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Detection of Mollicutes in bioreactor samples by real-time transcription-mediated amplification
Article first published online: 6 APR 2010
DOI: 10.1111/j.1472-765X.2010.02846.x
© 2010 The Authors. Journal compilation © 2010 The Society for Applied Microbiology
Additional Information
How to Cite
Laborde, S., Degrave, A., Lehmann, D., Jouette, S., Rofel, C., Muller, T., Hertzog, N., Rook, M. and Ribault, S. (2010), Detection of Mollicutes in bioreactor samples by real-time transcription-mediated amplification. Letters in Applied Microbiology, 50: 633–638. doi: 10.1111/j.1472-765X.2010.02846.x
Publication History
- Issue published online: 7 MAY 2010
- Article first published online: 6 APR 2010
- 2009/2014: received 20 November 2009, revised 29 March 2010 and accepted 29 March 2010
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Keywords:
- bioreactor;
- cells;
- Mycoplasma;
- real-time TMA;
- sample preparation
Abstract
Aim: Contamination by Mollicutes is a significant challenge for research laboratories and biopharmaceutical industry. It leads to alteration of results or production quality as well as loss of time, materials and revenue. These organisms can czoriginate from mammalian, avian, insect, plant or fish cells. Culture-based methods may require 28 days to detect Mollicutes. Traditional microbiology could advantageously be replaced by nucleic acid testing for earlier detection.
Methods and Results: A membrane filtration-based concentration of the Mollicutes has been coupled to real-time transcription-mediated amplification (real-time TMA) to demonstrate these advantages. The eight species required by European Pharmacopoeia have been tested and were detected with sensitivity below 100 CFU per 20-ml sample. Co-culture experiments, in which Mollicutes are grown with CHO-S (suspension) or HEK 293 (adherent) cells, were also performed to respectively mimic a bioreactor or flask contamination. Despite the fact that Mollicutes can attach to or invade mammalian cells, they were consistently detected over multiple days.
Conclusions: the sample preparation and amplification method used in this study increases sensitivity and reduces time-to-result for detection of Mollicutes.
Significance and Impact of the Study: the described system allows real-time monitoring for microbial contamination of cell-based processes and products for the biopharmaceutical industry.