This protocol only provides experimental guidance. The actual application must be optimized and adjusted according to your research objectives, sample type, and detection platform [1-2].
1. Preparation of reagents and materials
Nitrocefin, DMSO, PBS, bacterial colonies, bacterial suspension or lysed cell samples, hydrolysis buffer.
2. Preparation of Nitrocefin solution
(1) Stock solution (1–10mg/mL): Dissolve 5–10mg Nitrocefin in DMSO, mix well, store in the dark, and store at –20℃ for several months.
(2) Working solution (0.5–1.0mg/mL): Dilute the stock solution in PBS or Assay Buffer to 0.5–1.0mg/mL. If the solution turns red (not yellow) after preparation, it means the concentration is too high and should be further diluted to a yellow solution. The working solution should be kept away from light, stored at -20℃, and used within 14 days or 2 months.
3. Rapid detection method for β-lactamase activity
(1) Slide test: Add one drop of working solution to a clean slide, inoculate a single colony and observe the appearance of red (within 20-30 minutes).
(2) Direct contact method: Drop the working solution on the colony and observe the color change to red within 20-30 minutes.
(3) Bacterial solution method: Add 3-5 drops of working solution to 1mL of bacterial solution. If the color changes to red within 20-30 minutes, it indicates a positive result.
(4) Cell lysis method: Ultrasonic lyse 1mL of bacterial solution and add 3-5 drops of working solution. If the color changes to red within 40 minutes, it indicates β-lactamase activity.
4. Photometric quantitative detection
(1) Standard solution and reaction system: Prepare the standard solution and standard gradient using Nitrocefin and Hydrolysis Buffer + DMSO.
(2) Sample processing: After centrifugation, resuspending, and ultrasonic lysis of the solid strain, the supernatant was collected for assay. Reaction mixture: Sample + Reaction Mix (e.g., Assay Buffer + Nitrocefin) was added to each well.
(3) Reading and calculation: Kinetic reading was performed at 490nm. It is recommended to read every minute for 30–60min. β-lactamase activity was calculated based on the standard curve and time changes.
(4) The unit is defined as the amount of enzyme that hydrolyzes 1µmol Nitrocefin per minute.
Operation precautions
(1) Avoid light during operation to prevent drug degradation.
(2) Do not use the working solution before it turns red to maintain the accuracy of the yellow formula.
(3) Experimental conditions should be optimized by gradient concentration and time for each sample type.
References:
[1]. Shannon K, Phillips I. ?-Lactamase detection by three simple methods: Intralactam, nitrocefin and acidimetric[J]. Journal of Antimicrobial Chemotherapy, 1980, 6(5): 617-621.
[2]. Allen C, Turner C, Kalsi S, et al. Development of a rapid phenotypic test on a microfluidic device for carbapenemase detection using the chromogenic compound nitrocefin[J]. Diagnostic Microbiology and Infectious Disease, 2020, 96(4): 114926.
