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Mycoplasma
Facts:
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- 35% of cell cultures all over the world are contaminated with
mycoplasma1, and 80% in some
countries2
- three major sources of mycoplasma: infected lab personnel, infected cells, infected media
- 80% of lab technician’s throat swabs are mycoplasma
positive3, Human-sourced species account for 50% of contaminated
cultures1
- sneeze, cough and pipetting can transmit mycoplasma easily
- present on lab technician’s hands and lab equipment
- survive on
the surface of laminar flow hood for 6 days
- do NOT cause visible changes of growth medium in turbidity and pH (medium color)
- do NOT kill the cells outright
- infected
cell still looks healthy and has unapparent change of growth rate and cell morphology, but hundreds of host protein expressions change and NFkB signaling pathway is activated
- invisible under microscope even at concentration more than
107 copies/ml
- resistant to
penicillin/streptomycin and other common antibiotics
- can NOT be removed by 0.2um filter “sterilization”
- a
single contaminated culture can quickly infect other cultures in the laboratory
- once detected, the best solution to eliminate mycoplasma is to discard the contaminated cells
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| Good habits to minimize mycoplasma
contamination: |
- follow stringent aseptic culture techniques
- do
NOT sneeze, cough and loudly talk in cell culture area
- sterilize hood surface every time (70% ethanol)
- always wear glove
- work with only one cell culture at a time
- designate separate media and reagents for each individual cell line
- sterilize lab equipment
(hemacytometer and disposal trays) routinely
- test mycoplasma when thawing a new cells
- test mycoplasma when obtaining a cells from outside
- test mycoplasma every 3 months
- test every month if sharing hood space or
CO2 space with other
person
- experiment results
are not consistent/reproducible
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Challenge
of Mycopolasma
detection:
-----A simple, sensitive and quick tool is unavailable |
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Mycoplasma has long been recognized as common contaminations of cells culture, but infected cells may go undetected even for years. Among the available mycoplasma detection methods (broth culture, fluorescence staining, bioluminescence, ELISA, and
PCR), PCR is the highly sensitive, specific and convenient method4. It is chosen by most of mycoplasma detection kits, such as: MycoSEQ (Thermo-Fisher), MycoQuick
(Systembio), LookOut (Sigma), VenorGem (Sigma), Universal (ATCC), etc. However, all of these PCR kits need DNA extraction or sample preparation.
Post-PCR gel electrophoresis is also required except MycoSEQ (real time
PCR). Therefore, all of the current kits have until now been a long and laborious process.
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1.
Drexler HG, Uphof CC (2002) Mycoplasma contamination of
celcultures: Incidence, sources, effects, detection,
elimination, prevention. Cytotechnology 39: 75–90
2. Koshimizu K, Kotani H (1981). In: Procedures for the
Isolation and Identification of Human, Animal and
Plant Mycoplasmas (Nakamura, M., ed.), Saikon, Tokyo,
87–102.
3. McGarrity GJ. (1976) Spread and control of
mycoplasmal infection of cell cultures. In Vitro.
12(9):643-8.
4. Young L, Sung J, Stacey G, Masters JR. (2110)Detection of
Mycoplasma in cell cultures. Nat Protoc.
5(5):929-34.
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