%0 Journal Article
%@ 1660-3796 (Druck-Ausg.), 1660-3818 (Online-Ausg.)
%A Janetzko, Karin
%A Rink, Gabi
%A Hecker, Andrea
%A Bieback, Karen
%A Klüter, Harald
%A Bugert, Peter
%C Basel ; Freiburg
%D 2014
%F heidok:27237
%I Karger
%J Transfusion Medicine and Hemotherapy
%K cell culture , cell therapeutics , mycoplasma , mollicutes , quality control
%N 1
%P 83-89
%R 10.11588/heidok.00027237
%T A Single-Tube Real-Time PCR Assay for Mycoplasma Detection as a Routine Quality Control of Cell Therapeutics
%U https://archiv.ub.uni-heidelberg.de/volltextserver/27237/
%V 41
%X BACKGROUND: Contamination of cell culture and biological material by mollicute species is an important safety issue and requires testing. We have developed a singletube real-time polymerase chain reaction (PCR) assay for rapid detection of Mollicutes species stipulated by the European Pharmacopeia.     METHODS: Primers and TaqMan probes (FAM- abeled) were deduced from 16S rDNA sequence alignment of 18 mollicutes species. A synthetic internal control (IC) DNA and an IC-specific TaqMan probe (VIC-labeled) were included. The analytical sensitivity of the assay was determined on DNA dilutions from 12 mollicute strains. Specificity was proven by the use of DNA from other bacteria.     RESULTS: Analytical sensitivities of the PCR assay were in the range of 405–2,431 genomes/ml for 11 of the 12 tested mollicute DNA samples. The lowest sensitivity was found for Ureaplasma urealyticum (19,239 genomes/ml). Negative results for DNA samples from 3 different ubiquitous bacteria demonstrated the specificity of the PCR assay for Mollicutes. Direct testing of cell culture supernatants spiked with Mycoplasma orale revealed similar sensitivity compared to isolated DNA.     CONCLUSION: Our single-tube real-time PCR assay with internal reaction control enables rapid and specific detection of mollicute contaminants. The test protocol is suitable for routine quality control of cell therapeutics.
%Z Dieser Beitrag ist aufgrund einer (DFG-geförderten) Allianz bzw. Nationallizenz frei zugänglich.    This publication is freely accessible due to an Alliance licence and a national licence (funded by the DFG, German Research Foundation) respectively.