Detection of mycoplasmas in cell cultures etc.: Culture and PCR
When to test
When presence of mycoplasmas in cell cultures and similar products is suspected; it should be noted that most mycoplasma infections of cell lines are silent and will be revealed only by specific assays. The culture and PCR method is particularly recommended for a thorough test of cell bank material.
Advantages of this assay include increased sensitivity due to the larger sample volume tested, species identification of contaminating organisms, as well as detection of "non-cultivable" organisms due to culture in a mycoplasma-free indicator cell line (Vero).
Preparation of the cell culture before submission
Cell cultures should generally be tested at the time of change of medium. Samples should be not contain cryo-preservative and should have undergone at least 3 passages in antibiotic free media prior to submission for mycoplasma testing to achieve the highest level of assay sensitivity. It is recommended that a back-up sample is kept at -70oC or below. Please contact us for guidelines for sample preparation, packaging and shipment of the material.
Samples may be kept at +4°C for up to a week before shipment by mail. For long-term storage, the samples should be kept at -70°C or below and will keep stable for more than a year. The samples should be shipped frozen on dry ice for testing.
Ordering
- Test code: 018
- Please enclose a completed Sample Submission Form with the sample.
- We request that you contact us before you ship the samples.
Reporting
Results of the culture method will be reported as Mycoplasma detected or not detected by culture with species identification for positive cultures.
Results of the PCR method will be reported as Mycoplasma DNA detected, Mycoplasma DNA not detected, or inconclusive. Inconclusive results will be reported in case of sample material inhibiting the PCR assay.
Total testing time
Negative results will be reported after 4 weeks. Positive results will be reported as soon as detected by either culture or PCR and a final report will be issued after completion of species identification.
Interpretation of test results
Detection of mycoplasma by culture is considered the reference method. A high sensitivity is ensured by culture in several formulations of mycoplasma agar and broth media. The Vero indicator cell line method ensures amplification of low-grade infection with “non-cultivable” mycoplasma species which can subsequently be detected by PCR.
The PCR method will detect live as well as dead mycoplasmas. Borderline positive results can be seen in cell cultures grown in certain batches of medium despite negative test results by the mycoplasma culture method, probably due to remnants of mycoplasma DNA in the animal serum. In such cases a repeat test after change of the medium is advised. Heavy growth of other bacteria than mycoplasma may lead to false positive results. Such cell cultures will, however, usually be macroscopically contaminated, and mycoplasma detection is rarely needed.
Assay principle
Culture is performed by inoculation into several formulations of mycoplasma agar and broth media as well as on standard blood-agar plates incubated in CO2. The Vero indicator cell line method ensures amplification of low-grade infection with “non-cultivable” mycoplasma species which can subsequently be detected by passage into mycoplasma media and by PCR.
Mycoplasma DNA in the sample is detected by PCR. The amplified gene codes for 16S ribosomal RNA, and primers have been selected so that all Mycoplasma ssp, Ureaplasma ssp, Spiroplasma spp, and Acholeplasma spp. are detected. The detection limit is < 50 bacteria for all examined species. The validity of a negative result is ensured by simultaneous amplification of an internal amplification control.