TY  - JOUR
TI  - A Single-Tube Real-Time PCR Assay for Mycoplasma Detection as a Routine Quality Control of Cell Therapeutics
PB  - Karger
N1  - 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.
VL  - 41
N2  - 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.
KW  - cell culture 
KW  -  cell therapeutics 
KW  -  mycoplasma 
KW  -  mollicutes 
KW  -  quality control
CY  - Basel ; Freiburg
Y1  - 2014///
EP  - 89
AV  - public
ID  - heidok27237
UR  - https://doi.org/10.1159/000357096
IS  - 1
JF  - Transfusion Medicine and Hemotherapy
A1  - Janetzko, Karin
A1  - Rink, Gabi
A1  - Hecker, Andrea
A1  - Bieback, Karen
A1  - Klüter, Harald
A1  - Bugert, Peter
SP  - 83
SN  - 1660-3796 (Druck-Ausg.), 1660-3818 (Online-Ausg.)
ER  -