CN106047797A - Reagent, kit and treatment for treating mycoplasma contamination - Google Patents
Reagent, kit and treatment for treating mycoplasma contamination Download PDFInfo
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- CN106047797A CN106047797A CN201610692668.6A CN201610692668A CN106047797A CN 106047797 A CN106047797 A CN 106047797A CN 201610692668 A CN201610692668 A CN 201610692668A CN 106047797 A CN106047797 A CN 106047797A
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- antibiotic
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- minocycline
- mycoplasma contamination
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2500/00—Specific components of cell culture medium
- C12N2500/30—Organic components
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Abstract
The present invention discloses a reagent, a kit and a treatment method for treating mycoplasma contamination. The reagent is a solution containing antibiotic tiamulin, minocycline and antibiotic ciprofloxacin, wherein a concentration ratio of the antibiotic tiamulin to the minocycline to the antibiotic ciprofloxacin is 4:1:3. According to the present invention, with the reagent, the kit and the treatment method, the combination comprising the antibiotic tiamulin, the minocycline and the antibiotic ciprofloxacin according to the specific ratio is used, such that the mycoplasma contamination treatment effect can be significantly increased; and the mycoplasma contamination problem can be simply, safely, rapidly and effectively treated.
Description
Technical field
The present invention relates to a kind of reagent processing mycoplasma contamination and test kit thereof and processing method, belong to biotechnology
Field.
Background technology
It is one of modal pollution in cell cultivation process that mycoplasma (Mycoplasma) is polluted, and particularly moves in suckling
In the incubation of thing cell, mycoplasma contamination problem is the most troubling, and main cause has following three points: 1) mycoplasma volume
The least, a diameter of 0.1 μm, general filtration sterilization cannot be removed, and is difficult to see clearly its morphosis under optical microscope；2) prop up
Substance vitality is indomitable, can survive, and penicillium sp is have Drug resistance under meta-alkali condition (pH 7.6～8.0)；3) cell is by propping up
At the mycoplasma contamination initial stage, part sensitive cells visible cell growth propagation is slack-off, form becomes round.But without bright after most cell contaminations
Aobvious change, or be slightly changed, if processing not in time, cross-contamination also can be produced.Mycoplasma eventually results in cell death, but
More conventional situation is that mycoplasma coexists with cell；In the middle of this process, mycoplasma is slowly bred, until producing bright to cell
Aobvious impact, such as interference cell migration, signal transduction, lymphocyte activation and cytokine-expressing.
For related scientific research worker, the most fearful is that mycoplasma contamination changes many parameters unconsciously,
The credibility making achievement in research is had a greatly reduced quality, and even causes heavy losses.
The cell suffering mycoplasma contamination is typically directly discarded；But in the case of having some special, need to retain cell；
Therefore, after confirming mycoplasma contamination, need to be processed to retain cell by some more special experiments.
It is thus desirable to a kind of place's treatment kits that can process mycoplasma contamination and contaminated cell can be saved.
Summary of the invention
In view of the problems referred to above and/or the other problems of correlation technique, one aspect of the present invention provides a kind of process mycoplasma
The reagent polluted, described reagent is to comprise the most wonderful rhzomorph of antibiotic, minocycline and the solution of antibiotic ciprofloxacin；Its
In, the most wonderful rhzomorph of described antibiotic, described minocycline and described minocycline, the concentration proportion of three is 4:1:
3。
Preferably, described reagent is that the most wonderful rhzomorph of described antibiotic, minocycline and antibiotic ciprofloxacin dissolve
In the solution that aseptic double-distilled water is formed.
Another aspect of the present invention provides a kind of test kit processing mycoplasma contamination, and described test kit includes above-mentioned examination
Agent.
Preferably, the concentration of the most wonderful rhzomorph of described antibiotic is 20mg/ml, concentration 5mg/ of described minocycline
Ml, the concentration of described antibiotic ciprofloxacin is 15mg/ml.
Preferably, in described test kit, the specification of described reagent is 100ml, 50ml, 20ml or 10ml.
Further aspect of the present invention provides a kind of method processing mycoplasma contamination, wherein, by above-mentioned reagent, or on
State the reagent in test kit to mix with fresh culture to obtain Antibiotic medium, described anti-in described Antibiotic medium
The concentration of the most wonderful rhzomorph of raw element is 20mg/ml, concentration 5mg/ml of described minocycline, described antibiotic ciprofloxacin
Concentration is 15mg/ml；Pending cell culture is added in described Antibiotic medium, after cultivating 3 days, give up old
Culture medium；The described Antibiotic medium the most more renewed, cultivates 3 days；So circulation, until can't detect mycoplasma contamination feelings
Condition.
Preferably, the condition of culture of described pending cell culture is as follows: cell culture density be about 50000～
100000/ml, temperature 37 DEG C and 5%CO2。
Compared with prior art, the reagent processing mycoplasma contamination of the present invention and test kit thereof and processing method, use
The combination of antibiotic the most wonderful rhzomorph, minocycline and minocycline, and inventor studies through lot of experiments
It was unexpectedly observed that the combination of the three of special ratios kinds of antibiotic, it is possible to the problem more effectively processing mycoplasma contamination, and three
The effect of the combination of person's special ratios is far superior to the effect of the most wonderful rhzomorph of antibiotic and minocycline combination, and this illustrates
There is certain cooperative effect between minocycline and the most wonderful rhzomorph of antibiotic, minocycline, can significantly improve and prop up
The effect that mycoplasma contamination processes；It addition, the reagent processing mycoplasma contamination of the present invention and test kit and processing method thereof also have
Have the advantage that 1) easy and simple to handle: can directly process the mycoplasma contamination phenomenon occurred in cell cultivation process, reagent treatment
Without re-dissolved；2) safety is used: design exclusively for killing mycoplasma, the maximized minimizing wound to cellar culture cell
Evil；3) the most effective: this test kit, for the biological nature of mycoplasma, uses maximally effective composition, to reach quick, effectively to locate
The purpose of reason mycoplasma contamination；Generally speaking, it is possible to easy, safety and the problem fast and effeciently processing mycoplasma contamination.
Accompanying drawing explanation
Fig. 1 a is the detection of mycoplasma result figure after using embodiment 1 agent treated mycoplasma contamination；
Fig. 1 b is the detection of mycoplasma result figure after using the Antibiotic medium of comparative example 1 to process mycoplasma contamination；
Fig. 1 c is the detection of mycoplasma result figure after using the Antibiotic medium of comparative example 2 to process mycoplasma contamination.
Detailed description of the invention
The present invention is further illustrated by the following examples, but the present invention is not limited to these specific embodiment parties
Formula.
Embodiment 1: process reagent and the preparation of test kit of mycoplasma contamination
As a example by the test kit of 10ml specification:
The most wonderful for 200mg rhzomorph, 50mg minocycline and 150mg antibiotic ciprofloxacin are dissolved in 10ml successively aseptic
In distilled water, fully mix, i.e. obtain the reagent processing mycoplasma contamination prepared；Then, then by this reagent it is assembled to examination
In agent box.
Embodiment 2: the method processing mycoplasma contamination
The reagent (reagent in test kit in other words) embodiment 1 prepared, according to volume ratio (that is, the dilution of 1:100
After 100 times of the reagent volume that volume is embodiment 1) (pending cell is to add fresh RMPI 1640 culture medium to
293T cell, it uses RMPI 1640 culture medium) middle acquisition Antibiotic medium, wherein, the concentration of the most wonderful rhzomorph of antibiotic is
0.2mg/ml, concentration 0.05mg/ml of minocycline, the concentration of antibiotic ciprofloxacin is 0.15mg/ml.
Pending cell culture is added in above-mentioned Antibiotic medium, after cultivating 3 days, give up old cultivation
Base；The Antibiotic medium the most more renewed, cultivates 3 days；So circulation, until can't detect mycoplasma contamination situation.
After can't detect mycoplasma contamination situation, normal cell cultivation or Secondary Culture can be carried out.
In the present embodiment, the condition of culture of pending cell culture is as follows: cell culture density is about 80000
Individual/ml, temperature 37 DEG C and 5%CO2。
In the present embodiment, after 3 circulate, substantially can't detect mycoplasma contamination situation.
Effect data
The reagent of embodiment 1 is with the fresh RMPI 1640 mixed Antibiotic medium of culture medium, and antibiotic is the most wonderful
The concentration of rhzomorph is 0.2mg/ml, concentration 0.05mg/ml of minocycline, and the concentration of antibiotic ciprofloxacin is
0.15mg/ml；Total antibiotic concentration is 0.4mg/ml；
Comparative example 1:
After the same method, the Antibiotic medium of preparation comparative example 1, wherein, the concentration of the most wonderful rhzomorph of antibiotic is
0.32mg/ml, concentration 0.08mg/ml of minocycline；Total antibiotic concentration is 0.4mg/ml；
Comparative example 2:
After the same method, the Antibiotic medium of preparation comparative example 2, wherein, the concentration of antibiotic ciprofloxacin is
0.4mg/ml；Total antibiotic concentration is 0.4mg/ml；
Pending cell culture is divided into 3 parts, and the antibiotic of the preparation of reagents being respectively added to embodiment 1 is cultivated
Base, the Antibiotic medium of comparative example 1 and the Antibiotic medium of comparative example 2, after cultivating 3 days, give up old culture medium；The most more
The respective Antibiotic medium renewed, cultivates 3 days；So circulation.
Often experience one cycle period, take respective culture and carry out detection of mycoplasma, testing result join Fig. 1 a, 1b and
1c。
See Fig. 1 a, use the reagent of embodiment 1, after experiencing 3 cycle periods, can't detect mycoplasma contamination situation
?；
But, see Fig. 1 b and 1c, comparative example 1 and comparative example 2, after 3 cycle periods of experience, still suffer from mycoplasma dirty
Dye situation.
It can thus be seen that same total antibiotic concentration and same processing method, the employing the most wonderful rhzomorph of antibiotic,
Minocycline is the most excellent with the mycoplasma contamination treatment effect of the reagent that the special ratios of the three of minocycline combines
More in the effect of the most wonderful rhzomorph of antibiotic with minocycline combination, the farthest it is superior to the process effect of minocycline
Really, this illustrates to there is certain cooperative effect, energy between minocycline and the most wonderful rhzomorph of antibiotic, minocycline
Significantly improve the effect that mycoplasma contamination processes.
It is to be understood that, although this specification is been described by according to embodiment, but the most each embodiment only comprises one
Individual independent technical scheme, this narrating mode of description is only that for clarity sake those skilled in the art should will say
Bright book is as an entirety, and the technical scheme in each embodiment can also be through appropriately combined, and forming those skilled in the art can
With other embodiments understood.
The a series of detailed description of those listed above is only for the feasibility embodiment of the present invention specifically
Bright, they also are not used to limit the scope of the invention, all equivalent implementations made without departing from skill of the present invention spirit
Or change should be included within the scope of the present invention.
Claims (7)
1. the reagent processing mycoplasma contamination, it is characterised in that: described reagent is for comprising the most wonderful rhzomorph of antibiotic, dimethylamine
Tetracycline and the solution of antibiotic ciprofloxacin；Wherein,
The most wonderful rhzomorph of described antibiotic, described minocycline and described minocycline, the concentration proportion of three is 4:
1:3.
2. reagent as claimed in claim 1, it is characterised in that: described reagent is the most wonderful rhzomorph of described antibiotic, dimethylamine four
Ring element and antibiotic ciprofloxacin are dissolved in the solution that aseptic double-distilled water is formed.
3. the test kit processing mycoplasma contamination, it is characterised in that: described test kit includes as appointed in claim 1 to 2
Anticipate a described reagent.
4. test kit as claimed in claim 3, it is characterised in that: the concentration of the most wonderful rhzomorph of described antibiotic is 20mg/ml, institute
Stating concentration 5mg/ml of minocycline, the concentration of described antibiotic ciprofloxacin is 15mg/ml.
5. test kit as claimed in claim 4, it is characterised in that: in described test kit, the specification of described reagent be 100ml,
50ml, 20ml or 10ml.
6. the method processing mycoplasma contamination, it is characterised in that:
By the reagent described in any one in described claim 1 to 2, or in described claim 3 to 5 described in any one
Test kit in reagent mix with fresh culture to obtain Antibiotic medium, described anti-in described Antibiotic medium
The concentration of the most wonderful rhzomorph of raw element is 20mg/ml, concentration 5mg/ml of described minocycline, described antibiotic ciprofloxacin
Concentration is 15mg/ml；
Pending cell culture is added in described Antibiotic medium, after cultivating 3 days, give up old culture medium；Again
The described Antibiotic medium more renewed, cultivates 3 days；So circulation, until can't detect mycoplasma contamination situation.
7. method as claimed in claim 6, it is characterised in that:
The condition of culture of described pending cell culture is as follows: cell culture density be about 50000～100000/
Ml, temperature 37 DEG C and 5%CO2。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610692668.6A CN106047797A (en) | 2016-08-19 | 2016-08-19 | Reagent, kit and treatment for treating mycoplasma contamination |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610692668.6A CN106047797A (en) | 2016-08-19 | 2016-08-19 | Reagent, kit and treatment for treating mycoplasma contamination |
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| Publication Number | Publication Date |
|---|---|
| CN106047797A true CN106047797A (en) | 2016-10-26 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610692668.6A Pending CN106047797A (en) | 2016-08-19 | 2016-08-19 | Reagent, kit and treatment for treating mycoplasma contamination |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106635950A (en) * | 2017-01-05 | 2017-05-10 | 南方医科大学珠江医院 | Treatment method of mycoplasma contamination |
| 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 |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1234397A (en) * | 1999-04-02 | 1999-11-10 | 中国科学院上海药物研究所 | Compounds of carbostyrils and their preparation and use |
| CN1952104A (en) * | 2006-11-08 | 2007-04-25 | 高林 | Product for prevention and control of pollution of cell culture |
| CN102397552A (en) * | 2010-09-10 | 2012-04-04 | 广州自远生物科技有限公司 | Medicinal composite preparation containing quinolones, preparation method thereof and application thereof |
| CN102409019A (en) * | 2011-06-23 | 2012-04-11 | 胡晓鹏 | Reagent for eliminating mycoplasma contamination in cell culture and use method thereof |
| CN103387512A (en) * | 2012-05-08 | 2013-11-13 | 成都睿智化学研究有限公司 | Preparation method and intermediate of minocycline |
-
2016
- 2016-08-19 CN CN201610692668.6A patent/CN106047797A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1234397A (en) * | 1999-04-02 | 1999-11-10 | 中国科学院上海药物研究所 | Compounds of carbostyrils and their preparation and use |
| CN1952104A (en) * | 2006-11-08 | 2007-04-25 | 高林 | Product for prevention and control of pollution of cell culture |
| CN102397552A (en) * | 2010-09-10 | 2012-04-04 | 广州自远生物科技有限公司 | Medicinal composite preparation containing quinolones, preparation method thereof and application thereof |
| CN102409019A (en) * | 2011-06-23 | 2012-04-11 | 胡晓鹏 | Reagent for eliminating mycoplasma contamination in cell culture and use method thereof |
| CN103387512A (en) * | 2012-05-08 | 2013-11-13 | 成都睿智化学研究有限公司 | Preparation method and intermediate of minocycline |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106635950A (en) * | 2017-01-05 | 2017-05-10 | 南方医科大学珠江医院 | Treatment method of mycoplasma contamination |
| 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 |
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