Mycoplasma Contamination Of Cell Lines Research Articles

A highly sensitive internally-controlled real-time PCR assay for mycoplasma detection in cell cultures

Mycoplasma contamination of cell lines is a common occurrence and may affect the cell line behaviour in a variety of ways. Contamination with mycoplasma is usually not obvious so cell lines should be frequently tested. Several commercially available kits for mycoplasma detection exist, however the Ph. Eur. culture method which can take several weeks to yield results is still considered to be the ‘gold standard’ method. There is therefore a need for rapid alternative methods with comparable sensitivity, specificity and species range. Here, we describe an internally-controlled Taqman-based real-time PCR assay for cell culture medium without the need for DNA extraction. The assay can detect less than 10 CFU of the most frequently encountered mycoplasma contaminants in mammalian cell cultures. The validated assay has the potential to be used as a routine test in the production of cell culture-based Biologicals.

Detection of Mycoplasma contamination in cell cultures.

Mycoplasma contamination of cell lines is a major problem in cell culture technology. This unit presents protocols involving either the polymerase chain reaction (PCR) or fluorescent in situ hybridization (FISH) to provide independent, fast, and sensitive techniques to monitor mycoplasma contamination in laboratory cultures. Special emphasis is placed on the integration of control reactions to prevent false-negative as well as false-positive results due to reaction inhibition or contamination and background staining, respectively.

Detection of mycoplasma contaminations.

Mycoplasma contamination of cell lines is one of the major problems in cell culture technology. The specific, sensitive, and reliable detection of mycoplasma contamination is an important part of mycoplasma control and should be an established method in every cell culture laboratory. New cell lines as well as cell lines in continuous culture must be tested in regular intervals. The polymerase chain reaction (PCR) methodology offers a fast and sensitive technique to monitor all cultures in a laboratory. The technique can also be used to determine the contaminating mycoplasma species. The described assay can be performed within 3 h, including sample preparation, DNA extraction, performing the PCR reaction, and analysis of the PCR products. Special precautions necessary to avoid false-negative results resulting from inhibitors of the Taq polymerase present in the crude samples and the interpretation of the results are also described.

Application of a Mycoplasma group-specific PCR for monitoring decontamination of Mycoplasma-infected Chlamydia sp. strains

Mycoplasma contamination of biological materials remains a major problem. Most contaminations are caused by the use of Mycoplasma-contaminated cell lines. We adapted a Mycoplasma group-specific PCR to detect Mycoplasma contamination in cell lines and demonstrate its use in monitoring decontamination procedures with Mycoplasma-contaminated suspensions of Chlamydia spp. Three different methods were investigated: the use of Mycoplasma-specific antiserum in cell culture, physical separation by the combined use of enzymatic treatment and differential centrifugation, and the use of detergents. With these methods only incubation with Triton X-100 resulted in decontamination of Mycoplasma-contaminated suspensions of several laboratory strains of Chlamydia pneumoniae, C. pecorum, and C. trachomatis. Only one C. pneumoniae strain, UZG-1, was sensitive to Triton X-100 treatment. Since 39 of 40 throat swabs from patients with symptoms of an upper respiratory tract infection had positive reactions in the Mycoplasma group-specific PCR, this procedure could also have clinical significance in attempts to propagate C. pneumoniae strains from clinical specimens.

Specifity and sensitivity of polymerase chain reaction (PCR) in comparison with other methods for the detection of mycoplasma contamination in cell lines

The polymerase chain reaction (PCR) amplification was used for the detection of mycoplasma contamination in 42 continuous cell lines. Using the microbiological cultivation on agar as the reference method, 29 cell lines were regarded as positive and 13 cell lines as negative. The double-step PCR analysis employed nested primers that anneal to gene sequences coding for the evolutionarily conserved 16 S rRNA of some 25 different mycoplasma species (including the ones most commonly found in cell cultures). In terms of the positivity or negativity of mycoplasma infection the results were identical for the agar assay and PCR amplification. All positive cell lines displayed distinct, unequivocal, objectively discernible bands on agarose gels while the non-infected specimens showed no DNA amplification. A simultaneously performed comparison with four other commonly used detection methods (DNA-RNA hybridization in solution, DAPI DNA fluorescence staining, immunostaining with a monoclonal antibody and an ELISA) showed that PCR produced significantly less false-negative or false-positive results than all the other methods. Furthermore, in dilution experiments, PCR correctly detected the infecting mycoplasmas at the lowest level of 1/10 4 whereas the other assays were less sensitive. It is concluded that double-step PCR employing nested primers is superior to other mycoplasma detection methods in many respects: simplicity and speed, high specificity and extreme sensitivity, objectivity and accuracy.

Isolation of Mycoplasmas from tissue cell lines and transplantable tumor cells.

A survey was made of mycoplasma contamination in cell lines and transplantable tumor lines maintained in laboratories in Japan. A total of 60 cell lines were tested and 52 (86.7%) of which were contaminated by mycoplasmas. These mycoplasmas were isolated from the cell lines of human origin 10/13 (77%), from monkeys 3/3 (100%), from rats 14/19 (74%) and from mice 25/25 (100%). Mycoplasmas were not found in any of the 99 samples of tumor materials. Mycoplasma colonies, upon primary isolation from cell lines, could be differentiated into three morphological types. However, all colonial types showed similar biological properties. All but one of the isolated mycoplasmas could be identified as Mycoplasma hominis type 1 by both growth inhibition tests and by fluorescent antibody technique.