CN101993910B - Detection method and detection test paper for beta-lactamase - Google Patents
Detection method and detection test paper for beta-lactamase Download PDFInfo
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Abstract
The invention relates to the field of medicaments and provides a detection method for beta-lactamase. The detection method comprises the following steps of: culturing a sample which may contain microorganisms for 2 to 24 hours; and performing beta-lactamase detection on the sample by a chromogenic cephalosporin method. The invention also provides a method for detecting substrate characteristics of the beta-lactamase based on a substrate advantage combination principle. The method of the invention can not only judge whether the sample contains bacteria for producing the beta-lactamase rapidly, but also judge the medicament resistance of the bacteria to beta-lactam antibiotics. The invention also provides detection test paper for the beta-lactamase and a kit comprising the test paper. Compared with the prior art, the detection method and the detection test paper of the invention have the advantages that: the operation is simple and convenient, the detection speed is high, the response is sensitive and the result is stable; therefore, the detection method and the detection test have an extensive clinical application prospect.
Description
Technical field
The present invention relates to a kind of detection method, be specifically related to a kind of detection method and Test paper of β-lactamase.
Background technology:
Day by day serious bacterial drug resistance has become the problem of a global facing.The mechanism of bacterial resistance has a lot of aspects, and β-lactamase is the most general mechanism of the anti-β-lactam antibitics of gram negative bacilli.β-lactamase is applied to find that just it is not that bacterial growth is necessary before clinical that its unique function is the hydrolysis beta-lactam nucleus as far back as β-lactam antibitics.
So far thousands of kinds of the β-lactamases of finding extensively exist in the clinical drug-resistant bacterium.The beta-lactam enzyme classification is many, can be divided into five classes according to its biochemical character:
The first kind is cynnematin lytic enzyme (being the Amp-C enzyme), and producing bacterium mainly is gram-negative bacteria, such as pseudomonas, enterobacteria, acinetobacter calcoaceticus and klebsiella spp etc.The AmpC enzyme can be divided into induction type, structure-type and plasmid-mediated type.Its characteristic is: do not suppressed by clavulanic acid, Sulbactam, Tazobactam Sodium, can be suppressed by cloxacillin; The preferential substrate of selecting is cephalosporins; The high-level resistance of correct mould carbapenem antibiotic (such as cefoxitin); Part Amp-C expression of enzymes can be induced by clavulanic acid, third generation cephalosporin.The medicine that AmpC enzyme bacterium infects is produced in treatment, mainly contains carbapenems (imipenum), the 4th generation cynnematin (cefepime, cefpirome) etc.
Equations of The Second Kind is super wide spectrum enzyme (comprising the non-metal carbon penem enzyme that is produced by Serratia and enterobacter).Its characteristic is: energy hydrolyzing penicillin class, one or two third-generation cephalosporins, monobactams (such as aztreonam), can be suppressed by beta-lactamase inhibitor.The medicine that super wide spectrum enzyme bacterium infection is produced in treatment has carbapenems (except the non-metal carbon penem enzyme), penems, mould alkene class and beta-lactam/beta-lactamase inhibitor.The cefepime considerable part is stable, and the most effective medicine is the blue or green enzyme alkene of carbon class.Should avoid the third generation cephalosporin single therapy.For non-metal carbon penem enzyme, generally avoid selecting the β-lactam antibitics treatment.
The 3rd class is metalloenzyme, by Rhodopseudomonas, bacteroides fragilis genus, Flavobacterium, Serratia and have a liking for the Fructus Hordei Germinatus Xanthomonas and produce.Various beta-lactam antibioticss be can be hydrolyzed, penicillins, cephalosporins and carbapenems comprised; Do not suppressed by existing enzyme inhibitors.Generally avoid β-lactam antibitics when selecting medicine to treat.
The 4th class is Inhibitor-resistant β-lactamase (IR-BLs), is characterized in amoxycilline Trihydrate bp, ticarcillin and enzyme inhibitors compound formulation resistance thereof, can select the treatment of wide spectrum beta-lactam antibiotics.
The 5th class is penicillinase and wide spectrum enzyme, is characterized in penicillin and cephalo one or two generation β-lactam antibitics resistance.Can select wide spectrum beta-lactam antibiotics and ihibitors for treatment.
It is to select suitable antibacterials to avoid chemical sproof generation that the control resistant organism spreads a very important aspect.In view of the extensive existence of β-lactamase in bacterial resistance, and dissimilar β-lactamases has different principle of medication, necessary β-lactamase carried out rapid detection and classification, to select suitable antibacterials.
The beta-lactam enzyme assay method that uses now mainly contains microbial method, iodimetry,iodometry, scraps of paper acidity quantitative method and chromogenic cephalosporin method.Microbial method seldom is used now basically owing to poor specificity, sensitivity are low; Iodimetry,iodometry is usually used in detecting gonococcus; Scraps of paper acidity quantitative method is generally for detection of gonococcus, hemophilus influenzae and staphylococcus; Chromogenic cephalosporin method is to detect at present the most frequently used method of β-lactamase.Chromogenic cephalosporin method is that its color judges with this whether bacterium produces β-lactamase by the light yellow pink that becomes after utilizing the beta-lactam nucleus that produces look cynnematin such as Nitrocefin (nitrocefin) to be destroyed by β-lactamase.
The method that detects beta-lactam enzyme substrates profile mainly contains three kinds of bacterial cultivation, spectrophotometry and association reaction competition laws.More loaded down with trivial details, the consuming time length of front two kinds of operations is only used in the research department.Application number is that 2,003,801,071 85.6 patent describes rear a kind of method in detail, after namely substrate just to be checked and chromogenic substrate mix, the method of reacting with β-lactamase again, because substrate to be checked is lower than chromogenic substrate with the avidity of β-lactamase with the avidity overwhelming majority of β-lactamase, thereby sensitivity is low, false negative rate is high, and the result's is with a low credibility.
When all these detection methods are for detection of clinical samples at present, at least need 72h through bacterium separation, amplification to detecting the result from collecting clinical samples.There is no so far method and product that β-lactamase and the substrate characteristic thereof of clinical samples bacterium are carried out rapid detection.
Summary of the invention
The objective of the invention is to overcome prior art and detect the defective that the β-lactamase false negative rate is high, elapsed time is long, a kind of highly sensitive, detection method of β-lactamase conveniently is provided.
In order to realize the foregoing invention purpose, the present invention by the following technical solutions:
A kind of detection method of β-lactamase may further comprise the steps:
Step (1): the sample that may contain microorganism was cultivated 2-24 hour;
Step (2): sample is carried out the detection of β-lactamase with chromogenic cephalosporin method.
Preferably, also comprise step (3): the substrate characteristic that detects described β-lactamase with substrate advantage combined techniques.
Described step (1) is preferably cultivated under 16 ℃-38 ℃, 0-300rpm condition.
Described step (1) can add the medicine that suppresses the certain micro-organisms growth and carry out the selectivity cultivation.
The medicine of described inhibition microorganism growth can be penicillin, the amoxycilline Trihydrate bp, penicilline g potassium, Benzylpenicillin sodium, the long-acting Amoxicillin, Prostaphlin, Cloxacilin, penbritin, amoxycillin, Pyocianil, Pipril, furbenicillin, close XiLin, pivmecillinam, cefoxitin, the cephalo thiazole, Cephazolin, cefacetrile, Cephapirin, ceftezole, Cephaloglycin, Cephalexin Monohydrate Micro/Compacted, S 578, Cephradine, cefatrizine, cefroxadine, cefaclor, Loracarbef, Cefamandole, cefotiam, cefonicid, cephalofruxin, the combination of one or more in the cefoxitin.
Described drug level is preferably 0.5～32ug/ml.
Detection method is described in the step (3): make β-lactamase first in conjunction with substrate to be checked, be combined again colour developing with chromogenic substrate, the concentration of described substrate to be checked should be higher than described chromogenic agent, and the concentration of preferred described detection substrate is more than 10 times of described chromogenic agent.
The described substrate to be checked of step (3) is preferably a kind of or arbitrarily two or more combination in Penicillin antibiotics, first generation cephalosporin, second generation cephalosporin, third generation cephalosporin, the 4th generation cynnematin, penems antibiotics, monocycle beta-lactam antibiotics, monocycle beta-lactam antibiotics, metal ion chelation agent, the cloxacillin.
To the detected result of step (3) by judging shown in the table 1, thereby select suitable antibacterials.Substrate to be checked is any or multiple among A, B, C, D, the E, and the combination of any mode.Detected result is classified according to table 1:
The classification of table 1. β-lactamase
Annotate :+, medicine to be measured is the substrate of β-lactamase;-, medicine to be measured is not the substrate of β-lactamase.
Microbiotic A can be any one or more Penicillin antibiotics, such as penicillin, amoxycilline Trihydrate bp, penicilline g potassium, Benzylpenicillin sodium, long-acting Amoxicillin, Prostaphlin (new blue or green II number), Cloxacilin, penbritin, amoxycillin etc., Pyocianil, Pipril, furbenicillin, mecillinam and ester pivmecillinam thereof etc.
Also can for any one or more the one or two generation cynnematin, such as cefoxitin, cephalo thiazole, Cephazolin, cefacetrile, Cephapirin, ceftezole, Cephaloglycin, Cephalexin Monohydrate Micro/Compacted, S 578, Cephradine, cefatrizine, cefroxadine, cefaclor, Loracarbef, Cefamandole, cefotiam, cefonicid, cephalofruxin, cefoxitin, cefmetazole, cephalofruxin acetyl oxygen methyl esters, cefotiam hexamethylene oxygen carbonyl oxygen base ethyl ester etc.
Can also for Penicillin antibiotics and the one or two generation cynnematin any combination.
Microbiotic B can for any one or more the 3rd, four generation cynnematin, such as cefotaxime, ceftizoxime, cefmenoxime, ceftriaxone, cefuzonam, cefodizime, ceftazime, U-63196E, cefoperazone, cefpiramide, cefpirome, cefepime, cefotetan, cefbuperazone, cefminox, latamoxef, flomoxef, Cefixime Micronized, cephalo cloth spit of fland, Cefdinir, cefteram pivaloyl oxygen base methyl esters, Cefpodoxime isopropyl oxygen carbonyl oxygen base ethyl ester, cefetamet pivaloyl oxygen base methyl esters, cephalo kobold pivaloyl oxygen base methyl esters, cefepime, Wincef, cefpirome, cefuzonam sodium, Cefozopran etc.
Also can be any one or more other wide spectrum beta-lactam antibiotics, comprise: carbapenem antibiotic such as imipenum, imipenum/cilastatin, panipenem, panipenem/N-benzoyl beta Alanine, meropenem, Ou Tapeinan etc.; Penems antibiotics such as faropenem; Monocycle beta-lactam antibiotics such as aztreonam and carumonam etc.
Microbiotic C can be any one or more beta-lactamase inhibitor, such as clavulanic acid, Sulbactam, Tazobactam Sodium etc.
Also can be any combination of one or more beta-lactamase inhibitors with any other β-lactam antibitics.
Medicine D can be any one or more metal ion chelation agent, such as tripoly phosphate sodium STPP, sodium polyphosphate, Sodium hexametaphosphate 99, trisodium phosphate, ethylenediamine tetraacetic acid (EDTA) (ED-TA), nitrilotriacetic acid (NTA), diethylene-triamine pentaacetic acid (DTPA), N-hydroxyethyl ethamine nitrilotriacetic (HEDTA), ethylene glycol-two-(the amino ether of B-)-N, N-tetraacethyl (EGTA), Amino Trimethylene Phosphonic Acid (ATMP), 1-hydroxyl ethylidene-1,1-di 2 ethylhexyl phosphonic acid (HEDP), ethylene diamine tetra methylene phosphonic acid (EDTMP), diethylene triamine pentamethylene phosphonic (DTPMP), Amino Trimethylene Phosphonic Acid (ATP), gluconic acid, polyacrylic acid (PAA), toxilic acid (MAO) etc.
Described microbiotic E can be cloxacillin.
Detect result and the table 1 of the substrate characteristic of β-lactamase according to step (3), β-lactamase is classified, thereby select suitable antibacterials to avoid chemical sproof generation, instruct clinical application.
The described chromogenic substrate of step (3) comprises acceptable salt on Nitrocefin and the physiology thereof; or be the derivative of Nitrocefin; namely comprise 3-[2; 4-dinitrobenzene vinyl]-7-(2-thienyl ethanoyl)-3-cephalo ring-4-carboxylic acid; 7-[2-(thiazolamine-4-yl)-2-(1-carboxyl-1-methyl ethoxy imino-) kharophen]-3-(2; 4-dinitrobenzene vinyl)-3-cephalo ring-4-carboxylic acid; 7-[2-(thiazolamine-4-yl)-2-(1-carboxyl-1-methyl ethoxy imino-) kharophen]-3-(2; 6-dinitrobenzene vinyl)-3-cephalo ring-4-carboxylic acid; 7-[2-(thiazolamine-4-yl)-2-(1-carboxyl-1-methyl ethoxy imino-) kharophen]-3-(4-nitrostyrolene base)-3-cephalo ring-4-carboxylic acid; 7-[2-(thiazolamine-4-yl)-2-(1-carboxyl-1-methyl ethoxy imino-) kharophen]-3-(2; 4-dicyano styrene base)-3-cephalo ring-4-carboxylic acid; 7-[2-(thiazolamine-4-yl)-2-(1-carboxyl-1-methyl ethoxy imino-) kharophen]-3-(4-cyano-styrene base)-3-cephalo ring-4-carboxylic acid; 7-[2-(thiazolamine-4-yl)-2-(1-carboxyl-1-methyl ethoxy imino-) kharophen]-3-(2-cyano-styrene base)-3-cephalo ring-4-carboxylic acid; 7-[2-(1-carboxyl-1-methyl ethoxy imino-)-2-(thiazole-4-yl) kharophen]-3-(2; 4-dinitrobenzene vinyl)-3-cephalo ring-4-carboxylic acid; 7-[2-(thiazolamine-4-yl)-2-(1-carboxyl-1-methyl ethoxy imino-) kharophen]-3-(2; 4-dinitrobenzene vinyl)-3-cephalo ring-4-carboxylic acid-1-oxide compound; 7-[2-(thiazolamine-4-yl)-2-methoxyimino kharophen]-3-(4-nitrostyrolene base)-3-cephalo ring-4-carboxylic acid; 7-[2-(thiazolamine-4-yl)-2-methoxyimino kharophen]-3-(2; 4-dinitrobenzene vinyl)-3-cephalo ring-4-carboxylic acid; 7-[2-(thiazolamine-4-yl)-2-methoxyimino kharophen]-3-(2; 6-dinitrobenzene vinyl)-3-cephalo ring-4-carboxylic acid; 7-[2-(thiazolamine-4-yl)-2-methoxyimino kharophen]-3-(2 cyano-styrene base)-3-cephalo ring-4-carboxylic acid; 7-[2-(thiazolamine-4-yl)-2-carboxyl methoxyimino kharophen]-3-(2; 4-dinitrobenzene vinyl)-3-cephalo ring-4-carboxylic acid; 7-[2-(thiazolamine-4-yl)-2-carboxyl methoxyimino kharophen]-3-(2; 6-dinitrobenzene vinyl)-3-cephalo ring-4-carboxylic acid; 7-[2-(thiazolamine-4-yl)-2-carboxyl methoxyimino kharophen]-3-(4-nitrostyrolene base)-3-cephalo ring-4-carboxylic acid; 7-[2-(thiazolamine-4-yl)-2-carboxyl methoxyimino kharophen]-3-(2-nitrostyrolene base)-3-cephalo ring-4-carboxylic acid; 7-[2-(thiazolamine-4-yl)-2-carboxyl methoxyimino kharophen]-3-(2; 4-dicyano styrene base)-3-cephalo ring-4-carboxylic acid; 7-[2-(thiazolamine-4-yl)-2-carboxyl methoxyimino kharophen]-3-(4-cyano-styrene base)-3-cephalo ring-4-carboxylic acid; 7-[2-(thiazolamine-4-yl)-2-carboxyl methoxyimino kharophen]-3-(2-cyano-styrene base)-3-cephalo ring-4-carboxylic acid; 7-[2-2-carboxyl methoxyimino-2-(thiazole-4-yl)-kharophen]-3-(2; 4-dinitrobenzene vinyl)-3-cephalo ring-4-carboxylic acid; 7-[2-(thiazolamine-4-yl)-2-carboxyl methoxyimino kharophen]-3-(2,4-dinitrobenzene vinyl)-3-cephalo ring-4-carboxylic acid-1 oxide compound.
The present invention also provides a kind of β-lactamase Test paper, comprises following part: sample application zone, detection zone and colour developing district.Chromogenic substrate is contained in described colour developing district; Described detection zone contains substrate to be checked.
Described substrate to be checked is preferably a kind of in Penicillin antibiotics, first generation cephalosporin, second generation cephalosporin, third generation cephalosporin, the 4th generation cynnematin, penems antibiotics, monocycle beta-lactam antibiotics, metal ion chelation agent, the cloxacillin.
Described chromogenic substrate comprises acceptable salt on Nitrocefin and the physiology thereof, or is the derivative of Nitrocefin, and these compounds have detailed description in front.
The present invention also provides a kind of β-lactamase to detect the multi-arm test paper, comprises two the above β-lactamase Test papers, and described β-lactamase Test paper shares a sample application zone.
Described β-lactamase detects the multi-arm test paper, and preferably the substrate to be checked that contains of every detection paper district is different.
The present invention also provides a kind of beta-lactamase detection kit, comprises above-mentioned β-lactamase Test paper or β-lactamase and detects the multi-arm test paper.
The present invention mixes or the selectivity cultivation by clinical samples being carried out in short-term (about 2-24 hour), thereby Rapid Detection produces the β-lactamase resistant organism.Then after nutrient solution being carried out in short-term emulsification, by the method for diffusion, make β-lactamase first with the Binding Capacity to be checked of high density, with the chromogenic substrate contact, greatly improved the susceptibility that detects again.In addition, carry out at colour developing filter paper, the result observes easily, also helps to improve the sensitivity of detection.
Detection method of the present invention, with respect to existing detection method, its advantage is:
1) adopt diffusion principle, β-lactamase contacts the detection method of low concentration chromogenic substrate more first in conjunction with higher concentration substrate to be checked, has improved the sensitivity that detects.
2) a plurality of substrates detect and merge on a multi-arm test strip, and an application of sample is just finished the detection of substrate characteristic, and strong operability is convenient to the comparison between each arm, have improved the accuracy that detects.
3) the method for the invention is easy and simple to handle, detection is quick, the fastest only need 5 hours, and the result is stable, has widely potential applicability in clinical practice.
Description of drawings
Fig. 1 shows the β-lactamase test strip;
1 is propping material, and 2 is color test paper.
Fig. 2 is the synoptic diagram of 6 arm Test papers of β-lactamase of the present invention.
A-F: the numbering of six arms; 1 district is the label area, in order to mark arm number and substrate; 2 districts contain chromogenic substrate for the colour developing district; 3 districts substrate zone to be tried contains substrate to be checked on the filter paper; 4 districts are central sample application zone.
Embodiment
Embodiment 1: the preparation of β-lactamase test strip
Structure as shown in Figure 1.1 is propping material, paste on it 2 for color test paper, pack and labelled with the lucifuge material outward.
The making of color test paper: it is 0.01～1mg/ml that developer becomes final concentration with diluent preparing.After filter paper is cut into suitable size, color test paper can for different shape such as circle, rectangle etc., area is 0.2～1.5cm
2Be advisable.Wetting with nitrite ion, drying.Drying means can be dried the most handy lyophilization in short-term for 30-80 ℃.The dried scraps of paper are attached on the propping material, the lucifuge packing, and low-temperature dark is preserved.Use in 6 months.
Color test paper can for different shape such as circle, rectangle etc., area is 0.2～1.5cm
2Be advisable.
Embodiment 2: the preparation of β-lactamase multi-arm Test paper (take 6 arms as example)
Structure is as shown in Figure 2: A-F: the numbering of six arms.
Every arm divides 4 districts: 1 for the label area, in order to mark arm number and substrate; 2 districts contain the cephalo nitrophenol for the colour developing district; 3 districts substrate zone to be tried contains substrate to be checked on the filter paper; 4 districts are the central area sample application zone.
Size: every brachium 2～4cm, wide 0.5～1.5cm.
4 districts: diameter 0.5～1.0cm; Wide 0.5～1.5cm.
3 districts: long 0.5～1.5cm; Wide 0.5～1.5cm.
2 districts: long 0.3～1.5cm, wide 0.5～1.5cm.
1 district: long 0.5～1.5cm, wide 0.5～1.5cm.
Content is indicated in 1 district: arm number, these arm 3 district's medicine Chinese or english abbreviations.
Step 1: make the pastille scraps of paper:
All detect the drug concentration requirement 3 districts: be higher than used chromogenic agent.The concentration of preferred detection substrate is more than 10 times of chromogenic agent, and more preferably multiple is 100～500 times.
After medicine became the concentration of above-mentioned requirements with diluent preparing, 1 described method preparation contained medicine test-paper to be checked with reference to embodiment.
Step 2: make and detect the multi-arm test strip
1) supporting dielectric such as plastic film are made six arms;
2) stick double faced adhesive tape on six arms;
3) adhesive label paper (1 district) successively, interval 2～5mm, paste the colour developing scraps of paper (2 district), cover 2～4mm place and paste drug paper disk (3 district), circular filter paper sheet in the middle of sticking at last is (for application of sample, 4 districts), the circular filter paper sheet will cover drug paper disk (3 district) 2～4mm.Drug paper disk (3 district) does not cover 2 districts and 4 districts part is 3～8mm.
4) press mold packing (center well can not coating), film is transparent.
5) with wherein the positive contrast of an arm, these arm 3 district's scraps of paper do not contain medicine to be checked, so to become pink expression multi-arm test strip effective by pale yellow for the colour developing scraps of paper (2 district) color.Other five arms, 3 district's scraps of paper should comprise one or more of medicine to be checked as far as possible.
Step 3: the determining of application of sample amount
After testing: the water regain of test strip is approximately 50-100ul/cm
2. the application of sample amount requires liquid can just be diffused into 2 districts, and 1/3～3/4 being advisable of moistening 2 districts of energy.
Embodiment 3: the preparation of test kit of the present invention
The content that test kit comprises:
1, bacterium amplification bottle includes bacterium rapid amplifying culture medium powder, 4-8 ℃ of preservation.
2, the EP of 1.0mL pipe (including one or more in emulsifying agent N,O-Diacetylmuramidase, Tyrothricin, the polymyxin), 4-8 ℃ of preservation.
3, embodiment 1 preparation β-lactamase test strip ,-20 ℃ keep in Dark Place.
4, the β-lactamase type of embodiment 2 preparations detects the multi-arm test paper, and-20 ℃ keep in Dark Place.
Embodiment 4: the fast culture of microorganism
Step 1: clinical samples to be checked is that solid (such as ight soil), semi-solid (such as sputum, fester) and a small amount of liquid are (such as secretory product, prostatic fluid) time, get an amount of aquae destillata or two aquae destillata or ultrapure water in culturing bottle (including culture medium powder such as high nutrient broth), shake up to culture medium powder and dissolve fully, then put into clinical samples, shake up.
When clinical samples to be checked is large quantity of fluid (such as urine, drainage-fluid), then directly get an amount of sample in culturing bottle, shake up to culture medium powder and dissolve fully.
Step 2: above-mentioned clinical samples is through 16 ℃-38 ℃, and 0-300rpm shakes or do not shake and cultivated 2-24 hour.
The minimum bacterial concentration that detects the beta-lactam enzyme require is 10
5CFU/mL.Result of study shows: 37 ℃, the 150-200rpm concussion was cultivated 5 hours, and 24 hours detect is consistent with cultivating.If 37 ℃ of static cultivations then needed to cultivate 18 hours, bacterial concentration can reach 10
5CFU/mL.
Embodiment 5: the fast selective of microorganism is cultivated
For some research needs, need sift out anti-certain or some antibiotic infected patient or Resistant strain, then can in substratum, add this kind microbiotic, suitable concentration is 0.5～32ug/ml.
For example: the clinical study of carrying out to ceftazime/Sulbactam compound preparation, need in the clinical study to filter out the ceftazime resistance, and to the infected patient of ceftazime/Sulbactam sensitivity as research object.Adoptable method is: to gathering the clinical samples from the experimenter, be divided into two parts.First part of employing contains ceftazime, and (0.5～32ug/ml) culture medium culturing, ((then 2～32ug/ml) culture medium culturing detects β-lactamase to 0.5～32ug/ml) coupling Sulbactam to second part of employing ceftazime.The First specimen β-lactamase detects positive, and rear second part of sample β-lactamase detects negative person as MethodsThe cases enrolled simultaneously.
Embodiment 6: the detection of β-lactamase
Get 1 of embodiment 4 gained bacterium liquid, drip and producing on the look cynnematin colour developing filter paper, observe filter paper colour-change, as follows judged result under the room temperature:
If filter paper color in 30min does not change, then β-lactamase detects negative.If filter paper in 30min color by light yellow become pink or red, β-lactamase test positive then, the detection of then carrying out embodiment 7 to be judging the substrate characteristic of β-lactamase, and β-lactamase is classified.
Embodiment 7: the detection of the substrate characteristic of β-lactamase and the classification of β-lactamase
Get the 0.5-1mL nutrient solution and join in the EP pipe that includes the emulsifying agent polymyxin, fully mixing dissolving, 37 ℃ of temperature are incubated 10min, obtain bioavailability liquid.Emulsifying agent also can be selected a kind of or any combination of Tyrothricin, N,O-Diacetylmuramidase.
Get an amount of above-mentioned bioavailability liquid and join the central sample application zone (4 district) of multi-arm Test paper (seeing Fig. 1), liquid spreads to filter paper 2 districts gradually.Observations in the 30min.
Detected result is carried out the beta-lactam enzyme classification according to table 1:
The classification of table 1. β-lactamase
+, medicine to be measured is the substrate of β-lactamase;-, medicine to be measured is not the substrate of β-lactamase.
The result judges:
1) detecting effective condition is: each arm 2 district's test paper color is pale yellow, and A arm 2 district's colors are by the pale yellow pink that becomes in detecting simultaneously.
2) be judged to the standard of "+": be contrast with A arm 2 district's color changes, other arm color is constant or change and be starkly lower than the variation of A arm 2 districts and then be judged to "+".
3) be judged to the standard of "-": be contrast with A arm 2 district's color changes, other arm colour-change is similar or obviously be better than A arm 2 district's colour-change and then be judged to "-".
Embodiment 8: the method for the invention detects beta-lactam enzyme substrates characteristic and ordinary method compares
Experimental technique is as follows:
1) collect infected patient sample 31 examples, the sample type has sputum, urine, puncture fluid, fester, secretory product, prostatic fluid.Every part of sample is divided into two parts.
2) first part of fast culture process that adopts this test kit, 37 ℃, the 150rpm concussion was cultivated 5 hours, and cephalo nitrothiophene development process detects β-lactamase.For the β-lactamase positive sample, adopt the described method of this patent to detect the substrate characteristic of β-lactamase.
3) second part of specimen transfer Clinical microorganism section office cultivate routinely, isolated strains, adopt 200380107185.6 described methods (abbreviation Microdilution plate method) to detect the substrate characteristic of β-lactamase.Simultaneously the pathogenic bacterium that separate are increased, extract β-lactamase according to sonioation method, adopt determined by ultraviolet spectrophotometry beta-lactam enzyme substrates characteristic.
4) take the ultraviolet spectrophotometry detected result as the criterion Microdilution plate method with sensitivity and the accuracy of the described method of this patent.All experiments repeat 5 times, with the repeatability of investigation method.
Result of study: ultraviolet spectrophotometry detects 6 samples and contains product ESBLs bacterial strain, duplicate detection five times, and the result is in full accord, detects required time and is about 84 hours.Microdilution plate method detects 2 samples and contains product ESBLs bacterial strain, and 2 samples are suspicious, and poor repeatability detects required time and is about 72 hours.The detected result of this patent method is in full accord with ultraviolet spectrophotometry, needs 5 hours and only detect.In addition, the ultraviolet spectrophotometry complex operation, experimental technique requires high, and the method for the invention is simple to operate.Compare the advantage that the method for the invention has is easy and simple to handle, detect fast, be quick on the draw, the result is stable with prior art.
Claims (14)
1. the detection method of a β-lactamase may further comprise the steps:
Step (1): the sample that may contain microorganism was cultivated 2-24 hour;
Step (2): sample is carried out the detection of β-lactamase with chromogenic cephalosporin method;
Step (3): the substrate characteristic that detects described β-lactamase with substrate advantage combined techniques, described substrate advantage combined techniques is for making β-lactamase preferentially in conjunction with substrate to be checked, be combined colour developing with chromogenic substrate, the concentration of described substrate to be checked is higher than described chromogenic agent again.
2. detection method according to claim 1 is characterized in that, the described cultivation of step (1) is to cultivate under 16 ℃-38 ℃, 50-300rpm condition.
3. detection method according to claim 1 is characterized in that, step (1) adds the medicine that suppresses microorganism growth and carries out the selectivity cultivation.
4. detection method according to claim 3, it is characterized in that, described medicine is penicillin, the amoxycilline Trihydrate bp, penicilline g potassium, Benzylpenicillin sodium, the long-acting Amoxicillin, Prostaphlin, Cloxacilin, penbritin, amoxycillin, Pyocianil, Pipril, furbenicillin, mecillinam, pivmecillinam, cefoxitin, the cephalo thiazole, Cephazolin, cefacetrile, Cephapirin, ceftezole, Cephaloglycin, Cephalexin Monohydrate Micro/Compacted, S 578, Cephradine, cefatrizine, cefroxadine, cefaclor, Loracarbef, Cefamandole, cefotiam, cefonicid, cephalofruxin, the combination of one or more in the cefoxitin.
5. according to claim 3 or 4 described detection methods, it is characterized in that described drug level is 0.5～32ug/ml.
6. detection method according to claim 1 is characterized in that, the concentration of the described detection substrate of step (3) is more than 10 times of described chromogenic agent.
7. detection method according to claim 1, it is characterized in that described substrate to be checked is a kind of or arbitrarily two or more combination in Penicillin antibiotics, first generation cephalosporin, second generation cephalosporin, third generation cephalosporin, the 4th generation cynnematin, penems antibiotics, monocycle beta-lactam antibiotics, metal ion chelation agent, the cloxacillin.
8. detection method according to claim 1 is characterized in that, described chromogenic substrate comprises acceptable salt on Nitrocefin and the physiology thereof, and the derivative of Nitrocefin.
9. a β-lactamase Test paper sequentially comprises following part: sample application zone, detection zone, colour developing district successively; Chromogenic substrate is contained in described colour developing district; Described detection zone contains substrate to be checked, and β-lactamase is combined colour developing more preferentially in conjunction with substrate to be checked in the testing process with chromogenic substrate.
10. β-lactamase Test paper according to claim 9, it is characterized in that described substrate to be checked is a kind of in Penicillin antibiotics, first generation cephalosporin, second generation cephalosporin, third generation cephalosporin, the 4th generation cynnematin, penems antibiotics, monocycle beta-lactam antibiotics, metal ion chelation agent, the cloxacillin.
11. β-lactamase Test paper according to claim 9 is characterized in that, described chromogenic substrate comprises acceptable salt on Nitrocefin and the physiology thereof, and the derivative of Nitrocefin.
12. a β-lactamase detects the multi-arm test paper, comprises each the described β-lactamase Test paper of claim 9-11 more than two, described β-lactamase Test paper shares a sample application zone.
13. β-lactamase according to claim 12 detects the multi-arm test paper, it is characterized in that, the substrate to be checked that every detection paper district is contained is not identical.
14. a beta-lactamase detection kit is characterized in that, comprises each described β-lactamase Test paper of claim 9-11.
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| CN106404831B (en) * | 2016-12-20 | 2020-01-14 | 西北大学 | Rapid detection method for sensitivity of beta-lactam antibiotics |
| GB201712671D0 (en) * | 2017-08-07 | 2017-09-20 | Mast Group Ltd | Chromogenic Carbapenemase detection method |
| CN107703130A (en) * | 2017-09-19 | 2018-02-16 | 淮海工学院 | Suitable for the acetes chinensis method for the beta lactamase for detecting Residues in Milk |
| CN107607719B (en) * | 2017-11-02 | 2019-08-02 | 北京中生金域诊断技术股份有限公司 | Beta-lactam enzyme method in a kind of measurement sample |
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| JP2000224998A (en) * | 1999-02-04 | 2000-08-15 | Kokuritsu Kansenshiyou Kenkyusho | Discrimination of metallo-beta-lactamase producing bacterium |
| CN1729299A (en) * | 2002-10-29 | 2006-02-01 | 昭和药品化工株式会社 | Reagent composition for detecting beta-lactamase, detection kit and detection method |
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| JP2000224998A (en) * | 1999-02-04 | 2000-08-15 | Kokuritsu Kansenshiyou Kenkyusho | Discrimination of metallo-beta-lactamase producing bacterium |
| CN1729299A (en) * | 2002-10-29 | 2006-02-01 | 昭和药品化工株式会社 | Reagent composition for detecting beta-lactamase, detection kit and detection method |
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