Search

CN-122012668-A - Preparation method and application of flat plate for detecting minimum inhibitory concentration of fosfomycin by simple agar dilution method

CN122012668ACN 122012668 ACN122012668 ACN 122012668ACN-122012668-A

Abstract

The invention discloses a preparation method and application of a flat plate for detecting the minimum inhibitory concentration of fosfomycin by a simple agar dilution method. The method comprises the following steps of pre-dissolving fosfomycin and glucose-6-phosphate dry powder in a small amount of sterile water according to a target concentration, quantitatively coating the solution on the surface of solidified agar, and uniformly penetrating the dried fosfomycin into the agar to form a flat plate with a specific concentration gradient. The minimum inhibitory concentration of the fosfomycin is detected by the flat plate provided by the invention, and through verification of 100 clinical urinary tract infection escherichia coli strains, the basic consistency of the method is 100 percent, the classification consistency is 99 percent and the medicine distribution is uniform compared with the standard agar dilution method. The invention eliminates the complicated steps of preparing agar from scratch by the traditional method, has simple and convenient operation, is accurate and reliable, solves the technical problems of complicated operation and short shelf life of a flat plate of the traditional agar dilution method, and provides a high-efficiency and standardized fosfomycin drug-sensitive detection scheme for a clinical microbiological laboratory.

Inventors

  • LIANG SHIZHOU
  • Cai Wenpin
  • Kong Wanzhong
  • CHEN MENGQUAN
  • LIN YUHANG
  • YU JIANJIE

Assignees

  • 温州市中医院

Dates

Publication Date
20260512
Application Date
20260320

Claims (9)

  1. 1. A flat plate preparation method for detecting the minimum inhibitory concentration of fosfomycin by a simple agar dilution method is characterized by comprising the following steps: (1) Preparing a fosfomycin stock solution; (2) Preparing glucose-6-phosphate storage solution; (3) Mixing the fosfomycin stock solution with the glucose-6-phosphoric acid stock solution to obtain a working solution; (4) Pouring working solution directly on the surface of a commercial MH agar plate; (5) Uniformly coating the working solution on the surface of agar; (6) And (3) drying to enable the liquid to be completely absorbed, thus obtaining the fosfomycin drug sensitive plate.
  2. 2. The method according to claim 1, wherein in the step (1), the ratio N of the concentration of the fosfomycin stock solution to the final concentration of fosfomycin in the fosfomycin drug sensitive plate in the step (6) is calculated according to the volume V 1 of the fosfomycin stock solution in the working solution in the step (4) and the agar volume V 2 of the commercial MH agar plate, and the value of N is 。
  3. 3. The method for preparing a plate according to claim 2, wherein in the step (6), the final concentration of glucose-6-phosphate in the fosfomycin drug sensitive plate is 25 mg/L and the final concentration of fosfomycin is one or more of 0.03125-2048 mg/L.
  4. 4. The method according to claim 3, wherein the mixing ratio of the fosfomycin stock solution and the glucose-6-phosphate stock solution in the step (3) is 5:2.
  5. 5. The method of claim 3, wherein in the step (6), 17 fosfomycin-sensitive plates are provided, and the final fosfomycin concentration in the 17 fosfomycin-sensitive plates is between 0.03125 and 2048 mg/L, which is a total of 17 times concentration gradient distribution.
  6. 6. The method for producing a flat plate according to claim 2, wherein the drying treatment in the step (6) is to place the coated flat plate in a 37 ℃ incubator for 2 hours.
  7. 7. Use of a fosfomycin drug sensitive tablet prepared by the tablet preparation method of any one of claims 1-6 for detecting a minimal inhibitory concentration of escherichia coli on fosfomycin.
  8. 8. Use of a kit for detecting a minimum inhibitory concentration of fosfomycin based on a simple agar dilution method based on a plate preparation method according to any one of claims 1-6, characterized by comprising fosfomycin lyophilized powder, G-6-P, a plurality of commercially available finished MHA plates for configuring a plurality of fosfomycin drug sensitive plates of a series of final concentrations and instructions.
  9. 9. The use according to claim 7, wherein the fosfomycin freeze-dried powder is packaged according to gradient concentration, and the fosfomycin freeze-dried powder is mixed with a specific volume of dispersion solvent to obtain fosfomycin solution with 60 times of target final concentration; The G-6-P split charging is independent package corresponding to the split charging quantity of the fosfomycin freeze-dried powder.

Description

Preparation method and application of flat plate for detecting minimum inhibitory concentration of fosfomycin by simple agar dilution method Technical Field The invention belongs to the technical field of clinical microbiology inspection, and particularly relates to a preparation method and application of a flat plate for detecting the minimum inhibitory concentration of fosfomycin by a simple agar dilution method. Background Fosfomycin has recently been newly assigned an important clinical position as an ancient antimicrobial drug due to the severe situation of multiple-resistant gram-negative bacterial infections, especially in the treatment of urinary tract infections. The reasonable application of the method depends on accurate Minimum Inhibitory Concentration (MIC) detection. Currently, agar Dilution (AD) recommended by the clinical laboratory and laboratory standards institute (CLSI) is a reference method for the MIC detection of fosfomycin. However, the conventional agar dilution method has the following technical drawbacks: 1. complicated operation, namely, the need to prepare agar from scratch, dissolve medicines and pour a flat plate, a large number of pipetting and diluting steps are involved, the time consumption is long, and the labor intensity is high; 2. the shelf life of the tablet is short, namely the valid period of the prepared tablet containing the medicine is usually only about 1 week, and the tablet needs to be prepared at present, so that reagent waste is caused; 3. Errors are easy to introduce, namely the multi-step operation increases pollution risks and human errors, and the reproducibility among different batches is difficult to ensure; 4. The defects are difficult to develop routinely, so that the clinical laboratory rarely adopts an agar dilution method in daily work, and instead uses a paper sheet diffusion method, a gradient diffusion method and the like, but the methods have reports of unstable results when the fosfomycin is detected. Therefore, the method for detecting the fosfomycin drug sensitivity is simple, convenient, accurate and reliable in operation, can be used for standardized preparation, and has important clinical practical value. Disclosure of Invention The invention aims to overcome the defects of complicated operation, complex plate preparation and short storage period of an agar dilution method in the prior art, and provides a plate preparation method for detecting the minimum inhibitory concentration of fosfomycin by a simple agar dilution method and application thereof. The application provides a preparation method of a flat plate for detecting the minimum inhibitory concentration of fosfomycin by a simple agar dilution method, which comprises the following steps: (1) Preparing a fosfomycin stock solution; (2) Preparing glucose-6-phosphate storage solution; (3) Mixing the fosfomycin stock solution with the glucose-6-phosphoric acid stock solution to obtain a working solution; (4) Pouring working solution directly on the surface of a commercial MH agar plate; (5) Uniformly coating the working solution on the surface of agar; (6) And (3) drying to enable the liquid to be completely absorbed, thus obtaining the fosfomycin drug sensitive plate. Further, in the step (1), the ratio N of the concentration of the fosfomycin stock solution in the step (1) to the final concentration of fosfomycin in the fosfomycin medicine sensitive plate in the step (6) is calculated according to the volume V 1 of the fosfomycin stock solution in the working solution in the step (4) and the agar volume V 2 of the commercial MH agar plate, and the value of N is。 Further, in the step (6), the final concentration of glucose-6-phosphate in the fosfomycin drug sensitive plate is 25 mg/L, and the final concentration of fosfomycin is one or more of 0.03125-2048 mg/L. Further, the mixing ratio of the fosfomycin stock solution and the glucose-6-phosphate stock solution in the step (3) is 5:2. Further, in the step (6), 17 fosfomycin medicine sensitive plates are arranged, and the final concentration of fosfomycin in the 17 fosfomycin medicine sensitive plates is 0.03125-2048 mg/L and is distributed in a concentration gradient mode by 17 times. Further, the drying treatment in the step (6) is to put the coated flat plate in a 37 ℃ incubator for 2 hours. The application of the fosfomycin drug sensitive plate prepared by the plate preparation method in detecting the minimum inhibitory concentration of escherichia coli on fosfomycin. The application of the kit for detecting the minimum inhibitory concentration of the fosfomycin based on the simple agar dilution method based on the flat preparation method comprises fosfomycin freeze-dried powder, G-6-P, a plurality of commercial finished product MHA flat plates for preparing a plurality of fosfomycin drug sensitive flat plates with serial final concentrations and instructions. Further, the fosfomycin freeze-dried powder is packaged according to gr