Cambio - Excellence in Molecular Biology

Cell Culture Contamination Control

Cell Culture Contamination Control: Detection

Mycoplasma Detection

As an efficient contamination control measure we recommend frequent routine testing of cell culture materials with Venor®Gem Mycoplasma Detection Kits. These PCR- and qPCR-based mycoplasma assays allow fast (within a few hours), simple, effective and sensitive detection of mycoplasma-contaminated cell cultures in any standard molecular biology laboratory.

Regular mycoplasma testing and control procedures can significantly help in avoiding the costly consequences of contamination ensuring safe biopharmaceuticals and reliable scientific results.  Many scientific journals currently do not accept submissions of cell-based studies without evidence that mycoplasma testing has been performed.

To meet different customer needs Minerva Biolabs has developed a range of Venor®GeM mycoplasma detection assays using conventional PCR or qPCR methods which enable rapid and reliable identification of mycoplasma in cell cultures, culture supernatants and tissue culture media.

Conventional PCR

  • Venor®GeM Classic allows fast, reliable and time-saving routine monitoring of mycoplasma contamination in cell culture supernatants, media and biopharmaceuticals in research and industry by conventional PCR.

  • Venor®GeM OneStep is a kit for direct detection. It requires minimal pipetting, the kit includes all reagents required for PCR in a ready-to-use lyophilized reaction mix. Only samples or controls need to be added prior to PCR.

  • Venor®GeM Advance is designed for direct detection. Designed to reduce the total assay time and the pipetting steps, the kit contains PCR strips of tubes, each pre-dispensed with all reagents necessary for each PCR reaction, including the polymerase. For additional convenience, the gel loading buffer and dye are already included in the reaction buffer. After PCR, the products can be loaded directly on an agarose gel.

Real-time PCR (qPCR)

Our qPCR kits are TaqMan®-based qPCR Assays with labelled probes:  FAM® channel (mycoplasma) and HEX® channel (internal control to check for PCR inhibition from contaminants in sample).  So not only does the qPCR approach remove a lot of hassle, but the internal controls also give you reassurance that your negatives are not false.

  • Venor®GeM qEP utilises quantitative, real-time PCR (qPCR) for fast (approx. 3 h) and reliable screening of cell culture supernatants for mycoplasma contamination. The kit can be used in combination with any type of real-time PCR cycler able to detect the fluorescence dyes FAM® and HEX®. For clinical diagnostics in which human samples are tested we recommend using this kit as it conforms to EP 2.6.7, USP <71> and JP G3. It is suitable for both research and industry where certified compliance is needed or desirable. 

  • Venor®GeM qOneStep is a kit based on a real-time PCR (qPCR) assay for rapid, robust and sensitive detection of mycoplasma contamination. The detection procedure can be performed within 3 hours. Primers, nucleotides, probes, polymerase and an internal amplification control are provided as a ready-to-use, lyophilized reaction mix. This kit is a very straightforward and is highly suitable for academic labs and industrial applications where certified compliance of kits is not required.


Venor®GeM Classic Mycoplasma PCR Detection Kit

Venor®GeM Mycoplasma PCR Detection Kit for rapid and reliable detection of mycoplasma in various in situ biologicals including cell cultures and virus stocks

Minerva Biolabs

Catalogue No.DescriptionPack SizePriceQty
  • Change to:
  • £
11-1025Venor®GeM Classic Mycoplasma PCR Detection Kit25 tests €172.80 Quantity Add to Order
11-1050Venor®GeM Classic Mycoplasma PCR Detection Kit50 tests €304.80 Quantity Add to Order
11-1100Venor®GeM Classic Mycoplasma PCR Detection Kit100 tests €535.20 Quantity Add to Order
11-1250Venor®GeM Classic Mycoplasma PCR Detection Kit250 tests €1,148.40 Quantity Add to Order
11-1905Venor®GeM extra internal control DNA4 vials €26.40 Quantity Add to Order

Description

Venor®GeM Classic

Venor®GeM Classic

Venor®GeM Classic allows fast, reliable and time-saving routine monitoring of mycoplasma contamination by PCR.

Type of PCR

For conventional, endpoint PCR

Recommended Use / Scope

Applicable in research and industry for direct testing of cell cultures and biologicals. Intended for research use only. Not recommended for clinical diagnostics.

Kit Components

Lyophilized primer sets and nucleotides
10x reaction buffer optimized for MB Taq DNA Polymerase
Lyophilized positive control DNA
Lyophilized internal amplification control

Package Sizes

Primer sets and nucleotides are prepared in aliquots of 25 tests.

  • Cat. No. 11-1025 25 Tests
  • Cat. No. 11-1050 50 Tests
  • Cat. No. 11-1100 100 Tests
  • Cat. No. 11-1250 250 Tests

Result evaluation

Gel electrophoresis at endpoint of PCR

Required Consumables

Polymerase. We highly recommend our reliable hot-start MB Taq DNA Polymerase, Cat.-No. 53-0050/100/200/250.
PCR reaction tubes

Optional for process validation and EP 2.6.7 compliant testing:
Internal Control DNA extra (4 vials for 300 µl each of internal amplification control; Cat. No. 11-1905)
10CFU™ Sensitivity Standards available for all EP listed mycoplasma species

Required lab devices

Regular PCR cycler
Agarose gel electrophoresis and DNA staining system
Pipetting equipment
Tube centrifuge

Shelf Life and Storage

Components are maintainable at 2 to 8 °C for at least 6 months. After rehydratisation the reagents must be stored at -18 °C.

EP 2.6.7 compliance

Use of Venor®GeM Classic for QA testing of biologicals like master and working cell banks, autologous cells, culture media, bulk harvest and final product testing according to EP 2.6.7 is applicable after appropriate sample preparation and process validation.

Fig. Amplified PCR products are visualized by standard gel electrophoresis.

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Protocols

You can find your Venor®GeM Classic protocols here:

Click to download:

Venor®GeM Classic Kit Full Protocol  

alternatively,

if you are in a rush click to view:

Venor®GeM Classic Kit Quick Visual Guide

 

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References

 

Venor®GeM Mycoplasma Detection Kits

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Brinzeu D.G.T. et al., (2008). Microbial and Fungal Contamination of Keratinocyte and Fibroblast Cell Cultures. Journal of Experimental Medical & Surgical Research, 3 / 123 – 128.

Chal J. et al., (2016). Generation of human muscle fibers and satellite-like cells from human pluripotent stem cells in vitro. Nature Protocols 11(10):1833-50. doi: 10.1038/nprot.2016.110.

Dzieran J. et al., (2013). Comparative Analysis of TGF-β/Smad Signaling Dependent Cytostasis in Human Hepatocellular Carcinoma Cell Lines. PLoS One, 8(8):e72252. doi: 10.1371/journal.pone.0072252.

Falagan-Lotsch P. et al., (2015). Performance of PCR-based and bioluminescent assays for mycoplasma detection. Journal of Microbiological Methods, 118:31-6. doi: 10.1016/j.mimet.2015.08.010.

Gutzeit C. et al., (2014). Exosomes derived from Burkitt's lymphoma cell lines induce proliferation, differentiation, and class-switch recombination in B cells. Journal of Immunology, 192(12):5852-62. doi: 10.4049/jimmunol.1302068.

Henrich B. et al., (2010). Mycoplasma salivarium detected in a microbial community with Candida glabrata in the biofilm of an occluded biliary stent. Journal of Medical Microbiology, 59, 239–241. doi: 10.1099/jmm.0.013110-0.

Loring J.F., Wesselschmidt R.L., and Schwartz P.H. (2007). Human Stem Cell Manual: A Laboratory Guide. Elsevier Inc.

Maass V. et al., (2011). Sequence homologies between Mycoplasma and Chlamydia spp. lead to false-positive results in chlamydial cell cultures tested for mycoplasma contamination with a commercial PCR assay. Journal of Clinical Microbiology, 49(10):3681-2. doi: 10.1128/JCM.01092-11.

Malenovska H. and Reichelova M., (2011). Elimination of mycoplasma contamination of virus stocks. Veterinarni Medicina, 56 (11): 547–550.

Mudduluru G. et al., (2015). A Systematic Approach to Defining the microRNA Landscape in Metastasis. Cancer Research, 75(15). doi: 10.1158/0008-5472.CAN-15-0997.

Nübling C.M. et al., (2015). World Health Organization International Standard to Harmonize Assays for Detection of Mycoplasma DNA. Applied and Environmental Microbiology, 81(17):5694-702. doi: 10.1128/AEM.01150-15.

Oksvold M.P. et al., (2012). Effect of cycloheximide on epidermal growth factor receptor trafficking and signaling. FEBS Letters, 586(20):3575-81. doi: 10.1016/j.febslet.2012.08.022.

Pandrangi S.L. et al., (2014). Establishment and characterization of two primary breast cancer cell lines from young Indian breast cancer patients: mutation analysis. Cancer Cell International, 14(1):14. doi: 10.1186/1475-2867-14-14.

Panosa C. et al., (2013). Development of an Epidermal Growth Factor Derivative with EGFR Blocking Activity. PLoS One, 8(7):e69325. doi: 10.1371/journal.pone.0069325.

Pérez-Garay M. et al., (2010). 2,3-Sialyltransferase ST3Gal III Modulates Pancreatic Cancer Cell Motility and Adhesion In Vitro. PLoS One, 5(9): e12524. doi: 10.1371/journal.pone.0012524.

Picco G. et al., (2017). Loss of AXIN1 drives acquired resistance to WNT pathway blockade in colorectal cancer cells carrying RSPO3 fusions. EMBO Molecular Medicine, 9(3):293-303. doi: 10.15252/emmm.201606773.

Pietrosi G. et al., (2015). Phases I–II Matched Case-Control Study of Human Fetal Liver Cell Transplantation for Treatment of Chronic Liver Disease. Cell Transplantation, 24(8):1627-38. doi: 10.3727/ 096368914X682422.

Pontarin G. et al., (2006). Mitochondrial DNA Depletion and Thymidine Phosphate Pool Dynamics in a Cellular Model of Mitochondrial Neurogastrointestinal Encephalomyopathy. The Journal of Biological Chemistry, 281(32):22720-8. doi: 10.1074/jbc.M604498200.

Rappa G. et al., (2015). Ethanol induces upregulation of the nerve growth factor receptor CD271 in human melanoma cells via nuclear factor-κB activation. Oncology Letters, 10: 815-821. doi: 10.3892/ol.2015.3343.

Sajadian S.O. et al., (2016). Vitamin C enhances epigenetic modifications induced by 5-azacytidine and cell cycle arrest in the hepatocellular carcinoma cell lines HLE and Huh7. Clinical Epigenetics, 8:46. doi: 10.1186/s13148-016-0213-6.

Samanta D. et al., (2012). Smoking attenuates Transforming growth factor-β-mediated tumor suppression function through down-regulation of Smad3 in lung cancer. Cancer Prevention Research, 5(3): 453–463. doi:10.1158/1940-6207.

Schmitt M. et al., (2009). High-throughput detection and multiplex identification of cell contaminations. Nucleic Acids Research, 37(18):e119. doi:10.1093/nar/gkp581.

Smith C. (2005). Trouble in the hood: culturing difficult cell types. Nature Methods 2, 385–391. doi: 10.1038/nmeth0505-385

Thies A. et al., (2007). Clinically proven markers of metastasis predict metastatic spread of human melanoma cells engrafted in scid mice. British Journal of Cancer, 96(4):609-16. doi: 10.1038/sj.bjc.6603594.

Vickovic S. et al., (2016). Massive and parallel expression profiling using microarrayed single-cell sequencing. Nature Communications, 7:13182. doi: 10.1038/ncomms13182.

Waddington R.J. and Sloan A.J. (2017). Tissue Engineering and Regeneration in Dentistry – Current Strategies. John Wiley & Sons, Ltd.

Winter S. et al., (2016). Methylomes of renal cell lines and tumors or metastases differ significantly with impact on pharmacogenes. Scientific Reports, 6:29930. doi: 10.1038/srep29930.

Wolff J. et al., (2013). GMP-level adipose stem cells combined with computer-aided manufacturing to reconstruct mandibular ameloblastoma resection defects: Experience with three cases. Annals of Maxillofacial Surgery, 3(2):114-25. doi: 10.4103/2231-0746.119216.

 

 

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Notes

 

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