Real-time qPCR Mycoplasma Contamination Detection Service

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Labcorp's  cell line testing division offers a reliable, quick and economically  priced real-time qPCR test for routine detection of contaminating Mycoplasma species in your continuous cell lines.

Appropriate positive and negative controls are included in every assay to ensure the validity of the test results.

Venor® GeM qEP (Minerva Biolabs):  Detection of at least 107 Mycoplasma species including the most common cell culture contaminants:  Mycoplasma  orale, M. hyorhinis, M. arginini, M. fermentans, M. salivarium, M.  hominis M. pneumoniae, Acholeplasma laidlawii, M. synoviae and M. Urea; detection of the highly conserved Mycoplasma 16S rRNA sequences present in genomic DNA samples with gene specific primers

• Highly sensitive:  Limit of detection determined to be equivalent to (1-2) Mycoplasma genomes
• Rapid results:  Results typically within 3-5 business days
• Easy sample submission:  send one sample for both mycoplasma and cell line authentication (STR profiling) either as a cell pellet or genomic DNA
• Internal control included to identify false negative results
• Cross Reactivity:  None with other bacterial and mammalian DNA
• Recommended for:  Continuous cell cultures
• Cell line species accepted:  All species
  

Price:  $48/sample  (DNA extraction charge will apply if sending cell pellets for myco only testing)

NOTE:  Mycoplasma detection services are FOR RESEARCH ONLY; not for clinical diagnostic purposes.

What are Mycoplasmas? 

Mycoplasmas  have become one of the most widespread and severe contaminants within  cell culture systems in research institutions.  Mycoplasma is considered  the smallest free-living organism capable of self-replication.  They  can be as small as 0.2-0.3µm in diameter, lack a cell wall, and are  known to freely pass through some filters used to remove bacteria from  cell culture.

How can I tell if my culture is contaminated with Mycoplasma?

Mycoplasma can attain high densities in cell cultures (i.e. 107 - 108mycoplasma/ml)  without ANY noticeable changes to the pH of the medium and without  causing increased turbidity.  Typically there is an absence of visible  morphological changes.  Mycoplasma contamination cannot be detected  using bright-field or phase microscopy, even at high magnification.  In  other words, you do not know mycoplasma is contaminating the cell  culture without testing.

How common is Mycoplasma contamination in cell culture systems?

Estimates  vary but research studies performed by different groups have shown  anywhere from 5-35% of all cell cultures are contaminated with  mycoplasma; the real percentage may be even higher because many  researchers don't know that their cultures are contaminated.  If other  types of bacterial contamination are a common occurrence in your lab,  you most likely have mycoplasma contamination as well and just may not  know it.

Why is Mycoplasma contamination bad for cell culture?

Due  to the typically high concentrations of mycoplasma in infected  cultures, mycoplasma often out-compete the host cells for essential  nutrients resulting in altered growth and protein production.  

Mycoplasma contamination has been shown to alter almost every cell culture property and characteristic measured including:

• Altered DNA, RNA, and protein synthesis
• Lowered ATP levels
• Altered enzyme expression and activity
• Variable growth rates and viability
• Nucleic acid synthesis which leads to aneuploidy and other chromosomal irregularities
• Cytotoxicity
• Decreased transfection efficiency and 
• Culture starvation. 
  

As  with using a misidentified cell line, using cell cultures contaminated  with mycoplasma calls into question the validity of the experiments  using that cell line.

I use antibiotics in my medium, shouldn't that take care of the Mycoplasma contamination?

No!  Actually, mycoplasma contamination rates are much higher than average  for cell lines that are routinely grown in medium containing antibiotics  (72% were contaminated) versus no antibiotics in the medium (7%  contaminated) (Barile, et al 1973).  This is due to the  mycoplasma developing partial or complete antibiotic-resistance to  antibiotics such as penicillin and streptomycin.  In medium containing  antibiotics, other microbial contaminants disappear allowing plenty of  room for antibiotic resistant mycoplasma to grow and spread through the  culture undetected.

How often should I be testing for Mycoplasma?

Just like human cell line authentication or mouse cell line authentication,  testing for mycoplasma should occur at a regular basis while the cell  culture is actively growing.  It is recommended that you test for  mycoplasma contamination, as applicable:

• When creating a master cell bank OR working cell bank
• Before beginning a new series of experiments
• When the cell line is not behaving as expected
• Every 2 months that the cell culture is actively growing
• Before publication of experimental data
  

These  steps are crucial to the success and repeatability of the research and  to the acceptance of the research in the scientific community.

•  Armstrong,  S.E., Mariano, J.A., and D.J. Lundin.  "The scope of myocplasma  contamination within the biopharmaceutical industry", Biologicals 38  (2010) 211-213.
• Barile,  M.F., et al. "The identification and sources of mycoplasmas isolated  from contaminated cell cultures".  Ann. N.Y. Acad. Sci.  225 (1973)  251-264.
• Young,  L., Sung, J., Stacey, G, and John R. Masters.  "Detection of Mycoplasma  in cell cultures", Nature Protocols, Vol. 5, No.5, (2010) 929-934.
• Venor® GeM qEP kit - Product Insert

•  We  recommend that you passage the cell line for 3-6 days (or 1-2 passages)  without antibiotics to get the best result (antibiotics can suppress a  mycoplasma contamination but not eliminate it).
• You can submit the SAME sample types as for the cell line authentication testing.  
• We can use ONE sample for both STR profiling (cell line authentication) AND qPCR mycoplasma testing; same minimum volume/cell requirements as for cell line authentication testing.