Mycoplasmas, the smallest and simplest prokaryotes, can contaminate cell cultures and cost biotechnology companies lost time and reduced profit. They are a unique group of microorganisms that fall within the category between the bacteria and viruses, and are causal agents of numerous diseases of animals and humans causing contamination in eukaryotic cell cultures. The presence of mycoplasmas in cellular lines may significantly influence the results of experiments because they may affect: 1) the cell growth rate which induces morphological changes which induces chromosomal aberrations, 2) the amino and nucleic acids’ metabolism inducing cell transformation, and 3) molecular transport through the cell membrane.
Currently, European and U.S. regulations demand that all cell lines used to make recombinant therapeutic proteins are routinely tested for mycoplasma, a process that can take four to six weeks using conventional approaches such as broth/agar cultures and DNA staining of indicator cell cultures.
Fast Mycoplasma Testing Services:
MQA offers state of the art technology to provide fast mycoplasma testing so our clients can make expedited product release decisions or clear cell banks for use.
Touchdown PCR Detection Assay
Microbiology & Quality Associates (MQA) developed a new method based on Polymerase Chain Reaction (PCR) that reduces the mycoplasma screening test to 1-2 days. The MQA method is GMP validated using 6 different mycoplasma species. In extracts of mycoplasma DNA and in the presence of CHO cells with antibiotic, the assay limit of detection for six different mycoplasma species is 10 genomic copies. The assay is very specific to detect Mycoplasma in the presence of other genomic DNA. This economical service is targeted to screen research cell banks prior to using them.
ABI MycoSeq Mycoplasma RT-PCR Detection Assay
MQA offers the MycoSEQ™ Mycoplasma Detection Assay, based on real-time PCR and Power SYBR® Green detection technology, designed for the routine screening and detection of Mycoplasma and other closely related species such as Acholeplasma laidlawii and Spiroplasma citri for detection of more than 90 Mycoplasma species. This GMP assay is validated with a sensitivity to detect less than 10 copies/reaction. The MycoSEQ™ Mycoplasma Detection Assay has been approved by regulatory agencies to replace the direct and indirect culture method for product release. MQA can develop a custom validation protocol for regulatory submission.
Cell line samples should be pre-cultured in the absence of mycoplasma active antibiotics for several days to maximize test sensitivity. Samples should be derived from cultures that are at 90-100% confluence (1.0 x 106 to 1.0 x 107cells). PCR inhibiting substances may accumulate in the medium of older cultures.
Traditional Mycoplasma Testing Methods
- Mycoplasma Testing: For Human Vaccines and Biologicals Compliant with 21 CFR 610.30
- Direct Culture Method: This test consists of using predefined media specific for growing mycoplasma in broth and agar plates. The test complies with FDA and USP standards and requires up to 28 days to complete. MQA can also perform this assay to comply with EU requirements.
- DNA Fluorochrome Staining Assay with Indicator Cell Line: This DNA fluorochrome assay with inoculation on an indicator cell line enhances the level of assay sensitivity by reducing background from genetically unstable cell lines (e.g. hybridomas, etc.) and amplifies the titer of mycoplasma contaminants. Developed for the detection of mycoplasma contamination in cell cultures and related samples, this test provides reliable and timely results. This is the best test for detecting mycoplasma contamination in cell cultures and is offered as a GMP service.
Other Mycoplasma Services Include:
- Reagent standards
- Mycoplasma DNA for assay development
- Mycoplasma identification service
- Positive and Negative Control Slides for in-house development
- Mycoplasmastasis assay
|Assay Description||Service Code|
|Mycoplasma screening by Touchdown PCR||MY01|
|Mycoplasma screening by RT-PCR ABI MycoSeQ™||MY02|
|Mycoplasma screening by Direct Culture (PTC/USP/EU compliant)||MY03|
|Mycoplasma screening by DNA Fluorochrome Assay (PTC/USP/EU compliant)||MY04|
|Mycoplasma ID by 16S Ribosomal Sequencing||MY05|
|Mycoplasmastasis Assay (PTC/USP/EU compliant)||MY06|
|Titered mycoplasma stocks (CFU/mL or GC/mL)||MY07|
|Mycoplasma Media (SP4 Broth/Agar or Hayflick Broth/Agar)||MY08|