GenieColor Mycoplasma Detection Kit (MORV0013)

Technology

The GenieColor kit utilizes an advanced isothermal amplification technology to detect mycoplasma contamination in cell cultures with a pink-to-yellow visual color change. It offers several benefits including ease of use, clear results, high sensitivity, strong specificity, and anti-contamination features. By adding 1 μl of cell culture supernatant to the reaction system and incubating at 65°C for one hour, results can be visually observed. If mycoplasma contamination is present, the reaction solution changes from pink to yellow, providing a clear color contrast. Additionally, the reagent incorporates an anti-contamination system to prevent false positives from amplification product aerosols, ensuring accurate and reliable results. The GenieColor Mycoplasma Detection kit can identify 40 mycoplasma species, including 8 commonly found in cell cultures, and is compatible with a wide range of adherent cells, suspension cells, media, and serum.

Components

COMPONENTS	10 Runs	25 Runs	50 Runs
GenieColor LAMP Mix	200 μl	500 μl	2 × 500 μl
Positive Control	10 μl	15 μl	2 × 15 μl
Mineral Oil	200 μl	500 μl	2 × 500 μl

Storage

Store at -30°-15°C and transport at ≤0°C.

1. Collect the cell culture supernatant
   • Adherent cells: directly collect the supernatant. Cells should remain in culture for at least 72 h undisturbed prior to screening and reach 90% confluence. After 72 h, mycoplasma content in supernatant is relatively high and can be detected easily.
   • Suspension cells: collect the supernatant after centrifugation at 2,300 rpm (500 × g) for 5 min. Cells should remain in culture for at least 72 h undisturbed prior to screening. After 72 h, mycoplasma content in supernatant is relatively high and can be detected easily.
2. Preparation of reaction system
   • Thaw the GenieColor LAMP Mix from -30°-15°C and mix thoroughly.
   • Prepare the reaction system according to the following table:

• Vortex and briefly centrifuge to collect at the bottom of the tube, ensuring no bubbles in the reaction system. Add 20 μl of mineral oil if using a water bath for incubation.

3. Reaction
   • Incubate at 65°C for 60 min in a PCR instrument or water bath (make sure water bath is calibrated prior to use).
4. Results
   • After the reaction is completed, observe the color in a bright environment. Pink represents negative and yellow represents positive.
   • Do not open the lid of detection tubes to prevent false positives due to aerosol contamination.

Q1: Do I need a PCR machine to run GenieColor?

• No. The reaction is isothermal, so a calibrated water bath at 65°C is enough; a PCR instrument works too. You read the result by eye, so no gel, plate reader or luminometer is needed.

Q2: How does GenieColor differ from a PCR mycoplasma kit?

• PCR kits amplify by thermal cycling and read the result on an agarose gel, which typically takes 2–3 hours including electrophoresis. GenieColor uses LAMP amplification with a colour indicator, so the result appears in the tube after 60 minutes with no gel, and the reagent is more tolerant of culture-medium inhibitors than PCR. Assay Genie also offers the MycoGenie Rapid Mycoplasma Detection Kit (MORV0011), the same 1-hour visual format covering 28 species, which is cited in Nature 2024 and Nature Metabolism 2024.

Q3: What is the sensitivity of the kit (minimum detection limit)?

• The kit can detect mycoplasma positive plasmid at a minimum concentration of 100 copies/μl.

Q4: Which mycoplasma species does it detect?

• The kit detects 40 mycoplasma species, including the eight most common cell-culture contaminants: M. hyorhinis, M. fermentans, M. arginini, M. orale, M. hominis, M. salivarium, M. pirum and A. laidlawii. The full list is in the last question below.

Q5: What sample do I use, and how should I prepare it?

• Use 1 μl of culture supernatant. Leave cells undisturbed in culture for at least 72 h and, for adherent lines, reach about 90% confluence so that mycoplasma in the supernatant is high enough to detect. For suspension cultures, centrifuge at 500 × g for 5 min and use the supernatant.

Q6: What does it mean if the reaction liquid color is between pink and yellow?

• A color between pink and yellow indicates a weak positive. It is recommended to repeat the test. If the repeated test is positive or weak positive, it is considered positive. If the repeated test is negative, it is considered negative.

Q7: How is accuracy ensured?

• The product includes both negative and positive controls to ensure accurate results. It has been tested against 40 different mycoplasma species, including 8 common mycoplasma contaminants.

Q8: What is the detection principle of this kit?

• The kit utilizes loop-mediated isothermal amplification (LAMP) technology. The reagent is pre-mixed with Bst isothermal amplification enzyme, mycoplasma-specific primers (inner and outer primers), and an optimized buffer. After adding the template, the LAMP reaction begins. If mycoplasma DNA is present, the reagent color changes from pink to yellow. A negative result remains pink, while a positive result turns yellow.

Q9: What sequence is used in the positive control?

• The positive control contains a plasmid with the M. hyorhinis sequence at a concentration of 10 ng/μl.

Q10: Does the positive control of the kit cause contamination?

• No, the positive control is a mycogenic plasmid, which does not cause contamination.

Q11: Why does the reaction solution change color after adding the supernatant of the medium?

• The medium might interfere with the color change. If it does, then use the following treatment method:

Q12: What 40 Mycoplasma species are detected using this kit?