White paper
An ounce of prevention
How to protect your cells
with fast and accurate
mycoplasma screening
White paper TaKaRa Mycoplasma qPCR Detection Kit
For cell culture-based research, rigorously ensuring the health and quality of each cell line
is essential for the success of downstream experiments. For over a decade, mycoplasma—
a widespread and notoriously undetectable cell culture contaminant—has gained recognition
as one of the greatest challenges for scientists performing cell culture.
Introduction
The term “mycoplasma” is colloquially used to facultative anaerobes, mycoplasma can survive a
describe any bacteria of the class Mollicutes that infect broad range of temperatures, oxygen levels, and other
humans, several animal species, and cultured cells harsh conditions, including exposure to liquid nitrogen
in laboratories. Mycoplasma infection in cell lines is commonly used for cell cryopreservation. Mycoplasma
remarkably common, with an estimated 62% of cell infections are unusually problematic to identify. Infected
cultures infected worldwide (Kazemiha et al. 2016). cultures do not produce any clear visual indicator such
While over 100 species of mycoplasma exist, only as color or turbidity change in culture media (Young,
eight species (M. arginini, M. fermentans, M. orale, Sung, and Masters 2010), enabling mycoplasma to
M. hyorhinis, M. hominis, M. salivarium, M. pirum thrive in cell cultures for extended time periods while
and Acholeplasma laidlawii) account for >95% of cell escaping detection.
line contaminations (Nikfarjam and Farzaneh 2012).
Surprisingly, this high infection rate is largely due to the In addition to being resilient and elusive, mycoplasma is
highly advantageous adaptations mycoplasma have also relatively ubiquitous. Cell culture contaminations
evolved and not to low-sterility laboratory practices. can originate from many sources, including cells from
Due to their minute size (300–800 nm diameter) and collaborators or commercial suppliers, improperly
absence of a cell wall, mycoplasma is capable of sterilized laboratory equipment, airborne particles from
easily passing through standard 0.2 µm media filter nearby surfaces or lab staff. As mycoplasma naturally
membranes and spreading by touch or aerosol from infects many plant and animal species, serums and
relatively distant contaminated surfaces. As robust supplements derived from infected animals can also
harbor mycoplasma contaminants.
Mycoplasma contaminations can arise from a variety of sources
TaKaRa Mycoplasma qPCR Detection Kit white paper 2 takarabio.com
White paper TaKaRa Mycoplasma qPCR Detection Kit
Impact of mycoplasma infection
Mycoplasma infection can severely impact laboratories,
cores, and companies alike. Cells can be adversely
affected by mycoplasma contamination in many ways,
including altered proliferation, survival, morphology,
gene expression, and functional characteristics.
As the number and severity of symptoms manifest
inconsistently, data produced from mycoplasma-
infected cells are considered unreliable. Research
predicated upon those results must be discarded
or—if already published—retracted. The potential
disruptions mycoplasma can cause are wide-
reaching. In the biopharmaceutical and biotechnology
industries, contaminations can lead to significant
financial losses and production delays, including costly
decontamination efforts and potential product recalls,
as well as compromised research data and regulatory
setbacks. Mycoplasma infection compromises the
quality and safety of cell-generated biological products
and undermines the validity of research studies or
drug screens conducted with infected cells (Armstrong,
Mariano, and Lundin 2010; Baronti et al. 2013). In
addition to wasting current batches of cells or products,
future productions must be paused until affected cell
culture facilities undergo thorough (and expensive)
decontamination. Biohazard decontamination can
cost between $1,500 and $5,000, with potentially
higher costs for larger facilities and more severe
contaminations (Carlson 2024). For laboratories and
companies dealing with mycoplasma contamination,
an ounce of prevention is worth more than a pound of
precious data, labor, time, and money.
Options to limit mycoplasma infections in cell culture
are scant and often inadequate. Post-infection
treatments with antibiotics are rarely successful, as
its lack of cell wall renders mycoplasma resistant to
most antibiotics (Lanao, Chakraborty, and Pearson-
Shaver 2023). Treating cells is recommended solely
as an attempt to salvage irreplaceable samples, and
cells undergoing treatment must be maintained in
strict quarantine to avoid spreading infection. In most
Mycoplasma infection of cell cultures can severely impact
cases, it is highly recommended to promptly dispose laboratories, cores, and companies. Any product generated
of infected cultures before they can infect other cells. from infected cultures must be discarded, potentially resulting
Timely identification and disposal of infected cells is in extensive losses of profits, data, time, labor, and more.
crucial to limit spread. Preventative measures like
sterile technique, UV irradiation, and quarantining can
reduce the risk of infection, but mycoplasma is robust
6
and pervasive—one mycoplasma cell can grow to 1 x 10 As a result, the most successful method to deal with
colony forming units per ml within three to five days mycoplasma is to regularly screen cell lines to identify
in an infected cell culture (Drexler and Uphoff 2002). and isolate potential infections as quickly as possible.
TaKaRa Mycoplasma qPCR Detection Kit white paper 3 takarabio.com
White paper TaKaRa Mycoplasma qPCR Detection Kit
Frequency and methods of mycoplasma testing
Mycoplasma testing of cell lines should be performed cell lines should immediately undergo mycoplasma
every 6 months at a minimum, with additional testing testing, remaining isolated from other cell lines until
recommended preceding large-scale experiments or deemed negative for infection.
upon receipt of new cell lines (Cell Culture Services -
Penn Genetics 2024). Cell banks are highly susceptible Considering the high impact of infection, mycoplasma
to mycoplasma spread—one study showed up to 87% screening methods must be rapid enough for effective
of cell lines in different cell banks were infected with decision-making, sensitive enough to detect low levels
mycoplasma (Kazemiha et al. 2016). As such, cells of contamination, and robust enough that results are
should always be purchased from trusted facilities with conclusive. Currently, three standard methods are
mycoplasma-free certification, and newly received primarily used to test for mycoplasma: culture testing,
DNA staining, and PCR-based detection (CDC 2024).
Comparing methods of mycoplasma detection
Culture testing DNA staining PCR-based detection
Bacterial culture media is inoculated Confluent cells are fixed and stained Mycoplasma-specific gene targets are
with cell culture samples and incubated with Hoechst or DAPI to mark nuclei. selectively amplified from cell culture
on a mycoplasma agar plate for four Upon examination with a fluorescence samples and subsequently run on a gel.
to five weeks. Samples that produce microscope, filamentous staining indicates The presence of a band of amplified
a colony indicate a positive result. a positive result. This method is fast bacterial DNA indicates a positive result.
This method is simple and definitive, and convenient but lacks the sensitivity This method is both definitive and fast,
but has an incredibly long turnaround to definitively detect infections in low- taking less than a day to generate results.
time, which reduces the relevance contamination cultures. Relying on PCR-based mycoplasma tests usually
dependability of a negative mycoplasma subjective morphological assessment cost around $20 to $30 per sample
test result, wastes valuable culture of polymorphic mycoplasma, DNA to run, making it the most affordable
resources during the accompanying staining analysis is used mainly for option of the three conventional
cell quarantine period, and precludes initial screening or in tandem with other methods. Still, the necessity of running
timely decision-making to limit infection detection methods. While costs for DNA gel electrophoresis post-PCR can
spread. The long incubation period also staining detection appear relatively low, increase sample contamination risk and
leads to high direct costs for culture- averaging $200 to $1,000 per analysis, reduces quantification sensitivity.
based detection, ranging from $400 initial equipment investment and ongoing
to $2,000 per test. training fees can be significant.
Cost Cost Cost
$400–$2,000/test $200–$1,000/test $20–$30/test
Timeline Timeline Timeline
4–5 weeks <1 day <1 day
Reliability Reliability Reliability
100% 50% 98%
Slower than PCR Less consistent than PCR
Of these three conventional options, PCR-based specificity, and accuracy compared to conventional
detection has historically proven to be the most PCR (Kazemiha et al. 2016). Without the need for gel
reliable, fast, and sensitive method for mycoplasma electrophoresis, qPCR also offers a more streamlined
detection (Uphoff and Drexler 2011). However, recent and cost-effective workflow, making it an ideal option
years have seen the emergence of an additional option for repeated routine screening. For facilities seeking
for mycoplasma detection—real-time PCR/qPCR. simple and rigorous preventative mycoplasma
Studies have shown that qPCR-based mycoplasma screening, consider the TaKaRa Mycoplasma qPCR
tests demonstrate additional detection sensitivity, Detection Kit today.
TaKaRa Mycoplasma qPCR Detection Kit white paper 4 takarabio.com
White paper TaKaRa Mycoplasma qPCR Detection Kit
References
Armstrong, S. E., Mariano, J. A. & Lundin, D. J. Kazemiha, V. M. et al. Real-time PCR assay is superior
The scope of mycoplasma contamination within to other methods for the detection of mycoplasma
the biopharmaceutical industry. Biologicals 38, contamination in the cell lines of the National Cell Bank
211–213 (2010). of Iran. Cytotechnology 68, 1063–80 (2016).
Baronti, C., Pastorino, B., Charrel, R. & de Lamballerie, X. Lanao, A. E., Chakraborty, R. K. & Pearson-Shaver, A. L.
Mycoplasma removal: Simple curative methods for Mycoplasma Infections. (StatPearls Publishing, 2023).
viral supernatants. J. Virol. Methods 187, 234–237 (2013). at <https://www.ncbi.nlm.nih.gov/books/NBK536927/>
Carlson, J. Average cost of biohazard cleanup. Nikfarjam, L. & Farzaneh, P. Prevention and Detection
HomeGuide (2024). at <https://homeguide.com/costs of Mycoplasma Contamination in CellCulture. Cell J.
biohazard-cleanup-cost#:~:text=Biohazard cleanup costs 13, 203 (2012).
%2425 to,level and type of contaminants.>
Uphoff, C. C. & Drexler, H. G. Detecting mycoplasma
CDC. Laboratory Testing for Mycoplasma pneumoniae contamination in cell cultures by polymerase chain
| M. pneumoniae | CDC. at <https://www.cdc.gov/ reaction. Methods Mol. Biol. 731, 93–103 (2011).
mycoplasma/php/laboratories/index.html>
Young, L., Sung, J. & Masters, J. R. Detection of
Cell Culture Services - Penn Genetics. at <https:// Mycoplasma in cell cultures. Nat. Protoc. 2010 55 5,
genetics.med.upenn.edu/cores/cell-center-services/ 929–934 (2010).
services/cell-culture-services/>
Drexler, H. G. & Uphoff, C. C. Mycoplasma contamination
of cell cultures: Incidence, sources, effects, detection,
elimination, prevention. Cytotechnology 39, 75–90 (2002).
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