PCR-based detection and eradication of mycoplasmal infections from various mammalian cell lines: a local experience

Abstract

A total of 200 cell lines including different human, monkey, mice, hamster and rat cell types were examined for mycoplasma infection status. PCR assay using generic-specific universal primers showed that 40 (20%) of the cell lines are contaminated with mycoplasma. Employment of species-specific primers within these infected cell lines revealed infection with M. hyorhinis (42.5%), M. fermentas (37.5%), M. arginini (37.5%), M. orale (12.5%) and A. laidlawii (7.5%). A number of the cultures were coinfected with 2 or 3 different species. Contaminated samples were treated with BM-Cyclin, Ciprofloxacin and mycoplasma removal agent (MRA). Mycoplasma eradication was subsequently checked by PCR following 2 weeks continuous culture of treated cells in antibiotic free culture medium. Mycoplasmal infections were eradicated in 100, 70 and 42% of infected cell lines when the samples were treated with BM-Cyclin, MRA and Ciprofloxacin, respectively. However, 12% (BM-Cyclin), 62.5% (MRA) and 82.5% (Ciprofloxacin) of mycoplasma regrowth was observed 4 months after the treatment. Notably, the risk of spontaneous culture death was 17.5, 12.5 and 0% for BM-Cyclin, MRA and Ciprofloxacin, respectively.

Sensitivity of biochemical test in comparison with other methods for the detection of mycoplasma contamination in human and animal cell lines stored in the National Cell Bank of Iran

“…The cell pellet was washed with ethanol (70 % v/v) and dried for 30 min at room temperature. Finally, the precipitated DNA was suspended in RNA-DNA free sterile deionized water and kept at -20°C (Molla Kazemiha et al 2009Kazemiha et al , 2011.…”

“…In order to detect the mycoplasma contamination of cell lines with the PCR method, a universal primer pair in addition to 11 species-specific primer pairs were designed based on the 16SrRNA of mollicutes according to the previously published reports (Molla Kazemiha et al 2009Kazemiha et al , 2011 (Table 2). PCR master mix was prepared by 2.5 ll PCR 10x Buffer, 1 ll dNTPs (50 lM), 3 ll forward and reverse primers (15 pmol), 1 ll Taq DNA polymerase enzyme (1 U), 1 ll magnesium chloride (1.5 mmol), 12.5 ll distilled water and 1 ll mycoplasma genomic DNA (0.1 lg/ll) or cell DNA (1 lg/ll).…”

Interference of Mycoplasma Infection in a Gene Expression Study: It Was the Environment and Not the Gene

“…It is, of course, general knowledge that regular testing of cell cultures is indispensable for good scientific practice, especially to detect insidious species that do not cause overt cytopathology. Nevertheless, even recent studies report infection rates of cell lines in the range of 15 to 35% (5,7,10). Our observations add a new perspective to the jigsaw puzzle of possible scientific errors caused by contamination of cell lines with mycoplasma and once more stress the importance of regular mycoplasma screening in tissue culture.…”

Real-time PCR assay is superior to other methods for the detection of mycoplasma contamination in the cell lines of the National Cell Bank of Iran

“…In addition to 11 species-specific primer pairs, a universal primer pair was designed based on 16S rRNA mollicutes to detect the mycoplasma contamination of cell lines with PCR method which is indicated in Table 2 (Molla Kazemiha et al 2009Kazemiha et al , 2011Kazemiha et al , 2014. Master mix of PCR was prepared by 2.5 ll PCR 10 9 Buffer, 1 ll Taq DNA polymerase enzyme (1 U), 1 ll dNTPs (50 lM), 1 ll magnesium chloride (1.5 mmol), 3 ll forward and reverse primers (15 pmol), 12.5 ll distilled water and 1 ll mycoplasma genomic DNA (0.1 lg/ll) or cell DNA (1 lg/ll).…”