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A sensible technique to detect mollicutes impurities in human cells cultured in GMP condition
Elisabetta Ugolotti1, Irene Vanni
1Molecular Medicine, Istituto Giannina Gaslini, Largo G. Gaslini 5, Genova, 16147, Italy, elisabettaugolotti@ospedale-gaslini.ge.it.
Abstract:
In therapeutic trials the use of manipulated cell cultures for clinical applications is often required. Mollicutes microorganism contamination of tissue cultures is a major problem because it can determine various and severe alterations in cellular function. Thus methods able to detect and trace cell cultures with Mollicutes contamination are needed in the monitoring of cells grown under good manufacturing practice conditions, and cell lines in continuous culture must be tested at regular intervals. We here describe a multiplex quantitative polymerase chain reaction assay able to detect contaminant Mollicutes species in a single-tube reaction through analysis of 16S-23S rRNA intergenic spacer regions and Tuf and P1 cytoadhesin genes. The method shows a sensitivity, specificity, and robustness comparable with the culture and the indicator cell culture as required by the European Pharmacopoeia guidelines and was validated following International Conference on Harmonization guidelines and Food and Drug Administration requirements.
Insights
A new multiplex quantitative polymerase chain reaction (qPCR) assay effectively detects Mollicutes contamination in cell cultures. This method ensures the safety and reliability of cell-based therapies and manufactured biological products.
Area of Science:
- Microbiology
- Cell Biology
- Molecular Diagnostics
Background:
- Cell cultures are crucial for therapeutic trials and clinical applications.
- Mollicutes contamination significantly alters cellular function and compromises cell culture integrity.
- Reliable detection methods are essential for monitoring cell cultures under Good Manufacturing Practice (GMP).
Purpose of the Study:
- To develop and validate a sensitive and specific assay for detecting Mollicutes contamination in cell cultures.
- To provide a robust method for routine testing of cell lines in continuous culture.
- To ensure the quality and safety of cells used in therapeutic applications.
Main Methods:
- A multiplex quantitative polymerase chain reaction (qPCR) assay was designed.
- The assay targets 16S-23S rRNA intergenic spacer regions and Tuf and P1 cytoadhesin genes.
- Detection is performed in a single-tube reaction for efficiency.
Main Results:
- The developed multiplex qPCR assay demonstrated high sensitivity, specificity, and robustness.
- Performance was comparable to traditional culture methods and indicator cell culture assays.
- The assay meets European Pharmacopoeia guidelines for Mollicutes detection.
Conclusions:
- The multiplex qPCR assay is a reliable tool for detecting Mollicutes contamination in cell cultures.
- This method supports GMP compliance and ensures the quality of cell-based products.
- Validation followed International Conference on Harmonization (ICH) and Food and Drug Administration (FDA) requirements.
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