title: A Single-Tube Real-Time PCR Assay for Mycoplasma Detection as a Routine Quality Control of Cell Therapeutics
creator: Janetzko, Karin
creator: Rink, Gabi
creator: Hecker, Andrea
creator: Bieback, Karen
creator: Klüter, Harald
creator: Bugert, Peter
subject: ddc-610
subject: 610 Medical sciences Medicine
description: 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.
publisher: Karger
date: 2014
type: Article
type: info:eu-repo/semantics/article
type: NonPeerReviewed
format: application/pdf
identifier: https://archiv.ub.uni-heidelberg.de/volltextserverhttps://archiv.ub.uni-heidelberg.de/volltextserver/27237/5/357096.pdf
identifier: DOI:10.11588/heidok.00027237
identifier: https://doi.org/10.1159/000357096
identifier: urn:nbn:de:bsz:16-heidok-272378
identifier:   Janetzko, Karin ; Rink, Gabi ; Hecker, Andrea ; Bieback, Karen ; Klüter, Harald ; Bugert, Peter  (2014) A Single-Tube Real-Time PCR Assay for Mycoplasma Detection as a Routine Quality Control of Cell Therapeutics.  Transfusion Medicine and Hemotherapy, 41 (1).  pp. 83-89.  ISSN 1660-3796 (Druck-Ausg.), 1660-3818 (Online-Ausg.)     
relation: https://archiv.ub.uni-heidelberg.de/volltextserver/27237/
rights: info:eu-repo/semantics/openAccess
rights: http://archiv.ub.uni-heidelberg.de/volltextserver/help/license_urhg.html
language: eng