CN109536568A - A kind of quick detection produces method, kit and its application of carbapenem enzyme bacterial strain - Google Patents
A kind of quick detection produces method, kit and its application of carbapenem enzyme bacterial strain Download PDFInfo
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- 238000001514 detection method Methods 0.000 title claims abstract description 40
- 238000000034 method Methods 0.000 title claims abstract description 36
- 108090000790 Enzymes Proteins 0.000 title abstract description 49
- 102000004190 Enzymes Human genes 0.000 title abstract description 46
- YZBQHRLRFGPBSL-RXMQYKEDSA-N carbapenem Chemical compound C1C=CN2C(=O)C[C@H]21 YZBQHRLRFGPBSL-RXMQYKEDSA-N 0.000 title abstract description 44
- 230000001580 bacterial effect Effects 0.000 title abstract description 26
- 241000894006 Bacteria Species 0.000 claims abstract description 73
- 229920001817 Agar Polymers 0.000 claims abstract description 66
- 239000008272 agar Substances 0.000 claims abstract description 66
- WKDDRNSBRWANNC-UHFFFAOYSA-N Thienamycin Natural products C1C(SCCN)=C(C(O)=O)N2C(=O)C(C(O)C)C21 WKDDRNSBRWANNC-UHFFFAOYSA-N 0.000 claims abstract description 64
- 229960002182 imipenem Drugs 0.000 claims abstract description 64
- ZSKVGTPCRGIANV-ZXFLCMHBSA-N imipenem Chemical compound C1C(SCC\N=C\N)=C(C(O)=O)N2C(=O)[C@H]([C@H](O)C)[C@H]21 ZSKVGTPCRGIANV-ZXFLCMHBSA-N 0.000 claims abstract description 64
- 239000002253 acid Substances 0.000 claims abstract description 37
- 108010068385 carbapenemase Proteins 0.000 claims abstract description 18
- 239000002696 acid base indicator Substances 0.000 claims description 73
- 239000003814 drug Substances 0.000 claims description 70
- 229940079593 drug Drugs 0.000 claims description 65
- 239000002609 medium Substances 0.000 claims description 55
- XJRPTMORGOIMMI-UHFFFAOYSA-N ethyl 2-amino-4-(trifluoromethyl)-1,3-thiazole-5-carboxylate Chemical compound CCOC(=O)C=1SC(N)=NC=1C(F)(F)F XJRPTMORGOIMMI-UHFFFAOYSA-N 0.000 claims description 54
- 241000193385 Geobacillus stearothermophilus Species 0.000 claims description 25
- 239000008223 sterile water Substances 0.000 claims description 19
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 19
- 238000011534 incubation Methods 0.000 claims description 11
- 230000001954 sterilising effect Effects 0.000 claims description 10
- 238000004519 manufacturing process Methods 0.000 claims description 9
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- 239000001963 growth medium Substances 0.000 claims description 8
- 238000002156 mixing Methods 0.000 claims description 8
- ZPLCXHWYPWVJDL-UHFFFAOYSA-N 4-[(4-hydroxyphenyl)methyl]-1,3-oxazolidin-2-one Chemical compound C1=CC(O)=CC=C1CC1NC(=O)OC1 ZPLCXHWYPWVJDL-UHFFFAOYSA-N 0.000 claims description 5
- 238000007790 scraping Methods 0.000 claims description 5
- 238000004659 sterilization and disinfection Methods 0.000 claims description 5
- BTJIUGUIPKRLHP-UHFFFAOYSA-N 4-nitrophenol Chemical compound OC1=CC=C([N+]([O-])=O)C=C1 BTJIUGUIPKRLHP-UHFFFAOYSA-N 0.000 claims description 4
- 244000063299 Bacillus subtilis Species 0.000 claims description 4
- 235000014469 Bacillus subtilis Nutrition 0.000 claims description 4
- 235000003392 Curcuma domestica Nutrition 0.000 claims description 4
- 244000008991 Curcuma longa Species 0.000 claims description 4
- WWAABJGNHFGXSJ-UHFFFAOYSA-N chlorophenol red Chemical compound C1=C(Cl)C(O)=CC=C1C1(C=2C=C(Cl)C(O)=CC=2)C2=CC=CC=C2S(=O)(=O)O1 WWAABJGNHFGXSJ-UHFFFAOYSA-N 0.000 claims description 4
- 235000003373 curcuma longa Nutrition 0.000 claims description 4
- CEQFOVLGLXCDCX-WUKNDPDISA-N methyl red Chemical compound C1=CC(N(C)C)=CC=C1\N=N\C1=CC=CC=C1C(O)=O CEQFOVLGLXCDCX-WUKNDPDISA-N 0.000 claims description 4
- 235000013976 turmeric Nutrition 0.000 claims description 4
- 241000588724 Escherichia coli Species 0.000 claims description 2
- 241000589517 Pseudomonas aeruginosa Species 0.000 claims description 2
- 238000002845 discoloration Methods 0.000 claims description 2
- 238000001035 drying Methods 0.000 claims 3
- OYCLSQDXZMROJK-UHFFFAOYSA-N 2-bromo-4-[3-(3-bromo-4-hydroxyphenyl)-1,1-dioxo-2,1$l^{6}-benzoxathiol-3-yl]phenol Chemical compound C1=C(Br)C(O)=CC=C1C1(C=2C=C(Br)C(O)=CC=2)C2=CC=CC=C2S(=O)(=O)O1 OYCLSQDXZMROJK-UHFFFAOYSA-N 0.000 claims 2
- PGSADBUBUOPOJS-UHFFFAOYSA-N neutral red Chemical compound Cl.C1=C(C)C(N)=CC2=NC3=CC(N(C)C)=CC=C3N=C21 PGSADBUBUOPOJS-UHFFFAOYSA-N 0.000 claims 2
- 238000007605 air drying Methods 0.000 claims 1
- 238000012258 culturing Methods 0.000 claims 1
- 239000000203 mixture Substances 0.000 claims 1
- 238000003805 vibration mixing Methods 0.000 claims 1
- 206010011409 Cross infection Diseases 0.000 abstract description 2
- 238000004458 analytical method Methods 0.000 abstract description 2
- MKNQNPYGAQGARI-UHFFFAOYSA-N 4-(bromomethyl)phenol Chemical compound OC1=CC=C(CBr)C=C1 MKNQNPYGAQGARI-UHFFFAOYSA-N 0.000 abstract 3
- 206010029803 Nosocomial infection Diseases 0.000 abstract 1
- 238000012360 testing method Methods 0.000 description 53
- 241000697872 Bactria Species 0.000 description 36
- 230000002053 acidogenic effect Effects 0.000 description 36
- 239000013642 negative control Substances 0.000 description 12
- 239000013641 positive control Substances 0.000 description 12
- 241000196324 Embryophyta Species 0.000 description 10
- 241000193830 Bacillus <bacterium> Species 0.000 description 9
- 244000005700 microbiome Species 0.000 description 7
- 238000011081 inoculation Methods 0.000 description 6
- 108090000623 proteins and genes Proteins 0.000 description 6
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 5
- 229910052799 carbon Inorganic materials 0.000 description 5
- 230000000694 effects Effects 0.000 description 5
- 150000001336 alkenes Chemical class 0.000 description 4
- 238000006243 chemical reaction Methods 0.000 description 4
- 238000002474 experimental method Methods 0.000 description 4
- 230000002779 inactivation Effects 0.000 description 4
- 241000588747 Klebsiella pneumoniae Species 0.000 description 3
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical group [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 description 3
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 3
- KVYRCBOUKXJXDK-UHFFFAOYSA-N 3,4-dimethylphenazine-1,2-diamine hydrochloride Chemical compound Cl.C1=CC=CC2=NC3=C(C)C(C)=C(N)C(N)=C3N=C21 KVYRCBOUKXJXDK-UHFFFAOYSA-N 0.000 description 2
- 230000003321 amplification Effects 0.000 description 2
- GDTBXPJZTBHREO-UHFFFAOYSA-N bromine Substances BrBr GDTBXPJZTBHREO-UHFFFAOYSA-N 0.000 description 2
- -1 bromine cresols Chemical class 0.000 description 2
- 229910052794 bromium Inorganic materials 0.000 description 2
- 239000003795 chemical substances by application Substances 0.000 description 2
- 230000007812 deficiency Effects 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- JEIPFZHSYJVQDO-UHFFFAOYSA-N iron(III) oxide Inorganic materials O=[Fe]O[Fe]=O JEIPFZHSYJVQDO-UHFFFAOYSA-N 0.000 description 2
- 210000002429 large intestine Anatomy 0.000 description 2
- 238000003199 nucleic acid amplification method Methods 0.000 description 2
- 238000011160 research Methods 0.000 description 2
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- 239000003643 water by type Substances 0.000 description 2
- WPDYPOJHZBBFGR-UHFFFAOYSA-N C1=C(C)C=CC(C(C)C)=C1O.[Br] Chemical compound C1=C(C)C=CC(C(C)C)=C1O.[Br] WPDYPOJHZBBFGR-UHFFFAOYSA-N 0.000 description 1
- 241000588697 Enterobacter cloacae Species 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- 241000726221 Gemma Species 0.000 description 1
- 241000234221 Klebsiella pneumoniae ATCC BAA-1705 Species 0.000 description 1
- 241001466580 Klebsiella pneumoniae ATCC BAA-2146 Species 0.000 description 1
- 206010035664 Pneumonia Diseases 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 230000000844 anti-bacterial effect Effects 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 229940041011 carbapenems Drugs 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 238000002651 drug therapy Methods 0.000 description 1
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- 238000009963 fulling Methods 0.000 description 1
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- HHXMXAQDOUCLDN-RXMQYKEDSA-N penem Chemical compound S1C=CN2C(=O)C[C@H]21 HHXMXAQDOUCLDN-RXMQYKEDSA-N 0.000 description 1
- 239000007793 ph indicator Substances 0.000 description 1
- 238000007711 solidification Methods 0.000 description 1
- 230000008023 solidification Effects 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
- C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
- G01N2333/32—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Bacillus (G)
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Abstract
本发明提供了一种快速检测产碳青霉烯酶菌株的方法、试剂盒及其应用。所述的快速检测方法包括：亚胺培南与待检测菌菌苔共同孵育，制备含有溴甲酚紫和产酸细菌的MH琼脂培养基，将与待检测菌菌苔孵育后的亚胺培南纸片贴在含有溴甲酚紫和产酸细菌的MH琼脂培养基上，孵育3.5～4.5小时，即可直接通过溴甲酚紫是否变色，进而判定待检测菌株是否为产碳青霉烯酶菌株。本发明的检测方法操作简单、分析快速、适用范围广、准确性和特异性高、结果易于判读。同时，本发明还提供基于所述快速检测方法建立的检测试剂盒，对于及早发现产碳青霉烯酶菌株并采取有效措施，对医院感染控制及临床流行病学检测具有重要意义，应用前景良好。
The present invention provides a method, kit and application for rapid detection of carbapenemase-producing strains. The rapid detection method includes: co-incubating imipenem with the bacterial fur to be detected, preparing MH agar medium containing bromocresol violet and acid-producing bacteria, and imipenem incubated with the bacterial fur to be detected. Paste the paper sheet on the MH agar medium containing bromocresol violet and acid-producing bacteria, and incubate for 3.5 to 4.5 hours, you can directly check whether the bromocresol violet changes color, and then determine whether the strain to be tested is carbapenem-producing. enzyme strains. The detection method of the invention has simple operation, rapid analysis, wide application range, high accuracy and specificity, and easy interpretation of results. At the same time, the present invention also provides a detection kit based on the rapid detection method, which is of great significance for early detection of carbapenemase-producing strains and effective measures for nosocomial infection control and clinical epidemiological detection, and has good application prospects .
Description
Technical field
The invention belongs to field of biotechnology, in particular to a kind of quickly detection produces the method for carbapenem enzyme bacterial strain, examination
Agent box and its application.
Background technique
Carbapenem antibiotic is when treatment is infected as caused by most of Gram-positives and gram-negative venereal bacteria
It is had very important effect with significant therapeutic effect, therefore clinically.However, as bacterium is constantly evolved, one
A little bacteriums can generate carbapenem enzyme, that is, with the appearance for the bacterium for producing carbapenem enzyme, cause many clinical use carbon green
The case of mould alkenes drug therapy failure occurs.
Currently, carbapenem enzyme method for deactivating (carbapenem inactivation method, CIM) is clinically to examine
A kind of common method for producing carbapenem enzyme bacterial strain is surveyed, it is easy to operate because its is cheap, therefore be widely used.But
The obvious deficiency of one of this method is to need 18~24 hours can just obtain testing result, and detection time is long.And Carba NP is tried
Test higher with the technical requirements of Carba NP direct test, the scope of application does not have CIM extensive, needs special reagent and specially
Industry personnel operation.
Discovery as early as possible produces carbapenem enzyme bacterial strain and adopts an effective measure, and examines to hospital infection control and clinical epidemiology
Measuring tool is significant.Therefore, easy to operate, quick, the applied widely, accuracy of analysis and specific high carbon are researched and developed
Penem enzyme assay method has urgent need.
Summary of the invention
The primary purpose of the present invention is that the shortcomings that overcoming the prior art and deficiency, provide a kind of quickly detect and produce carbon mould
The method of alkene enzyme bacterial strain, the method can quickly detect the bacterium for generating carbapenem enzyme in 3.5~4.5 hours.
A further object of the present invention is to provide a kind of quickly detections to produce carbapenem enzyme bacterial strain kit.
Another object of the present invention is to provide the application of the detection method or kit.
The purpose of the invention is achieved by the following technical solution:
A kind of method that quick detection produces carbapenem enzyme bacterial strain, includes the following steps:
(1) Imipenem drug sensitive test paper is placed in the centrifuge tube containing sterile water, is vortexed to centrifuge tube, make the scraps of paper
In medicaments uniformity be diffused in sterile water；Or Imipenem is dissolved in containing in sterile water, and sterilizing filter paper piece is added；
(2) the detected bacterium lawn for scraping an oese adds in the centrifuge tube that step (1) obtains is incubated at least 30 points jointly
Clock；
(3) acid-base indicator is added in MH agar medium after sterilization, it is cooling, and the production of culture to logarithmic phase is added
Acetic bacterial mixes well, and the MH agar medium containing acid-base indicator and acidogenic bactria is prepared；
(4) the MH agar medium containing acid-base indicator and acidogenic bactria is poured into sterile petri dish,
Naturally dry makes agar solidification, wherein the color of the MH agar medium at this time containing acidogenic bactria and acid-base indicator is soda acid
The alkaline color of indicator；
(5) Imipenem drug sensitive test paper or sterilizing filter paper piece after taking out step (2) and the incubation of detected bacterium lawn, and paste
In the sterile petri dish for the MH agar medium containing acid-base indicator and acidogenic bactria that step (4) obtains, incubation 3.5~
4.5 hour；
(6) result interpretation:
The color change interval of the acid-base indicator is pH 4.2~8, is sentenced by the color change of acid-base indicator
Determine whether detected bacterium is to produce carbapenem enzyme bacterial strain；
If the MH agar medium around Imipenem drug sensitive test paper becomes the sour color of acid-base indicator, determine that this is to be detected
Bacterium is to produce carbapenem enzyme bacterial strain；
If the MH agar medium around Imipenem drug sensitive test paper is non-discolouring, and remainder MH agar medium becomes
The sour color of acid-base indicator determines that the detected bacterium is not produce carbapenem enzyme bacterial strain.
The alkaline color refers to the color that acid-base indicator is presented under alkaline environment, and the sour color refers to that acid-base indicator exists
The color presented under acidic environment.
When the operation of step (1) is that Imipenem is dissolved in containing in sterile water, the final concentration of the Imipenem
Preferably 0.02~0.05 μ g/ μ L；The sterilizing filter paper piece is preferably the sterilizing filter paper piece of 6~6.35mm of diameter.
It is specific to grasp when the operation of step (1) is that Imipenem drug sensitive test paper is placed in the centrifuge tube containing sterile water
Make preferred are as follows: a piece of drug sensitive test paper for containing 10 μ g Imipenems is placed in containing in 500 μ L sterile water centrifuge tubes, to centrifuge tube whirlpool
Rotation 10~20 seconds.
The time of the vortex is more preferably 15 seconds.
The centrifuge tube is preferably the centrifuge tube of 2mL.
The Imipenem drug sensitive test paper can be prepared by commercially available approach or voluntarily.
The time being incubated for jointly described in step (2) is preferably 30 minutes.
Acidogenic bactria described in step (3) is the bacterium that can be produced acid and acid-base indicator is made to change colour, including stearothermophilus
Bacillus, bacillus subtilis etc..
Culture described in step (3) to logarithmic phase is preferably to cultivate to the concentration of acidogenic bactria to reach 108CFU/mL makes
It is higher to obtain acidogenic bactria activity.
The end of acidogenic bactria is dense in MH agar medium containing acid-base indicator and acidogenic bactria described in step (3)
Degree preferably 105~107CFU/mL；Further preferably 107CFU/mL。
The final concentration of acid-base indicator described in step (3) is preferably 0.01~0.02g/L；Further preferably
0.018g/L。
Acid-base indicator described in step (3) include methyl red, chlorophenol red, paranitrophenol, bromocresol purple, dibromophenolphthalein,
At least one of Bromothymol blue (bromothymol blue), turmeric, dimethyl diaminophenazine chloride；Further preferably bromocresol purple.
Cooling described in step (3) is preferably cooled to 40~45 DEG C；Further preferably 40 DEG C.
Mixing described in step (3), which is preferably vibrated, to be mixed.
Sterilizing described in step (3) is preferably high pressure sterilization.
The time of naturally dry described in step (4) is preferably at least 25 minutes；Further preferably 25~35 minutes；
More preferably 30 minutes.
The time of incubation described in step (5) is more preferably 3.5 hours.
The temperature of incubation described in step (5) is preferably the optimum growth temperature of acidogenic bactria.
When the acidogenic bactria is bacillus stearothermophilus, the temperature of incubation described in step (5) is preferably 60
±5℃。
The operation of taking-up described in step (5) preferably uses aseptic inoculation ring to take out.
A kind of quickly detection production carbapenem enzyme bacterial strain kit, is incubated altogether by detected bacterium and Imipenem drug sensitive test paper
It after educating, is added into the culture medium containing acidogenic bactria and acid-base indicator, judges acid-base indicator whether because acidogenic bactria produces
Acid and change colour, and then determine whether bacterial strain to be detected is production carbapenem enzyme bacterial strain；The kit includes Imipenem medicine
The quick scraps of paper, acidogenic bactria, acid-base indicator and acidogenic bactria culture medium.
The acid-base indicator includes methyl red, chlorophenol red, paranitrophenol, bromocresol purple, dibromophenolphthalein, bromine Thymol
At least one of blue (bromothymol blue), turmeric, dimethyl diaminophenazine chloride；Further preferably bromocresol purple.
The culture medium of the acidogenic bactria is preferably MH agar medium.
The application of the detection method or kit in the bacterial strain that detection produces carbapenem enzyme.
The bacterial strain is preferably Escherichia coli or pseudomonas aeruginosa.
The present invention has the following advantages and effects with respect to the prior art:
1. the present invention provides a kind of rapid detection methods of completely new production carbapenem enzyme bacterial strain
It is in contrast to the prior art, the method that quick detection disclosed in this invention generates the bacterium of carbapenem enzyme
It include: that Imipenem drug sensitive test paper is placed in the centrifuge tube containing sterile water, and is vortexed to centrifuge tube；Scrape an oese
Detected bacterium lawn be placed in centrifuge tube and be incubated for jointly；Acid-base indicator is added in autoclaved MH agar medium
(such as bromocresol purple), it is cooling, and the acidogenic bactria of culture to logarithmic phase is added, it fullys shake to mixing；It will be indicated containing soda acid
The MH agar medium of agent (such as bromocresol purple) and acidogenic bactria is poured into sterile petri dish, naturally dry, wherein containing at this time
Alkaline color (such as the bromine cresols of acid-base indicator is presented in the MH agar medium of acidogenic bactria and acid-base indicator (such as bromocresol purple)
Purple purple)；Imipenem drug sensitive test paper after being incubated for detected bacterium lawn is pulled out with aseptic inoculation ring, and is attached to sterile
On MH agar medium containing acid-base indicator (such as bromocresol purple) and acidogenic bactria in culture dish, it is incubated for 3.5~4.5 hours
(when acidogenic bactria is bacillus stearothermophilus, being preferably incubated at 60 DEG C)；If the MH around Imipenem drug sensitive test paper
Agar medium becomes the sour color (for example, when acid-base indicator is bromocresol purple, becoming yellow) of acid-base indicator, and determining should
Detected bacterium is to generate the bacterium of carbapenem enzyme.
2. detection method of the invention is easy to operate, quick, accuracy and specificity are high
It is compared to traditional CIM method, the detection time of the method disclosed in the present was by original 18~24 hours
It greatly shortens to 4~5 hours, meanwhile, the accuracy and specificity of detection method of the invention are suitable with CIM method.
Detailed description of the invention
Fig. 1 is the flow diagram for the method that the quick detection that embodiment 1 constructs generates carbapenem enzyme bacterium.
Fig. 2 is negative control group in embodiment 2 (negative sign "-" in figure), positive controls (being positive sign "+" in figure), experiment
The testing result photo figure of group (number 2).
Fig. 3 is negative control group in embodiment 3 (negative sign "-" in figure), positive controls (being positive sign "+" in figure), experiment
The testing result photo figure of group (number 3).
Specific embodiment
Present invention will now be described in further detail with reference to the embodiments and the accompanying drawings, but embodiments of the present invention are unlimited
In this.
The building of 1 detection method of embodiment
Fig. 1 is the flow diagram of the method for the bacterium that quickly detection of the invention generates carbapenem enzyme.This method includes
Following steps:
Step S101: Imipenem drug sensitive test paper is placed in the centrifuge tube containing sterile water, and carries out whirlpool to centrifuge tube
Rotation.
Imipenem drug sensitive test paper is the drug sensitive test paper of commercialization, and Imipenem drug sensitive test paper is placed on containing sterile water
Centrifuge tube be vortexed, mainly the medicaments uniformity in the scraps of paper is diffused in sterile water.
In the present embodiment, Imipenem drug sensitive test paper is placed in the centrifuge tube containing sterile water, and to centrifuge tube into
The step of row is vortexed includes: that the Imipenem drug sensitive test paper of a 10 μ g is placed in 2 milliliters of centrifugations containing 500 microlitres of sterile waters
Guan Zhong, and be vortexed 10~20 seconds to centrifuge tube.Imipenem drug sensitive test paper is placed on centrifuge tube inside vortex 10~20 seconds,
The medicaments uniformity in the scraps of paper is enabled to spread.
Preferably, it is vortexed 15 seconds to the centrifuge tube for being placed with Imipenem drug sensitive test paper.
Step S102: the detected bacterium lawn for scraping an oese is placed in centrifuge tube described in step S101 incubates jointly
It educates.
In the present embodiment, the detected bacterium lawn for scraping an oese is placed on the step of being incubated for jointly in centrifuge tube packet
Include: the detected bacterium lawn for scraping an oese is placed in centrifuge tube to be incubated at least 30 minutes jointly, mainly allows detected bacterium
The carbapenem enzyme of generation is sufficiently acted on Imipenem drug.
Preferably, detected bacterium lawn is placed in centrifuge tube and is incubated for jointly 30 minutes.
Step S103: being added acid-base indicator (such as bromocresol purple) in MH agar medium after autoclaving, cooling,
And the acidogenic bactria of culture to logarithmic phase is added, it fullys shake to mixing.
In the present embodiment, acidogenic bactria includes bacillus stearothermophilus, and bacillus stearothermophilus can produce acid, thermophilic
Acid caused by hot Bacillus stearothermophilus can make acid-base indicator discoloration, and (such as bromocresol purple itself is purple, stearothermophilus
Acid caused by bacillus can be such that bromocresol purple turns yellow).Certainly, in some embodiments, acidogenic bactria further includes
Bacillus subtilis, bacillus subtilis, which can also produce acid, makes acid-base indicator change colour.
It is worth noting that, when bacillus stearothermophilus and acid-base indicator (such as bromocresol purple) collective effect, and
Acid-base indicator will not be made to change colour (for example, turning yellow bromocresol purple) at once, bacillus stearothermophilus needs in temperature
Degree environment is incubated in the state of being 60 ± 5 DEG C and can just generate within 3.5~4.5 hours enough acid acid-base indicator is changed colour.
Further, the Imipenem drug of Imipenem drug sensitive test paper is able to suppress bacillus stearothermophilus growth,
Cause it that cannot produce acid.
In the present embodiment, acid-base indicator (such as bromocresol purple) is added in autoclaved MH agar medium, it is cold
But, and the quick acidogenic bactria cultivated to logarithmic phase is added, fulling shake to the step of mixing includes: in autoclaved MH fine jade
The acid-base indicator (such as bromocresol purple) of final concentration of 0.01~0.02g/L is added in rouge culture medium, is cooled to 40~45 DEG C, adds
The acidogenic bactria for entering culture to logarithmic phase, makes final concentration of the 10 of acidogenic bactria5~107CFU/mL fullys shake to mixing.
Preferably, the bromocresol purple of final concentration of 0.018g/L is added in autoclaved agar medium.
Preferably, 40 DEG C are cooled to, and the acidogenic bactria of culture to logarithmic phase is added, makes the final concentration of of acidogenic bactria
107CFU/mL。
Step S104: the MH agar medium containing acid-base indicator (such as bromocresol purple) and acidogenic bactria is placed in nothing
In bacterium culture dish, naturally dry.In this step, contain the MH of acid-base indicator (such as bromocresol purple) and quick acidogenic bactria at this time
Agar medium is the alkaline color of acid-base indicator.
In the present embodiment, the MH agar medium containing acid-base indicator (such as bromocresol purple) and acidogenic bactria is poured into
In sterile petri dish, the step of naturally dry includes: the MH that will contain acid-base indicator (such as bromocresol purple) and acidogenic bactria
Agar medium is poured into sterile petri dish, and naturally dry 30 minutes or more.
Preferably, the MH agar medium containing acid-base indicator (such as bromocresol purple) and acidogenic bactria is placed in sterile
In culture dish, naturally dry 30 minutes.
Step S105: the Imipenem drug sensitive test paper after being incubated for detected bacterium lawn is pulled out with aseptic inoculation ring, and
It is attached on the MH agar medium containing acid-base indicator (such as bromocresol purple) and acidogenic bactria in sterile petri dish, 60 ± 5 DEG C
It is incubated for 3.5~4.5 hours.
Preferably, it is attached to the agar culture for containing acid-base indicator (such as bromocresol purple) and acidogenic bactria in sterile petri dish
On base, and it is incubated for 3.5 hours in the environment that temperature is 60 DEG C.It is worth noting that, 60 DEG C of temperature is stearothermophilus bud
The optimum growth temperature of spore bacillus, and bacillus stearothermophilus is in room temperature or 37 DEG C do not grow.
Step S106: if the MH agar medium around Imipenem drug sensitive test paper becomes the sour color (example of acid-base indicator
Such as, bromocresol purple becomes yellow), determine the detected bacterium for the bacterium of generation carbapenem enzyme.
The drug it is worth noting that, bacterium for producing carbapenem enzyme can degrade in Imipenem drug sensitive test paper.
It, can be by Imipenem susceptibility after the incubation of step S102 if detected bacterium is to produce the bacterium of carbapenem enzyme
Drug degradation in the scraps of paper, therefore Imipenem drug sensitive test paper is attached to containing acid-base indicator (such as bromocresol purple) and thermophilic rouge
Bacillus stearothermophilus will not be inhibited to grow when on the MH agar medium of fat bacillus, it will not be inhibited to produce acid, then
MH agar medium containing acid-base indicator (such as bromocresol purple) and bacillus stearothermophilus can change colour and culture medium is caused to become
The sour color (for example, bromocresol purple becomes yellow) of acid-base indicator, therefore do not have colour developing in MH agar medium and iris out now.
Step S107: if the MH agar medium around Imipenem drug sensitive test paper is non-discolouring, and remainder MH agar
Culture medium becomes the sour color (for example, bromocresol purple becomes yellow) of acid-base indicator, determines that the detected bacterium is not generate carbon blueness
The bacterium of mould alkene enzyme.
MH agar medium around Imipenem drug sensitive test paper keeps the alkaline color of acid-base indicator (for example, bromine cresols
Purple keeps purple), and remainder MH agar medium becomes the sour color of acid-base indicator (for example, bromocresol purple becomes yellow
Color), then occur the colour developing of the alkaline color (for example, being purple when bromocresol purple) of acid-base indicator around Imipenem drug sensitive test paper
Circle.
If detected bacterium is not produce the bacterium of carbapenem enzyme, the drug in Imipenem drug sensitive test paper will not be dropped
Solution, therefore the Drug inhibition bacillus stearothermophilus in Imipenem drug sensitive test paper produces acid to make the color of acid-base indicator
It is constant, i.e., it can be because the presence of Imipenem drug makes containing acid-base indicator in a certain range around Imipenem drug sensitive test paper
The MH agar medium of (such as bromocresol purple) and bacillus stearothermophilus is non-discolouring, this range is outer because not by Imipenem medicine
The influence of object, then the MH agar medium containing acid-base indicator (such as bromocresol purple) and bacillus stearothermophilus can change colour and cause
Culture medium becomes the sour color (for example, bromocresol purple becomes yellow) of acid-base indicator, to make occur soda acid on agar medium
The colour developing circle (for example, colour developing circle that bromocresol purple is purple) of the alkaline color of indicator, and alkaline color (such as the purple of bromocresol purple)
Being then sour color (such as yellow when bromocresol purple) outside colour developing circle.
It is compared to traditional CIM method, the holding one of the accuracy and specificity and CIM method of method of the invention
It causes, detection time of the invention was foreshortened to 4~5 hours by original 18~24 hours, and can directly pass through pH indicator
It develops the color, it is easier to result interpretation.
Embodiment 2
(1) negative control group (negative sign "-" position in Fig. 2) is set, positive controls (positive sign "+" position in Fig. 2
Set), an experimental group (No. 2 positions in Fig. 2), wherein negative control group is one plant by large intestine of the PCR identification without producing enzyme gene
Bacillus reference culture ATCC25922 (is purchased from Chinese microorganism strain collection), and positive controls are one plant of kerekou pneumonia primary
Bacterium ATCC BAA-1705 (is purchased from Chinese microorganism strain collection), and experimental group is one plant of Klebsiella Pneumoniae
ATCCBAA2146 (is purchased from Chinese microorganism strain collection).
By CIM method (method reference literature Zwaluw K V D, Haan A D, Pluister G N, et al.The
Carbapenem Inactivation Method(CIM),a Simple and Low-Cost Alternative for the
Carba NP Test to Assess Phenotypic Carbapenemase Activity in Gram-Negative
Rods [J] .Plos One, 2015,10 (3): e0123690.) obtain experimental result: above-mentioned negative control group is not produce carbon mould
The bacterium of alkene enzyme, positive controls are to produce the bacterium of carbapenem enzyme, and experimental group is to produce the bacterium of carbapenem enzyme.
(2) a piece of Imipenem drug sensitive test paper (every contains 10 μ g Imipenems, is purchased from OXID company) is placed in containing 500
2 milliliters of centrifuge tube mesoscale eddies 15 seconds of microlitre sterile water, production three pipe, by positive controls, negative control group, experimental group
Lawn is scraped an oese and is respectively placed in three centrifuge tubes to be incubated for 30 minutes jointly.
(3) it is added eventually in autoclaved MH agar medium (buying from Huankai Microbes Tech Co., Ltd., Guangdong)
The bacillus stearothermophilus reference culture of culture to logarithmic phase is added in the bromocresol purple of concentration 0.018g/L when being cooled to 40 DEG C
ATCC7953 (be purchased from Chinese microorganism strain collection), make its final concentration of 107CFU/mL is poured into nothing after mixing well
In bacterium culture dish, naturally dry 30 minutes, the MH agar culture containing bromocresol purple and bacillus stearothermophilus is prepared
Base.
(4) it is attached to system respectively after pulling the Imipenem drug sensitive test paper after being incubated for 30 minutes with lawn with aseptic inoculation ring out
On the good MH agar medium containing bromocresol purple and bacillus stearothermophilus, 3.5 are incubated in 60 DEG C of constant incubators
Its colour developing circle size is observed after hour.
(5) PCR is identified
Carbapenem enzyme correlation drug resistant gene amplimer is synthesized by Qing Kexin industry Bioisystech Co., Ltd, see Table 1 for details.
1 carbapenem enzyme correlation drug resistant gene amplimer of table
Note: F indicates upstream primer；R indicates trip primer
The amplification of target gene: 25 μ L PCR reaction systems are prepared using rTaq enzyme, carbapenem enzyme drug resistant gene is carried out
Amplification, it is shown that reaction system such as table 2.
2 rTaq enzyme PCR reaction system of table
PCR response parameter is shown in Table 3.
3 carbapenem enzyme drug resistant gene PCR of table detects reaction condition
Experimental result as shown in Fig. 2, the Imipenem drug sensitive test paper of positive controls be attached to make contain bromocresol purple
With do not occur the colour developing circle of purple on the MH agar medium of bacillus stearothermophilus, i.e., the training of MH agar containing bromocresol purple
Feeding base can become yellow；The Imipenem drug sensitive test paper of negative control group be attached to make containing bromocresol purple and stearothermophilus bud
Occur the colour developing circle of purple on the MH agar medium of spore bacillus, i.e., the colour developing of purple occurs around Imipenem drug sensitive test paper
Circle；The Imipenem drug sensitive test paper of experimental group be attached to make containing the MH agar of bromocresol purple and bacillus stearothermophilus train
It supports without there is colour developing circle on base, i.e. MH agar medium around Imipenem drug sensitive test paper is yellow.
Pass through above-mentioned experiment, it was demonstrated that the bacterial strain of experimental group is the bacterium for producing carbapenem enzyme, contains producing enzyme by PCR identification
Gene is consistent with CIM experimental result.
Embodiment 3
(1) negative control group (negative sign "-" position in Fig. 3) is set, positive controls (positive sign "+" position in Fig. 3
Set), an experimental group (No. 3 positions in Fig. 3), wherein negative control group is one plant by large intestine of the PCR identification without producing enzyme gene
Bacillus ATCC25922 (is purchased from Chinese microorganism strain collection), and positive controls are one plant of Klebsiella Pneumoniae
ATCCBAA-1705 (is purchased from Chinese microorganism strain collection), and experimental group is one plant of Klebsiella Pneumoniae ATCCBAA-1706
(being purchased from Chinese microorganism strain collection).
Obtain experimental result by CIM method (with embodiment 2): above-mentioned negative control group is not produce the thin of carbapenem enzyme
Bacterium, positive controls are to produce the bacterium of carbapenem enzyme, and experimental group is not produce the bacterium of carbapenem enzyme.
(2) a piece of Imipenem drug sensitive test paper (every contains 10 μ g Imipenems) is placed in 2 containing 500 microlitres of sterile waters
Milliliter centrifuge tube mesoscale eddies 15 seconds, three pipe of production, scrapes an inoculation for the lawn of positive controls, negative control group, experimental group
Ring is respectively placed in three centrifuge tubes and is incubated for jointly 30 minutes.
(3) bromocresol purple of final concentration 0.018g/L is added in autoclaved MH agar medium, is cooled to 40 DEG C
When be added culture to logarithmic phase bacillus stearothermophilus reference culture ATCC7953, make its final concentration of 107CFU/mL fills
Divide after mixing and be poured into sterile petri dish, naturally dry 30 minutes, is prepared containing bromocresol purple and stearothermophilus gemma
The MH agar medium of bacillus.
(4) it is attached to system respectively after pulling the Imipenem drug sensitive test paper after being incubated for 30 minutes with lawn with aseptic inoculation ring out
On the good MH agar medium containing bromocresol purple and bacillus stearothermophilus, 3.5 are incubated in 60 DEG C of constant incubators
Its colour developing circle size is observed after hour.
Experimental result as shown in figure 3, the Imipenem drug sensitive test paper of positive controls be attached to make contain bromocresol purple
With do not occur the colour developing circle of purple on the MH agar medium of bacillus stearothermophilus, i.e., the training of MH agar containing bromocresol purple
Feeding base can become yellow；The Imipenem drug sensitive test paper of negative control group be attached to make containing bromocresol purple and stearothermophilus bud
Occur the colour developing circle of purple on the MH agar medium of spore bacillus, i.e., the colour developing of purple occurs around Imipenem drug sensitive test paper
Circle；The Imipenem drug sensitive test paper of experimental group be attached to make containing the MH agar of bromocresol purple and bacillus stearothermophilus train
It supports on base and the colour developing circle of purple occurs, i.e., occur the colour developing circle of purple around Imipenem drug sensitive test paper.
Pass through above-mentioned experiment, it was demonstrated that experimental group bacterial strain is the bacterium for not producing carbapenem enzyme, and (PCR is identified by PCR identification
Method is with embodiment 2) Klebsiella Pneumoniae without producing enzyme gene, it is consistent with CIM result.
4 clinical application of embodiment test
In order to further study the specificity and accuracy of detection method of the invention, the present embodiment is to from clinical sample
(bacterial strain is supported in acquisition in 2014~2018 years from Shandong, Guangdong, Guangxi by inventor team for isolated 92 plants of bacterial strains
Field is grown, is separated and is saved by pharmacology teaching and research room, College of Veterinary Medicine, South China Agricultural University) carry out detection research, wherein it include big
The uncommon bacterium of intestines angstrom, Klebsiella Pneumoniae, not formula citric acid fungus, enterobacter cloacae etc..
In this 92 plants of clinical strains, PCR result positive strain is 70 plants, and PCR result feminine gender strain is 22 plants.
Be respectively adopted Carbapenems antibacterials inactivation test (carbapenem inactivation method,
CIM, operate with embodiment 2) and embodiment 1 building detection method (CIMG.stearothermophilus) detected, testing result
As shown in table 4.
The accuracy of CIM is 98%, and specificity is 100%；CIMG.stearothermophilusAccuracy: 98%, specificity
100%.The detection method that the present invention constructs can correctly detect all bacterial strains, and no cross reaction is specific good.
4 clinical strains experimental result of table
The above embodiment is a preferred embodiment of the present invention, but embodiments of the present invention are not by above-described embodiment
Limitation, other any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present invention,
It should be equivalent substitute mode, be included within the scope of the present invention.
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| CN110117531A (en) * | 2019-06-13 | 2019-08-13 | 武汉市第四医院 | Drug-resistant phenotype quickly detects susceptibility plate and drug-resistant phenotype rapid detection method |
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