Test 131 – Mycoplasma species Multiplex Detection by Polymerase Chain Reaction
What is the best way to find out if my cells or biological materials are contaminated with Mycoplasma species?
Mycoplasma refers to a genus of bacteria that lack a cell wall with more than 120 species. Mycoplasma is the smallest known cell and is about 0.1 micron (μm) in diameter. Mycoplasmas generally possess a relatively small genome of 0.58-1.38 megabases, which results in drastically reduced biosynthetic capabilities and explains their dependence on a host. Mycoplasmas depend on sterols for the stability of their cytoplasmic membrane and they obtain sterols from the host’s cholesterol. They are known to have a low GC content in their genomes (23-40%).
The lack of a cell wall offers them drug resistance to commonly used antibiotics that inhibit cell wall synthesis such as penicillin or other beta-lactam antibiotics. Mycoplasma species have adapted over time to live as independent bacteria or parasitize the host. They have a wide host range that allows them to infect humans, animals, plants and other genera. While many species of Mycoplasma are considered commensals and are able to live in the host without causing disease or can be considered opportunistic in immunocompromised hosts, others are implicated in clinical conditions. Atypical or walking pneumonia is often caused by Mycoplasma pneumoniae or Mycoplasma hominis while non-specific urethritis is very commonly caused by Mycoplasma genitalium which is also thought to be involved in pelvic inflammatory disease. Several species are pathogenic in humans, including M. pneumoniae, which is an important cause of atypical pneumonia and other respiratory disorders, and M. genitalium, which is believed to be involved in pelvic inflammatory diseases.
Mollicutes have been reported to seriously interfere with life sciences research when cell lines or laboratory animals are infected. The bacteria can live inside and outside of the cells, deplete nutrients from the medium and change the biology of cells, disrupt metabolism, modify the immune response, inhibit or prevent viral replication or have unexplained findings in cell culture experiments.
Nocard and Roux reported the cultivation of the causative agent of Contagious Bovine Pleuro Pneumoniae (CBPP) in 1898, a disease that was widespread in cattle herds and was found to be caused by M. mycoides subsp. mycoides SC (small-colony type). In 1956, the first species of Mycoplasma were isolated in culture. The most commonly encountered species are Acholeplasma laidlawii, Mycoplasma arginini, M. orale, M. salivarium, M. fermentans, and M. hyorhinis. Species such as Ureaplasma urealyticum, M. pneumoniae and M. pirum are rarely present in cell cultures. It is anticipated that many of the cell lines created prior to 1990 were infected by Mycoplasma species that originated from contaminated animal serum and further disseminated by contaminated aerosols in the laboratory and laboratory technicians while they conducted their experiments.
A. laidlawii and M. arginini are of bovine origin; M. orale and M. salivarium are of a human origin (oropharynx) and M. hyorhinis originated from swine. It is hypothesized that presence of Mycoplasmas of swine origin in bovine serum is justified by the contamination of this product in mixed slaughterhouses.
It is estimated that as many as 30% of all cell lines generated prior to 1990 are contaminated with Mycoplasma species.
Having problems with Cell Lines contaminated with Mycoplasma? Check out our Mycoplasma Elimination Service (Protocol CB124)..Here
Invisible in cell culture but can cause serious damage!
It is always a good practice to check your cell lines on a regular basis for Mycoplasma contamination. Mycoplasma contamination could be devastating to a cell culture facility and could result in heavy costs and wasted time if not detected early. At Clongen Laboratories, we offer several ways to test cell lines (or any sample source) for the presence of Mycoplasma, Ureaplasma and Acholeplasma contamination. One of our assays is a Cell-Based Assay where the test material (cell line or other) is inoculated onto a Vero cell monolayer known to be negative for the presence of Mycoplasma, incubated for a few days, fixed and stained with e Hoechst stain (DNA fluorochrome 33258). This fluorescence pattern of DNA in the cytoplasm of indicator cells indicates the infection status of the cells. Our laboratory scientists have many years of experience in evaluating microscopic fluorescence patterns and the assay is fairly sensitive (can detect 75-150 Mycoplasmas per ml). There are rare occasions where lysed cells or artifacts in the tested material can produce a false positive readout. We recommend a confirmatory assay for positive cultures other than the cell-based assay.
We also offer a highly sensitive Multiplex PCR (Polymerase Chain Reaction) assay for the detection of low copy number. The PCR assay is capable of detecting any of the 20 or more Mycoplasma and Acholeplasma species detected in cell culture.
At Clongen Labs, we offer aerobic and anaerobic cultivation of Mycoplasma on highly enriched special media used for the cultivation of Mycoplasma. Our assays are performed under GMP conditions and if requested, we offer GLP testing for Mycoplasma (21CFR 610.30) intended mainly for vaccine products. This assay detects both the cultivable and non-cultivable Mycoplasmas using the cell-culture based technique and the enriched microbiological medium to attempt to grow Mycoplasmas. We offer Mycoplasma GLP testing (9CFR113.28) for cultivable Mycoplasmas in enriched medium and the Points to Consider 1993 testing for Virus Stocks using cell-based and enriched medium growth. Protocols will be provided to clients upon request.
Clongen Laboratories offer Mycoplamsa Elimination service that will clean up a cell line using a commercially available antibiotic cocktail (see protocol number and price at bottom of page). With this service, you will receive one PCR test at baseline for Mycoplasma presence, two PCR tests during the course of the treatment and one test five days after stopping the treatment to screen for the presence of Mycoplasma. You will receive a detailed 5-7 page report describing all work done with gel images and a certificate of quality along with a working cell bank of 25 vials at a minimum of 1 million cells per vial on dry ice.
We offer Mycoplasma testing in cell lines, biologicals and samples of human origin by PCR, cell-based fluorescence, according to 21CFR 610.30, 9CFR113.28 or the 1993 Points to Consider Guidelines To learn more about Mycoplasma Infections, Symptoms, Treatment and other related information, please visit helpful links on our website.