MycoStrip® 2.0 - Mycoplasma Detection Kit

MycoStrip® 2.0 detects mycoplasma using loop-mediated isothermal amplification (LAMP) PCR, a robust amplification method performed at a constant temperature of 65°C. The assay targets a conserved region of the 16S rRNA gene within mollicute genera using a proprietary reaction mix. Amplified DNA is then visualized as a band on an immunochromatographic strip, enabling rapid and easy result interpretation.

MycoStrip® 2.0 has been upgraded with an optimized, high-performance enzyme that enhances the overall efficiency of the amplification reaction. This improvement directly translates into better assay performance compared to the previous version of MycoStrip®. 

As a result, MycoStrip® 2.0 achieves detection of 10 - 100 genome copies (GC)/µl (~CFU/ml) in just 30 minutes, compared to 40 minutes required with the old version. Under optimal conditions and by extending the incubation time to 50 minutes, MycoStrip® 2.0 can detect as little as 1 GC/µl.

The updated reaction mix also provides a broader temperature tolerance, making the assay less sensitive to temperature fluctuations and therefore more robust across different laboratory conditions. As a result, the assay can be performed using a variety of standard heating devices, such as a water bath, dry bath, or thermocycler.

In addition, MycoStrip® 2.0 offers expanded detection coverage, including additional mollicute species, such as Ureaplasma and Spiroplasma genera, allowing detection of a wider range of potential contaminants. 
 

The assay is highly sensitive and can detect little as 1 GC/µl when incubated for 50 minutes. Longer incubation will not improve detection.

Sensitivity is now expressed in genome copies per µl (GC/µl) in alignment with updated edition 12.2 of the European Pharmacopoeia, which favors molecular quantification methods over culture-based measurements.

While CFU/ml (colony-forming units) reflects the number of viable organisms capable of forming colonies, GC/µl measures the amount of mycoplasma DNA present in a sample. This molecular approach provides a more direct and sensitive assessment of contamination, including both live and dead organisms.

To relate these two units, InvivoGen performed rigorous in-house studies using digital PCR (dPCR) on the most common mycoplasma contamination species. These results, together with external data, show that the ratio between GC and CFU varies depending on the species and method used, but is generally in the same order of magnitude.

Based on these internal validations, InvivoGen considers that 1 CFU/ml is approximately equivalent to 1 GC/µl for the most common mycoplasma contaminants, providing a practical reference for comparing molecular and culture-based measurements.

No. MycoStrip® 2.0 is highly specific for mycoplasma and does not detect phylogenetically related bacteria, such as Clostridium, Salmonella, or Streptococcus, nor waterborne organisms like Achromobacter. The assay specificity has been validated using DNA from Mollicutes, non-Mollicute bacteria, and eukaryotic cells/tissues, with no cross-reactivity observed with non-target DNA.

No. The presence of mammalian cells or their DNA does not interfere with mycoplasma detection.

Only a heating device is needed (e.g., thermocycler, heat block, dry bath, or water bath).

You can use a heat block, dry bath, or water bath. However, water baths may increase contamination risk.

• Hands-on time: 15 minutes
• Total time: 45 minutes

During the sample preparation step, heating the supernatant at 95°C for 5 minutes is optional. Although it does not increase sensitivity, it is recommended to inactivate mycoplasma and minimize contamination risks.

While not required for every single sample, it is strongly recommended to include positive and negative controls on a regular basis. This helps ensure the reliability of results and facilitates troubleshooting if needed.

The kit is shipped at room temperature.
Upon receipt, store all components at -20°C.
All components, including the strips, remain stable for 18 months when properly stored.

Samples can be stored at different stages of the workflow:

• Cell culture supernatant (before sample preparation):
  Can be stored at -20°C. Before use, thaw completely at 37°C and vortex to ensure proper mixing and salt dissolution.
• Prepared sample (after steps 1–4 of sample preparation):
  Can be stored at -20°C until needed.
• Processed sample (after steps 1–4 of the detection assay):
  Can be stored at -20°C for up to 3 months.

• Extend the 65°C incubation to 50 minutes for increased sensitivity. If a faint band remains faint after 50 minutes, it is considered negative.
• Alternatively, grow cells for 48 hours to achieve 100% confluency and re-test.
• Do not vortex the mix before the 65°C incubation step, as this can inhibit the reaction.

• Avoid repeated freeze–thaw cycles. The positive control as well as the Reaction Mix should be aliquoted upon first use and stored appropriately to minimize degradation.

• Do not vortex the reaction mix vigorously (it contains the enzyme).

• Do not leave the assembled reaction at room temperature for too long. Since the system is not warm-start, the reaction can still begin at room temperature. Keep this time below 15 minutes and transfer the reaction to 65°C immediately.

• Ensure the heating device is set to 65°C (±5°C).

No, the use of antibiotics on your cells will not interfere with the assay.

The positive control is not a source of mycoplasma contamination; it only contains a fragment of DNA.

The MycoStrip® 2.0 test requires 1 mL of cell culture supernatant from suspension or adherent cell cultures prior to passaging, or cell culture media constituents such as fetal bovine serum (FBS).

MycoStrip® 2.0 is a robust, sensitive, and extremely simple method for the detection of mycoplasma. The speed and convenience of MycoStrip® 2.0 allows for routine testing of cells in culture and commonly used constituents of complete media (e.g. FBS). It requires minimal hands-on-time with rapid results which are easily interpreted – no need for calculation, distinguishing similar colours, or running of an agarose gel.

Yes, it is possible to use nuclease free water instead of PBS.

No. Exceeding the recommended incubation time (maximum 60 minutes at 65°C) can significantly affect assay performance. We therefore do not recommend using samples that have been incubated for longer than specified.

No. Reagents from MycoStrip® and MycoStrip® 2.0 are not interchangeable.

Yes. MycoStrip® 2.0 detects both live and dead mycoplasma. This provides a more comprehensive assessment of contamination, as methods that detect only viable organisms may underestimate its presence. Detecting dead mycoplasma can still indicate a recent contamination event or the persistence of residual DNA. This is particularly important for quality control, where all traces of contamination must be taken into account.

This typically indicates contamination. To prevent this, use filtered tips and handle the positive control last.