CN101851603B - Mycoplasma clearing reagent and application thereof - Google Patents
Mycoplasma clearing reagent and application thereof Download PDFInfo
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- CN101851603B CN101851603B CN2010101480281A CN201010148028A CN101851603B CN 101851603 B CN101851603 B CN 101851603B CN 2010101480281 A CN2010101480281 A CN 2010101480281A CN 201010148028 A CN201010148028 A CN 201010148028A CN 101851603 B CN101851603 B CN 101851603B
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- 241000204031 Mycoplasma Species 0.000 title claims abstract description 59
- 239000003153 chemical reaction reagent Substances 0.000 title claims abstract description 30
- 241000204003 Mycoplasmatales Species 0.000 claims abstract description 13
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- 239000012530 fluid Substances 0.000 claims description 16
- 238000000034 method Methods 0.000 claims description 10
- 238000004113 cell culture Methods 0.000 claims description 9
- 238000012258 culturing Methods 0.000 claims description 5
- 239000001963 growth medium Substances 0.000 claims description 3
- 239000006143 cell culture medium Substances 0.000 claims description 2
- 230000010261 cell growth Effects 0.000 claims 1
- 239000004098 Tetracycline Substances 0.000 abstract description 9
- 239000003120 macrolide antibiotic agent Substances 0.000 abstract description 9
- 235000019364 tetracycline Nutrition 0.000 abstract description 9
- 150000003522 tetracyclines Chemical class 0.000 abstract description 9
- 239000003242 anti bacterial agent Substances 0.000 abstract description 8
- 229940088710 antibiotic agent Drugs 0.000 abstract description 8
- 229940040944 tetracyclines Drugs 0.000 abstract description 8
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- 239000002245 particle Substances 0.000 abstract description 4
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- AGOYDEPGAOXOCK-KCBOHYOISA-N clarithromycin Chemical compound O([C@@H]1[C@@H](C)C(=O)O[C@@H]([C@@]([C@H](O)[C@@H](C)C(=O)[C@H](C)C[C@](C)([C@H](O[C@H]2[C@@H]([C@H](C[C@@H](C)O2)N(C)C)O)[C@H]1C)OC)(C)O)CC)[C@H]1C[C@@](C)(OC)[C@@H](O)[C@H](C)O1 AGOYDEPGAOXOCK-KCBOHYOISA-N 0.000 description 2
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- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention relates to a mycoplasma clearing reagent and application thereof. The mycoplasma clearing reagent of the invention comprises the following components in percentage by weight: 1.3 to 1.5 percent of fluoroquinolones antibiotics, 0.3 to 0.5 percent of tetracyclines derivatives, 0.1 to 0.3 percent of macrolides antibiotics and 97.7 to 98.3 percent of buffer solution, can most effectively inhibit and kill mycoplasmas, and has a short period, namely, all the mycoplasmas can be killed in only one week without influencing the metabolism of cells per se; and the treated cells have smooth surfaces, substantially have no black particles and are difficult to be re-infected by the mycoplasmas.
Description
Technical field
The present invention relates to mycoplasma clearing reagent and application thereof.
Background technology
Mycoplasma be the most common in the cell cultures, be difficult for being discovered the pollutent with the interference experiment result, have approximately in 30.3%～50.5% the cell culture mycoplasma contamination arranged.The source of mycoplasma contamination comprises in the culturing cell: some products of employed animal-origin such as serum or by the cell strain system of mycoplasma contamination.The concentration of mycoplasma can reach 107/ml in the cell culture supernatant, and cultured cells seems very normal, under visual inspection or ordinary optical microscope, observes, and contaminated cell can not have considerable change.Because competition nutrition and the deleterious meta-bolites of mycoplasma, but the physiologically active of mycoplasma contamination remarkably influenced culturing cell descends the verity of result of study and safety greatly.
Most mycoplasmas are suitable for existence (pH7.6-8.0) under the inclined to one side alkali condition, and are poor to sour tolerance, responsive to ratio of specific heat, insensitive to general microbiotic.The most outstanding constitutional features of mycoplasma is not have cell walls, and is insensitive fully like beta-lactam, vancomyein etc. to acting on the biosynthetic microbiotic of cell walls, to polymyxin, the general resistance of sulfonamide.The tool of mycoplasma is suppressed active, the microbiotic quinolones of clinical interference albumen synthetic antibiotic tetracycline class commonly used, Macrolide and prevention dna replication dna; Aminoglycoside, paraxin have less restraining effect; Di-terpene class-Tiamulin, its activity is superior to other microbiotic; Antitumor type of ametycin, dactinomycin also have outstanding restraining effect.But the laboratory cell cultures is different from clinical application, and in the cell cultures, mycoplasma is easy to repeated infection, and the important cells strain for testing is necessary to remove as early as possible mycoplasma.
Summary of the invention
The object of the invention is a kind of mycoplasma clearing reagent of exploitation, can remove mycoplasma effectively, does not influence the metabolism of cell itself.
Mycoplasma clearing reagent of the present invention comprises following components in weight percentage:
Fluoroquinolone antibiotics 1.3-1.5%
Tetracyclines verivate 0.3-0.5%
Macrolide antibiotics 0.1-0.3%
Damping fluid 97.7-98.3%.
Preferable, said fluoroquinolone antibiotics is selected from one or more in Moxifloxacin, PD 127391 and the SB 265805, best, said fluoroquinolone antibiotics is a SB 265805.
Preferable, said tetracyclines verivate is selected from one or more in vibra-, Minocycline HCl, rolitetracycline, the WAY-GAR 936, best, said tetracyclines verivate is a Minocycline HCl.
Preferable, said macrolide antibiotics is selected from one or more in Azythromycin, clarithromycin, the Roxithromycin, best, said macrolide antibiotics is an Azythromycin.
Preferably, said damping fluid comprises following components in weight percentage:
NaOH 0.2-0.6％
NaCl 0.9-1％
Glucose 5-8%
H
2The O surplus
Mycoplasma clearing reagent of the present invention is the yellow liquid shape at normal temperatures, to light and thermo-responsive, uses with preserve must lucifuge, avoids multigelation, melts the back if be normal phenomenon during crystallization, shakes memotron to crystallization and disappears and can normally use.Mycoplasma clearing reagent of the present invention uses in a short time needs lucifuge to preserve in 4 ℃ of refrigerators; Its highest tiring can be kept 2-3 week under this temperature; Prolonged preservation is used if desired needs lucifuge frozen in-20 ℃ of refrigerators, and its highest tiring can be kept more than 1 year under this temperature.
The present invention also further discloses the purposes that mycoplasma clearing reagent of the present invention is used to remove mycoplasma and the growth of prevention mycoplasma, the purposes of especially removing and preventing the mycoplasma in the cell cultures.
Mycoplasma clearing reagent of the present invention is when being used for removing the mycoplasma of cell cultures; Only need it is added in the unparalleled anti-cell culture medium; When going down to posterity subsequently, use this culture medium culturing; Continue a week can remove mycoplasma basically, suitably the repeat function one-period is to reach best effect for serious pollution cell, and recommended work concentration is 10ug/mL.In order to keep the removing effect, the concentration of available 2～5ug/ml is kept prevention.
Mycoplasma clearing reagent of the present invention, wherein the main body microbiotic belongs to FQNS, also comprises tetracyclines verivate and part macrolide antibiotics in addition; Mix with special damping fluid, can the most effectively suppress and kill mycoplasma, short only needs of action period just can all be killed mycoplasma at a week; Do not influence the metabolism of cell itself; And treated cell surface is smooth, and the black powder particle disappears basically, and cell can not infected by mycoplasma easily again.
Description of drawings
Fig. 1: mycoplasma clearing reagent uses synoptic diagram
Fig. 2: use mycoplasma clearing reagent cross-reference figure:
A: DLD-1 cell white light visual field A ' before handling: handle the DLD-1 cell white light visual field, back
B: DLD-1 cell fluorescence visual field B ' before handling: handle the DLD-1 cell fluorescence visual field, back
C: Hela cell white light visual field C ' before handling: handle the Hela cell white light visual field, back
D: Hela cell fluorescence visual field D ' before handling: handle the Hela cell fluorescence visual field, back
E: MG-63 cell white light visual field E ' before handling: handle the MG-63 cell white light visual field, back
F: MG-63 cell fluorescence visual field F ' before handling: handle the MG-63 cell fluorescence visual field, back
H: SGC-7901 cell white light visual field H ' before handling: handle the SGC-7901 cell white light visual field, back
I: SGC-7901 cell fluorescence visual field I ' before handling: handle the SGC-7901 cell fluorescence visual field, back
J: SMMC-7721 cell white light visual field J ' before handling: handle the SMMC-7721 cell white light visual field, back
K: SMMC-7721 cell fluorescence visual field K ' before handling: handle the SMMC-7721 cell fluorescence visual field, back
L: Panc-1 cell white light visual field L ' before handling: handle the Panc-1 cell white light visual field, back
M: Panc-1 cell fluorescence visual field M ' before handling: handle the Panc-1 cell fluorescence visual field, back
The nest-type PRC detected result of Fig. 3: embodiment 3
Fig. 4: mycoplasma clearing is the nest-type PRC detected result relatively
Appearance explanation on the swimming lane
M：DL250+
1: the cell conditioned medium of pollution does not add medicine and removes
2: the cell conditioned medium after SB 265805 is removed
3: the cell conditioned medium after Minocycline HCl is removed
4: the cell conditioned medium after Azythromycin is removed
Cell conditioned medium after 5:Mycoplasma-OUT removes
6: do not add IC and PC
7: only add IC
8: only add PC
Embodiment
Below enumerate specific examples with further elaboration the present invention, should be understood that instance is not to be used to limit protection scope of the present invention.
The configuration of embodiment 1 mycoplasma clearing reagent
Press following formulated 250ul reagent (each component is by weight percentage):
Prescription 1: SB 265805 1.4%
Minocycline HCl 0.4%
Azythromycin 0.2%
Damping fluid 98%
Buffer formulation:
NaOH 0.4％
Nacl 0.9％
H
2O 93.7％
Prescription 2: Moxifloxacin 1.3%
Vibra-0.5%
Clarithromycin 0.1%
Damping fluid 98.1%
Buffer formulation:
NaOH 0.2％
Nacl 1.0％
H
2O 90.8％
Prescription 3: PD 127391 1.5%
WAY-GAR 936 0.3%
Roxithromycin 0.3%
Damping fluid 97.9%
Buffer formulation:
NaOH 0.6％
Nacl 0.9％
H
2O 93.5％
Collocation method
1. the configuration of damping fluid
1) ultrapure water 103.4kpa vp temperature reaches 121.3 ℃, and it is subsequent use to keep 15-20 minute moist heat sterilization
2) according to each component preparation of above prescription weighing
3) dissolve above preparation in 4 ℃ of preservations with ultrapure water
2. by filling a prescription weighing fluoroquinolone antibiotics, tetracyclines verivate and macrolide antibiotics in the aseptic centrifuge tube of 50ml, dissolve with the damping fluid lucifuge
3. filter packing with millipore 0.22um aperture filter
4. concentration 20mg/mL, 250ul/ box, 4 ℃ of preservations
Adopt the mycoplasma clearing reagent of embodiment 1 prescription 1 preparation, method of use such as Fig. 1 are said, comprise the steps:
1. the recovery cell carries out kind of a plate after the adjustment, plants 1x10 in the 6cm dish
6Individual cell;
2. after treating cell attachment next day, in the 2000x ratio mycoplasma clearing reagent is diluted to and contains in the unparalleled anti-substratum of 5%FBS, when going down to posterity subsequently, use this culture medium culturing;
3. act on 7 days, note to observe in this process and the record cell state, change liquid or keep with the substratum that contains mycoplasma clearing reagent when going down to posterity;
Do not contain antibiotic perfect medium cultivation 2 days normally 4.7 change into after it, the long-pending 4-5ml that adds to of every disk body;
5.2 culture supernatant 500ul is drawn in it back, carries out PCR (detect step and see lucky triumphant gene GC-PCR Mycoplasma TestKit operational manual for details);
6. the cell of detected result after negative enlarges frozen guarantor and plants or directly carry out follow-up cytologic experiment.
Process object be the laboratory clearly by the DLD-1 cell of mycoplasma contamination, Hela cell, MG-63 cell, SGC-7901 cell, SMMC-7721 cell and Panc-1 cell, the cellular form of handling before and after 7 days is as shown in Figure 2 with efficiency of infection comparison photo.
Visible by photo: cell surface has fragment before handling, the black particle shape material, and background is very dirty, the part cell rounding, efficiency of infection is lower; Handle back cell surface fragment and black particle shape material and disappear, background is clean, and cell spreads out fully, and efficiency of infection obviously increases.
Experiment equipment:
The negative 80 degree refrigerator PCR appearance electrophoresis chamber gel imaging appearance electrophoresis apparatuses of ultra dressing table water-bath whizzer
Aseptic PCR manages the aseptic rifle head of aseptic 1.5ml EP pipe PCR pipe support cotton balls
Pipettor (1ml 200ul 10ul 2ul)
Experiment reagent:
Aseptic ultrapure water sample 75% ethanol for disinfection to be checked goes mycoplasma sprays (embodiment 1 preparation)
Germ-free?test?medium
The quick detection kit of nest-type PRC mycoplasma (Shanghai Ji Sheng drugmaker)
2% agarose gel EB electrophoretic buffer Mark (DL250+) 6x Loading Buffer
Experimental procedure:
The method of reference implementation 2 is used mycoplasma clearing reagent.
Adopt following method to use the quick detection kit of nest-type PRC mycoplasma:
1. collection needs the sample of detection, and negative 80 degree refrigerators are preserved
2.95 boil sample 5～10min to be checked in the degree boiling water bath, 12000 rpms of centrifugal 2min
3. with 75% all experiment equipments of ethanol for disinfection wiping and material, spend the mycoplasma sprays again and spray important material and equipment, evenly wipe surfaces is with the mycoplasma in the thorough removal environment
4. it is some to take out the PCR pipe, and 1.5ml EP pipe is some, places subsequent use
5. dilution primer; (effect of IC is and the dna sequence dna formation competitive relation of mycoplasma, when not having mycoplasma contamination, can amplifies band PC (Positive Control) with IC (Internal Control) plasmid; When polluting, it is suppressed not by amplification) to proper concn
6. dispose first round PCR system house steward, every then pipe 48ul branch installs in the cut-and-dried PCR pipe, wherein need add the water contrast and contrast with IC
7. add sample to be checked, carry out first round PCR on the vibrator behind the mixing
8. after first round PCR finished, PCR system house steward was taken turns in configuration second, and every then pipe 48ul branch installs in the cut-and-dried PCR pipe, wherein need add the PC contrast
9. add first round PCR product, carry out second on the vibrator behind the mixing and take turns PCR
10. finish the back and add Loading Buffer, dispose 2% agarose gel, last appearance is carried out electrophoresis, the PCR system of taking pictures:
H
2O 32ul
5X?Buffer(Mg2+) 10ul
dNTP 3ul
Primer?MIX 2.5ul
Taq enzyme 0.5ul
Template 2ul
Circulation is taken turns in first round circulation second
94 ℃ of for 2min of 94 ℃ of for 2min of 1 circulation, 1 circulation
94 ℃ of for 30sec of 94 ℃ of for 30sec of 30 circulation cycle, 30 circulation e
62℃?for?1min 53℃?for?30sec
72℃?for?1min 72℃?for?30sec
72 ℃ of for 10min of 72 ℃ of for 10min of 1 circulation, 1 circulation
Be cooled to 4 ℃ and be cooled to 4 ℃
Experimental result and analysis:
The result is as shown in Figure 3
Last appearance numbering:
1～3:293T cell (recovery back); 293T cell (before the dosing); 293T cell (after the dosing)
4～6:panc-1 cell (recovery back); Panc-1 cell (before the dosing); Panc-1 cell (after the dosing)
7～9:SGC-7901 cell (recovery back); SGC-7901 cell (before the dosing); SGC-7901 cell (after the dosing)
10～12:H1299 cell (recovery back); H1299 cell (before the dosing); H1299 cell (after the dosing)
13～15：IC；IC+PC；PC
The sample of " recovery back " is the residue supernatant of last round of PCR;
The sample of " before the dosing " is after cell recovery has passed 4-5 and passes, and cultivates density and reaches 80～90% cells and supernatant with containing two anti-perfect mediums;
The sample of " after the dosing " is a treated with medicaments after one week, changes Germ-free test medium into and cultivates that density reaches 80～90% cells and supernatant after 2 days.
PCR result: before handling with handle after cell conditioned medium form sharp contrast, get a desired effect
1. this experimental selection 293T, panc-1, SGC-7901, H1299 four strain cells detect; Every strain cell is provided with 3 groups (before recovery back, dosings, after the dosing); Purpose is exactly to want to look at cell in the go down to posterity pollution degree of depth situation of process of recovery, with the cell after the antibiotic treatment whether also have mycoplasma contamination maybe.
2. can find out from PCR result that first round detection has the cell of pollution to receive mycoplasma contamination really, cell all changes into feminine gender after dosing is handled, and explains that mycoplasma clearing agent of the present invention can be used for removing fast the first-selected reagent of mycoplasma.
3. the cell after mycoplasma clearing agent of the present invention is handled, the outside surface fragment disappears, and the cell entire body is smooth, and diopter is good, and cell attachment is more opened, and the speed of growth significantly improves, and preliminary experiment proof transfection and efficiency of infection improve.
To supposeing 1:
SB 265805 4.2%
The damping fluid surplus
Prescription: NaOH 0.4%
Nacl 0.9％
H
2O 93.7％
To supposeing 2:
Minocycline HCl 1.2%
The damping fluid surplus
Prescription: NaOH 0.4%
Nacl 0.9％
H
2O 93.7％
To supposeing 3:
Azythromycin 0.6%
The damping fluid surplus
Prescription: NaOH 0.4%
Nacl 0.9％
H
2O 93.7％
To supposeing 4:
The prescription 1 of embodiment 1
Experimentation:
1. have the cell of pollution to carry out kind of a plate (6 orifice plate) detection, cultivated 1-2 days adherent back
2. the method by embodiment 2 adds equal volume analogy 1-4 is removed separately in orifice plate
3. after acting on a week, go culture supernatant to carry out PCR according to the method for embodiment 3 and detect, the result is as shown in Figure 4.
Experimental analysis:
All can not well remove totally the mycoplasma of contamination of cells when composition uses separately separately among the 1-3 to analogy; And the 5th swimming lane is negative to supposeing that 4 cells and supernatant after removing detect; After fluoroquinolone antibiotics, tetracyclines verivate and macrolide antibiotics are share in explanation; On the removing mycoplasma synergy having taken place, makes the effect of removing mycoplasma all be superior to using separately each medicine.And the damping fluid that adopts in this reagent has played the effect of better protection cell than conventional damping fluid.This result shows, after prescription of the present invention share fluoroquinolone antibiotics, tetracyclines verivate and macrolide antibiotics, on the removing mycoplasma, synergy has taken place, and makes the effect of removing mycoplasma all be superior to using separately each medicine.And the damping fluid that adopts in this reagent has played the effect of better protection cell than conventional damping fluid.
Claims (3)
1. mycoplasma clearing reagent comprises following components in weight percentage:
The weight percent of said damping fluid consists of:
2. mycoplasma clearing reagent is used for removing and prevent the purposes of the mycoplasma growth of cell cultures according to claim 1.
3. the method for use of mycoplasma clearing reagent when being used for removing the mycoplasma of cell cultures according to claim 1; Comprise the steps: said mycoplasma clearing reagent is added in the unparalleled anti-cell culture medium; When going down to posterity subsequently, use this culture medium culturing; Continue a week removing mycoplasma, for serious pollution cell repeat function one-period more then.
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| CN2010101480281A CN101851603B (en) | 2010-04-15 | 2010-04-15 | Mycoplasma clearing reagent and application thereof |
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| CN102409019B (en) * | 2011-06-23 | 2013-03-27 | 胡晓鹏 | Reagent for eliminating mycoplasma contamination in cell culture and use method thereof |
| CN105950719A (en) * | 2016-04-28 | 2016-09-21 | 上海源培生物科技股份有限公司 | Mycoplasma detecting and removing method |
| CN106282105A (en) * | 2016-08-13 | 2017-01-04 | 浙江译美生物科技有限公司 | A kind of mescenchymal stem cell culture medium |
| CN107714710A (en) * | 2017-07-13 | 2018-02-23 | 江西农业大学 | A kind of antibacterial combination of ROX and fortimicin |
| CN111117962A (en) * | 2019-12-24 | 2020-05-08 | 武汉博士德生物工程有限公司 | Method for effectively removing mycoplasma pollution |
| CN113373105A (en) * | 2021-05-18 | 2021-09-10 | 广东乾晖生物科技有限公司 | Mycoplasma clearing compositions and methods of treating cultured cells in vitro |
| CN113652387A (en) * | 2021-07-16 | 2021-11-16 | 上海佐润生物科技有限公司 | Hemicellae removing reagent and preparation method thereof |
| CN114196633B (en) * | 2021-11-29 | 2023-07-07 | 湖南亚大丰晖新材料有限公司 | Kit for clearing and detecting mycoplasma and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1298706A (en) * | 1999-12-24 | 2001-06-13 | 国家药品监督管理局四川抗菌素工业研究所 | Kelarmycin eye drops and its preparing process |
| US20040127403A1 (en) * | 2002-09-06 | 2004-07-01 | Francesco Parenti | Methods for treating and preventing Gram-positive bacteremias |
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| US7994225B2 (en) * | 2004-03-17 | 2011-08-09 | Rempex Pharmaceuticals, Inc. | Bacterial efflux pump inhibitors for the treatment of ophthalmic and otic infections |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1298706A (en) * | 1999-12-24 | 2001-06-13 | 国家药品监督管理局四川抗菌素工业研究所 | Kelarmycin eye drops and its preparing process |
| US20040127403A1 (en) * | 2002-09-06 | 2004-07-01 | Francesco Parenti | Methods for treating and preventing Gram-positive bacteremias |
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