disruption of the spatial conformation of pathogen antigens, while also assisting in the dissolution of plasma protein interfering substances; saponins synergistically enhance the erythrocyte lysis effect with Triton X-100, while simultaneously promoting the release of pathogens encapsulated within blood cells, thus improving antigen recovery rate; polyethylene glycol 6000 (PEG) is also present. 6000) specifically precipitates interfering proteins (such as albumin and globulin) in plasma, reducing the impact of interfering substances on immune binding; PC300 acts as a preservative to prevent microbial contamination during sample processing and avoid antigen degradation; EDTA-Na2 chelates metal ions in the sample to avoid the inhibition of antibody activity by metal ions and stabilizes the structure of pathogen antigens; bovine serum albumin (BSA) acts as an anti-interference blocking agent to block non-specific binding sites in the sample in advance, avoiding non-specific binding of impurities in plasma to detection antibodies, and also helps stabilize pathogen antigens and reduce the false positive rate; sodium citrate acts as an anti-hemolytic and anti-jaundice interference agent to chelate free hemoglobin in hemolyzed samples and bilirubin in jaundice samples, avoiding their interference with immune binding and colorimetric reactions.

[0021] The sample processing solution described in this invention forms a synergistic system of "dual lysis + dual anti-interference + specific precipitation + stabilization" through optimized formulation. Specifically, Triton X-100 and saponins construct a dual lysis synergistic system, increasing erythrocyte lysis efficiency by 15% and pathogen antigen release rate to over 97%. BSA and sodium citrate, as dual anti-interference components, address interference issues in blood samples with hemolysis, lipemia, and jaundice, reducing false positive and false negative rates to 0. PEG 6000 and EDTA-Na2 synergistically achieve interference removal and antigen stabilization, forming a synergistic process that can detect pathogens as low as 3 CFU / mL, with processing time shortened to 10-15 minutes, suitable for emergency needs. The sample processing solution described in this invention is not only suitable for routine blood sample testing but also for testing patients with sepsis in the emergency department.

[0022] The coating solution of this invention uses carbonate buffer as a base and, by weight / volume percentage (w / v), contains 0.12%~0.14% trehalose, 0.095%~0.105% SDS, 0.03%~0.05% methanol, 0.02%~0.03% BSA, 0.01%~0.015% PC300, and 0.003%~0.005% proline. Preferably, it contains 0.13% trehalose, 0.1% SDS, 0.04% methanol, 0.025% BSA, 0.012% PC300, and 0.004% proline. The concentration of the carbonate buffer is 0.05 mol / L, and the pH value is 9.5~9.8.

[0023] The preparation method of the coating solution of the present invention includes: sequentially adding trehalose, SDS, methanol, BSA, PC300, and proline to a carbonate buffer solution, magnetically stirring for 20-30 minutes until completely dissolved, adjusting the pH, and aseptically filtering; the aseptic filtration is preferably performed using a 0.22 μm filter membrane. The coating solution of the present invention is sealed and stored at 4°C for later use.

[0024] In the coating solution of this invention, trehalose acts as a stabilizer to maintain the native conformation of the antibody and prevent denaturation and reduced activity during coating and storage; SDS assists in antibody adsorption onto the surface of the detection plate, enhancing coating stability and reducing antibody detachment; methanol improves the uniformity of antibody distribution on the detection plate surface and increases coating efficiency; bovine serum albumin (BSA) blocks non-specific binding sites on the detection plate surface and simultaneously helps maintain antibody activity, reducing false positives caused by non-specific binding; PC300 enhances the binding force between the antibody and the detection plate surface, further improving coating stability and extending the shelf life of the kit; proline acts as an anti-denaturing stabilizer, working synergistically with trehalose and BSA to prevent antibody denaturation under extreme conditions and enhance the binding affinity between the antibody and the antigen.

[0025] The coating solution of this invention forms a synergistic system of "triple synergistic stabilization + adsorption promotion + solubilization" through optimized formulation. Proline, trehalose, and BSA form a triple synergistic stabilizing system, increasing antibody activity maintenance by more than 20%. SDS and PC300 synergistically enhance antibody coating stability, while methanol improves antibody distribution uniformity, extending the kit's storage period at 4°C to 15 months and at room temperature to 8 months. It can be stably transported under extreme conditions ranging from -20°C to 35°C. The ionic strength of the coating solution described in this invention is compatible with mainstream 96-well microplates (polystyrene, polyvinyl chloride materials), exhibiting strong compatibility.

[0026] The kit described in this invention also includes a detection plate, monoclonal antibody, enzyme-labeled secondary antibody, washing solution, colorimetric solution, and stop solution.

[0027] The detection plate described in this invention can be a 96-well ELISA plate (made of polystyrene or polyvinyl chloride), with each well coated with a specific monoclonal antibody against common bloodstream pathogens and drug-resistant strains. The coating conditions can be selected as incubation at 37°C for 1.5 hours, followed by blocking overnight at 4°C. The monoclonal antibodies described in this invention include any one or more of the following: Escherichia coli monoclonal antibody, Klebsiella pneumoniae monoclonal antibody, Acinetobacter baumannii monoclonal antibody, Pseudomonas aeruginosa monoclonal antibody, Staphylococcus aureus monoclonal antibody, and Candida albicans monoclonal antibody. The monoclonal antibodies described in this invention can be from conventional commercial sources.

[0028] The enzyme-labeled secondary antibody described in this invention is a horseradish peroxidase-labeled goat anti-mouse IgG antibody, which can be selected from conventional commercial sources.

[0029] The washing solution described in this invention uses PBS buffer as a base and, by weight / volume percentage (w / v), comprises 0.05% Tween-20, 0.01% PC300, and 0.005% EDTA-Na2. The concentration of the PBS buffer is 0.01 mol / L, and the pH value is 7.2%~7.4. The washing solution can chelate residual metal ions, avoiding interference with the colorimetric reaction.

[0030] The colorimetric solution described in this invention is a TMB colorimetric solution, which can prolong colorimetric stability and improve colorimetric sensitivity.

[0031] The terminating solution described in this invention is a 2 mol / L H2SO4 solution, which can quickly terminate the colorimetric reaction and ensure stable results.

[0032] The technical solutions of this invention will be clearly and completely described below with reference to the embodiments thereof. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0033] In specific embodiments of this invention, the commercially available monoclonal antibodies were purchased from the following sources: Escherichia coli monoclonal antibody (catalog number: 130001-MM05) and Klebsiella pneumoniae monoclonal antibody (catalog number: 130002-MM08) were purchased from Beijing Yiqiao Shenzhou Technology Co., Ltd.; Acinetobacter baumannii monoclonal antibody (catalog number: D160375) and Pseudomonas aeruginosa monoclonal antibody (catalog number: D121044) were purchased from Shanghai Sangon Biotech Co., Ltd.; Staphylococcus aureus monoclonal antibody (catalog number: 66200-1-Ig) and Candida albicans monoclonal antibody (catalog number: 12110-1-AP) were purchased from Wuhan Sanying Biotechnology Co., Ltd.; all antibodies were murine monoclonal antibodies with a purity ≥95% and a titer of ≥1:10000. HRP-labeled goat anti-mouse IgG antibody (catalog number: bs-0296G-HRP) was purchased from Beijing Bio-Sen Biotechnology Co., Ltd., with a titer of ≥1:50000.

[0034] Unless otherwise specified, the following embodiments are all conventional methods.

[0035] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0036] Example 1 Kit for detecting bloodstream pathogens: sample processing solution, coating solution, detection plate, monoclonal antibody, enzyme-labeled secondary antibody, washing solution, chromogenic solution and stop solution.

[0037] The sample processing solution used PBS buffer (0.01 mol / L, pH 7.3) as a base, containing, by weight / volume percentage: 0.2% Triton X-100, 0.1% saponin, 0.4% polyethylene glycol 6000, 0.03% PC300, 0.06% EDTA-Na2, 0.015% BSA, and 0.008% sodium citrate. Preparation method: Triton X-100, saponin, PEG 6000, PC300, EDTA-Na2, BSA, and sodium citrate were added sequentially to the PBS buffer. The mixture was magnetically stirred for 35 min until completely dissolved. The pH was adjusted, and the solution was aseptically filtered through a 0.22 μm filter membrane and stored sealed at 4°C for later use.

[0038] The coating buffer, based on carbonate buffer (0.05 mol / L, pH 9.6), contained 0.13% trehalose, 0.1% SDS, 0.04% methanol, 0.025% BSA, 0.012% PC300, and 0.004% proline by weight / volume percentage. Preparation method: Trehalose, SDS, methanol, BSA, PC300, and proline were added sequentially to the carbonate buffer. The mixture was magnetically stirred for 25 min until completely dissolved. The pH was adjusted, and the solution was aseptically filtered through a 0.22 μm membrane and stored sealed at 4°C for later use.

[0039] The assay plate is a 96-well ELISA plate (made of polystyrene or polyvinyl chloride). Each well is coated with monoclonal antibodies against Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Staphylococcus aureus, and Candida albicans. The plates are incubated at 37°C for 1.5 hours and then blocked overnight at 4°C.

[0040] The enzyme-labeled secondary antibody is horseradish peroxidase-labeled goat anti-mouse IgG antibody, with a dilution ratio of 1:4000-1:6000 (adjustable to adapt to different concentrations of antigen detection).

[0041] The washing buffer was based on PBS buffer (0.01 mol / L, pH 7.3) and contained 0.05% Tween-20, 0.01% PC300, and 0.005% EDTA-Na2 by mass-volume percentage.

[0042] The colorimetric solution is TMB colorimetric solution.

[0043] The stop solution was a 2 mol / L H2SO4 solution.

[0044] Example 2 The kit for detecting bloodstream pathogens differs from Example 1 only in the preparation of the sample processing solution and coating solution: The sample processing solution was based on PBS buffer (0.01 mol / L, pH 7.2) and contained, by weight / volume percentage: 0.18% Triton X-100, 0.09% saponins, 0.35% polyethylene glycol 6000, 0.02% PC300, 0.05% EDTA-Na2, 0.01% BSA, and 0.005% sodium citrate. The preparation method was the same as in Example 1.

[0045] The coating solution was based on carbonate buffer (0.05 mol / L, pH 9.5) and contained 0.12% trehalose, 0.095% SDS, 0.03% methanol, 0.02% BSA, 0.01% PC300, and 0.003% proline by weight / volume percentage. The preparation method was the same as in Example 1.

[0046] Example 3 The kit for detecting bloodstream pathogens differs from Example 1 only in the preparation of the sample processing solution and coating solution: The sample processing solution was based on PBS buffer (0.01 mol / L, pH 7.4) and contained, by weight / volume percentage: 0.22% Triton X-100, 0.11% saponins, 0.45% polyethylene glycol 6000, 0.04% PC300, 0.07% EDTA-Na2, 0.02% BSA, and 0.01% sodium citrate. The preparation method was the same as in Example 1.

[0047] The coating solution was based on carbonate buffer (0.05 mol / L, pH 9.8) and contained 0.14% trehalose, 0.105% SDS, 0.05% methanol, 0.03% BSA, 0.015% PC300, and 0.005% proline by weight / volume percentage. The preparation method was the same as in Example 1.

[0048] Example 4 The kit for detecting bloodstream pathogens differs from Example 1 only in the preparation of the sample processing solution: The sample processing solution was based on PBS buffer (0.01 mol / L, pH 7.4) and contained, by weight / volume percentage: 0.22% Triton X-100, 0.11% saponins, 0.4% polyethylene glycol 6000, 0.03% PC300, 0.06% EDTA-Na2, 0.015% BSA, and 0.008% sodium citrate. The preparation method was the same as in Example 1.

[0049] Example 5 Instructions for use of the kits in Examples 1-4: 1. Blood sample processing Routine blood sample processing: Take 100 μL of clinical venous blood sample, add 200 μL of sample processing solution (Example 1, Example 2 or Example 3), vortex for 5 min, incubate at 37℃ for 10 min, centrifuge at 8000 r / min for 5 min, and take the supernatant as the test sample.

[0050] Blood sample processing for patients with emergency sepsis: Take 100 μL of clinical venous blood sample, add 200 μL of sample processing solution (Example 4), vortex for 5 min, incubate at 37°C for 5 min, centrifuge at 8000 r / min for 5 min, and take the supernatant as the test sample.

[0051] 2. Wrapped Take a 96-well microplate made of polystyrene or polyvinyl chloride and add 100 μL of antibody-coating working solution to each well, ensuring the liquid evenly covers the bottom of the well without air bubbles. Incubate the microplate at 37°C for 1.5 h to allow the antibody to fully adsorb onto the solid phase surface of the microplate. After coating, discard the liquid in the wells, add 200 μL of PBS to each well, let stand for 30 s, then shake dry. Repeat the washing process 3 times. Finally, invert the microplate on absorbent paper and gently pat dry to remove any residual liquid. Add 200 μL of 5% BSA blocking solution to each well, covering the entire solid phase surface and avoiding edge effects. Seal the microplate and incubate it overnight at 4°C to block any unbound sites on the microplate and reduce subsequent non-specific adsorption. The next day, remove the blocked microplate, discard the blocking solution, wash 3 times with PBS, and pat dry.

[0052] 3. Sample addition and incubation Add 100 μL of test sample to each well of the test plate, incubate at 37°C for 30 min, wash three times with washing solution for 30 seconds each time, and pat dry.

[0053] 4. Incubation with enzyme-labeled secondary antibody Dilute the enzyme-labeled secondary antibody, add 100 μL of enzyme-labeled secondary antibody (2 ng / mL) to each well, incubate at 37°C for 20 min, wash 5 times with washing buffer for 30 seconds each time, and pat dry.

[0054] 5. Color Development and Termination Add 100 μL of colorimetric solution to each well, incubate at 37°C in the dark for 15 min, add 50 μL of stop solution, and gently shake to mix.

[0055] 6. Result Determination The absorbance (OD value) of each well was measured using an ELISA reader at a wavelength of 450 nm. An OD value ≥ 0.3 was considered positive (corresponding pathogen present), and an OD value < 0.3 was considered negative (corresponding pathogen absent). Semi-quantitative criteria: OD values ​​of 0.3–0.5 indicated low viral load infection, 0.5–1.0 indicated medium viral load infection, and > 1.0 indicated high viral load infection, providing a preliminary assessment of the severity of the infection.

[0056] Comparative Example 1 The kit for detecting bloodstream pathogens differs from Example 1 only in that BSA and sodium citrate are removed from the sample processing solution.

[0057] Comparative Example 2 The kit for detecting bloodstream pathogens differs from Example 1 only in that the addition of saponins is removed from the sample processing solution.

[0058] Comparative Example 3 The kit for detecting bloodstream pathogens differs from Example 1 only in that polyethylene glycol 6000 (PEG 6000) is removed from the sample processing solution.

[0059] Comparative Example 4 The kit for detecting bloodstream pathogens differs from that in Example 1 only in that proline is removed from the coating solution.

[0060] Comparative Example 5 The kit for detecting bloodstream pathogens differs from Example 1 only in that the addition of SDS and PC300 is removed from the coating solution.

[0061] Experimental Example 1 1. Experimental Samples Routine blood samples: 50 negative blood samples (clinically confirmed to be free of bloodstream infection), 60 positive blood samples (containing Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Staphylococcus aureus, and Candida albicans, 10 samples of each), with concentration gradients of 3 CFU / mL, 5 CFU / mL, 10 CFU / mL, and 100 CFU / mL; Abnormal blood samples: 20 hemolytic blood samples, 20 lipemic blood samples, and 20 jaundice blood samples (all clinically confirmed negative blood samples, with corresponding interfering substances artificially added; hemoglobin concentration 10 g / L, lipid concentration 15 mmol / L, and bilirubin concentration 300 μmol / L). Cross-reactive samples: 20 samples (containing non-target pathogens such as Haemophilus influenzae and Streptococcus, at a concentration of 100 CFU / mL).

[0062] 2. Experimental Methods Following the method in Example 5, each experimental group was set up with 3 replicates. The data were taken as mean ± standard deviation (x ± SD). The t-test was used to analyze the significance of differences, and P < 0.05 was considered significant. The results were judged as follows: OD value ≥ 0.3 was positive, and OD value < 0.3 was negative.

[0063] (1) Validation of the optimization effect of the sample processing solution Routine positive blood samples (3 CFU / mL) and abnormal blood samples were detected using the kits from Examples 1 and Comparative Examples 1-3. Example 1 was designated as Experimental Group 1, Comparative Example 1 as Control Group 1, Comparative Example 2 as Control Group 2, and Comparative Example 3 as Control Group 3. A blank control group was also included, using only PBS buffer instead of the sample processing solution. The OD value of each well was measured at 450 nm using a microplate reader. Antigen recovery rate, false negative rate, and false positive rate were calculated. Data from each group were statistically analyzed, and significant differences were compared between the experimental groups and each control group. The results are as follows: Table 1. Antigen recovery rate, false negative rate, and false positive rate for each treatment group.

[0064] The results showed that experimental group 1 could effectively detect pathogens with low viral loads of 3 CFU / mL, with an antigen recovery rate of 97.3%, a false negative rate of only 1.7%, and a false positive rate of 0 for abnormal blood samples, proving that the sample processing solution of the present invention can achieve efficient antigen release and thorough removal of interference. The false negative and false positive rates of control group 1 were significantly increased, proving that BSA and sodium citrate can effectively block non-specific binding sites and eliminate interference from abnormal blood samples such as hemolysis and jaundice. Control group 2 had the lowest antigen recovery rate and the highest false negative rate, proving that the synergistic effect of Triton X-100 and saponins can significantly improve the efficiency of erythrocyte lysis and the release rate of pathogen antigens. The antigen recovery rate and false negative / false positive rate of control group 3 were worse than those of experimental group 1, proving that PEG 6000 can effectively precipitate interfering proteins and improve detection accuracy. The OD value of the blank group was significantly lower than that of experimental group 1, proving that there was no background interference in the experiment and the data was reliable, further verifying the inventiveness and effectiveness of the synergistic system of the sample processing solution of the present invention.

[0065] (2) Validation of the optimization effect of the coating solution The kits of Example 1 and Comparative Examples 4-5 were used. Example 1 was designated as Experimental Group 2, Comparative Example 4 as Control Group 4, and Comparative Example 5 as Control Group 5. Another Control Group 6 was set up, which used carbonate buffer instead of coating solution. Another Blank Control Group was set up, which used carbonate buffer instead of coating solution and did not perform antibody coating (containing no antibody).

[0066] Repeatability testing: Take 100 CFU / mL positive blood samples, process them with the sample processing solution of this invention, add them to each group of detection plates, with 3 replicate wells in each group, measure the OD value according to the standard detection procedure, and calculate the coefficient of variation (CV).

[0067] Storage stability: Each group of test plates was stored at 4°C for 24 months, at 25°C for 6 months, and at 37°C for 14 days. The OD value of 100 CFU / mL positive blood samples was measured, and the decrease in OD value and the maintenance rate of antibody activity were calculated.

[0068] Adaptability to extreme conditions: After freezing and thawing each group of test plates five times at -20℃, the OD value and antibody activity retention rate were measured.

[0069] Data from each group were statistically analyzed, and a significance analysis was performed to compare the differences between the experimental group and each control group. The results are as follows: Table 2. Repeatability data for each treatment group.

[0070] Table 3. Storage stability and extreme condition adaptability data for each treatment group.

[0071] The results showed that experimental group 2 had excellent repeatability (CV=3.5%), which was significantly better than all control groups, proving that it could improve the uniformity of antibody distribution and that the adsorption-promoting component could enhance the coating consistency. The antibody activity maintenance rate of control group 4 was significantly lower than that of experimental group 2, proving that the three-synergistic stabilizing system formed by proline, trehalose, and BSA could effectively maintain the antibody conformation and prevent antibody denaturation. The antibody activity maintenance rate and repeatability of control group 5 were both poor, proving that SDS and PC300 could enhance the binding force between the antibody and the detection plate and improve the coating stability.

[0072] Experimental group 2 can be stably stored at 4℃ for 24 months and at room temperature for 6 months. Even under extreme conditions, it can still maintain high antibody activity, which is significantly better than control group 6 (common commercial level). This proves that the coating liquid system of the present invention can significantly extend the storage period of the kit. The blank group has extremely low OD value, which proves that there is no background interference. The experimental data are reliable and verify the effectiveness of the coating liquid synergistic system of the present invention.

[0073] (3) Exploration of multi-scenario adaptation and device compatibility Routine blood samples (10 CFU / mL) were collected. One group (Experimental Group 3) was processed using the kit from Example 1 according to the routine blood sample processing method in Example 5. The other group (Experimental Group 4) was processed using the kit from Example 4 according to the routine blood sample processing method for emergency sepsis patients in Example 5. The remaining steps were performed according to the standard procedure. Processing time and OD values ​​were recorded to verify the suitability of the testing scenario. Data from each group were statistically analyzed, and the results are as follows: Table 4. Detection data for each treatment group

[0074] The results showed that the processing time for experimental group 4 (emergency scenario) was shortened to 10 minutes, and the OD value and positive detection rate were not significantly different from those for experimental group 3 (routine scenario), proving that the emergency sample processing solution can meet the needs of rapid emergency testing. Compared with commercially available kits (usually ≥20 min), the kit of this invention has a shorter processing time, better detection effect, and is suitable for more scenarios, solving the problems of poor adaptability and insufficient compatibility of existing kits.

[0075] (4) Validation of the overall effectiveness of the kit (comprehensive validation) The kit from Example 1 (referred to as experimental group 5, using the method of Example 5) and the Hotgen Biotech Upconversion Luminescent Bloodstream Infection Kit (catalog number: BJ-2022-BSI) (referred to as control group 7, using its instructions) were compared to verify the overall performance (sensitivity, specificity, storage stability, and anti-interference ability) of the kit of the present invention. A blank group was set up and operated according to the same procedure, and the background OD value was measured to eliminate background interference.

[0076] Sensitivity verification: Low-load positive blood samples of 3, 5, and 10 CFU / mL were taken and tested using three sets of kits according to the standard procedure. Each set had 3 replicate wells. The OD value was recorded and the limit of detection was calculated.

[0077] Specificity verification: Take cross-reactive samples (non-target pathogens) and negative blood samples, and test them with three sets of kits. Record the false positive rate to verify specificity.

[0078] Anti-interference verification: Blood samples with abnormalities such as hemolysis, lipemia, and jaundice were collected and tested using three sets of reagent kits. The false positive rate and OD value changes were recorded to verify the anti-interference ability.

[0079] Storage stability verification: The three kits were stored at 4°C. After 18 months, the OD value of 100 CFU / mL positive blood samples was measured, and the antibody activity retention rate was calculated.

[0080] The overall accuracy data for each group was statistically analyzed. The results are as follows: Table 5. Comparison of the overall detection performance of the three reagent kits.

[0081] The results showed that: (1) The sensitivity of the present invention is significantly superior: the detection limit of experimental group 5 is 3 CFU / mL, which is significantly better than that of control group 7 (10 CFU / mL), proving that the kit of the present invention can effectively detect low-load bloodstream infection pathogens and solve the problem of insufficient sensitivity of existing kits; (2) Excellent specificity and anti-interference: the false positive rate of experimental group 5 is 0 and the anti-interference rate is 100%, which can completely eliminate the interference of non-target pathogens, hemolysis and other abnormal blood samples, while the control group has a certain false positive rate and weak anti-interference ability, which verifies the effectiveness of the anti-interference design of the present invention; (3) Outstanding storage stability: experimental group 5 can be stably stored at 4℃ for 18 months, and the antibody activity maintenance rate is 99.8%, which is significantly higher than that of commercially available kits, greatly extending the storage period of the kit and reducing storage and transportation costs; (4) Higher detection efficiency and accuracy: the comprehensive accuracy of experimental group 5 is 99.5%, which is significantly better than that of commercially available kits, and can provide detection results quickly and accurately to meet the needs of rapid clinical diagnosis. Based on a comprehensive comparison of key indicators such as sensitivity, specificity, anti-interference ability, storage stability, and detection efficiency, the reagent kit of this invention significantly outperforms commercially available reagent kits in all aspects and has good clinical application value.

[0082] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A kit for detecting bloodstream pathogens, characterized in that, Includes sample processing solution and coating solution; The sample processing solution, based on PBS buffer, contains, by weight and volume percentage: 0.18%–0.22% Triton X-100, 0.09%–0.11% saponins, 0.35%–0.45% polyethylene glycol 6000, 0.02%–0.04% PC300, 0.05%–0.07% EDTA-Na2, 0.01%–0.02% BSA, and 0.005%–0.01% sodium citrate. The coating solution is based on carbonate buffer and contains, by mass-volume percentage, 0.12%~0.14% trehalose, 0.095%~0.105% SDS, 0.03%~0.05% methanol, 0.02%~0.03% BSA, 0.01%~0.015% PC300 and 0.003%~0.005% proline.

2. The reagent kit according to claim 1, characterized in that, The concentration of the PBS buffer is 0.01 mol / L, and the pH value is 7.2~7.

4.

3. The reagent kit according to claim 1, characterized in that, The carbonate buffer solution has a concentration of 0.05 mol / L and a pH value of 9.5~9.

8.

4. The reagent kit according to claim 1, characterized in that, It also includes a detection plate, monoclonal antibody, enzyme-labeled secondary antibody, washing solution, colorimetric solution, and stop solution.

5. The reagent kit according to claim 4, characterized in that, The monoclonal antibodies include any one or more of the following: Escherichia coli monoclonal antibody, Klebsiella pneumoniae monoclonal antibody, Acinetobacter baumannii monoclonal antibody, Pseudomonas aeruginosa monoclonal antibody, Staphylococcus aureus monoclonal antibody, and Candida albicans monoclonal antibody.

6. The reagent kit according to claim 4, characterized in that, The enzyme-labeled secondary antibody is horseradish peroxidase-labeled goat anti-mouse IgG antibody.

7. The reagent kit according to claim 4, characterized in that, The washing solution is based on PBS buffer and contains, by weight and volume percentage, 0.05% Tween-20, 0.01% PC300, and 0.005% EDTA-Na2.

8. The reagent kit according to claim 4, characterized in that, The colorimetric solution is TMB colorimetric solution.

9. The reagent kit according to claim 4, characterized in that, The terminating solution is a 2 mol / L H2SO4 solution.

10. A method for preparing the reagent kit according to any one of claims 1 to 9, characterized in that, The preparation method of the sample processing solution includes: adding Triton X-100, saponin, PEG 6000, PC300, EDTA-Na2, BSA and sodium citrate sequentially to PBS buffer, stirring magnetically for 30-40 minutes until completely dissolved, adjusting the pH, and filtering aseptically. The preparation method of the coating solution includes: adding trehalose, SDS, methanol, BSA, PC300 and proline sequentially to a carbonate buffer solution, stirring magnetically for 20-30 minutes until completely dissolved, adjusting the pH, and aseptically filtering.

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