Development and Evaluation of a New qPCR Assay for the Detection of <i>Mycoplasma</i> in Cell Cultures
2023
José A. Carrillo-Ávila | Amanda de la Fuente | Rocío Aguilar-Quesada | Gertrudis Ligero | Juan Manuel del Río-Ortiz | Purificación Catalina
In recent years, cell culture has become an important tool not only in research laboratories, but also in diagnostic and biotechnological development laboratories. <i>Mycoplasma</i> contamination is present in up to 35% of cell cultures used in research and in cell therapies. This fact represents a significant problem since such contamination can cause disastrous effects on eukaryotic cells by altering their cellular parameters, which, in turn, can lead to unreliable experimental results. For this reason, it is mandatory to carry out continuous testing for the presence of <i>Mycoplasma</i> in cell culture and the development of appropriate methodologies for this purpose. An ideal detection methodology should be fast, sensitive, and reliable. In this study, we propose an alternative detection method based on real-time PCR in conjunction with a novel combination of primers and probes that have been improved to increase their efficiency. The new PCR method demonstrates 100% sensitivity and specificity results in the detection of common <i>Mycoplasma</i> species that contaminate cell cultures. Whilst 11 of 45 tested supernatants were positive for <i>Mycoplasma</i> (24.4%) using the new PCR method (corresponding to 5 of the 14 lines tested (35.71%)), only 10 of 45 supernatants showed positive results with the commercial Venor<sup>®</sup>GeM qEP and Plasmotest<sup>®</sup> kit. In addition, the new PCR method exhibits a high capacity to detect less-frequent <i>Mycoplasma</i> species, such as those related to the <i>M. mycoides</i> cluster. The use of an alternative <i>Mycoplasma</i>-detection method in cell culture labs can guarantee the detection of <i>Mycoplasma</i> contamination, especially in cases when dubious results are recorded.
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