CN106701888A - Enzyme linked immunosorbent assay instrument testing method for penicillium marneffei mycelia-phase in-vitro susceptibility testing - Google Patents
Enzyme linked immunosorbent assay instrument testing method for penicillium marneffei mycelia-phase in-vitro susceptibility testing Download PDFInfo
- Publication number
- CN106701888A CN106701888A CN201611221144.5A CN201611221144A CN106701888A CN 106701888 A CN106701888 A CN 106701888A CN 201611221144 A CN201611221144 A CN 201611221144A CN 106701888 A CN106701888 A CN 106701888A
- Authority
- CN
- China
- Prior art keywords
- penicillium marneffei
- eliasa
- drug sensitive
- sensitive test
- linked immunosorbent
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- 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
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Analytical Chemistry (AREA)
- Toxicology (AREA)
- Immunology (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Biotechnology (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The invention discloses an enzyme linked immunosorbent assay instrument testing method for penicillium marneffei mycelia-phase in-vitro susceptibility testing. The light transmittance of a bacterium-containing diluent with an antibiotic medicinal liquid is tested by using an enzyme linked immunosorbent assay instrument in the penicillium marneffei mycelia-phase in-vitro susceptibility testing process. The densities are different in different bacterium growth situations, the light transmittances are also different, results are judged by using the enzyme linked immunosorbent assay instrument, apparent phenomena can be converted into values, and the operation can be relatively convenient and accurate; secondly, as an enzyme linked immunosorbent assay instrument pore plate is used, material consumption can be reduced; only a solution less than 1ml is needed in each pore, so that the amount of medicines is small, and waste can be greatly reduced; in addition, penicillium marneffei has infectivity, so that the pollution property can be further reduced because of the small use amount. Finally, normal growth of the penicillium marneffei can be not affected by the enzyme linked immunosorbent assay instrument, the operation is simple, convenient and rapid, and precise in-vitro susceptibility data results can be obtained.
Description
Technical field
The invention belongs to penicillium Marneffei detection technique field, more particularly to a kind of penicillium Marneffei Hyphal form
The ELIASA determination method of In vitro chemo-drug sensitive test.
Background technology
Infection of penicillium Marneffei is to cause south China area especially Guangxi, In Guangdong Province AIDS patient death
Main infection factor.Penicillium Marneffei can cause fungi deep infection when body immunity is low, and invasion and attack whole body is more
Organ, if cannot correctly treat in time, can cause death.At present, the primary treatment medicine of penicilliosis marneffei is
Antibiotic, amphotericin B (AmB), Fluconazole, Flucytosine etc. are the common drugs for treating penicilliosis marneffei.
Penicillium Marneffei is the only pathogenic bacteria in temperature biphasic or bipolar type in Penicillium notatum, is in bacterium when temperature is 25 DEG C
Silk state, for yeast state and with infectiousness at 37 DEG C.The In vitro chemo-drug sensitive test Main Basiss CLSI M38- of penicillium Marneffei
A2Method operated and result judgement, but because it is using visually carrying out result judgement, so its break is difficult to judge,
Individual judges that difference is also difficult to obtain accurate result, and this all brings certain error to experimental result.Heilongjiang Province is wild
Shi Luyi, Zhang Hanyuan of Institute of Botany et al. be in vitro on the basis of drug sensitive experiment micro-dilution method, using ELIASA to knot
Fruit carries out interpretation, largely avoided the deficiency in reading, but its method is only applied to Escherichia coli, and other bacterial strains do not appear in the newspapers
Road.Zhou Zixiong of Chinese medicine institute of Guangdong Pharmaceutical University et al. also determines antibacterial material bacteriostatic activity to ELIASA and has carried out research and side
The foundation of method, but its method is only applied to staphylococcus aureus.
The content of the invention
The technical problem to be solved in the present invention is to provide a kind of simple and efficient, accurately and reliably penicillium Marneffei mycelia
The ELIASA determination method of phase In vitro chemo-drug sensitive test.
In order to solve the above technical problems, the present invention uses following technical scheme：
The ELIASA determination method of penicillium Marneffei Hyphal form In vitro chemo-drug sensitive test, it is luxuriant and rich with fragrance to Marni blue or green using ELIASA
The light transmittance containing bacterium dilution of antibiotic liquid is added to be measured during mould In vitro chemo-drug sensitive test.
The ELIASA determination method of above-mentioned penicillium Marneffei Hyphal form In vitro chemo-drug sensitive test, comprises the following steps：
<1>Configuration antibiotic liquid, is dissolved using dimethylsulfoxide solvent and is made antibiotic liquid；
<2>Antibiotic liquid is diluted using RPMI RPMI-1640s, is then filtered using aseptic microporous barrier, it is standby；
<3>By penicillium Marneffei stroke spore in RPMI RPMI-1640s；
<4>Using the special orifice plate of ELIASA, antibiotic liquid is added in orifice plate, be subsequently adding dilution containing bacterium, be placed in body
Outer drug sensitive experiment incubator；
<5>Using the light transmittance values after ELIASA detection addition antibiotic liquid 15min and after 3 days, and by computing formula
Calculate bacterium rate long.
Step<1>In by mass volume ratio 1: 1-10 using dimethylsulfoxide solvent dissolve be made antibiotic liquid.
Step<3>Middle penicillium Marneffei reaches 0.4-5 × 10 in nutrient solution miospore number4CFU/mL。
Step<4>It is middle to control In vitro chemo-drug sensitive test incubator temperature for 25 DEG C.
For the problem that traditional visual method identification penicillium Marneffei In vitro chemo-drug sensitive test result is present, inventor sets up
A kind of ELIASA determination method of penicillium Marneffei Hyphal form In vitro chemo-drug sensitive test, using ELIASA to Penicillium marneffei
The light transmittance containing bacterium dilution of antibiotic liquid is added to be measured during bacterium In vitro chemo-drug sensitive test.The method is anti-by control
Preparation, the regulation of the spore count of dilution containing bacterium and the setting of cultivation temperature of raw element liquid, make penicillium Marneffei normally give birth to
It is long.Cause density different because bacterium situation long is different, light transmittance is different, judges result using ELIASA, makes apparent
Phenomenon can change into numerical value, more facilitate accurate；Secondly, using ELIASA orifice plate, consumptive material is less, only needs to be less than in each hole
1ml bulk solutions, drug dosage is few, greatly reduces waste, and penicillium Marneffei has infectivity, using more on a small quantity
Reduce contaminative.Finally, the present invention does not interfere with the normal growth of penicillium Marneffei using ELIASA, simple and efficient, has
Help obtain accurate external susceptibility data result.
Brief description of the drawings
Fig. 1 be in penicillium Marneffei In vitro chemo-drug sensitive test bacterium rate long with antibiotic concentration variation diagram.
In figure：On concentration axle 1-12 represent 4 respectively, 3,2,1,0.5,0.25,0.125,0.062,0.031,0.016,
0.008、0.004μg/mL。
Specific embodiment
Embodiment 1
<1>Configuration antibiotic liquid, after precise ketoconazole 1.0mg, is made anti-using the dissolving of 2mL dimethylsulfoxide solvents
Raw element liquid；
<2>Antibiotic liquid is diluted to 4 μ g/mL using RPMI RPMI-1640s, then uses aseptic microporous barrier (0.22 μ
M) filtered, it is standby；
<3>
The purifying culture of penicillium Marneffei：Penicillium Marneffei freeze-dried powder is used into method of dilution butteron on plate, in PDA trainings
Support and carry out culture purified on base；
The preparation of spore liquid：Penicillium Marneffei spore after to be purified uses aseptic liquid relief after maturation long on flat board
Rifle pipette tips draw 100~500 μ L RPMI RPMI-1640s carried out on flat board roll collect spore, and by liquid collect to
It is stand-by in 10mL aseptic plastic pipes；
The cell count of spore liquid：Liquid in plastic tube containing spore is added into 10mL, after mixing, 1~5 μ L liquid is drawn
To blood counting chamber, cover slide and counted under the microscope；Make spore by adding spore or dilution nutrient solution
Quantitative range in a liquid is controlled in 0.4-5 × 104CFU/mL；
<4>Using special 96 orifice plate (flat) of ELIASA, added in orifice plate 1-12 holes certain volume antibiotic liquid (plus
Dose be not more than orifice plate maximum volume 1/2), be subsequently adding spore suspension make its final volume for 200 μ L (in 1-12 holes, ketone
The concentration of health azoles is respectively 2,1,0.5,0.25,0.125,0.062,0.031,0.016,0.008,0.004,0.002,0.001 μ
G/mL, while doing blank；) it is placed in In vitro chemo-drug sensitive test incubator；It is 25 DEG C to control In vitro chemo-drug sensitive test incubator temperature
(penicillium Marneffei Hyphal form growth temperature is 25 DEG C), makes its normal growth；
<5>Using the light transmittance values after ELIASA detection (wavelength is 540nm) addition antibiotic liquid 15min and after 3 days,
And by computing formula (bacterium value (%)=sample sets (OD long3d-OD15min)/blank group (OD3d-OD15min) × 100%) calculates bacterium long
Rate.
Influence of the ketoconazole concentration of table 1 to penicillium Marneffei bacterium rate long
As can be seen from the table, with the increase of antibiotic concentration, bacterium rate long is gradually decreased, that is, show antibiotic to Ma Er
Buddhist nun's phenanthrene Penicillium notatum generates suppression.
Embodiment 2
<1>Configuration antibiotic liquid, after precise amphotericin B 1.0mg, is dissolved using 2mL dimethylsulfoxide solvents and made
Into antibiotic liquid；
<2>Antibiotic liquid is diluted to 4 μ g/mL using RPMI RPMI-1640s, then uses aseptic microporous barrier (0.22 μ
M) filtered, it is standby；
<3>
The purifying culture of penicillium Marneffei：Penicillium Marneffei freeze-dried powder is used into method of dilution butteron on plate, in PDA trainings
Support and carry out culture purified on base；
The preparation of spore liquid：Penicillium Marneffei spore after to be purified uses aseptic liquid relief after maturation long on flat board
Rifle pipette tips draw 100~500 μ L RPMI RPMI-1640s carried out on flat board roll collect spore, and by liquid collect to
It is stand-by in 10mL aseptic plastic pipes；
The cell count of spore liquid：Liquid in plastic tube containing spore is added into 10mL, after mixing, 1~5 μ L liquid is drawn
To blood counting chamber, cover slide and counted under the microscope；Make spore by adding spore or dilution nutrient solution
Quantitative range in a liquid is controlled in 0.4-5 × 104CFU/mL；
<4>Using special 96 orifice plate (flat) of ELIASA, added in orifice plate 1-12 holes certain volume antibiotic liquid (plus
Dose be not more than orifice plate maximum volume 1/2), be subsequently adding spore suspension make its final volume for 200 μ L (in 1-12 holes, two
The concentration of property mycin B is respectively 2,1,0.5,0.25,0.125,0.062,0.031,0.016,0.008,0.004,0.002,
0.001 μ g/mL, while doing blank；) it is placed in In vitro chemo-drug sensitive test incubator；Control In vitro chemo-drug sensitive test incubator temperature
It is 25 DEG C (penicillium Marneffei Hyphal form growth temperature is 25 DEG C), makes its normal growth；
<5>Using the light transmittance values after ELIASA detection (wavelength is 540nm) addition antibiotic liquid 15min and after 3 days,
And by computing formula (bacterium value (%)=sample sets (OD long3d-OD15min)/blank group (OD3d-OD15min) × 100%) calculates bacterium long
Rate.
Influence of the amphotericin B concentration of table 2 to penicillium Marneffei bacterium rate long
Claims (5)
1. a kind of ELIASA determination method of penicillium Marneffei Hyphal form In vitro chemo-drug sensitive test, it is characterised in that use ELIASA
The light transmittance containing bacterium dilution to adding antibiotic liquid during penicillium Marneffei In vitro chemo-drug sensitive test is measured.
2. the ELIASA determination method of penicillium Marneffei Hyphal form In vitro chemo-drug sensitive test according to claim 1, it is special
Levy is to comprise the following steps：
<1>Configuration antibiotic liquid, is dissolved using dimethylsulfoxide solvent and is made antibiotic liquid；
<2>Antibiotic liquid is diluted using RPMI RPMI-1640s, is then filtered using aseptic microporous barrier, it is standby；
<3>By penicillium Marneffei stroke spore in RPMI RPMI-1640s；
<4>Using the special orifice plate of ELIASA, antibiotic liquid is added in orifice plate, be subsequently adding dilution containing bacterium, be placed in external medicine
Quick experiment incubator；
<5>Using the light transmittance values after ELIASA detection addition antibiotic liquid 15min and after 3 days, and calculated by computing formula
Bacterium rate long.
3. the ELIASA determination method of penicillium Marneffei Hyphal form In vitro chemo-drug sensitive test according to claim 2, it is special
It is step to levy<1>In by mass volume ratio 1: 1-10 using dimethylsulfoxide solvent dissolve be made antibiotic liquid.
4. the ELIASA determination method of penicillium Marneffei Hyphal form In vitro chemo-drug sensitive test according to claim 2, it is special
It is step to levy<3>Middle penicillium Marneffei reaches 0.4-5 × 10 in nutrient solution miospore number4CFU/mL。
5. the ELIASA determination method of penicillium Marneffei Hyphal form In vitro chemo-drug sensitive test according to claim 2, it is special
It is step to levy<4>It is middle to control In vitro chemo-drug sensitive test incubator temperature for 25 DEG C.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201611221144.5A CN106701888A (en) | 2016-12-26 | 2016-12-26 | Enzyme linked immunosorbent assay instrument testing method for penicillium marneffei mycelia-phase in-vitro susceptibility testing |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201611221144.5A CN106701888A (en) | 2016-12-26 | 2016-12-26 | Enzyme linked immunosorbent assay instrument testing method for penicillium marneffei mycelia-phase in-vitro susceptibility testing |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN106701888A true CN106701888A (en) | 2017-05-24 |
Family
ID=58896356
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201611221144.5A Pending CN106701888A (en) | 2016-12-26 | 2016-12-26 | Enzyme linked immunosorbent assay instrument testing method for penicillium marneffei mycelia-phase in-vitro susceptibility testing |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106701888A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110079462A (en) * | 2019-04-30 | 2019-08-02 | 广西医科大学第一附属医院 | A method of improving the luxuriant and rich with fragrance basket bacterium gene knockout conversion ratio of Marni |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101413876A (en) * | 2008-11-21 | 2009-04-22 | 天津科技大学 | Micropore plate biological detection method for rapidly detecting bacteriostatics activity |
| CN102759513A (en) * | 2012-07-25 | 2012-10-31 | 山东农业大学 | Method for quickly testing sensitivity of Botrytis cinerea to bactericide |
| RU2491348C2 (en) * | 2011-10-06 | 2013-08-27 | Государственное бюджетное образовательное учреждение высшего профессионального образования "Ростовский государственный медицинский университет" Министерства здравоохранения Российской Федерации (ГБОУ ВПО РостГМУ Минздрава России) | Method of determining minimal inhibitory concentration of antimicrobial preparation |
-
2016
- 2016-12-26 CN CN201611221144.5A patent/CN106701888A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101413876A (en) * | 2008-11-21 | 2009-04-22 | 天津科技大学 | Micropore plate biological detection method for rapidly detecting bacteriostatics activity |
| RU2491348C2 (en) * | 2011-10-06 | 2013-08-27 | Государственное бюджетное образовательное учреждение высшего профессионального образования "Ростовский государственный медицинский университет" Министерства здравоохранения Российской Федерации (ГБОУ ВПО РостГМУ Минздрава России) | Method of determining minimal inhibitory concentration of antimicrobial preparation |
| CN102759513A (en) * | 2012-07-25 | 2012-10-31 | 山东农业大学 | Method for quickly testing sensitivity of Botrytis cinerea to bactericide |
Non-Patent Citations (4)
| Title |
|---|
| 周子雄 等: "酶标仪快速测定抗菌物质抑菌活性方法的建立", 《微生物前沿》 * |
| 施路一 等: "应用微量酶标仪检测药物体外抑菌作用方法的建立及应用", 《黑龙江畜牧兽医》 * |
| 陆永绥 等: "《临床检验管理与技术规程（第二版）下册》", 31 December 2014, 浙江大学出版社 * |
| 韦高 等: "采用微量稀释法对双相马尔尼菲青霉菌体外药敏试验观察", 《临床皮肤科杂志》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110079462A (en) * | 2019-04-30 | 2019-08-02 | 广西医科大学第一附属医院 | A method of improving the luxuriant and rich with fragrance basket bacterium gene knockout conversion ratio of Marni |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN110846377B (en) | Drug sensitivity kit, preparation method thereof and bacterial drug sensitivity detection method | |
| CN107937453B (en) | Preparation method and antibacterial activity application of dichloro-substituted II-type halogenated polyketone compound | |
| CN106868094A (en) | The method for quick of antibiotic residue in a kind of raw milk | |
| CN106701888A (en) | Enzyme linked immunosorbent assay instrument testing method for penicillium marneffei mycelia-phase in-vitro susceptibility testing | |
| CN103173517B (en) | Microdetermination method for measuring minimum inhibitory concentration and minimal bactericidal concentration of cationic antimicrobial peptide | |
| CN105136505A (en) | Method for testing mould removing effect of air purification product | |
| CN108315384A (en) | The microbial limit tests of Nifuratel nystatin expandable vaginal soft capsule | |
| CN108926559A (en) | Wedelolactone is preparing the application in anti-candida albicans drug | |
| CN108498784A (en) | Application of the Radix pseudostellariae cyclic peptides B in preparing anti-candida albicans drug | |
| CN110951822A (en) | Bacterial drug sensitivity detection method suitable for drug sensitivity plate | |
| CN108785291A (en) | Application of the hypocrellin in the product for preparing anti-candida albicans | |
| CN104293661A (en) | Quick antibacterial test method and kit | |
| CN104188962A (en) | Application of magnolol and azole medicines to preparation of antifungal combined medicines | |
| CN105663126A (en) | Antifungal product combining ambroxol hydrochloride with fluconazole and application thereof | |
| CN101880705A (en) | Microbial screening model for screening anti-tumor medicament | |
| CN111956672A (en) | Extraction and application of cordyceps militaris antibacterial active substance | |
| CN105079007A (en) | Application of salicylic acid in preparing medicine for inhibiting staphylococcus xylosus biofilm | |
| CN108653277A (en) | Application of the kawain derivative in preparing anti-candida albicans drug | |
| CN106978467A (en) | The qualitative checking method of yeast and mold in a kind of eye sticker | |
| CN110403943A (en) | CC-115 is preparing application and its pharmaceutical composition in antifungal drug | |
| CN108578400A (en) | Application of the nevadensin in preparing anti-candida albicans drug | |
| CN108743575A (en) | Application of the butyl p-hydroxybenzoate in preparing anti-candida albicans drug | |
| CN106729610A (en) | Purposes of the N15 polypeptides in staphylococcus aureus inhibitor is suppressed | |
| CN107412227A (en) | Application and antifungal composition of a kind of anthraquinone analog compound in antifungal drug is prepared | |
| CN108836976B (en) | Application of ginsenoside RG5 in preparation of anti-Candida albicans medicine |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20170524 |