MycoGenie Rapid Mycoplasma Detection Kit
MycoGenie Rapid Mycoplasma Detection Kit is designed for rapid detection of mycoplasma contamination in cell culture. It’s easy to use: after adding 1 μl of the cell culture supernatant to the reaction system and incubated at 60℃ for 1 hr, the results can be determined by visual observation. Detection can be easily completed in cell room without doing PCR, qPCR, or
electrophoresis. Compared with conventional PCR methods, MycoGenie Mycoplasma Detector is more resistant to the inhibitor in the culture supernatant, which avoids weak positive and false negative. There’s no need to do electrophoresis after reaction, which avoids false positive from aerosol of amplification product. The result is highly consistent with the most sensitive and accurate PCR method.
It has been validated that MycoGenie Rapid Mycoplasma Detection Kit could detect up to 28 kinds of mycoplasma, including 8 kinds commonly found in cell culture. This kit is suitable for mycoplasma detection in many kinds of suspension and adherent cells, including CHO, Vero, hybridoma, Sf9, HEK293, etc. This kit has a wide range of media and serum compatibility. It’s
suitable for routine mycoplasma detections in biopharmaceutical companies, vaccine/monoclonal antibody manufacturers, cell therapy/embryo laboratories, and other scientific research laboratories.
|Components||MORV0193 – 20 test||MORV0194 – 50 tests|
*Contains chromogenic reagent.
Store at -20°C
Materials Needed But Not Supplied
PCR instrument or water bath.
MycoGenie Rapid Mycoplasma Detection Kit is suitable for many kinds of suspension and adherent cells with a wide range of media and serum compatibility, which include but do not limit to:
Suspension cells: CHO, NS0, 293F, mouse hybridomas, Sf9, BHK21, etc.
Adherent cells: Vero, MDCK, SP2/0, 293T, HepG2. HeLa, A549, MB-MDA231, L929, MEF, etc.
Medium: CD FortiCHO, CDM4, Expi 293 Medium, CD Hybridoma, Grace, DMEM, 1640, F12, etc.
Serum: Fetal bovine/calf serum, horse serum, Gibco KSR serum replacement, etc.
1. Collect the cell culture supernatant
For adherent cells: directly collect the supernatant. It is recommended to collect the sample when the cells are passed or medium is exchanged for more than 3 days with a cell confluence of about 90%. At this moment, mycoplasma content in supernatant is relatively high and easy to be detected.
For suspension cells: collect the supernatant after centrifugation at 500 × g for 5 min. It is recommended to collect the sample when the cells are passed or medium is exchanged for more than 3 days. At this moment, mycoplasma content in supernatant is relatively high and easy to be detected.
2. Preparation of reaction system
Thaw the MycoGenie Buffer and mix thoroughly. Prepare following reaction system in a microcentrifuge tube:
|Components||Volume for a Single Reaction|
24 µl x Number of Samplesa x 1.1b
Note: a. Set a negative control and a positive control for each experiment, if necessary.
b. The extra 10% volume of solution is necessary to ensure that sufficient quantities can be divided into each tube, due to pipetting errors.
Gently mix by pipetting then aliquot 25 μl of solution to each PCR tube or microcentrifuge tube.
3. Adding Samples
For the first reaction tube, add no sample or add 1 μl of sterile water as a negative control.
For each of other tubes, add 1 μl of supernatant to be detected, respectively.
For the last reaction tube, add 1 μl of Positive Control.
Note: If the reaction is carried out in a water bath, add 25 μl of Paraffin Oil to each tube to prevent liquid evaporation. When adding paraffin oil, please change tips between samples to prevent cross-contamination.
Note: If the reaction is carried out in a PCR instrument with a hot lid, it is not necessary to add paraffin oil.
Incubate at 60℃ for 60 min in a PCR instrument or water bath.
Note: It is not recommended to use an oven for this reaction
Observe the solution color in a bright environment. It is recommended to use a white paper as background. Purple solution color represents mycoplasma negative and sky blue color represents mycoplasma positive (as
shown in figure on the right). In a few cases (i.e. low mycoplasma content), the color may be between purple and sky blue. Under this circumstance, extend the reaction time to 75 min-90 min and observe the color again. The negative control or positive control can also be used as references.
Note: The reaction tube should not be opened, due to possible false positives in the subsequent detection resulted from aerosol. Wrap the reaction tube in a plastic bag and discard it into trash in another room and clean up on time.
1. What is the sensitivity of MycoGenie Mycoplasma Detector? How to ensure the detection sensitivity?
MycoGenie Rapid Mycoplasma Detection Kit is suitable for most cell culture experiments. It can accurately detect at least 500 cfu of mycoplasma from 1 μl of culture supernatant (5 × 105cfu/ml), while typically, the mycoplasma content in culture supernatant is between 106 -108 cfu/ml. As validated in published literatures, one single mycoplasma in cell culture can grow to 106 cfu/ml in 3-5 days. Therefore, it is highly recommended to detect after the third day from passing or replacing
2. Reaction solution color turns as soon as adding the supernatant. Or the solution turns to other clolors other than purple and blue after reaction.
In rare cases, ingredients of the medium interfere with the color of the MycoGenie reagent. For example, the Cell Boost 5 (Hyclone) makes the MycoGenie reagent appears pink. To avoid this,
(1) Collect a small amount of culture supernatant or cell suspension and centrifuge at 500 × g for 5
min. Collect the supernatant.
(2) Centrifuge again with a high speed (> 12,000 × g) for 5 min to precipitate mycoplasma in the supernatant. Discard most of the supernatant and leave about 50μl in the tube. Add 950μl of sterile water and mix gently by pipetting.
(3)Repeat the Step (2) for three times. Discard most of the supernatant and leave about 50μl in the tube.
(4)Take 1μl of supernatant for detection.
3. How to save my cells from mycoplasma contamination?
If mycoplasma contamination occurs, it is recommended to discard the cells to prevent other cells from contamination. If mycoplasma positive is detected, the same batch of cells should be discarded.
4. How to avoid false positives?
Generally, no false positives will occur during proper operations. DO NOT open the tubes after reaction, due to possible false positives in the subsequent detection resulted from aerosol. Change tips between samples, and the positive control should be added at last. These operations can further reduce the risk of false positives.
5. How many kinds of mycoplasma can be detected by MycoGenie Mycoplasma Detector?
There are 28 kinds of mycoplasma that can be detected accurately by MycoGenie Mycoplasma
M. fermentans* M. hyorhinis*
M. salivarium* M. pirum*
A. granularum A. pleciae
M. neophronis M. timone
M. leopharyngis M. maculosum
* More than 95% mycoplasma contaminations in cell culture are caused by these 8 kinds of mycoplasma