CN105403640A - Method for detecting ampicillin resistance of bacteria, and detection kit thereof - Google Patents
Method for detecting ampicillin resistance of bacteria, and detection kit thereof Download PDFInfo
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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Abstract
The invention discloses a method for detecting the ampicillin resistance of bacteria, and a detection kit thereof. The principle of the method is characterized in that plasmid mediation or chromosome mutation makes the bacteria generate beta-lactamase, and the beta-lactamase can rapidly hydrolyze ampicillin into metabolites in order to inactivate the ampicillin. In drug resistance situations, the concentration of ampicillin in bodies reduces with the passage of time, and the concentration of the metabolites in the bodies increases with the passage of time. In sensitive situations, the bacteria cannot generate beta-lactamase under the inhibition of the ampicillin, the ampicillin drug concentration keeps unchanged in a certain time, and no metabolites are detected. The detection method has the advantages of good stability, high accuracy and sensitivity, simple operation and short detection unit; and the detection time of the method is 90min and is shorter than the detection time of traditional drug sensitivity detection methods of 18-24h, so the method disclosed in the invention is helpful for clinic doctors to rapidly diagnose, and the treatment scheme can be improved, thereby abuse of antibiotic drugs is reduced.
Description
Technical field
The invention belongs to biological technical field, be specifically related to detection method and the kit of bacterium ampicillin-resistant.
Background technology
Microbiotic is most widely used antibacterials in hospital clinical, irreplaceable effect is had in control, the various infectious diseases of prevention and therapy, but along with antibiotic extensively, continue and excessively use, the drug resistance of pathogen to common antibacterials constantly increases.Effectively and reasonably antibiotic therapy depends on antibacterial drug sensitive test promptly and accurately.Doctor more and more payes attention to selecting medication according to the result of drug sensitive test, produces antibody-resistant bacterium to avoid inappropriate medication.
At present, the method that the sensitization test of antibacterials is conventional has minimal inhibitory concentration (MIC) method, disk diffusion method etc.Wherein MIC is owned by France in goldstandard, can show the susceptibility of bacterium to medicine, thus guiding clinical treatment.But higher to Test Condition Requirements, step is comparatively loaded down with trivial details, provide report time longer, from taking clinical samples, need within 3-5 days, just can take result accurately, even calculate from point pure colonies, also result could be obtained after needing 18-24h, this causes clinician can only adopt empirical medication under many circumstances, or adopt the microbiotic of wide spectrum to treat, antibiotic choice and operation tool bears the character of much blindness, cause antibiotic abuse, affect treatment and the rehabilitation of patient.
Beta-lactam class microbiotic is a most popular class in existing microbiotic, comprises clinical the most frequently used penicillin and cynnematin, and other atypia beta-lactam antibiotics such as the cephamycin-type of new development, thiomycin class, monobactams.This type of microbiotic has the advantage that bactericidal activity is strong, toxicity is low, indication is wide and clinical efficacy is good.The mechanism of bacterium to beta-lactam Hang Sheng element resistance is considered to three aspects at present: the permeability of adventitia declines; By beta-lactam enzyme hydrolysis; Can not effectively be combined by the PBP on inner membrance.The bacterium of clinical separation is carried out to the sensitivity Detection of beta-lactamase, find the strain enzyme-producing of 78.9%, the bacterial strain of 70.2% is to ampicillin-resistant.Show that domestic clinically isolated bacterium is all comparatively general in product enzyme and resistance to ampicillin, be worth the parties concerned to pay attention to.The resistance to ampicillin of bacterium producing multi enzyme preparation of research discovery 84.2%, the only resistance to ampicillin of non-bacterium producing multi enzyme preparation of 17.7%.Show to produce the main path that beta-lactamase is the resistance to ampicillin of bacterium, and the beta-lactam ring structure that this enzyme can be hydrolyzed rapidly ampicillin forms metabolin, thus make its inactivation.When resistance, As time goes on the drug concentration of ampicillin in human body can reduce, and the concentration of metabolin then can increase.Otherwise the suppression that bacterium is subject to ampicillin cannot produce beta-lactamase, ampicillin drug concentration can maintain a balance at short notice, and cannot metabolin be detected.Can judge that whether bacterium is to ampicillin-resistant and kit according to ampicillin and metabolin peak area with the variation tendency of incubation time.
Summary of the invention
The technical problem to be solved in the present invention is the ampicillin-resistant bacterium method for quick that a kind of accuracy is high, detection time is short.
For solving the problems of the technologies described above, the present invention adopts following technical scheme:
A detection method for bacterium ampicillin-resistant,
(1) sample preparation: get testing sample, centrifugal collecting precipitation, uses physiological saline washing and precipitating; In the precipitation of having cleaned, add the physiological saline that ampicillin concentration is 0.100 μ g/mL, mix, after being placed in 37 DEG C of hatching 5 ~ 90min, sample analysis at set intervals;
(2) testing conditions:
Liquid chromatographic detection condition:
Chromatographic column: ACQUITYUPLC
tMhSST3,2.1*50mm, 1.8 μm;
Mobile phase: by volume ratio be 5: 1 A liquid and B liquid form, A liquid is the volume fraction of formic acid is 0.1% aqueous solution, and B liquid is methyl alcohol: water: the volume ratio of formic acid is the mixed liquor of 95: 5: 0.1;
Flow velocity is 0.4mL/min, is isocratic elution during wash-out;
Sample size: 0.020mL;
Mass Spectrometry Conditions:
Positive ion mode, uses electron spray ion (ESI) source, detects in multiple-reaction monitoring (MRM) mode; The ionic reaction of quantitative test is respectively: ampicillin: m/z350.2 → 160.0Da; Metabolin: m/z368.2 → 324.0Da, described metabolin is ampicillin thiazole acid;
(3) determination methods: take incubation time as horizontal ordinate, peak area is ordinate mapping, if the detected peaks area of ampicillin reduces in time gradually, and the peak area of metabolin increases gradually, illustrate that bacterium has ampicillin resistance, if the detected peaks area of ampicillin remains unchanged, illustrate that bacterium does not have ampicillin resistance.
Wherein, the concrete grammar of sample preparation is as follows:
Get 0.500mL bacterium liquid or 5mL urine sample, the centrifugal 10min of 14000rpm, abandons supernatant; Add 5mL physiological saline, the centrifugal 10min of 14000rpm after mixing, abandons supernatant, repeats this step twice; Ampicillin solution 0.016mL and the physiological saline 0.784mL of 0.005mg/mL is added in the precipitation of having cleaned, ampicillin final concentration is made to be 0.100 μ g/mL, respectively get 0.200mL be placed in 37 DEG C of hatchings 5,15,30,60, after 90min, get the analysis of 0.020mL Liquid sample introduction.
Wherein, described testing sample is blood plate or urine.
A kind of bacterium ampicillin-resistant quick detection kit, comprises ampicillin standard items, physiological saline, mobile phase and chromatographic column.
Wherein, described mobile phase is volume ratio is the A liquid of 5: 1 and the potpourri of B liquid, and described A liquid formic acid volume fraction is the aqueous solution of 0.1%, and described B liquid is methyl alcohol: water: the volume ratio of formic acid is the mixed liquor of 95: 5: 0.1.
Wherein, described physiological saline to be massfraction be 0.9% sodium-chloride water solution.
Wherein, described chromatographic column is ACQUITYUPLC
tMhSST3 (2.1*50mm, 1.8 μm, Waters, US).
Beneficial effect:
Compared with prior art, bacterium ampicillin-resistant method for quick of the present invention, bacterium producing multi enzyme preparation is adopted to produce beta-lactamase, the principle that hydrolysis beta-lactam ring structure forms metabolin detects, can directly be applied on high performance liquid chromatography-tandem mass instrument, simple to operate, detection time is short, and result is accurate.The microbiotic that original shape is hydrolyzed into metabolin inactivation for Production by Bacteria enzyme by any resistance base reason all can adopt this principle to detect.Therefore, the method can need to widen according to clinical detection.The advantage that the method for the invention is outstanding is simple to operate, and detection time is short, and whole process only needs 90min, and doctor can determine medication in time according to testing result.The stabilization of kit be based upon in this detection method is good, accuracy and precision high, make drug sensitive experiment more accurate, simple and easy and quick.
Accompanying drawing explanation
Fig. 1 is the typical color spectrogram after the rear bacterium liquid sample introduction of ampicillin standard solution and process, and the retention time of ampicillin absorption peak is 2.5min, and the retention time of metabolin absorption peak is 1.3min.1-a is the chromatogram after the ampicillin solution sample introduction of 0.100 μ g/mL; 1-b is the chromatogram of sample introduction after the bacterium pre-service to ampicillin sensitive; 1-c is the chromatogram of sample introduction after the bacterium pre-service to ampicillin-resistant.
Fig. 2 is the testing result figure of 2 routine clinical blood plate samples, and horizontal ordinate is incubation time, and ordinate is peak area.2-a is 1 routine testing result is the blood plate of ampicillin sensitive, and " ■ " represents ampicillin, metabolin do not detected; 2-b is 1 routine testing result is the blood plate of ampicillin-resistant, and " ■ " represents ampicillin, and " ▲ " represents metabolin.
Fig. 3 is the testing result figure of 2 routine clinical human's urine samples, and horizontal ordinate is incubation time, and ordinate is peak area.3-a is 1 routine testing result is the urine sample of ampicillin sensitive, and " ■ " represents ampicillin, metabolin do not detected; 3-b is 1 routine testing result is the urine sample of ampicillin-resistant, and " ■ " represents ampicillin, and " ▲ " represents metabolin.
Embodiment
According to following embodiment, the present invention may be better understood.But those skilled in the art will readily understand, the content described by embodiment only for illustration of the present invention, and should can not limit the present invention described in detail in claims yet.
The invention discloses a kind of bacterium ampicillin-resistant quick detection kit and method, person skilled can use for reference present disclosure, suitably improves experiment parameter according to actual needs.The method can need to widen according to clinical detection.The reagent do not mentioned in following embodiment, operation steps, experimental apparatus and operating parameter etc. are all selected according to this area routine.
(1) key instrument
This kit is used on high performance liquid chromatography-tandem mass instrument and operates, the preferred ShimadzuLC-20A liquid chromatographic system of high performance liquid chromatography part (comprises DGU-20A3R type degasser, LC-20ADXR type binary infusion pump, SRL-20ACXR type injector and RESERVOIRTRAY type column oven), Japanese Shimadzu Corporation; Tandem mass spectrometer part preferred API4000 type triple quadrupole bar tandem mass spectrometer, is furnished with electron spray ion (ESI) source and Analyst1.6.1 data handling system, AppliedBiosystems company of the U.S.;
(2) reagent consumptive material
Ampicillin provided by the invention reference substance, acetonitrile, sodium dihydrogen phosphate, phosphoric acid, sodium chloride etc. all can be buied by market.Wherein, ampicillin reference substance is purchased from Nat'l Pharmaceutical & Biological Products Control Institute, and lot number is: 130410-200706; Acetonitrile is purchased from Sigma Co., USA, and article No. is: 34851-4L; Sodium dihydrogen phosphate is purchased from amresco company of the U.S., and article No. is: 0823-1kg; Phosphoric acid is purchased from Fluka company, and article No. is 79606-00M; Sodium chloride is purchased from Chemical Reagent Co., Ltd., Sinopharm Group, and article No. is 10019318.Chromatographic column ACQUITYUPLC
tMhSST3 (2.1*50mm, 1.8 μm) is purchased from Waters, US, and article No. is 186004055.
Bacterium ampicillin-resistant method for quick of the present invention realizes by the following technical solutions:
Adopt high performance liquid chromatography-tandem mass method to determine the chromatographic retention of ampicillin, optimize chromatographic mass spectrometry condition;
The clinical sample pre-treating method set up according to the present invention carries out pre-treatment to clinical sample;
High performance liquid chromatography-tandem mass method sample introduction is adopted by clinical sample after pre-treatment to analyze;
Judge that whether bacterium is to ampicillin-resistant according to ampicillin and metabolin peak area with the variation tendency of incubation time.
Wherein, described clinical samples can be blood plate or urine specimen.
Ampicillin reference substance can select Sigma company or Nat'l Pharmaceutical & Biological Products Control Institute to produce, the standard items of what this example was selected is Nat'l Pharmaceutical & Biological Products Control Institute.Acetonitrile, sodium dihydrogen phosphate and phosphoric acid preferentially select the product of Sigma company.
In the present embodiment, described high performance liquid chromatography testing conditions is as follows: chromatographic column: ACQUITYUPLC
tMhSST3 (2.1*50mm, 1.8 μm); Mobile phase: by volume ratio be 5: 1 A liquid and B liquid form, A liquid to be formic acid volume fraction be 0.1% aqueous solution, B liquid is methyl alcohol: water: the volume ratio of formic acid is the mixed liquor of 95: 5: 0.1, and flow velocity is 0.4mL/min, is isocratic elution during wash-out; Sample size: 0.020mL.
In the present embodiment, described Mass Spectrometry Conditions is as follows: positive ion mode, uses electron spray ion (ESI) source, detects in multiple-reaction monitoring (MRM) mode.Wherein, gas curtain gas CUR is 15psi, heat air Gs1 is 25psi, and auxiliary heating gas Gs2 is 35psi, and heat air temperature is 450 DEG C, and collision gas is the high pure nitrogen of 6psi, and electron spray pin voltage is 4500V.Adopt many reaction detection ion (MRM) to scan, the ionic reaction for quantitative test is respectively: ampicillin: m/z350.2 → 160.0Da; Metabolin: m/z368.2 → 324.0Da; Sweep time is 200ms, and corresponding mass spectrometry parameters is in table 1.
Below in conjunction with embodiment, the present invention is described in detail.The experimental technique used in following embodiment if no special instructions, is conventional method.
The preparation method of embodiment 1 kit.
(1) ampicillin standard items: be purchased from Nat'l Pharmaceutical & Biological Products Control Institute, room temperature keeps in Dark Place.Adopt 1mL water-soluble solution 1mg ampicillin reference substance during use, after eddy current mixing, obtain the mother liquor of 1mg/mL;
(2) physiological saline: the sodium-chloride water solution of 0.9%, 4 DEG C of preservations.Sodium chloride is purchased from Chemical Reagent Co., Ltd., Sinopharm Group;
(3) mobile phase: A liquid is 0.1% formic acid water, and formic acid is purchased from Fluka company of the U.S.; B liquid is methyl alcohol: water: formic acid (95: 5: 0.1), and methyl alcohol is purchased from Sigma Co., USA, can use, 4 DEG C of preservations after A liquid and B liquid being mixed according to volume ratio 5: 1;
(4) chromatographic column: ACQUITYUPLC
tMhSST3 (2.1*50mm, 1.8 μm) is purchased from Waters, US, room temperature preservation.
The using method of embodiment 2 kit of the present invention.
(1) preparation of ampicillin working solution: adopt the mother liquor of mobile phase or normal saline dilution 1mg/mL to obtain corresponding ampicillin working fluid;
(2) balance instrument: open high performance liquid chromatography-tandem mass instrument, A liquid and B liquid are mixed to get the mobile phase needed for experiment according to volume ratio 5: 1, adopt the flow velocity balance ACQUITYUPLC of 0.4mL/min
tMhSST3 (2.1*50mm, 1.8 μm) chromatographic column; The sample (clinical sample pre-treating method is shown in shown in following (3)) processed is after liquid phase systems is separated, be separated with mobile phase in mass spectrum part, after being ionized, separated by mass number by fragment ion through mass spectrographic mass analyzer, device obtains mass spectrogram after testing.
Mass spectrum part adopts the API4000 Tandem Mass Spectrometry Analysis instrument with electron spray ionisation source (ESI) to analyze as detecting device.The Mass Spectrometry Conditions adopted is: positive ion mode detects; Heat air temperature is 450 DEG C, ion source gas 1 (N
2) pressure is 25psi, ion source gas 2 (N
2) pressure is 35psi, collision gas is the high pure nitrogen of 6psi, and gas curtain gas CUR is 15psi, and electron spray pin voltage is 4500V; Scan mode is multiple-reaction monitoring (MRM), m/z350.2 → 160.0Da (ampicillin) and m/z368.2 → 324.0Da (metabolin); Collision energy (CE) is 20eV; Sweep time is 200ms.
(3) clinical sample pre-treatment: blood plate and urine sample adopt following methods respectively:
Blood plate: the appropriate bacterium colony physiological saline solution of picking, gets 0.500mL liquid in the centrifugal 10min of 14000rpm, abandons supernatant; Sediment fraction adds 5mL physiological saline, and the centrifugal 10min of 14000rpm after mixing, abandons supernatant, repetitive operation twice; Sediment fraction adds ampicillin solution 0.016mL and the physiological saline 0.784mL of 0.005mg/mL, makes ampicillin final concentration be 0.100 μ g/mL, respectively get 0.200mL be placed in 37 DEG C hatching 5,15,30,60,90min after, get the analysis of 0.020mL Liquid sample introduction;
Urine sample: get 5.00mL urine in the centrifugal 10min of 14000rpm, abandon supernatant; Sediment fraction adds 5mL physiological saline, and the centrifugal 10min of 14000rpm after mixing, abandons supernatant, repetitive operation twice; Sediment fraction adds ampicillin solution 0.016mL and the physiological saline 0.784mL of 0.005mg/mL, makes ampicillin final concentration be 0.100 μ g/mL, respectively get 0.200mL be placed in 37 DEG C hatching 5,15,30,60,90min after, get the analysis of 0.020mL Liquid sample introduction;
(4) sample introduction analysis: by each increment product sample introduction one by one after ampicillin solution and process, chromatographic retention when recording each sample introduction and peak area;
(5) result is resolved: according to the method described above after sample introduction, it is characterized in that: high performance liquid chromatograph occurs the time of ampicillin absorption peak is 2.5min, the time of metabolin absorption peak is 1.3min.In resistance situation, As time goes on the drug concentration of ampicillin in human body can reduce, and the concentration of metabolin then can increase.Otherwise under sensitive situations, the suppression that bacterium is subject to ampicillin cannot produce beta-lactamase, and ampicillin drug concentration can remain unchanged within a certain period of time, and cannot metabolin be detected.Can judge that whether bacterium is to ampicillin-resistant according to ampicillin and metabolin peak area with the variation tendency of incubation time.
Kit of the present invention is used for 30 routine clinical samples ampicillin-resistants to detect, testing result and minimal inhibitory concentration method result are compared, and testing result is in table 2, and two kinds of methodology testing results of all samples are consistent; Table 3 is the typical datas after process after blood plate sample feeding; Table 4 is the typical datas after process after urine sample sample introduction.Table 3 and the metabolin described in table 4 are ampicillin thiazole acid.Typical color spectrogram after ampicillin standard solution and clinical sample sample introduction is as shown in Figure 1: wherein 1-a is the chromatogram after the ampicillin solution sample introduction of 0.100 μ g/mL; 1-b is the chromatogram of sample introduction after the bacterium pre-service to ampicillin sensitive; 1-c is the chromatogram of sample introduction after the bacterium pre-service to ampicillin-resistant.Be horizontal ordinate by table 3 data with incubation time, peak area is ordinate mapping, and obtain the typical relation figure (see Fig. 2) of incubation time and peak area, 2-a represents that bacterium is to ampicillin sensitive, and 2-b represents that bacterium is to ampicillin-resistant; Table 4 data mapped in the same manner (see Fig. 3), 3-a represents that bacterium is to ampicillin sensitive, and 3-b represents that bacterium is to ampicillin-resistant.Result shows: for the bacterium of ampicillin sensitive, and along with the prolongation of incubation time, ampicillin peak area remains unchanged substantially, and does not monitor metabolin (ampicillin thiazole acid); And for the bacterium of ampicillin-resistant, ampicillin peak area reduces along with the prolongation of incubation time, metabolin (ampicillin thiazole acid) peak area then increases along with the prolongation of incubation time, and explanation can judge bacterium whether ampicillin-resistant according to the peak area variation tendency of hatching rear ampicillin and metabolin (acid of ampicillin thiazole).
Table 1 reaction detection ion scan MRM condition
Table 2 clinical sample detects comparison result
Table 3 hatches the peak area measurement result (blood plate) of rear different time points ampicillin and metabolin
Table 4 hatches the peak area measurement result (urine) of rear different time points ampicillin and metabolin
In sum, it is good that this kit has specificity, and accuracy is high, fast simple to operate, the feature such as with low cost, can realize bacterium ampicillin-resistant detects fast, contribute to clinician and carry out quick diagnosis, improve therapeutic scheme, thus reduce the abuse of antibiotic medicine.The microbiotic that original shape is hydrolyzed into metabolin inactivation for Production by Bacteria enzyme by any resistance base reason all can adopt this principle to detect.Therefore, the method can need to widen according to clinical detection, is easy to promote, has good application prospect.
Claims (7)
1. a detection method for bacterium ampicillin-resistant, is characterized in that:
(1) sample preparation: get testing sample, centrifugal collecting precipitation, uses physiological saline washing and precipitating; In the precipitation of having cleaned, add the physiological saline that ampicillin concentration is 0.100 μ g/mL, mix, after being placed in 37 DEG C of hatching 5 ~ 90min, sample analysis at set intervals;
(2) testing conditions:
Liquid chromatographic detection condition:
Chromatographic column: ACQUITYUPLC
tMhSST3,2.1*50mm, 1.8 μm;
Mobile phase: by volume ratio be 5: 1 A liquid and B liquid form, A liquid to be formic acid volume fraction be 0.1% aqueous solution, B liquid is methyl alcohol: water: formic acid volume ratio is the mixed liquor of 95: 5: 0.1;
Flow velocity is 0.4mL/min, is isocratic elution during wash-out;
Sample size: 0.020mL;
Mass Spectrometry Conditions:
Positive ion mode, uses electron spray ion (ESI) source, detects in multiple-reaction monitoring (MRM) mode; The ionic reaction of quantitative test is respectively: ampicillin: m/z350.2 → 160.0Da; Metabolin: m/z368.2 → 324.0Da, described metabolin is ampicillin thiazole acid;
(3) determination methods: take incubation time as horizontal ordinate, peak area is ordinate mapping, if the detected peaks area of ampicillin reduces in time gradually, and the peak area of metabolin increases gradually, illustrate that bacterium has ampicillin resistance, if the detected peaks area of ampicillin remains unchanged, illustrate that bacterium does not have ampicillin resistance.
2. the detection method of bacterium ampicillin-resistant according to claim 1, is characterized in that, the concrete grammar of sample preparation is as follows:
Get testing sample, the centrifugal 10min of 14000rpm, abandons supernatant; Add 5mL physiological saline, the centrifugal 10min of 14000rpm after mixing, abandons supernatant, repeats this step twice; Ampicillin solution 0.016mL and the physiological saline 0.784mL of 0.005mg/mL is added in the precipitation of having cleaned, ampicillin final concentration is made to be 0.100 μ g/mL, respectively get 0.200mL be placed in 37 DEG C of hatchings 5,15,30,60, after 90min, get the analysis of 0.020mL Liquid sample introduction.
3. the detection method of bacterium ampicillin-resistant according to claim 1 and 2, is characterized in that, described testing sample is blood plate or urine.
4. a bacterium ampicillin-resistant quick detection kit, is characterized in that, comprises ampicillin standard items, physiological saline, mobile phase and chromatographic column.
5. bacterium ampicillin-resistant quick detection kit according to claim 4, it is characterized in that, described mobile phase is volume ratio is the A liquid of 5: 1 and the potpourri of B liquid, the volume fraction of described A liquid formic acid is 0.1% aqueous solution, and described B liquid is methyl alcohol: water: the volume ratio of formic acid is the mixed liquor of 95: 5: 0.1.
6. bacterium ampicillin-resistant quick detection kit according to claim 4, is characterized in that, described physiological saline is the sodium-chloride water solution of 0.9%.
7. bacterium ampicillin-resistant quick detection kit according to claim 4, is characterized in that, described chromatographic column is ACQUITYUPLC
tMhSST32.1*50mm, 1.8 μm.
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| CN112051321A (en) * | 2020-08-24 | 2020-12-08 | 复旦大学 | Rapid antibiotic sensitivity testing method combining deuterium water culture and matrix-assisted laser desorption ionization time-of-flight mass spectrometry |
| CN112051321B (en) * | 2020-08-24 | 2022-02-15 | 复旦大学 | Rapid antibiotic sensitivity testing method combining deuterium water culture and matrix-assisted laser desorption ionization time-of-flight mass spectrometry |
| CN113552259A (en) * | 2021-07-21 | 2021-10-26 | 上海市东方医院（同济大学附属东方医院） | Kit and detection method for rapidly detecting A/B-type carbapenemase-producing bacteria in enterobacteriaceae |
| CN114019055A (en) * | 2021-11-09 | 2022-02-08 | 中国科学院城市环境研究所 | Kit for evaluating drug resistance effect of bacterial aminoglycoside antibiotics and application thereof |
| CN114019055B (en) * | 2021-11-09 | 2024-03-01 | 中国科学院城市环境研究所 | Kit for evaluating drug resistance effect of bacterial aminoglycoside antibiotics and application thereof |
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