The characterization and certification of a quantitative reference material for L egionella detection and quantification by qPCR

Aims The characterization and certification of a L egionella DNA quantitative reference material as a primary measurement standard for L egionella 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 ( LDA s). 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 L egionella 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 q PCR . Significance and Impact of the Study The use of this primary standard will improve the accuracy of L egionella q PCR 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 q PCR methods and ensuring the reliability of these methods.