In-house abbreviated qualification of a real-time polymerase chain reaction method and strategies to amplify mycoplasma detection in human mesenchymal stromal cells

Mathews, Smitha; Rabani, R.; Rasti, Mozhgan; Viswanathan, Sowmya

doi:10.1016/j.jcyt.2021.07.002

Cited by 1 publication

(1 citation statement)

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“…Our experiment detected a minimum of 10 CFU/mL for the nine species tested, with Cq values between 29.4 for S. citri and 36.74 for M. pneumoniae and M. arginini . Other PCR tests have been previously evaluated and proven to be sensitive to at least 10 CFU/mL for detecting Mycoplasma in culture supernatants [ 30 ], while the sensitivity of other published assays has been comparatively lower [ 22 , 31 , 32 ].…”

Section: Discussionmentioning

confidence: 99%

Development and Evaluation of a New qPCR Assay for the Detection of Mycoplasma in Cell Cultures

Carrillo-Ávila,

de la Fuente,

Aguilar-Quesada

et al. 2023

CIMB

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In recent years, cell culture has become an important tool not only in research laboratories, but also in diagnostic and biotechnological development laboratories. Mycoplasma 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 Mycoplasma 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 Mycoplasma species that contaminate cell cultures. Whilst 11 of 45 tested supernatants were positive for Mycoplasma (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®GeM qEP and Plasmotest® kit. In addition, the new PCR method exhibits a high capacity to detect less-frequent Mycoplasma species, such as those related to the M. mycoides cluster. The use of an alternative Mycoplasma-detection method in cell culture labs can guarantee the detection of Mycoplasma contamination, especially in cases when dubious results are recorded.

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