Detecting Mycoplasma Contamination in Cell Cultures by Polymerase Chain Reaction

Abstract

The detection of mycoplasmas in human and animal cell cultures is mandatory for every cell culture laboratory, because these bacteria are common contaminants, persist unrecognized in cell cultures for many years, and affect research results as well as the purity of cell culture products. The reliability of the mycoplasma detection depends on the sensitivity and specificity of the method and should also be convenient to be included in the basic routine of cell culture quality assessment. Polymerase chain reaction (PCR) detection is one of the acknowledged methodologies to detect mycoplasmas in cell cultures and cell culture products. Although the PCR offers a fast and simple technique to detect mycoplasmas, the method is also susceptible to errors and can produce false positive as well as false-negative results. Thus, the establishment and the routine application of the PCR assay require optimization and the inclusion of the appropriate control reactions. The presented protocol describes sample preparation, DNA extraction, PCR run, the analysis of the PCR products, and speciation of the contaminant. It also provides detailed information on how to avoid artifacts produced by the method. Established properly, PCR is a reliable, fast, and sensitive method and should be applied regularly to monitor the contamination status of cell cultures.

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

“…Therefore, interpretation of the biological tests on these cells will be affected by incorrect results. In order to increase the quality and safety of the biological products prepared in animal cell cultures, US Food and Drug Administration (FDA) strongly recommends the application of mycoplasmafree cells approved by proper detection tests (Cheng et al 2007;Dabrazhynetskaya et al 2011;Huang et al 2008;Uphoff and Drexler 2011). It has been reported (in different studies) that between 5 and 87 % of the cell lines have been contaminated by mycoplasmas.…”

Knockdown of microRNA-1323 restores sensitivity to radiation by suppression of PRKDC activity in radiation-resistant lung cancer cells

“…Cell lines in the maximal range of up to 20 passages were used for the present study. All cell lines tested negative for mycoplasma contamination by polymer chain reaction (PCR) methods (16). Cell lines were authenticated using the short tandem repeats (STR) testing.…”

DNA-dependent protein kinase catalytic subunit inhibitor reverses acquired radioresistance in lung adenocarcinoma by suppressing DNA repair

“…A maximum of 20 passages were used. To avoid mycoplasma contamination, the cell line was assessed monthly using polymerase chain reaction (PCR) (23). For PCR, the ATCC Universal Mycoplasma Detection Kit (ATCC) was used.…”