Search

CN-115820789-B - Pseudomonas aeruginosa detection method and kit

CN115820789BCN 115820789 BCN115820789 BCN 115820789BCN-115820789-B

Abstract

The invention provides a pseudomonas aeruginosa detection method and a kit. The method comprises the steps of S1, mixing a sample to be detected and galactose modified magnetic nano particles in a dispersion system to obtain a mixed dispersion system, S2, placing the mixed dispersion system in a magnetic field, and performing magnetic separation to obtain a magnetic substance, S3, placing the magnetic substance in the step S2 and gelatin enzyme sensitive particles carrying fluorescent molecules in a sterile liquid culture medium for culture to obtain a culture solution, S4, centrifuging the culture solution in the step S3 to obtain a supernatant, S5, measuring the fluorescence intensity of the supernatant in the step S4, and judging whether the sample to be detected contains pseudomonas aeruginosa according to the fluorescence intensity. The detection method provided by the invention realizes the rapid detection of the pseudomonas aeruginosa based on the characteristic of the specific interaction of galactose and the pseudomonas aeruginosa surface and the metabolism of the gelatin enzyme by the pseudomonas aeruginosa.

Inventors

  • PENG QINGZHI
  • LU ZHENTAN
  • ZHANG LI
  • GUO YINLI
  • WANG MINGQIU
  • LIN JIN
  • YU TINGTING
  • LI SHIYAO

Assignees

  • 湖北省食品质量安全监督检验研究院

Dates

Publication Date
20260508
Application Date
20221018

Claims (9)

  1. 1. The method for detecting the pseudomonas aeruginosa for non-diagnostic purposes is characterized by comprising the following steps of: s1, placing a sample to be tested and galactose modified magnetic nanoparticles in a dispersion system for mixing to obtain a mixed dispersion system; s2, placing the mixed dispersion system in the step S1 in a magnetic field, and obtaining a magnetic substance through magnetic separation; S3, placing the magnetic substance and the gelatinase-sensitive particles carrying fluorescent molecules in the step S2 in a sterile liquid culture medium for culturing to obtain a culture solution, wherein the gelatinase-sensitive particles are gelatinase particles, and if the pseudomonas aeruginosa exists in a sample to be detected, gelatinase produced by the metabolism of the pseudomonas aeruginosa can decompose the gelatinase-sensitive particles, so that the fluorescent molecules are released into the culture solution; s4, centrifuging the culture solution obtained in the step S3 to obtain a supernatant; s5, measuring the fluorescence intensity of the supernatant in the step S4, and judging whether the sample to be detected contains pseudomonas aeruginosa according to the fluorescence intensity.
  2. 2. The method according to claim 1, wherein the galactose-modified magnetic nanoparticles in step S1 are present in a concentration of 0.08-0.15mg/mL in the mixed dispersion.
  3. 3. The method according to claim 1, wherein the magnetic field in step S2 has a strength of 0.4 to 0.8 tesla.
  4. 4. The method according to claim 1, wherein the concentration of the fluorescent molecule-loaded gelatinase-sensitive particles in the culture solution in step S3 is 0.35-0.5mg/mL.
  5. 5. The method according to claim 1, wherein the sterile liquid medium of step S3 comprises tryptone, yeast extract, naCl and water.
  6. 6. The method according to claim 1, wherein the temperature of the culture in step S3 is 35-37℃and the time of the culture is 5-10 hours.
  7. 7. The method according to claim 1, wherein the centrifugation in step S4 is performed for 2-4min at 4000-6000 rpm.
  8. 8. The method according to claim 1, wherein the criteria for the determination in step S5 include: When the fluorescence intensity of the sample group to be detected is more than or equal to 2 times of that of the negative control group, judging that the sample to be detected contains pseudomonas aeruginosa; And when the fluorescence intensity of the sample group to be detected is 2 times of that of the negative control group, judging that the sample to be detected does not contain pseudomonas aeruginosa.
  9. 9. The method of any one of claims 1-8, wherein the method of detection comprises any one of the following applications: (1) The sample to be tested in the step S1 is a diet product and is used for detecting whether the diet product contains pseudomonas aeruginosa or not; (2) The sample to be detected in the step S1 is a cosmetic and is used for detecting whether the cosmetic contains pseudomonas aeruginosa or not; (3) The sample to be detected in the step S1 is in-vitro environment sampling and is used for detecting whether pseudomonas aeruginosa is contained in-vitro environment.

Description

Pseudomonas aeruginosa detection method and kit Technical Field The invention relates to the technical field of microorganism detection, in particular to a pseudomonas aeruginosa detection method and a kit. Background Pseudomonas aeruginosa is a common conditional pathogenic bacterium in natural environment, is also one of main pathogenic bacteria in hospitals, and can cause wound surface, respiratory tract and urinary system infection. The quality detection standards of the current foods and cosmetics clearly require that pseudomonas aeruginosa cannot be detected in samples. The existing detection method for pseudomonas aeruginosa is complex. Taking GB 8538-2022 national Standard for food safety as an example of a method for testing natural mineral Water for drinking, the current national Standard for detecting Pseudomonas aeruginosa comprises the steps of filtering 250mL of water sample by using a bacterial filter membrane, attaching the filter membrane to the surface of a solid culture medium, culturing for 20-48h in a professional laboratory constant temperature environment, selecting suspicious colonies, counting respectively, and further identifying whether Pseudomonas aeruginosa exists through a pyocin test, an ammonia production test, a 42 ℃ growth test, an oxidase test and a fluorescence test. The traditional national standard detection method for pseudomonas aeruginosa has high accuracy and strong specificity. However, the problem of long detection time also exists, the storage cost of the food industry is increased, and the time-consuming requirement in the food safety guarantee of important social activities is difficult to adapt. Disclosure of Invention In view of the technical problems in the background art, the invention provides a pseudomonas aeruginosa detection method. The method realizes the rapid detection of the pseudomonas aeruginosa based on the characteristic of the specific interaction of galactose and the pseudomonas aeruginosa surface and the metabolism of gelatin enzyme by the pseudomonas aeruginosa. The invention provides a non-diagnostic pseudomonas aeruginosa detection method, which comprises the following steps: s1, placing a sample to be tested and galactose modified magnetic nanoparticles in a dispersion system for mixing to obtain a mixed dispersion system; s2, placing the mixed dispersion system in the step S1 in a magnetic field, and obtaining a magnetic substance through magnetic separation; S3, placing the magnetic substance and the gelatinase sensitive particles carrying fluorescent molecules in the step S2 into a sterile liquid culture medium for culture to obtain a culture solution; s4, centrifuging the culture solution obtained in the step S3 to obtain a supernatant; s5, measuring the fluorescence intensity of the supernatant in the step S4, and judging whether the sample to be detected contains pseudomonas aeruginosa according to the fluorescence intensity. Preferably, the concentration of galactose-modified magnetic nanoparticles in the mixed dispersion of step S1 is 0.08-0.15mg/mL. Preferably, the magnetic field in step S2 has a strength of 0.4 to 0.8 Tesla. Preferably, after the magnetic substance is obtained in step S2, the magnetic substance is washed with sterile water. Preferably, the concentration of the gelatinase-sensitive particles loaded with fluorescent molecules in the step S3 in the culture solution is 0.35-0.5mg/mL. Preferably, the sterile liquid medium of step S3 comprises tryptone, yeast extract, naCl and water. Preferably, the temperature of the culture in step S3 is 35-37 ℃, and the time of the culture is 5-10 hours. Preferably, the centrifugation parameters in step S4 are 4000-6000 rpm and 2-4min. Preferably, the criteria for the determination in step S5 include: When the fluorescence intensity of the sample group to be detected is more than or equal to 2 times of that of the negative control group, judging that the sample to be detected contains pseudomonas aeruginosa; And when the fluorescence intensity of the sample group to be detected is 2 times of that of the negative control group, judging that the sample to be detected does not contain pseudomonas aeruginosa. Preferably, the detection method comprises any one of the following applications: (1) The sample to be tested in the step S1 is a diet product and is used for detecting whether the diet product contains pseudomonas aeruginosa or not; (2) The sample to be detected in the step S1 is a cosmetic and is used for detecting whether the cosmetic contains pseudomonas aeruginosa or not; (3) The sample to be detected in the step S1 is in-vitro environment sampling and is used for detecting whether pseudomonas aeruginosa is contained in-vitro environment. The invention also provides a kit for detecting pseudomonas aeruginosa, which comprises the galactose modified magnetic nanoparticle, the gelatinase sensitive particle loaded with fluorescent molecules and the sterile liquid culture medium.