Detection of mycoplasmas in cell cultures etc.: 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 method is recommended for regular screening and for screening of cell cultures before they are accepted in the laboratory.

Preparation of the cell culture before submission

Cell cultures should generally be tested at the time of change of medium. It is not necessary to passage the cell culture without antibiotics before testing.

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 -20°C or below and will keep stable for more than a year. The samples should be shipped frozen or immediately after thawing.

Sample preparation

a. Cells growing in suspension:

1 - 10 ml of the cell-suspension should be collected at the time of medium change.

b. Adhering cells:

At the time of medium change, the adhering cells are harvested by scraping into the spent medium, and 1-10 ml of the cell-suspension is collected.

Other types of specimens can be tested after previous consultation with the laboratory. Please note: Samples exceeding BSL 2 or containing HIV are NOT accepted.

Packaging

The cell suspension should be shipped in containers that can be firmly closed, and for shipment by mail, dual casing with absorbant material wrapping the tube with the cell-suspension is required. 

Note. In case of leakage during shipment SSI will ask for a new sample. Samples that show signs of leakage will not be tested.

Ordering

Reporting

Results 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

The analysis is performed twice a week. A negative result will be reported on the first working-day after testing by mail, fax or e-mail as requested.

Positive test results will be confirmed after repeated extraction of the sample and a secondary confirmatory PCR assay on the next working-day. A test report will be sent immediately after the result of the confirmatory assay is available.

Samples with inconclusive results at the initial test will be repeated on the following working-day.

Interpretation of test results

The 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

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.

Accreditation

Testing performed according to the current list of test methods, cf. www.danak.dk.

The company complies with the criteria in EN ISO/IEC 17025:2005 – “General requirements for the competence of testing and calibration laboratories” and demonstrates technical competence for the defined scope and the operation of a quality management system in general compliance with the principles in EN ISO 9001 (refer joint ISO-ILAC-IAF Communiué dated April 2017 document).