MycoGenie Rapid Mycoplasma Detection Kit

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MycoGenie Rapid Mycoplasma Detection Kit - Introduction

The MycoGenie Rapid Mycoplasma Detection Kit enables the rapid and simple detection of mycoplasma contamination in cell culture. This novel system detects mycoplasma in 1 hour from 1µl of cell culture supernatant by visual determination eliminating the requirement for PCR, qPCR, electrophoresis or ELISA. Compared with PCR, the MycoGenie Rapid Mycoplasma Detection Kit is more resistant cell culture inhibitors thus avoiding false positive and false negative results. The results are highly consistent with the most sensitive and accurate PCR kits for Mycoplasma detection.

The MycoGenie Rapid Mycoplasma Detection Kit can detect up to 28 mycoplasma species, including 8 of the most common species associated with cell culture contamination. It is suitable for mycoplasma detection in a wide range of suspension and adherent cells including CHO, Vero, hybridoma, Sf9, HEK293 and many more. Furthermore, the kit is compatible with a comprehensive suite of cell culture media and serum.

The MycoGenie Rapid Mycoplasma Detection Kit is an excellent choice for routine mycoplasma detection in biopharma, vaccine/monoclonal antibody production, cell therapy/embryo laboratories and other scientific research laboratories.

Visual Mycoplasma Detection

Figure 1: Visual detection of Mycoplasma contamination using the MycoGenie Mycoplasma detection kit ( MORV0011). ES cultured cell cultures had no detectable Mycoplasma 3 days post-treatment with the with MycoPlasma Elimination Reagent ( MORV0012). Tube 1: Positive Control | Tube 2: Day 0 | Tube 3: Day 3 | Tube 4: Negative Control.

Excellent Sensitivity

Mycoplasma Copy No. Visual Result Interpretation









Table 1: The MycoGenie Rapid Mycoplasma Detection kit enables the detection of as little as 3.07x102 copies of Mycoplasma. This is more sensitive than in-house PCR analysis (3.07x103 copies) & the same as in-house QPCR analysis at 3.07x102 copies of Mycoplasma.

Mycoplasma Detection Kit - Components

Components MORV0011 - 20 tests MORV0011 - 50 tests

MycoGenie Buffer*

480 µl

1.2 ml

MycoGenie Enzyme

20 µl

50 µl

Positive Control

10 µl

25 µl

Paraffin Oil

500 µl

1.25 ml

*Contains chromogenic reagent


Store at -20°C

Materials Required but not Supplied

PCR instrument (heating function only), heated dry-bath or water bath.

Mycoplasma Detection Kit - Applications

MycoGenie Rapid Mycoplasma Detection Kit is suitable for many kinds of suspension and adherent cells with a wide range of media and serum compatibility. These include but are not limited to:

Application Example

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.


CD FortiCHO, CDM4, Expi 293 Medium, CD Hybridoma, Grace, DMEM, 1640, F12, etc.


Fetal bovine/calf serum, horse serum, Gibco KSR serum replacement, etc.

Mycoplasma Detection Kit Protocol

1. Collect the cell culture supernatant
Adherent cells: directly collect the supernatant. It is recommended to collect the sample 3 days after the cells were passed or the culture media was exchanged and there is a cell confluence of about 90%. Mycoplasma content in the supernatant is relatively high and easy to detect at this stage.
Suspension cells: centrifuge cells at 500 × g for 5 min and collect the supernatant. It is recommended to collect the sample 3 days after the cells were passed or the culture media was exchanged.

2. Preparation of reaction mixture
Thaw the MycoGenie Buffer and mix thoroughly. Prepare following reaction system in a microcentrifuge tube:

Components Volume for a Single Reaction

MycoGenie Buffer

24 µl x Number of Samplesa x 1.1b

MycoGenie Enzyme

1 µl

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 and then aliquot 25 μl of solution to a PCR tube or microcentrifuge tube.

3. Sample Addition

To each reaction mixture, add the following as per experimental design:

    • Negative Control: Add 1 μl of sterile water as a negative control
    • Experimental Samples: Add 1 μl of the cell culture media to be assayed
    • Positive Control: Add 1 μl of the Positive Control (to be determined by end-user)

Mix thoroughly with a pipette or vortex.

Note: If the reaction is carried out in a water bath or PCR machine without heated lid, add 25 μl of Paraffin Oil to each tube to prevent liquid evaporation. When adding paraffin oil, change tips between samples to prevent cross-contamination.

4. Reaction

Incubate at 60℃ for 60 min in a PCR instrument, heated dry-bath or water bath.
Note: It is not recommended to use an oven for this reaction

5. Results

To determine the results, observe the color change in a bright environment and use white paper as a background.

Mycoplasma Negative Culture: Purple color solution
Mycoplasma Positive Culture: Sky blue color solution (as shown in figure on the right).

In rare circumstances (i.e. very low mycoplasma content), the color may be between purple and sky blue. In such cases, extend the reaction time to 75 min-90 min at 60℃ and observe the color again. The negative control or positive control can also be used as references.

Note: The reaction tube should never be reopened, due to possible false positives resulting from possible aerosol contamination.


1. What is the sensitivity of the MycoGenie Rapid Mycoplasma Detection Kit?
The 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 × 105 cfu/ml). Typically, the mycoplasma content in culture supernatant is between 106 -108 cfu/ml. According to published literature, 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 after cell passage or after replacing media.

2. What to do if the detection color changes immediately after supernatant is added or different colors (not blue or purple) appear during the reaction?
In rare cases, ingredients in the media interfere with the color of the MycoGenie reagent. For example, Cell Boost 5 (Hyclone) makes the MycoGenie reagent appear 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 at 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 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 protect cells from mycoplasma contamination?
If mycoplasma contamination occurs, it is recommended to discard the cells to prevent other cells from contamination. If a mycoplasma positive result is detected, the same batch of cells should be discarded. Also, consider MycoGenie MycoPlasma Elimination Kit (Cat. No. MORV0012) as an excellent choice to remove mycoplasma from cell culture.

4. How to avoid false positives?
Generally, no false positives will occur during proper usage. The reaction tube should never be reopened, due to false positives resulting from aerosol contamination. Change tips between samples and always add the positive control last.

5. How many mycoplasma species can be detected by MycoGenie Rapid Mycoplasma Detection Kit?
There are 28 species of mycoplasma that can be detected accurately by MycoGenie Mycoplasma Detector:

A. laidlawii*

M. hominis*

M. arginini*

M. fermentans* M. hyorhinis*

M. salivarium* M. pirum*

M. orale*

A. granularum A. pleciae

M. neophronis M. timone

M. caviae

M. alvi

M. bovis

M. primatum

M. leopharyngis M. maculosum

A. oculi

M. iners

M. gallinarum

M. sphenisci

M. bovigenitalium

M. auris

M. columbinum

M. lipophilum

M. falconis

M. alkalescens

* More than 95% mycoplasma contaminations in cell culture are caused by these 8 species.