title: A sensible technique to detect mollicutes impurities in human cells cultured in GMP condition.
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.
authors.affiliation: Molecular Medicine, Istituto Giannina Gaslini, Largo G. Gaslini 5, Genova, 16147, Italy, elisabettaugolotti@ospedale-gaslini.ge.it.
authors.forename: Elisabetta
authors.initials: E
authors.lastname: Ugolotti
authors.forename: Irene
authors.initials: I
authors.lastname: Vanni
mesh_terms: D018929:Cell Culture Techniques / Q000295:instrumentation / Q000379:methods* / Q000592:standards; D004269:DNA, Bacterial / Q000032:analysis / Q000235:genetics / Q000302:isolation & purification; D004866:Equipment Contamination; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*; D008973:Tenericutes / Q000235:genetics* / Q000302:isolation & purification*
chemical_list: D004269:DNA, Bacterial