阅读说明：本技术 一种快速微生物抗生素敏感性检测方法 (Rapid detection method for microbial antibiotic sensitivity ) 是由 唐明忠 张元珍 于 2018-07-27 设计创作，主要内容包括：本发明提供的一种快速微生物抗生素敏感性检测方法,采用不同浓度的待测抗生素分别与待测微生物悬浮液混合并孵育,与不含待测抗生素的阳性对照相比,当活体待测微生物受抗生素抑制细胞个数降低的百分率达到20％以上时,对应的所述待测抗生素的浓度为待测微生物的最小抑菌浓度；本发明提出的上述的快速微生物抗生素敏感性检测方法,所述方法可在3小时内即确定待测微生物的最小抑菌浓度(MIC),且与常规的方法测定的结果一致,测定的结果稳定可靠,可以在短时间内测定微生物对抗生素的敏感性,适合于临床实践,解决了现有技术中的微生物抗生素敏感性检测方法时间长,不适于临床实践的缺陷,且成本低,易操作,应用范围广。(According to the rapid detection method for the microbial antibiotic sensitivity, antibiotics to be detected with different concentrations are mixed with a suspension of the microbes to be detected respectively and are incubated, compared with a positive control without the antibiotics to be detected, when the percentage of the number of cells of the living microbes to be detected, which are inhibited by the antibiotics, is reduced to more than 20%, the corresponding concentration of the antibiotics to be detected is the minimum inhibitory concentration of the microbes to be detected; the method for rapidly detecting the antibiotic sensitivity of the microorganism can determine the Minimum Inhibitory Concentration (MIC) of the microorganism to be detected within 3 hours, has the same determination result with the conventional method, has stable and reliable determination result, can determine the sensitivity of the microorganism to the antibiotic within short time, is suitable for clinical practice, overcomes the defects that the method for detecting the antibiotic sensitivity of the microorganism in the prior art has long time and is not suitable for clinical practice, and has low cost, easy operation and wide application range.)

1. A rapid detection method for microbial antibiotic sensitivity is characterized in that antibiotics to be detected with different concentrations are adopted to be respectively mixed with a suspension of the microorganisms to be detected and incubated, and when the percentage of the number of cells of the living microorganisms to be detected, which are inhibited by the antibiotics, is reduced to more than 20% compared with a positive control containing no antibiotics to be detected, the corresponding concentration of the antibiotics to be detected is the minimum inhibitory concentration of the microorganisms to be detected.

2. The method for rapidly detecting the antibiotic susceptibility of microorganisms according to claim 1, wherein when the percentage of the number of cells of the microorganisms to be detected in vivo, which are inhibited by the antibiotic, is reduced to more than 40%, the corresponding concentration of the antibiotic to be detected is the minimum inhibitory concentration of the microorganisms to be detected.

3. The method for rapidly detecting the antibiotic sensitivity of microorganisms according to claim 1 or 2, wherein when the percentage of the number of cells of the microorganisms to be detected in vivo, which are inhibited by the antibiotic, is reduced to more than 50%, the corresponding concentration of the antibiotic to be detected is the minimum inhibitory concentration of the microorganisms to be detected. Preferably, when the percentage of the number of cells of the living microorganism to be tested, which are inhibited by the antibiotic, is reduced to more than 60%, the corresponding concentration of the antibiotic to be tested is the minimum inhibitory concentration of the microorganism to be tested.

4. The rapid microbial antibiotic susceptibility test method of any of claims 1-3, wherein the incubation time is between 30 minutes and 180 minutes.

5. The rapid microbial antibiotic susceptibility test method of any of claims 1-4, wherein the incubation time is between 60 minutes and 120 minutes. Preferably, the incubation time is 90 minutes.

6. The method for rapid sensitivity to microbial antibiotics according to any one of claims 1-5, wherein the concentration of the suspension of the microorganism to be tested is 0.4-0.6 McLee's unit. Preferably, the concentration of the suspension of the microorganism to be tested is 0.5 McLee units.

7. The method for rapid sensitivity test of microbial antibiotics according to any one of claims 1-6, wherein the bacterial content of the antibiotics to be tested is (4-6). times.10 after mixing with the suspension of the microbes to be tested respectively with different concentrations⁵cfu/ml. Preferably, the bacteria content is 5X 10⁵cfu/ml。

8. The rapid microbial antibiotic susceptibility test method of any of claims 1-7, wherein the number of viable test microbial cells is measured using a flow cytometer or a fluorescence microscopy instrument.

9. The rapid microbial antibiotic susceptibility test method of any of claims 1-8, wherein the incubation temperature is 34-36 ℃. Preferably, the incubation temperature is 35 ℃.

Technical Field

The invention belongs to the field of microbial drug resistance detection, and particularly relates to a rapid microbial antibiotic susceptibility detection method.

Background

The response characteristics of microorganisms under external environmental stress such as antibiotics are important for ecological safety evaluation and the like, and have been receiving attention from people. The problem of microbial resistance is becoming more serious and rapidly widespread worldwide due to abuse of antibiotics, and therefore, in response characteristics, microbial resistance is currently the most interesting. Rational use of antibiotics is the most central task to cope with microbial resistance, and rapid detection of microbial antibiotic susceptibility is central. Therefore, various rapid detection methods have been developed and applied for determining antibiotic sensitivity or Minimum Inhibitory Concentration (MIC).

The essence of antibiotic sensitivity detection is to observe the influence of antibiotic on the growth, metabolism and reproduction of bacteria, and to deduce the effectiveness of future medication according to the condition of the influence of antibiotic on the growth, metabolism and reproduction of bacteria observed in vitro tests and the clinical and pharmacokinetic conditions. The traditional method comprises a diffusion method and a dilution method, wherein antibiotics with different concentrations are added into a culture medium by the dilution method, and the inhibition characteristic of the microorganisms on the antibiotics is evaluated by observing the turbidity of a liquid culture medium or the growth condition of colonies on a solid culture medium by naked eyes. The disadvantages of the above method are that it takes a long time, it generally takes overnight for the culture, and the consumption of the sample or reagent is large. Besides the method, an E-test antibacterial test strip can be adopted, antibiotics with concentration gradient are fixed on the test strip, a plate culture method is combined, and an MIC value is read through the intersection point of an antibacterial ring and the test strip. The bacteriostatic test strip has the defect of long culture time. From the above, the conventional antibiotic sensitivity detection methods have long detection time, and cannot rapidly provide effective treatment schemes for patients, so that the application universality is limited.

In order to shorten the detection time of antibiotic sensitivity, a great deal of research is carried out at home and abroad, and various antibiotic sensitivity detection methods such as a mass spectrometry method, a vibrating cantilever microbial cell weighing method, an isothermal micro-mass heating method, a magnetic bead rotation method, a microdroplet detection method, a real-time PCR method, a microarray method, an RNA sequencing method, a phage method and the like are explored. However, the above methods are currently only in the research stage, only small sample analysis can be performed, and all the methods require professional technical personnel to operate, and the instruments are expensive, non-traditional professional equipment, complex to operate, unstable in performance, high in cost, inconvenient to use, and not widely applicable. At present, a full-automatic antibiotic sensitivity detection method is adopted clinically, wherein a VITEK system of French Merrier company and a Phoenix system of American BD company are the fastest detection systems, the principle of the detection system is an optical turbidimetry or a colorimetry, the reliability and the accuracy of the two systems are proved, the average detection time of the VITEK system is 9.8 hours, and the average detection time of the Phoenix system is 12.1 hours. Although the detection time of the two systems is greatly shortened, the doctor can only pertinently select the medicine according to the detection result in the next day in consideration of the work flow and the work and rest time of the doctor, and the empirical broad-spectrum antibiotic treatment cannot be switched to the targeted treatment as soon as possible, so that the illness state is easily delayed, the death of the patient is caused, and the medical cost is increased. In view of the wide clinical application of mass spectrometry and nucleic acid technology, the rapidity of bacterial identification has been substantially realized, and therefore, the antibiotic sensitivity detection time of the two systems limits clinical practice.

Therefore, the invention provides a rapid detection method for the antibiotic sensitivity of the microorganism, which can determine the sensitivity of the microorganism to the antibiotic in a short time and is suitable for clinical practice.

Disclosure of Invention

Therefore, the technical problem to be solved by the invention is to overcome the defects that the drug sensitivity detection time of the microorganism is long and the microorganism is not suitable for clinical practice in the prior art, so that the invention provides a rapid detection method for the antibiotic sensitivity of the microorganism, which can determine the sensitivity of the microorganism to the antibiotic in a short time and is suitable for clinical practice.

The invention provides a rapid detection method for the antibiotic sensitivity of microorganisms, which adopts antibiotics to be detected with different concentrations to be mixed and incubated with a suspension of the microorganisms to be detected respectively, and when the percentage of the number of cells of the microorganisms to be detected, which are inhibited by the antibiotics, of a living body is reduced to more than 20 percent compared with a positive control containing no antibiotics to be detected, the corresponding concentration of the antibiotics to be detected is the minimum inhibitory concentration of the microorganisms to be detected.

According to the rapid detection method for the microbial antibiotic susceptibility, when the percentage of the number of cells of a living microorganism to be detected, which are inhibited by the antibiotic, is reduced to more than 40%, the corresponding concentration of the antibiotic to be detected is the minimum inhibitory concentration of the microorganism to be detected.

According to the rapid detection method for the microbial antibiotic susceptibility, when the percentage of the number of cells of a living microorganism to be detected, which are inhibited by the antibiotic, is reduced to more than 50%, the corresponding concentration of the antibiotic to be detected is the minimum inhibitory concentration of the microorganism to be detected. Preferably, when the percentage of the number of cells of the living microorganism to be tested, which are inhibited by the antibiotic, is reduced to more than 60%, the corresponding concentration of the antibiotic to be tested is the minimum inhibitory concentration of the microorganism to be tested.

The quick detection method for the antibiotic sensitivity of the microorganisms has the incubation time of 30-180 minutes.

The quick detection method for the antibiotic sensitivity of the microorganisms has the incubation time of 60-120 minutes. Preferably, the incubation time is 90 minutes.

According to the rapid detection method for the microbial antibiotic sensitivity, the concentration of the microbial suspension to be detected is 0.4-0.6 McLee unit. Preferably, the concentration of the suspension of the microorganism to be tested is 0.5 McLee units.

The quick detection method for the microbial antibiotic sensitivity adopts the method that after antibiotics to be detected with different concentrations are respectively mixed with a microbial suspension to be detected, the bacteria content is (4-6) multiplied by 10⁵cfu/ml. Preferably, the bacteria content is 5X 10⁵cfu/ml。

The rapid detection method for the antibiotic sensitivity of the microorganisms adopts a flow cytometer or a fluorescence microscopy instrument to detect the cell number of the microorganisms to be detected in a living body.

The quick detection method for the antibiotic sensitivity of the microorganisms has the incubation temperature of 34-36 ℃. Preferably, the incubation temperature is 35 ℃.

The technical scheme of the invention has the following advantages:

1. according to the rapid detection method for the microbial antibiotic sensitivity, antibiotics to be detected with different concentrations are mixed with a suspension of the microbes to be detected respectively and are incubated, and when the percentage of reduction of the number of the cells of the living microbes to be detected reaches more than 20% compared with a positive control without the antibiotics to be detected, the corresponding concentration of the antibiotics to be detected is the minimum inhibitory concentration of the microbes to be detected; the research of the invention finds that the number of living cells of the microorganism has the antibiotic effect change with statistical significance after the microorganism suspension is added with the antibiotic and incubated for 6 minutes, and when the percentage of the number of the cells of the microorganism to be detected, which are inhibited by the antibiotic, of the living body is reduced by more than 20 percent compared with the positive control without the antibiotic to be detected, the corresponding concentration of the antibiotic to be detected is equivalent to the Minimum Inhibitory Concentration (MIC) determined by the prior VITEK and test methods, so the invention provides the rapid detection method of the antibiotic sensitivity of the microorganism, the method can determine the Minimum Inhibitory Concentration (MIC) of the microorganism to be detected within 3 hours, and the result determined by the conventional method is consistent, the determined result is stable and reliable, the sensitivity of the microorganism to the antibiotic can be determined within a short time, the method is suitable for clinical practice, and the problem that the detection method of the antibiotic sensitivity in the prior art is long in time is solved, is not suitable for clinical practice, and has the advantages of low cost, easy operation and wide application range.

2. According to the rapid detection method for the antibiotic sensitivity of the microorganisms, when the percentage of the number of cells of the microorganisms to be detected in a living body, which are inhibited by the antibiotics, is reduced to more than 60%, the corresponding concentration of the antibiotics to be detected is the minimum inhibitory concentration of the microorganisms to be detected, the minimum inhibitory concentration is completely consistent with the minimum inhibitory concentration detected by a conventional method, and the accuracy rate is 100%.

3. According to the rapid detection method for the microbial antibiotic susceptibility provided by the invention, the number of the cells of the living to-be-detected microorganism is detected after the incubation time is selected to be 90 minutes, the incubation time for different microorganisms to reduce the number of the cells of the living to-be-detected microorganism to 60% is different, but common microorganisms can achieve the effect that the reduction percentage of the number of the cells of the to-be-detected microorganism is more than 60% compared with a positive control after the incubation time is 90 minutes, and the time for detecting the antibiotic susceptibility is short.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.

FIG. 1 is a histogram of the change in the number of viable bacteria of Escherichia coli at different times in Experimental example 1 of the present invention;

FIG. 2 is a histogram of turbidity changes of Escherichia coli at different times in Experimental example 1 of the present invention;

FIG. 3 shows the results of ampicillin sensitivity test of E.coli in Experimental example 2 of the present invention.

Detailed Description

The following examples refer to the main instruments: flow cytometry (FACSCAntoII, BD Co.), fluorescence microscopy (olympus fluorescence microscope BX43), turbidimetry (PhoenixSpec nephelometer, BD Co.) was used to determine the cell count of the live test microorganisms.

Antibiotics such as ampicillin, bouillon such as AST bouillon or MH bouillon, fluorescent dyes, referred to in the following examples, are all commercially available products, and the technical solution of the present invention does not make any significant difference with the above products of different manufacturers or models.

Preparation of the antibiotic susceptibility testing strains referred to in the following examples: the strain was inoculated on blood agar medium and incubated at 35 ℃ for 18 hours for use. The strains are respectively ATCC25922 escherichia coli, ATCC25923 staphylococcus aureus and ATCC27853 pseudomonas aeruginosa.

Preparation of broth for antibiotic sensitivity assay: 12 drug sensitive test tubes are prepared, each drug sensitive test tube contains an equal amount of drug sensitive test broth such as AST broth or MH broth, wherein 10 drug sensitive test tubes sequentially contain antibiotics to be tested with concentration gradients, and specific concentration indexes of different types of antibiotics to be tested are carried out according to the antibiotic concentration requirements described in US CLSL standard document M100. Such as the antibiotic ampicillin, at respective concentrations of 0.5, 1, 2, 4, 8, 16, 32, 64, 128 and 256, in units of mug/ml. The 1 drug sensitive test tube does not contain antibiotics to be tested and is used as a positive control, and the 1 drug sensitive test tube does not add the microorganism suspension to be tested and is used as a negative control during detection.