eprintid: 27237
rev_number: 20
eprint_status: archive
userid: 1249
dir: disk0/00/02/72/37
datestamp: 2019-10-16 13:57:46
lastmod: 2020-02-19 07:56:40
status_changed: 2019-10-16 13:57:46
type: article
metadata_visibility: show
creators_name: Janetzko, Karin
creators_name: Rink, Gabi
creators_name: Hecker, Andrea
creators_name: Bieback, Karen
creators_name: Klüter, Harald
creators_name: Bugert, Peter
title: A Single-Tube Real-Time PCR Assay for Mycoplasma Detection as a Routine Quality Control of Cell Therapeutics
subjects: ddc-610
divisions: i-63400
keywords: cell culture , cell therapeutics , mycoplasma , mollicutes , quality control
note: 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.
abstract: 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.
date: 2014
publisher: Karger
id_scheme: DOI
id_number: 10.11588/heidok.00027237
official_url: https://doi.org/10.1159/000357096
ppn_swb: 1680086545
own_urn: urn:nbn:de:bsz:16-heidok-272378
language: eng
bibsort: JANETZKOKAASINGLETUB2014
full_text_status: public
publication: Transfusion Medicine and Hemotherapy
volume: 41
number: 1
place_of_pub: Basel ; Freiburg
pagerange: 83-89
issn: 1660-3796 (Druck-Ausg.), 1660-3818 (Online-Ausg.)
citation:   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.)     
document_url: https://archiv.ub.uni-heidelberg.de/volltextserver/27237/5/357096.pdf