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Updated: May 1, 2026
Optimized PCR-based Detection of Mycoplasma
Published on: June 20, 2011
A single-tube real-time PCR assay for Mycoplasma detection as a routine quality control of cell therapeutics
Karin Janetzko1, Gabi Rink1, Andrea Hecker1
1Institute of Transfusion Medicine and Immunology, Medical Faculty Mannheim, Heidelberg University; German Red Cross Blood Service Baden-Württemberg - Hessen, Mannheim, Germany.
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-labeled) 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.
Conclusions:
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.
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