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Original Article
The characterization and certification of a quantitative reference material for Legionella detection and quantification by qPCR
Article first published online: 11 MAR 2013
DOI: 10.1111/jam.12172
© 2013 The Society for Applied Microbiology
Additional Information
How to Cite
Baume, M., Garrelly, L., Facon, J.P., Bouton, S., Fraisse, P.O., Yardin, C., Reyrolle, M. and Jarraud, S. (2013), The characterization and certification of a quantitative reference material for Legionella detection and quantification by qPCR. Journal of Applied Microbiology, 114: 1725–1733. doi: 10.1111/jam.12172
Publication History
- Issue published online: 17 MAY 2013
- Article first published online: 11 MAR 2013
- Accepted manuscript online: 25 FEB 2013 05:45AM EST
- Manuscript Revised: 15 FEB 2013
- Manuscript Accepted: 15 FEB 2013
- Manuscript Received: 18 OCT 2012
- Abstract
- Article
- References
- Cited By
Keywords:
- detection;
- Legionella ;
- PCR ;
- quality control;
- water quality
Abstract
Aims
The characterization and certification of a Legionella DNA quantitative reference material as a primary measurement standard for Legionella qPCR.
Methods and Results
Twelve laboratories participated in a collaborative certification campaign. A candidate reference DNA material was analysed through PCR-based limiting dilution assays (LDAs). The validated data were used to statistically assign both a reference value and an associated uncertainty to the reference material.
Conclusions
This LDA method allowed for the direct quantification of the amount of Legionella DNA per tube in genomic units (GU) and the determination of the associated uncertainties. This method could be used for the certification of all types of microbiological standards for qPCR.
Significance and Impact of the Study
The use of this primary standard will improve the accuracy of Legionella qPCR measurements and the overall consistency of these measurements among different laboratories. The extensive use of this certified reference material (CRM) has been integrated in the French standard NF T90-471 (April 2010) and in the ISO Technical Specification 12 869 (Anon 2012 International Standardisation Organisation) for validating qPCR methods and ensuring the reliability of these methods.