A rapid detection of bacterial energy metabolism kit and its preparation method and application

Abstract

The application discloses a kit for rapidly detecting bacterial energy metabolism and a preparation method and application thereof, and belongs to the technical field of microorganism analysis, drug screening and detection. The kit comprises reaction premixed powder, anaerobic reaction liquid and optional positive control; the reaction premixed powder comprises the following components in parts by weight: 1 part of benzyl viologen; 0.43-0.64 parts of sodium pyruvate; 0.054-0.082 parts of coenzyme A; 0.037-0.055 parts of MgCl2.7H2O; the anaerobic reaction liquid is a buffer solution containing sodium sulfide-9 hydrate; and the positive control is nitazoxanide or glychalcone A. The application further discloses a preparation method and application of the kit, and the application comprises the following steps: rapidly detecting bacterial energy metabolism activity; and / or screening substances for inhibiting bacterial energy metabolism. The kit can rapidly, sensitively and at low cost evaluate the physiological activity of bacteria, and can also be used for rapidly screening and evaluating anaerobic microorganism energy metabolism inhibitors.

Description

Technical Field

[0001] This invention belongs to the field of microbial analysis, drug screening, and detection technology, specifically relating to a rapid detection kit for bacterial energy metabolism activity based on benzyl viologen (BV) as a core redox indicator, its preparation method, and applications. This kit can not only be used for rapid, sensitive, and low-cost assessment of the physiological activity status of bacteria, but more importantly, it is specifically optimized for the rapid screening and evaluation of anaerobic microbial energy metabolism inhibitors. This invention also creatively introduces nitrozonide as a preferred positive control compound for this system. This kit can be widely used in high-throughput primary screening of novel antimicrobial drugs (especially those targeting energy metabolism), environmental microbial activity monitoring, and basic research. Background Technology

[0002] With the increasing severity of resistance to traditional antibiotics, the development of antibacterial drugs with novel mechanisms of action, especially those targeting bacterial energy metabolism pathways, has become a research hotspot. However, screening and evaluation technologies for such drugs are severely lagging behind. Currently, the mainstream method still relies on minimum inhibitory concentration (MIC) assays. While this technique is mature, it is time-consuming (typically requiring 16-24 hours) and its mechanism of action is ambiguous: it can only determine whether bacterial growth is inhibited, but cannot distinguish whether the drug works by inhibiting energy metabolism or by interfering with other pathways such as cell wall synthesis and protein translation. This lack of functional differentiation makes it difficult for researchers to quickly identify lead molecules that truly target energy metabolism from a vast pool of candidate compounds. Therefore, there is an urgent need in this field for a functional assay tool that can rapidly and intuitively reflect the state of bacterial energy metabolism, especially for inhibitor screening.

[0003] Benzyl violetine (BV) is a synthetically produced low-potential electron transport mediator. In biological redox systems, BV can be reduced by various dehydrogenases (such as hydrogenases and NADH dehydrogenases), transforming from a colorless or pale yellow state into a deep purple free radical cation (BV). + • Based on this characteristic, chlorophyll (BV) is often used in basic research as an artificial electron acceptor or chromogenic indicator to detect the reducing activity of microorganisms or enzyme preparations, with sporadic applications, particularly in the study of enzymes related to anaerobic energy metabolism. Although BV theoretically possesses the potential to serve as a chromogenic indicator of bacterial energy metabolism activity, its development from a laboratory research tool into a stable, reliable, and reproducible commercial reagent kit, and its application in practical scenarios with extremely high requirements for consistency and sensitivity, such as high-throughput inhibitor screening and efficacy evaluation, still faces the following insurmountable technical obstacles: First, the aqueous solution stability of the core reagent is extremely poor: BV itself, especially when formulated into a working solution with buffer salts and reaction substrates, is chemically unstable in aqueous solution, easily undergoing degradation or non-specific redox reactions. This results in a very short shelf life for liquid reagent kits, making it difficult to meet the basic commercial requirements for storage, transportation, and sales. In fact, there are currently no commercially available BV-based energy metabolism assay kits, directly reflecting the lack of breakthroughs in stability for this technology. Existing literature reports mostly describes laboratory-prepared methods, with varying formulations and proportions, and all are prepared on-demand, making cross-sectional data comparisons between different research teams difficult, severely hindering the standardization and widespread application of this method.

[0004] Secondly, the complexity of the operational procedures introduces unavoidable human error. Since premixed, ready-to-use stable reagents are unavailable, users must weigh milligram or even sub-milligram levels of BV powder before each experiment, then dissolve and mix it sequentially with other components. This process is not only tedious and time-consuming, but more importantly, the accuracy of micro-weighing, the completeness of dissolution, and the order of reagent addition are all highly dependent on the operator's skill, easily introducing intra-batch and inter-batch variations. For high-throughput screening, this operational uncertainty directly translates into data noise, severely impacting the reliability and reproducibility of screening results. Furthermore, subtle fluctuations in pH, ionic strength, and dissolved oxygen concentration in hastily prepared reagents can further interfere with reaction kinetics, leading to unstable colorimetric signals.

[0005] More critically, existing BV-based detection systems generally lack standardized quality control methods. Neither literature reports nor laboratory-developed methods provide known energy metabolism inhibitors as positive controls. This makes it difficult for researchers to verify whether the reagent system is functioning correctly, and to determine whether the observed signal attenuation is a genuine inhibitory effect of the analyte or a false positive due to reagent inactivation, operational errors, or other accidental factors. In modern in vitro diagnostics and drug screening systems, the lack of positive controls means that experimental results lack a benchmark, significantly reducing their scientific explanatory power and data reliability, making it difficult to meet the stringent requirements of high-throughput screening for data quality and traceability.

[0006] In summary, existing BV-based detection systems suffer from several problems, including poor stability of core reagents leading to difficulties in commercialization, complex operating procedures that are prone to human error, a lack of standardized positive controls resulting in ineffective quality control, and difficulty in applying them to high-throughput inhibitor screening.

[0007] Therefore, providing a rapid detection kit for bacterial energy metabolism activity based on benzyl viologen, which features high component stability, simple and quick operation, good repeatability of detection results, and built-in standardized positive control, to solve the technical problems of existing BV-based detection systems that cannot be commercialized, are cumbersome to operate, and lack effective quality control, thus making them difficult to apply to high-throughput screening, has become an urgent technical problem to be solved by those skilled in the art. Summary of the Invention

[0008] One of the objectives of this invention is to provide a rapid detection kit for bacterial energy metabolism, which can rapidly, sensitively, and cost-effectively assess bacterial physiological activity and can also be used for the rapid screening and evaluation of anaerobic microbial energy metabolism inhibitors.

[0009] A second objective of this invention is to provide a method for preparing the reagent kit.

[0010] A third objective of this invention is to provide applications for the reagent kit.

[0011] To achieve the above objectives, the technical solution adopted by the present invention is as follows: The first aspect of this invention discloses a rapid detection kit for bacterial energy metabolism, comprising the following materials: a reaction premix powder, an anaerobic reaction solution, and an optional positive control. The reaction premixed powder comprises the following components in parts by weight: 1 part benzyl viologen; 0.43~0.64 parts sodium pyruvate; 0.054~0.082 parts coenzyme A; and 0.037~0.055 parts MgCl2·7H2O. The anaerobic reaction solution is a buffer solution containing sodium sulfide-9 hydrate, wherein the concentration of sodium sulfide-9 hydrate is 15~25 μg / mL; The amount of reaction premix powder added per 100 mL of anaerobic reaction solution is 0.30~0.40 g; The positive control is either nitrozonide or glycyrrhizin A.

[0012] In some embodiments of the present invention, the reaction premixed powder comprises the following components in parts by weight: 1 part benzyl viologen; 0.537 parts sodium pyruvate; 0.068 parts coenzyme A; and 0.046 parts MgCl2·7H2O. And / or, the concentration of sodium sulfide-9 hydrate in the anaerobic reaction solution is 20 μg / mL; And / or, the amount of reaction premix powder added per 100 mL of anaerobic reaction solution is 0.3395 g.

[0013] In some embodiments of the present invention, the anaerobic reaction solution is a phosphate buffer solution containing sodium sulfide-9 hydrate; preferably, the pH value of the phosphate buffer solution is 7.2.

[0014] In some embodiments of the present invention, the concentration of phosphate in the anaerobic reaction solution is: K2HPO4 0.698 g / 100mL, KH2PO4 0.135 g / 100 mL.

[0015] In some embodiments of the present invention, the positive control nitrozonide is provided in the form of a solid powder or a DMSO solution; when used, its working concentration is ≥8 μg / mL; And / or, the positive control, glycyrrhizin A, is provided in solid powder form or as a DMSO solution; when used, its working concentration is 32–256 μg / mL.

[0016] In some embodiments of the present invention, both the reaction premix powder and the anaerobic reaction solution are stored under anaerobic conditions.

[0017] The second aspect of this invention discloses a method for preparing the above-mentioned rapid detection kit for bacterial energy metabolism, which includes the following steps: S1. Preparation of reaction premix powder: Weigh each component of the reaction premix powder according to the proportion, mix them evenly, dispense them into brown glass bottles, anaerobic gas equilibration treatment to remove oxygen, and seal. S2. Anaerobic reaction solution: Under inert gas protection, phosphate and sodium sulfide-9 hydrate are added to deoxygenated water, mixed and dissolved to obtain a mixed reaction solution; the mixed reaction solution is dispensed into sealed containers, sterilized, and then kept sealed and cooled to room temperature to obtain an anaerobic reaction solution. Steps S1 and S2 are not sequential.

[0018] In some embodiments of the present invention, in step S2, phosphate and sodium sulfide-9 hydrate stock solution are added to the deoxygenated water and mixed and dissolved.

[0019] In some embodiments of the present invention, when the positive control is provided in the form of a solid powder, an appropriate amount is weighed and sealed for storage. When the positive control is provided in the form of DMSO solution, prepare the DMSO solution of the positive control and store it in a sealed container.

[0020] When using the kit of the present invention, the premixed powder and the anaerobic reaction solution are thoroughly mixed in a certain proportion to obtain the colorimetric solution.

[0021] The third aspect of this invention discloses the application of the above-mentioned rapid detection kit for bacterial energy metabolism, the application including: (1) Rapid detection of bacterial energy metabolism activity; and / or (2) Screening for substances that inhibit bacterial energy metabolism.

[0022] Compared with the prior art, the present invention has the following beneficial effects: This invention is scientifically designed and ingeniously conceived. Addressing the problems of complex operation, poor aqueous solution stability, difficulty in standardizing reaction conditions, and lack of effective quality control methods in existing microbial energy metabolism detection systems, this invention provides an integrated and standardized detection solution. The kit uses a stabilized premixed powder form containing BV, coenzyme, and antioxidant protectant. Users do not need to prepare complex reagents on-site; simple reconstitution is sufficient to conduct experiments, significantly simplifying the operation process and greatly improving the operability of the detection process and the batch-to-batch reproducibility of experimental results. Simultaneously, the kit introduces a standard positive control for the first time, preferably a known energy metabolism inhibitor, enabling effective monitoring and signal verification of the detection system's performance, ensuring the reliability and comparability of the detection results.

[0023] This invention's kit is not only suitable for basic analysis of microbial energy metabolism activity and research on antibacterial mechanisms, but also extends to a highly efficient platform for screening energy metabolism inhibitors. Its screening principle is based on a metabolic indicator system: the test compound is introduced into a mixture of bacteria and reagents; if the compound exhibits energy metabolism inhibitory activity, the microbial electron transport chain is disrupted, resulting in a blue BV (biochemical oxygen demand) color. + • When the formation of the compound is inhibited or weakened, its inhibitory efficacy can be quickly and intuitively determined by visually observing color changes or measuring absorbance values. This design upgrades the kit from a single detection tool to a fully functional drug screening system.

[0024] In summary, this invention effectively overcomes key technical challenges such as the instability of aqueous solutions, large operational errors, uncontrollable reaction conditions, and lack of standardized controls in traditional detection systems. It realizes the transformation from laboratory principle exploration to standardized commercial reagents, and has good detection stability, convenient operation, and significant prospects for industrial application. Attached Figure Description

[0025] Appendix Figure 1 The image shows the colorimetric reaction results of the colorimetric solution of the present invention in various bacteria based on energy metabolism activity in Experiment Example 3; Appendix Figure 2 The figure shows the results of the investigation on the inhibitory effect of nitrozonide on the energy metabolism of various bacteria in Experiment Example 4.

[0026] Appendix Figure 3 This is a visualization of the screening results of Clostridium difficile energy metabolism inhibitors based on the chromogenic kit of the present invention in Experiment Example 5; Appendix Figure 4 The figure shows the results of the investigation of Experiment Example 6, in which Figure 4Figure A shows the effect of the positive control drug NTZ on the energy metabolism of Clostridium difficile. Figure 4 B shows the dynamic inhibition results of the screening compound LCA on ATP production in Clostridium difficile; Figure 4 The vertical axis represents ATP bioluminescence intensity. Detailed Implementation

[0027] The technical solutions in some embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments provided by the present invention, all other embodiments obtained by those skilled in the art are within the scope of protection of the present invention.

[0028] The Clostridium difficile 630, Clostridium difficile 1870, Clostridium difficile 43255, Escherichia coli, Propionibacterium acnes, Bacteroides fragilis, Pseudomonas aeruginosa, Bifidobacterium, Bacillus coagulans, Lactococcus lactis, Lactobacillus plantarum, and Lactobacillus helveticus mentioned in the embodiments of this invention are all derived from the American Type Culture Collection Center (ATCC).

[0029] The composition of the reaction premix powder and anaerobic reaction solution of the detection kit in this embodiment of the invention is shown in Table 1: Table 1

[0030] Example 1 This embodiment discloses a method for preparing the bacterial energy metabolism detection kit of the present invention. The kit of this embodiment includes a reaction premixed powder and an anaerobic reaction solution, and its preparation method is as follows: 1. Prepare the reaction premix powder. Taking the preparation of the premix powder required for 100 mL of anaerobic reaction solution as an example, the specific steps are as follows: (1) Weighing and grinding: Weigh the basic salts as the main mixture. Accurately weigh the following components into a mortar: BV 0.205 g, sodium pyruvate 0.110 g, coenzyme A 0.015 g, MgCl2·7H2O 0.0095 g, totaling approximately 0.3395 g. Grind each component thoroughly until a uniform and fine powder is obtained, and then further mix using a vortex mixer to obtain the reaction premix powder.

[0031] (2) Dispensing and deoxygenation: Weigh the reaction premixed powder obtained in step 1 according to the actual needs. For example, 0.0679 g is needed to prepare 20 mL of reaction solution. Dispense the weighed powder into brown glass bottles and seal them. Then, place the dispensed bottles into an anaerobic glove box and perform anaerobic gas equilibration treatment for 2 hours to remove oxygen and ensure that the system is in an anaerobic environment.

[0032] (3) Storage: After processing, tighten the bottle cap and store in a dark and dry place at -20℃ for later use.

[0033] 2. Preparation of anaerobic reaction solution (1) Preparation of stock solution: Accurately weigh 0.698 g of K2HPO4 and 0.135 g of KH2PO4 for later use. Also prepare a stock solution of sodium sulfide-9 hydrate (Na2S·9H2O) with a concentration of 1 g / L.

[0034] (2) Dissolution and deoxygenation: 98 mL of ultrapure water was measured and bubbled with nitrogen for 10 min to remove most of the dissolved oxygen in the water. Then, under nitrogen protection, the weighed K2HPO4 and KH2PO4, as well as 2 mL of the above 1 g / L sodium sulfide-9 hydrate stock solution (to make the final concentration of sodium sulfide-9 hydrate in the reaction solution 20 μg / mL), were added and stirred to dissolve, thus obtaining the reaction solution.

[0035] (3) Dispensing and sterilization: Dispense the prepared reaction solution into 20 mL portions into transparent sealed bottles and autoclave at 121°C for 20 min. After sterilization, remove the bottles, allow them to cool naturally to room temperature, and keep them sealed to prevent oxygen from re-dissolving and to ensure an anaerobic state.

[0036] In this embodiment, the reagent kit is prepared by thoroughly mixing the premixed powder with the anaerobic reaction solution in a certain proportion to obtain the colorimetric solution.

[0037] Example 2 This embodiment discloses a method for preparing the bacterial energy metabolism detection kit of the present invention. The kit of this embodiment includes a reaction premixed powder and an anaerobic reaction solution, and its preparation method is as follows: 1. Prepare the reaction premix powder. Taking the preparation of the premix powder required for 100 mL of anaerobic reaction solution as an example, the specific steps are as follows: (1) Weighing and grinding: Weigh the basic salts as the main mixture. Accurately weigh the following components into a mortar: BV 0.205 g, sodium pyruvate 0.088 g, coenzyme A 0.011 g, MgCl2·7H2O 0.0076 g, totaling approximately 0.3116 g. Grind each component thoroughly until a uniform and fine powder is obtained, and then further mix using a vortex mixer to obtain the reaction premix powder.

[0038] (2) Dispensing and deoxygenation: Weigh the reaction premixed powder obtained in step 1 according to the actual needs. For example, 0.0623 g is needed to prepare 20 mL of reaction solution. Dispense the weighed powder into brown glass bottles and seal them. Then, place the dispensed bottles into an anaerobic glove box and perform anaerobic gas equilibration treatment for 2 hours to remove oxygen and ensure that the system is in an anaerobic environment.

[0039] (3) Storage: After processing, tighten the bottle cap and store in a dark and dry place at -20℃ for later use.

[0040] 2. Preparation of anaerobic reaction solution (1) Preparation of stock solution: Accurately weigh 0.698 g of K2HPO4 and 0.135 g of KH2PO4 for later use. Prepare a stock solution of sodium sulfide-9 hydrate (Na2S·9H2O) with a concentration of 0.75 g / L.

[0041] (2) Dissolution and deoxygenation: 98 mL of ultrapure water was measured and bubbled with nitrogen for 10 min to remove most of the dissolved oxygen in the water. Then, under nitrogen protection, the weighed K2HPO4 and KH2PO4, as well as 2 mL of the above 1 g / L sodium sulfide-9 hydrate stock solution (to make the final concentration of sodium sulfide-9 hydrate in the reaction solution 15 μg / mL) were added and stirred to dissolve, thus obtaining the reaction solution.

[0042] (3) Dispensing and sterilization: Dispense the prepared reaction solution into 20 mL portions into transparent sealed bottles and autoclave at 121°C for 20 min. After sterilization, remove the bottles, allow them to cool naturally to room temperature, and keep them sealed to prevent oxygen from re-dissolving and to ensure an anaerobic state.

[0043] In this embodiment, the reagent kit is prepared by thoroughly mixing the premixed powder with the anaerobic reaction solution in a certain proportion to obtain the colorimetric solution.

[0044] Example 3 This embodiment discloses a method for preparing the bacterial energy metabolism detection kit of the present invention. The kit of this embodiment includes a reaction premixed powder and an anaerobic reaction solution, and its preparation method is as follows: 1. Prepare the reaction premix powder. Taking the preparation of the premix powder required for 100 mL of anaerobic reaction solution as an example, the specific steps are as follows: (1) Weighing and grinding: Weigh the basic salts as the main mixture. Accurately weigh the following components into a mortar: BV 0.205 g, sodium pyruvate 0.131 g, coenzyme A 0.017 g, MgCl2·7H2O 0.0113 g, totaling approximately 0.3643 g. Grind each component thoroughly until a uniform and fine powder is obtained, and then further mix using a vortex mixer to obtain the reaction premix powder.

[0045] (2) Dispensing and deoxygenation: Weigh the reaction premixed powder obtained in step 1 according to the actual needs. For example, 0.0729 g is needed to prepare 20 mL of reaction solution. Dispense the weighed powder into brown glass bottles and seal them. Then, place the dispensed bottles into an anaerobic glove box and perform anaerobic gas equilibration treatment for 2 hours to remove oxygen and ensure that the system is in an anaerobic environment.

[0046] (3) Storage: After processing, tighten the bottle cap and store in a dark and dry place at -20℃ for later use.

[0047] 2. Preparation of anaerobic reaction solution (1) Preparation of stock solution: Accurately weigh 0.698 g of K2HPO4 and 0.135 g of KH2PO4 for later use. Prepare a stock solution of sodium sulfide-9 hydrate (Na2S·9H2O) with a concentration of 0.75 g / L.

[0048] (2) Dissolution and deoxygenation: 98 mL of ultrapure water was measured and bubbled with nitrogen for 10 min to remove most of the dissolved oxygen in the water. Then, under nitrogen protection, the weighed K2HPO4 and KH2PO4, as well as 2 mL of the above 1.25 g / L sodium sulfide-9 hydrate stock solution (to make the final concentration of sodium sulfide-9 hydrate in the reaction solution 25 μg / mL) were added and stirred to dissolve, thus obtaining the reaction solution.

[0049] (3) Dispensing and sterilization: Dispense the prepared reaction solution into 20 mL portions into transparent sealed bottles and autoclave at 121°C for 20 min. After sterilization, remove the bottles, allow them to cool naturally to room temperature, and keep them sealed to prevent oxygen from re-dissolving and to ensure an anaerobic state.

[0050] In this embodiment, the reagent kit is prepared by thoroughly mixing the premixed powder with the anaerobic reaction solution in a certain proportion to obtain the colorimetric solution.

[0051] Experimental Example 1 This experiment investigated whether the addition of sodium sulfide-9 hydrate helps maintain the anaerobic state of the anaerobic reaction solution.

[0052] 1. Grouping Control group: Contains only K2HPO4 and KH2PO4, without sodium sulfide-9 hydrate, and was prepared according to the method for preparing the anaerobic reaction solution in Example 1: Experimental group: the anaerobic reaction solution of Example 1, i.e. containing K2HPO4, KH2PO4 and sodium sulfide-9 hydrate.

[0053] 2. After sterilization and cooling, take one bottle of reaction solution from each group, add the anaerobic indicator resazurin (final concentration 0.001‰) under aseptic conditions, mix immediately, and observe the color change. Resazurin is blue / pink under aerobic conditions and colorless under anaerobic conditions.

[0054] 3. Results The color change results are shown in Table 2. The reaction solution of the control group without sodium sulfide-9 hydrate turned light blue after the addition of resazurin, indicating that trace amounts of dissolved oxygen remained in the system; while the reaction solution of the experimental group with sodium sulfide-9 hydrate was colorless, proving that it was under strict anaerobic conditions. The results indicate that the addition of sodium sulfide-9 hydrate helps maintain an anaerobic environment in the reaction solution during sterilization and cooling.

[0055] Table 2. Sodium sulfide in the reaction solution promotes the formation of an anaerobic state in the reaction solution.

[0056] Experimental Example 2: Investigation of the promoting effect of sodium sulfide-9 hydrate on the colorimetric reaction To investigate the role of sodium sulfide-9 hydrate in the colorimetric reaction and verify whether it catalyzes or promotes the colorimetric process, this experiment included an experimental group and a control group. The experimental group used the anaerobic reaction solution containing sodium sulfide-9 hydrate prepared in Example 1 to prepare the colorimetric solution, while the control group used an anaerobic reaction solution without sodium sulfide-9 hydrate.

[0057] The test method is as follows: (1) Preparation of colorimetric solution: Take 20 mL of anaerobic reaction solution containing sodium sulfide-9 hydrate (experimental group) and 0.0679 g of reaction premix powder prepared in Example 1 respectively, and mix thoroughly to obtain the corresponding colorimetric solution.

[0058] (2) Colorimetric reaction determination: Take a 96-well plate and add a suspension of Clostridium difficile strain 1870 (OD200) to each well. 600 = 0.5) 100 μL, then add 100 μL of the above colorimetric solution, and mix immediately to start the colorimetric reaction. Monitor the absorbance (OD value) at a wavelength of 546 nm, and record the colorimetric time and the final OD value. The larger the OD value, the deeper the color.

[0059] The results are shown in Table 3: Compared with the control group, the color development time of the experimental group containing sodium sulfide-9 hydrate was significantly shortened, and the final color was deeper (larger OD value).

[0060] The above results indicate that sodium sulfide-9 hydrate has a significant catalytic or promoting effect in the colorimetric reaction, and its mechanism may involve providing a reducing environment or participating in the substrate reaction, thereby accelerating the colorimetric process.

[0061] Table 3. Sodium sulfide is used in the preparation of the colorimetric solution to promote the colorimetric reaction.

[0062] Experimental Example 3: Strain-specific analysis of colorimetric reactions To evaluate the responsiveness of the chromogenic solution of the present invention to the metabolic activities of different bacteria and to verify its feasibility for high-throughput screening of inhibitors, a variety of representative bacteria were selected for detection.

[0063] The colorimetric solution used in this experiment was prepared according to the method of experimental group in Experiment Example 2.

[0064] Clostridium difficile (strains 630, 1870, and 43255), Escherichia coli, Propionibacterium acnes, Bacteroides fragilis, Pseudomonas aeruginosa, Bifidobacterium, Bacillus coagulans, Lactococcus lactis, Lactobacillus plantarum, and Lactobacillus helveticus were cultured to the logarithmic growth phase, and the bacterial concentration was adjusted to OD0.05. 600 =0.5. Add 100 μL of bacterial culture to the corresponding well of a 96-well plate, then add 100 μL of colorimetric solution to each well, mix well, and incubate under suitable conditions in the dark until color development occurs. Take a picture to record the results.

[0065] The layout of the perforated plate is shown in the attached figure. Figure 1 As shown: A1, A2, and A3 are Clostridium difficile 630, 1870, and 43255, respectively; A4 is Lactobacillus helveticus; B1 is Escherichia coli; B2 is Propionibacterium acnes; B3 is Bacteroides fragilis; B4 is Pseudomonas aeruginosa; C1 is Bifidobacterium; C2 is Bacillus coagulans; C3 is Lactococcus lactis; C4 is Lactobacillus plantarum; A5, B5, and C5 are blank controls (containing only culture medium and chromogenic solution).

[0066] The results showed that after incubation with the chromogenic solution, all wells of each bacterial strain showed obvious color development, while the blank control wells showed no obvious coloration. This indicates that the chromogenic solution can be catalytically reduced to produce color changes under the action of bacterial energy metabolism, and the reaction depends on the metabolic activity of live bacteria. These results confirm that this chromogenic system can be used to detect the energy metabolic status of various bacteria, and can be combined with positive inhibitors to achieve high-throughput rapid screening of candidate compounds that inhibit bacterial energy metabolism by observing the weakening or disappearance of the color intensity.

[0067] Experimental Example 4: Inhibitory effect of the positive control compound nitrozonide on the colorimetric reaction of bacterial energy metabolism. To verify the broad-spectrum nature of the positive control compound nitazoxanide as an energy metabolism inhibitor, representative strains covering different oxygen requirements were selected for testing.

[0068] The colorimetric solution used in this experiment was prepared according to the method of experimental group in Experiment Example 2.

[0069] Each test strain was cultured to the logarithmic growth phase, and the bacterial suspension concentration was adjusted to OD200. 600 = 0.5. Take a 96-well plate, add 100 μL of bacterial suspension to each well, then add 100 μL of chromogenic solution, and mix well. At the same time, add nitrozonidine to the positive control wells to a final concentration of 100 μg / mL to evaluate its inhibitory effect on bacterial metabolic activity. For each strain, set up a normal chromogenic group (without nitrozonidine) and a positive control group (with nitrozonidine) in triplicate wells to ensure the reproducibility and reliability of the experimental results.

[0070] The perforated plate layout is shown in the attached figure. Figure 2 As shown, each grid represents three duplicate holes: A1–A3: Clostridium difficile 630 ( Clostridioides difficile 630) Normal color development group; A4–A6: Clostridium difficile 630 positive control group (+ nitrozonide). B1–B3: ​​Escherichia coli ( Escherichia coli Normal colorimetric group; B4–B6: Escherichia coli positive control group (+ nitrozonide). C1–C3: Bifidobacteria ( Bifidobacterium spp. Normal colorimetric group; C4–C6: Bifidobacterium positive control group (+ nitrozonide); D1–D3: Bacillus coagulans ( Bacillus coagulans Normal colorimetric group; D4–D6: Bacillus coagulans positive control group (+ nitrozonide). E1–E3: Bacteroides ( Bacteroides spp. Normal colorimetric group; E4–E6: Bacteroides positive control group (+ nitrozonide); F1–F3: Propionibacterium acnes ( Cutibacterium acnes Normal colorimetric group; F4–F6: Propionibacterium acnes positive control group (+ nitrozonidine).

[0071] The selected strains cover strict anaerobes (Clostridium difficile, Bifidobacterium, Bacteroides), facultative anaerobes (Escherichia coli, Bacillus coagulans), and skin-resident microaerophiles (Propionibacterium acnes), and are broadly representative in terms of metabolic type and oxygen requirement.

[0072] After adding the sample, the 96-well plate was incubated in the dark under suitable conditions for 7 hours. During this time, the color development changes were observed and photographed. Color intensity (OD) 546 This reflects the bacterial energy metabolism activity.

[0073] The results show that ( Figure 2Compared to the normal chromogenic group, the addition of nitrozonide significantly inhibited the chromogenic reactions of all tested strains, manifested as a marked decrease or complete disappearance of chromogenic intensity. This phenomenon indicates that nitrozonide can effectively interfere with the core energy metabolism pathways of various bacteria, possessing broad-spectrum metabolic inhibitory properties, and can be used as a positive control in relevant screening experiments.

[0074] Example 5: Screening Method for Bacterial Energy Metabolism Inhibitors Based on Colorimetric Reaction This experimental example uses Clostridium difficile 630 as a representative strain to illustrate the specific procedure for screening bacterial energy metabolism inhibitors using the kit of this invention.

[0075] 1. Preparation of bacterial suspension: Culterate Clostridium difficile 630 to the logarithmic growth phase, and adjust the bacterial suspension concentration to OD. 600 = 0.5, spare.

[0076] 2. Sample addition and compound treatment: see attached. Figure 3 As shown, under anaerobic conditions, the following were added to a 96-well plate: Blank control group (A1, B1, C1): 100 μL of culture medium (sterile-free) was added to each well; Positive control group (A2, B2, C2): 100 μL of bacterial suspension was added to each well, and the positive control compound (nitrozonide) was added to make the final concentration 100 μg / mL; Experimental group (the remaining wells): 100 μL of bacterial suspension was added to each well, and the candidate compounds to be screened were added separately. The final concentration of each compound was uniformly set to 128 μg / mL.

[0077] After adding the sample, incubate the 96-well plate at a suitable temperature for 1 hour to allow the compound to fully interact with the bacteria.

[0078] 3. Color Development and Interpretation: Add 100 μL of the color development solution prepared according to the method in Experimental Group 2 of Example 2 to each well, mix well, and continue incubation. Observe and record the color development results. If the color depth of the experimental well is significantly weaker than that of the normal bacterial culture control well (parallel settings can be made), the candidate compound is determined to be a potential positive, possessing activity in inhibiting bacterial energy metabolism. This method achieves high-throughput, visualized initial screening for bacterial energy metabolism inhibitors.

[0079] Example 6: Confirmation of the energy metabolism inhibitory activity of screened compounds To confirm the inhibitory effect of the screened compounds on bacterial energy metabolism and to further verify the authenticity and reliability of the kit in screening functional inhibitors, we conducted verification experiments based on the core principle that "impaired energy metabolism directly affects ATP production." Given that ATP is a key end product of energy metabolism, and its intracellular content directly reflects the metabolic activity and vitality of microorganisms, we selected a commercially available ATP assay kit (Promega BacTiter-Glo™ Microbial Cell Viability Assay) as an independent verification method. By detecting changes in bacterial ATP levels after compound treatment, we assessed its impact on Clostridium difficile's energy metabolism.

[0080] 1. Experimental Grouping Blank control group (NC): Clostridium difficile bacterial suspension.

[0081] Positive control group: Clostridium difficile suspension, co-incubated with nitrozonidine (NTZ, final concentration 32 µg / mL).

[0082] LCA group: Clostridium difficile bacterial suspension, added with glycyrrhizin chalcone A (LCA, final concentration 64 µg / mL) obtained through screening, and incubated for 1 h, 3 h, and 6 h respectively. 2. Test Methods Clostridium difficile 630 bacterial culture in the logarithmic growth phase (OD) 600 = 0.5) were co-incubated with NTZ or LCA, respectively, with a blank control group without the drug. After a certain reaction time, BacTiter-Glo reagent was added to lyse the bacteria and initiate the luciferase reaction. The intracellular ATP level was quantified by detecting the bioluminescence intensity. Each group had three replicates, and the experiment was repeated three times.

[0083] 3. Experimental Results Compared with the blank control group, the intracellular ATP luminescence value of Clostridium difficile decreased significantly after NTZ (32 µg / mL) treatment for 1 h, indicating that the detection system has a good response to energy metabolism inhibitors. Figure 4 A).

[0084] After treatment with LCA (64 µg / mL), the bacterial ATP level decreased rapidly within 1 h and further decreased with prolonged incubation time (3 h, 6 h), showing a significant time-dependent inhibitory effect. Figure 4 B).

[0085] 4. Experimental Conclusions LCA rapidly depletes the intracellular ATP pool of Clostridium difficile and inhibits its energy metabolism pathway, demonstrating a clear antibacterial mechanism. This result is highly consistent with the colorimetric reaction trend of the energy metabolism chromogenic kit, validating the accuracy and reliability of this kit in screening energy metabolism inhibitors. Therefore, LCA can be used as a positive control compound for this kit in subsequent functional screening studies.

[0086] The above description is merely a preferred embodiment of the invention. It should be understood that the invention is not limited to the forms disclosed herein and should not be construed as excluding other embodiments. It can be used in various other combinations, modifications, and environments, and can be altered within the scope of the concept described herein through the above teachings or related technical or knowledge. Any modifications and variations made by those skilled in the art that do not depart from the spirit and scope of the invention should be protected within the scope of the appended claims.

Claims

1. A rapid detection kit for bacterial energy metabolism, characterized in that, The materials include: reaction premix powder, anaerobic reaction solution, and optional positive control; The reaction premixed powder comprises the following components in parts by weight: 1 part benzyl viologen; 0.43~0.64 parts sodium pyruvate; 0.054~0.082 parts coenzyme A; and 0.037~0.055 parts MgCl2·7H2O. The anaerobic reaction solution is a buffer solution containing sodium sulfide-9 hydrate, wherein the concentration of sodium sulfide-9 hydrate is 15~25 μg / mL; The amount of reaction premix powder added per 100 mL of anaerobic reaction solution is 0.30~0.40 g; The positive control is either nitrozonide or glycyrrhizin A.

2. The rapid detection kit for bacterial energy metabolism according to claim 1, characterized in that, The reaction premixed powder comprises the following components in parts by weight: 1 part benzyl viologen; 0.537 parts sodium pyruvate; 0.068 parts coenzyme A; and 0.046 parts MgCl2·7H2O. And / or, the concentration of sodium sulfide-9 hydrate in the anaerobic reaction solution is 20 μg / mL; And / or, the amount of reaction premix powder added per 100 mL of anaerobic reaction solution is 0.3395 g.

3. The rapid detection kit for bacterial energy metabolism according to claim 1, characterized in that, The anaerobic reaction solution is a phosphate buffer solution containing sodium sulfide-9 hydrate; preferably, the pH value of the phosphate buffer solution is 7.

2.

4. A rapid detection kit for bacterial energy metabolism according to claim 3, characterized in that, The concentrations of phosphates in the anaerobic reaction solution were: K₂HPO₄ 0.698 g / 100 mL and KH₂PO₄ 0.135 g / 100 mL.

5. A rapid detection kit for bacterial energy metabolism according to claim 1, characterized in that, The positive control, nitrozanide, is provided in solid powder form or as a DMSO solution; when used, its working concentration is ≥8 μg / mL. And / or, the positive control, glycyrrhizin A, is provided in solid powder form or as a DMSO solution; when used, its working concentration is 32–256 μg / mL.

6. A rapid detection kit for bacterial energy metabolism according to any one of claims 1-5, characterized in that, Both the reaction premix powder and the anaerobic reaction solution were stored under anaerobic conditions.

7. A method for preparing a rapid detection kit for bacterial energy metabolism according to any one of claims 1-6, characterized in that, Includes the following steps: S1. Preparation of reaction premix powder: Weigh each component of the reaction premix powder according to the proportion, mix evenly, dispense into brown glass bottles, anaerobic gas equilibration treatment to remove oxygen, and seal. S2. Anaerobic reaction solution: Under inert gas protection, phosphate and sodium sulfide-9 hydrate are added to deoxygenated water, mixed and dissolved to obtain a mixed reaction solution; the mixed reaction solution is dispensed into sealed containers, sterilized, and then kept sealed and cooled to room temperature to obtain an anaerobic reaction solution. Steps S1 and S2 are not sequential.

8. The method for preparing a rapid detection kit for bacterial energy metabolism according to claim 7, characterized in that, In step S2, phosphate and sodium sulfide-9 hydrate stock solution are added to the deoxygenated water and mixed to dissolve.

9. The method for preparing a rapid detection kit for bacterial energy metabolism according to claim 7, characterized in that, When the positive control is provided in solid powder form, weigh an appropriate amount and store it in a sealed container. When the positive control is provided in the form of DMSO solution, prepare the DMSO solution of the positive control and store it in a sealed container.

10. The application of a rapid detection kit for bacterial energy metabolism according to any one of claims 1-6, characterized in that, The applications include: (1) Rapid detection of bacterial energy metabolism activity; and / or (2) Screening for substances that inhibit bacterial energy metabolism.

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