title: A single-tube real-time PCR assay for Mycoplasma detection as a routine quality control of cell therapeutics.
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-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.
authors.affiliation: Institute of Transfusion Medicine and Immunology, Medical Faculty Mannheim, Heidelberg University; German Red Cross Blood Service Baden-Württemberg - Hessen, Mannheim, Germany.
authors.forename: Karin
authors.initials: K
authors.lastname: Janetzko
authors.affiliation: Institute of Transfusion Medicine and Immunology, Medical Faculty Mannheim, Heidelberg University; German Red Cross Blood Service Baden-Württemberg - Hessen, Mannheim, Germany.
authors.forename: Gabi
authors.initials: G
authors.lastname: Rink
authors.affiliation: Institute of Transfusion Medicine and Immunology, Medical Faculty Mannheim, Heidelberg University; German Red Cross Blood Service Baden-Württemberg - Hessen, Mannheim, Germany.
authors.forename: Andrea
authors.initials: A
authors.lastname: Hecker
authors.affiliation: Institute of Transfusion Medicine and Immunology, Medical Faculty Mannheim, Heidelberg University; German Red Cross Blood Service Baden-Württemberg - Hessen, Mannheim, Germany.
authors.forename: Karen
authors.initials: K
authors.lastname: Bieback
authors.affiliation: Institute of Transfusion Medicine and Immunology, Medical Faculty Mannheim, Heidelberg University; German Red Cross Blood Service Baden-Württemberg - Hessen, Mannheim, Germany.
authors.forename: Harald
authors.initials: H
authors.lastname: Klüter
authors.affiliation: Institute of Transfusion Medicine and Immunology, Medical Faculty Mannheim, Heidelberg University; German Red Cross Blood Service Baden-Württemberg - Hessen, Mannheim, Germany.
authors.forename: Peter
authors.initials: P
authors.lastname: Bugert
keywords: Cell culture; Cell therapeutics; Mollicutes; Mycoplasma; Quality control