CN102288586B - Method for determining minimal inhibitory concentration of drug - Google Patents
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Abstract
The invention discloses a method capable of rapidly, simply, conveniently and quantitatively detecting minimal inhibitory concentration, and the method comprises the following steps of: in accordance with the standard of CLSI (Clinical and Laboratory Standards Institute) (Version 2010), adding a fresh enterococcus suspension of a certain concentration into a sterile 96-well plate containing concentration gradient antibacterials for co-incubation; upon the ending of the 4-hour incubation, adding a fixed amount of fluorescent dyes SYTOX Green and DAPI (4,6-diamino-2-phenyl indole) to all the wells, protecting the wells from light for 15 minutes at room temperature, reading the fluorescent intensity of the two dyes in the wells by use of a fluorescent microplate reader respectively; after relevant background fluorescence is deducted, drawing a corresponding curve between bacterial fluorescence intensity ratio (Pdead/livel) and concentration of drug (CDrug), and determining the minimal concentration of drug corresponding to the moment the Pdead/livel is no longer fluctuated as the MIC (Minimal Inhibitory Concentration) of the antibiotic to the bacterium. The detection method provided by the invention has the characteristics of being simple, convenient, rapid and objective and being capable of performing mathematic statistics and analysis directly on detected data, becomes a new detection method for determining the minimal inhibitory concentration of antibacterials, and has a wide application prospect.
Description
Technical field
The present invention relates to a kind of method of measuring Mlc, specifically a kind ofly based on fluorescent dye the medicine minimum inhibitory concentration is carried out method for measuring.
Background of invention
The assay method of antibacterials minimum inhibitory concentration (MIC) commonly used comprising: broth dilution method, agar dilution and E-test.Above several method all is according to NCCLS antibacterials sensitivity tests operation standard, selection comprises the drug concentration scope (special circumstances exception) of resistance, intermediary and responsive boundary point value, utilize bacterium long-time difference of cultivating the back growth conditions in the medium that contains the variable concentrations medicine (meat soup or agar), judge the lowest drug concentration of bacteria growing inhibiting.General incubation time is 20～24h, and the time of obtaining the result is longer relatively, this waits for too long, delay treatment easily for emergency treatment patient or critical days patient.And for the relatively poor bacterium of some suspensions, the difference that the range estimation sentence read result exists inevitable human factor to cause.
Enterococcus is the part of humans and animals normal intestinal flora, but can cause serious infection under pathologic condition.Though enterococcus is of a great variety, have only minority can cause the human infection.Along with the continuous growth of antibody-resistant bacterium, enterococcus becomes important hospital pathogenic bacteria gradually.In time, microbiotic application effectively and reasonably is the key that the treatment enterococcus infects.This research selects for use enterococcus type strain and clinical separation strain as research object, investigates clinical three kinds of microbiotic commonly used to its inhibiting effect.Minimum inhibitory concentration is the important indicator that clinical labororatory determines bacterial resistance and detection new drug antibacterial activity.It not only is used for determining patient's dosage, has also determined application method, and this has vital role for the generation that reduces antibody-resistant bacterium.Usually, with reference to CLSI, standards such as BSAC or EUCAST, MIC can utilize agar dilution or broth dilution method to measure.But their unified disadvantage is that Measuring Time is longer, and in the time of especially need providing related drugs information rapidly for critical situation, classic method is slightly inadequate.The fluorescent dye of two kinds of high-qualitys is adopted in this research, utilizes three kinds of microbiotic of fluorescence spectrometry to enterococcal minimum inhibitory concentration, successfully will foreshorten to 4h detection time.
Existing broth dilution method utilizes naked eyes range estimations interpretation to cultivate result behind the 20h, does not have the corresponding concentration in muddy growth hole as the medicine minimum inhibitory concentration with naked eyes.And the enterococcus spp bacterium of investigating among the present invention has the characteristic of long-time cultivation free settling, during the ocular estimate interpretation, is the main source that causes result difference between the laboratory for not having muddy growth but bottom the orifice plate interpretation of the critical hole of bacterial sediment being arranged.
Summary of the invention
The purpose of this invention is to provide a kind of quick, easy, to bacterium poison little, and can objective, quantitative, accurately determine the detection method of MIC.
Another object of the present invention is to propose a kind of assay method of the enterococcus minimum inhibitory concentration based on said method.
To achieve these goals, the present invention adopts following concrete technical scheme:
A kind of method of measuring the medicine minimum inhibitory concentration, described method step is as follows:
(1) preparation final concentration the best is 5 * 10
6The bacteria suspension of CFU/ml; Did a series of concentration gradients in the experimentation, 2.5 * 10
6CFU/ml～5 * 10
6The CFU/ml scope all can, selected this concentration is from the reasonable angle Selection of fluorescence intensity read-around ratio.
(2) prepare the medicine working fluid of variable concentrations gradient respectively, concentration range comprises among 1024,512,256,128,64,32,16,8,4,2,1,0.5,0.25, the 0.125 μ g/ml; Medicine working fluid concentration provided by the invention is the most comprehensive concentration, bacterial strain for the known roughly MIC of relevant criterion concentration ranges such as reference CLSI, can the reference standard scope can comprise the break of fluorescence intensity ratio to high concentration and low concentration direction 3 concentration gradients that respectively extend respectively again, select for use many group gradients to make trend more obvious in the research, more complete, figure is more attractive in appearance.In addition, for the persister in the clinical separation strain, in order still to provide clear and definite MIC concentration, the maximum concentration that we select for use is very high, and 1024 μ g/ml are optional concentration;
(3) bacteria suspension is joined respectively in the medicine working fluid of above-mentioned concentration gradient, mixing, is hatched 4～6h by 35 ℃; Described bacteria suspension is 1: 1 with medicine working fluid volume ratio.It is more convenient that bacteria suspension and working fluid volume are elected calculating in 1: 1 as in fact, also can be other ratios, considering under the actual volume situation of micropore, needs only the normal growth that guarantees final concentration and do not influence bacterium, need not be strict with proportional range.
(4) add the mixed liquor of dyestuff SYTOX Green and DAPI in bacteria suspension and the medicine working fluid mixed liquor, make two kinds of dyestuff final concentrations be respectively 1.5 μ M and 300nM.(two kinds of fluorescent dyes have been selected different concentration combination for use in the experiment, though can draw identical trend as a result after testing, but selected final concentration is the best effort concentration of thinking herein, can be so that the fluorescence intensity level of two kinds of dyestuffs that read be unlikely to have big difference, and can influence fine differentiation with the background of fluorescent dye self.) room temperature lucifuge effect 15min;
(5) under the fluorescence microplate reader, read the fluorescence intensity level of each hole SYTOX Green and two kinds of dyestuffs of DAPI respectively;
(6) draw corresponding curve map between fluorescence intensity ratio and each drug concentration.
In the described step (1), the best final concentration of bacteria suspension is 5 * 10
6CFU/ml.
Maximum excitation wavelength and the emission wavelength of described two kinds of dyestuffs are respectively: DAPI 358/461nm; SYTOXGreen 504/535nm.
Corresponding curve map between the fluorescence intensity ratio that described step (6) is drawn and each drug concentration, the corresponding medicine working fluid of first flex point concentration in curve map behind the peak is this medicine to the minimum inhibitory concentration of this bacterium.
Described medicine is ampicillin, lavo-ofloxacin and vancomycin.
Described bacterium is enterococcus faecalis ATCC 29212.
Beneficial effect of the present invention:
MIC method for quick of the present invention and prior art broth dilution method compare, and incubation time shortens to 4h～6h by the 20h～24h of prior art.The inventor has proposed through drawing corresponding curve map between fluorescence intensity ratio and each drug concentration, the corresponding medicine working fluid of first flex point concentration behind the peak is this medicine to the minimum inhibitory concentration of this bacterium, can directly draw MIC value after the inventive method is drawn, and pass through this flex point value of experimental verification and be MIC value accurately.The present invention is quick and precisely easy to be with low cost, has good application prospects and market outlook.
Exploitativeness of the present invention and outstanding substantive distinguishing features and good effect can be embodied by following example, but do not limit its scope.
Description of drawings
Fig. 1 fluorescence method detects 3 kinds of microbiotic to the MIC of ATCC 29212, and incubation time is respectively 4h and 20h (vancomycin 24h).
Fig. 2 is that ATCC 29212 finishes two kinds of fluorescent dyes of back adding with the finite concentration drug effect, the picture under the different fluorescence channels of the same visual field.A only shows blue-fluorescence under the DAPI passage; B only shows green fluorescence under the SYTOX Green passage; C is the fusion picture of A and B.
The corresponding relation (calibration curve) of Fig. 3 dead bacterium percentage and fluorescence intensity ratio (dead bacterium/work bacterium) for fluorescence method detects.
Fig. 4 is for being depicted as with respect to broth dilution method, and fluorescence method detects 3 kinds of microbiotic to the accuracy of the MIC of 90 strain clinical separating sausage coccus.Area under curve is respectively 0.85 (lavo-ofloxacin), 0.765 (ampicillin) and 0.832 (vancomycin).
Embodiment
Raw material sources in the embodiment:
DAPI dyestuff: Ivitrogen, Molecular Probes, D21490.
SYTOX Green dyestuff: Ivitrogen, Molecular Probes, S7020.
Vancomycin, lavo-ofloxacin, ampicillin standard items are all available from National Institute for Food and Drugs Control's enterococcus faecalis (ATCC 29212): available from ATCC, i.e. and the biological product of Unite States Standard (USS) collecting center.
TECANinfinite 200 fluorescence microplate readers
The described rapid fluorescence method of embodiment 1 detects ampicillin, lavo-ofloxacin, vancomycin to the MIC value of enterococcus faecalis ATCC 29212
(1) microbiotic preparation: each microbiotic that provides with reference to CLSI (2010 editions) is at enterococcal MIC reference value, and setting the drug concentration gradient is 1024,512,256,128,64,32,16,8,4,2,1,0.5,0.25,0.125 μ g/ml.The antibacterials of variable concentrations behind the doubling dilution are joined respectively in the aseptic 96 hole polystyrene plates, every hole 100 μ l, each gradient is done 3 multiple holes.Negative control hole (70% ethanol treatment groups) and not dosing hole are set simultaneously as growth control (positive control).
(2) bacteria suspension preparation: with the bacteria suspension of 0.5 Maxwell than turbid standard, through MH meat soup (Mueller-Hinton Broth OXOID) after 10 times of the dilutions, joins respectively in each hole of containing the variable concentrations medicine, 100 μ l/ holes, final concentration is 5 * 10
6CFU/ml.The rearmounted 35 ℃ of normal air incubators of sealing are hatched 4h (but vancomycin reads to 6h for enterococcal effect proper extension).
(3) hatch the end back adds 100 μ l SYTOX Green dyestuffs and mixed liquor from DAPI dyestuff to each hole, the final concentration of two kinds of dyestuffs is respectively 1.5 μ M and 300nM, room temperature lucifuge reaction 15min.
(4) reacted microwell plate is placed TECANinfinite 200 fluorescence microplate readers, read the fluorescence intensity level of two kinds of dyestuffs under corresponding exciting light and emission light action respectively, note is made FDAPI and FSYTOX Green respectively.
Excitation wavelength and the emission wavelength of described two kinds of dyestuffs are respectively: DAPI 358/461nm; SYTOXGreen 504/535nm.
(5) data analysis, after deducting corresponding background fluorescence, draw the corresponding curve of bacterial fluorescence strength ratio (Pdead/live) and drug concentration (CDrug), corresponding minimum drug concentration (CDrug) was this microbiotic to the MIC of this bacterium when Pdead/live no longer fluctuateed.[annotating: Pdead/live=FSYTOX Green/FDAPI]
The result as shown in Figure 1, the corresponding relation of variable concentrations medicine and fluorescence intensity ratio.A, B, when C figure is respectively 4h and 20h/24h, the fluorescence intensity ratio of the lavo-ofloxacin of variable concentrations, ampicillin, vancomycin correspondence (Pdead/live=FSYTOX Green/FDAPI).Arrow is depicted as fluorescence method (4h and 20h/24h) the MIC value of surveying among the figure.Arrow is depicted as the minimum inhibitory concentration (MIC of two kinds of situations mensuration of figure C differs 1 concentration gradient, illustrates that vancomycin needs proper extension to 6h to enterococcal effect) of certain medicine under different action times
As seen lavo-ofloxacin is 1 μ g/ml to the MIC value of ATCC 29212, and the ampicillin is 2 μ g/ml to the MIC value of ATCC 29212, and vancomycin is 2 μ g/ml to the MIC value of ATCC 29212, and embodiment 3 is seen in experimental verification.These three kinds of microbiotic are clearly arranged to the MIC scope of ATCC29212 type strain in the CLSI file 2010 editions, because selected type strain is sensitive strain, the lavo-ofloxacin of demarcating among the CLSI, ampicillin and/or vancomycin are respectively≤2 μ g/ml the scope of its MIC, ≤ 8 μ g/ml ,≤4 μ g/ml.Three kinds of medicines that the present invention adopts the fluorescence method gained to the MIC value of type strain all in controlled range.
Embodiment 2
(1) microbiotic preparation: each microbiotic that provides with reference to CLSI (2010 editions) is at enterococcal MIC reference value, and setting the drug concentration gradient is 512,256,128,64,32,16,8,4,2,1,0.5,0.25,0.125 μ g/ml.The antibacterials of variable concentrations behind the doubling dilution are joined respectively in the aseptic 96 hole polystyrene plates, every hole 100 μ l, each gradient is done 3 multiple holes.Negative control hole (70% ethanol treatment groups) and not dosing hole are set simultaneously as growth control (positive control).
(3) bacteria suspension preparation: with the bacteria suspension of 0.5 Maxwell than turbid standard, through MH meat soup (Mueller-Hinton Broth OXOID) after 10 times of the dilutions, joins respectively in each hole of containing the variable concentrations medicine, 100 μ l/ holes, final concentration is 2.5 * 10
6CFU/ml.The rearmounted 34.5 ℃ of normal air incubators of sealing are hatched 4h (but vancomycin reads to 6h for enterococcal effect proper extension).
(3) hatch the end back adds 50 μ l SYTOX Green dyestuffs and mixed liquor from DAPI dyestuff to each hole, the final concentration of two kinds of dyestuffs is respectively 1.5 μ M and 300nM, room temperature lucifuge reaction 15min.
(4) reacted microwell plate is placed TECANinfinite 200 fluorescence microplate readers, read the fluorescence intensity level of two kinds of dyestuffs under corresponding exciting light and emission light action respectively, note is made FDAPI and FSYTOX Green respectively.
Excitation wavelength and the emission wavelength of described two kinds of dyestuffs are respectively: DAPI 358/461nm; SYTOXGreen 504/535nm.
(5) data analysis, after deducting corresponding background fluorescence, draw the corresponding curve of dead bacterial fluorescence strength ratio (Pdead/live) and drug concentration (CDrug), corresponding minimum drug concentration (CDrug) was this microbiotic to the MIC of this bacterium when Pdead/live no longer fluctuateed.[annotating: Pdead/live=FSYTOX Green/FDAPI]
The result is identical with embodiment 1, and lavo-ofloxacin is 1 μ g/ml to the MIC value of ATCC 29212, and the ampicillin is 2 μ g/ml to the MIC value of ATCC 29212, and vancomycin is 2 μ g/ml to the MIC value of ATCC 29212.
Embodiment 3 is applied to the enterococcus of the clinical separation of 90 strains with the inventive method, and compares with the parallel micro-dilution method gained MIC result who carries out, and checking rapid fluorescence method detects the feasibility of MIC.
One, materials and methods
1. bacterial strain and microbiotic
1.1 bacterial strain: enterococcus faecalis type strain ATCC 29212; 90 strain clinical separating sausage coccus are enterococcus through identifying 90 strains, and wherein 75 strains are that enterococcus faecalis and 15 strains are Enterococcus faecium.
1.1 microbiotic: lavo-ofloxacin, ampicillin, vancomycin standard items are all available from National Institute for Food and Drugs Control.With aseptic deionized water 3 kinds of pharmaceutical standards product are made liquid storage, packing ,-70 ℃ are frozen.
2. method
2.1MIC determine
This studies the micro-broth dilution method of parallel employing (MIC/R) and fluorescence method (MIC/F) is measured 3 kinds of microbiotic to the minimum inhibitory concentration of enterococcus (type strain and 90 strain clinical separation strains).
2.1.1 micro-broth dilution method MIC (reference method, MIC/R)
Broth dilution method is considered the vitro detection microbiotic usually to the basic experiment chamber method of microorganism susceptibility.It is generally acknowledged with agar diffusion method and compare that the meat soup system is more near internal milieu, and can draw MIC intuitively and need not conversion or return to calculate.And this method can be further used for the mensuration of MBC.This research selects for use micro-broth dilution method as the reference method of standard, also revises a little with reference to the CLSI standard and carries out.Soon the microbiotic behind the doubling dilution adds 96 orifice plates, 100 μ l/ holes successively.Every hole adds the enterococcus suspension of 100 μ l fresh cultured respectively subsequently, and final concentration is 5 * 10
6/ hole.After application of sample is finished,, put 35 ℃ of incubators and hatch 20h (vancomycin and enterococcus make the time spent incubation time and extend to 24h) pore plate by sealing with masking foil.Terminal point interpretation: choose the black background bore hole and observe each hole bacterial growth situation and penetrability.With the antibiotic concentration of first hole correspondence of turbidity disappeared as corresponding MIC.
2.1.2 measuring MIC (MIC/F), the inventive method rapid fluorescence method sees embodiment 1.
Two, result
1. two kinds of fluorescent dyes have perfect discrimination
1.1 observe the discrimination of two kinds of fluorescent dyes under the fluorescent microscope
The present invention selects two kinds of nucleic acid dyes for use, the ability difference of the two permeates cell membranes.SYTOX Green only can penetrate the damaged cells film, and DAPI can penetrate the cell membrane of all cells.When two kinds of dyestuffs act on the cell of damaged membrane simultaneously, only show the color of SYTOX Green.As shown in Figure 2, can checkmate bacterium and the bacterium that lives of two kinds of fluorescent dyes distinguished well.
Add two kinds of fluorescent dyes behind ATCC 29212 and the finite concentration drug effect, the picture under the different fluorescence channels of the same visual field.A only shows blue-fluorescence under the DAPI passage; B only shows green fluorescence under the SYTOX Green passage; C is the fusion picture of A and B.
1.2 calibration curve further two kinds of fluorescent dyes of proof is applicable to that the MIC based on microwell plate measures.
Can accurately reflect the ratio variation of dead bacterium and bacterium alive for the fluorescence intensity variation of two kinds of fluorescent dyes in the proof microwell plate, we with the bacterium of fresh cultured (bacterium lives) with handle through propyl alcohol after bacterium (extremely bacterium) mix in varing proportions, make the suspension of different proportion, concrete as table 1.Measure the fluorescence intensity level of each two kinds of dyestuff in hole of different proportion respectively, draw the corresponding relation figure (Fig. 3) of fluorescence intensity ratio (dead bacterium/work bacterium) and dead bacterium proportion.
The different proportion relations of the dead bacterium of table 1. and work bacterium
Fig. 3 is example with ATCC29212, and fluorescence method detects the corresponding relation of dead bacterium percentage and fluorescence intensity ratio (dead bacterium/work bacterium).As seen from the figure, along with increasing of dead bacteria content, it is big that its corresponding fluorescence intensity ratio also becomes gradually, and the two is proportionate.Each data point is the mean value of 3 independent experiments among the figure.
2. fluorescence method detects three kinds of microbiotic to the MIC of ATCC 29212
2.1 the foundation of experimental system
Through a series of preliminary experiment, we have optimized whole experimental system, and subsequent experimental is all undertaken by following scheme: the suitableeest inoculum density of each micropore is 5 * 10
6CFU/ml.A large amount of pre-stage tests show, the time that medicine and bacterium are hatched altogether extends to 24h from 4h, gained drug concentration and the corresponding curvilinear trend of fluorescence intensity ratio (Fig. 1) in full accord.Therefore, in the follow-up test, the incubation time of fluorescence method is 4h.Simultaneously, parallel carry out micro-broth dilution method as with reference to the contrast.
Fig. 1 fluorescence method detects 3 kinds of microbiotic to the MIC of ATCC 29212, and incubation time is respectively 4h and 20h (vancomycin 24h).As seen from the figure, under two kinds of incubation time conditions, drug concentration is in full accord with the change curve trend of corresponding fluorescence intensity ratio.The corresponding antibiotic concentration of first flex point in the curve behind the peak is this medicine to the MIC of this bacterium, and the result is identical with reference method.
2.2 the explanation of above-mentioned fluorescence intensity ratio and antibiotic concentration corresponding relation figure
As Fig. 1, when antibiotic concentration was not enough to bacteria growing inhibiting, along with the increase of drug concentration, fluorescence intensity ratio was in rising trend, until peak.In case and drug concentration is enough big, then the bacterial growth in the system can effectively be suppressed, and the increase again of drug concentration is little to its fluorescence intensity ratio influence, and it is constant substantially to show as fluorescence intensity ratio.This phenomenon and reference method are cultivated the muddy phenomenon of effective antibacterial each Kong Junwu of back in 20h (the vancomycin group needs 24h) back, and the visual inspection indifference is similar.Among the present invention, the corresponding relation figure of drug concentration and fluorescence intensity ratio shows as the curve of a rising, arrive behind the corresponding peak suddenly to descend, and be one section more level and smooth curve afterwards.The corresponding drug concentration of first node on the smooth curve is MIC.It is to be noted when fluorescence method detects vancomycin to enterococcal MIC, gradient of the measurement result deviation behind result and the 24h, this may need the proper extension incubation time relevant with the vancomycin effect, such as 6h.
3. the rapid fluorescence method is measured the MIC of 90 strain clinical separating sausage coccus.
In order to verify that rapid fluorescence method detection of antibiotics of the present invention is to the validity of enterococcus MIC, the enterococcus of the clinical separation of picked at random 90 strains (75 strain enterococcus faecalis and 15 Enterococcus faecalis), adopt fluorescence method (MIC/F) and micro-broth dilution method (MIC/R) to detect simultaneously, the results are shown in Table 2.Two kinds of method detection lavo-ofloxacins, ampicillin, vancomycins have higher correlativity to the MIC result of 90 strain clinical separating sausage coccus, and related coefficient is 0.834～0.953.And by comparing with reference method, the influence of fluorescence method and reference method sentence read result gradient difference, gradient difference does not all cause the difference of interpretation character as a result.
Table 2. fluorescence method, detect three kinds of microbiotic to 90 strain clinical separating sausage coccus MIC results' comparison with reference to method
4. fluorescence method detects the accuracy of MIC
The ROC curve is usually in order to assess the accuracy of diagnostic test.Area under curve (AUC) is closely related with the accuracy of diagnostic test.Particularly: the 0.90-1.00=accuracy is high; 0.80-0.90=accuracy is very high; 0.70-0.80=accuracy is higher; 0.60-0.70=poor accuracy; 0.50-0.60=failure.It has been generally acknowledged that AUC＜0.70 expression diagnostic accuracy is relatively low; AUC＞0.90 expression diagnostic accuracy is quite high; And during AUC=0.70-0.90, expression has the accuracy of moderate.This research adopts ROC tracing analysis rapid fluorescence method to detect 3 kinds of microbiotic to the accuracy of 90 strain clinical separation strain MIC.As shown in Figure 4, the ROC area under curve represents fluorescence method respectively and detects the accuracy that 3 kinds of microbiotic detect clinical separating sausage coccus MIC, and it is 0.85 (lavo-ofloxacin) that the result is respectively, 0.765 (ampicillin) and 0.832 (vancomycin).This shows that perhaps the rapid fluorescence method detects 3 kinds of microbiotic to the accuracy that the MIC of 90 strain clinical separating sausage coccus has moderate, can be used as the alternative in order to the MIC of quick mensuration microbiotic to clinical strains, to instruct the rational use of medicines of broth dilution method.
Fluorescence method detects 3 kinds of microbiotic to the accuracy of the MIC of 90 strain clinical separating sausage coccus as shown in Figure 4.Area under curve is respectively 0.85 (lavo-ofloxacin), 0.765 (ampicillin) and 0.832 (vancomycin).
Three, discuss
We have set up and have utilized two kinds of nucleotide fluorescent dye fast detecting microbiotic to the new method of enterococcus minimum inhibitory concentration.Different with classic method, this research is not the number of direct counting bacterium alive, but the existence of bacterium in the fluorescence intensity investigation system of passing through to analyze dead bacterium and live bacterium recently.This is a kind of quick, easy-operating detection method, and can objectively obtain the fluorescence intensity of two kinds of dyestuffs simultaneously, by the automatic analysis result of computer.Because two kinds of dyestuffs have good discrimination, live bacterium and dead bacterium are respectively by exclusive mark blueness or green fluorescence.And, advantage such as dead bacterial dye PI compares with tradition, and SYTOX Green has no autofluorescence, and specificity fluorescent intensity is big.The fluorescence method of initial invention is the metabolic activity for detection of environmental samples or microorganism, and all these are used all is to rely on flow cytometer to finish, and cost is high and can not robotization.MIC under the following several situations of this study tour, and compared.Particularly be: reference method is that micro-broth dilution method detects MIC (MIC/R); Fluorescence method detects MIC (MIC/L), operation and the same MIC/R of incubation time, the just final fluorescent dye sentence read result that adds; The rapid fluorescence method detects MIC (MIC/F), adds the fluorescent dye sentence read result after hatching 4h.
And follow-up 3 kinds of microbiotic show also that to the MIC result of 90 strain clinical separating sausage coccus the rapid fluorescence method is applicable to that in fact clinical fast detecting microbiotic to the MIC of certain isolated strains, has practical value.
Traditional direct viable bacteria counting method is based on the integrality of metabolic activity or the after birth of bacterium.But the utilization of metabolic activity only is suitable for the application of part bacterium, and the many difficulties of appraisal procedure of dependence film integrality overcome strong background fluorescence.Two kinds of fluorescent dyes that the present invention selects for use do not have autofluorescence substantially, so even if the excessive slightly fluorescent dye of adding also can not influence result's interpretation in order to guarantee to dye fully.When being used for the detection of clinical separating sausage coccus, owing to directly read fluorescence intensity level, be not subjected to enterococcus easily to precipitate the influence of characteristics.By table 2 we as can be seen, the rapid fluorescence method is compared with reference method has higher correlativity, related coefficient is 0.834～0.953.In a word, fluorescence method detects for MIC and has advantage quick, easy, that easily be automated.And this method should be applicable to also that detection of antibiotics to the detection of other kind bacterium MIC, has broad application prospects.
Claims (5)
1. a method of measuring the medicine minimum inhibitory concentration is characterized in that, described method step is as follows:
(1) the preparation final concentration is 2.5 * 10
6CFU/ml～5 * 10
6The bacteria suspension of CFU/ml;
(2) prepare the medicine working fluid of variable concentrations gradient respectively, concentration range comprises 512,256,128,64,32,16,8,4,2,1,0.5,0.25,0.125 μ g/ml;
(3) bacteria suspension is joined respectively in the medicine working fluid of above-mentioned concentration gradient, mixing, is hatched 4～6h by 35 ℃ ± 0.5 ℃;
(4) add the mixed liquor of dyestuff SYTOX Green and DAPI in bacteria suspension and the medicine working fluid mixed liquor, final concentration is respectively 1.5 μ M and 300nM, room temperature lucifuge effect 15min;
(5) under the fluorescence microplate reader, read the fluorescence intensity level of each hole SYTOX Green and two kinds of dyestuffs of DAPI respectively;
(6) draw corresponding curve map between fluorescence intensity ratio and each drug concentration; Described fluorescence intensity ratio is SYTOX Green fluorescence intensity level and DAPI fluorescence intensity level ratio; Corresponding curve map between described fluorescence intensity ratio and each drug concentration, the corresponding medicine working fluid of first flex point concentration in curve map behind the peak is this medicine to the minimum inhibitory concentration of this bacterium.
2. method according to claim 1 is characterized in that, in the described step (1), the bacteria suspension final concentration is 5 * 10
6CFU/ml.
3. method according to claim 1 and 2 is characterized in that, excitation wavelength and the emission wavelength of described two kinds of dyestuffs are respectively: DAPI358/461nm; SYTOX Green504/535nm.
4. method according to claim 3 is characterized in that, described medicine is ampicillin, lavo-ofloxacin and/or vancomycin.
5. method according to claim 4 is characterized in that, described bacterium is enterococcus faecalis ATCC29212.
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| CN111458313A (en) * | 2020-04-07 | 2020-07-28 | 上海交通大学医学院附属仁济医院 | Antibacterial drug sensitivity test detection method based on fluorescent D-type amino acid metabolism marker |
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