Use of Accuracy Profile Procedure to Validate a Real-Time PCR Method to Quantify Bacteria in Feces

Manuel J Saint-Cyr (1) , Agnès Perrin-Guyomard (1, 2) , Paméla Houée (1) , Maleck V. Vasseur (1, 3) , Michel Laurentie (1)

1 ANSES - Agence nationale de sécurité sanitaire de l'alimentation, de l'environnement et du travail
2 Unité de Toxicologie des Contaminants
3 ToxAlim - ToxAlim

This study describes a novel validation procedure of real-time PCR based on accuracy profile to estimate bacterial concentrations in fecal samples. To assess the performance of the method, measurements of axenic fecal samples spiked with a measured quantity of known bacterial species (Bacteroides fragilis, Bifidobacterium adolescentis, Enterococcus faecium, and Escherichia coli) were performed under repeatability and intermediate precision conditions. Data collected were used to compute a tolerance interval that was compared to a defined acceptance interval. It is concluded that the method is valid and relevant for the studied validation range of 8.20-10.24 and 7.43-9.47 log10 CFU/g of feces to ensure proper measurement of B. fragilis and E. coli, respectively. The LOQ is 8.20 and 7.43 log10 CFU/g of feces. In contrast, the method is not valid for the quantification of E. faecium and B. adolescentis, but by applying a correction factor of +0.63 log10 CFU/g, it can be considered valid for E. faecium. This correction is included in the final results. In conclusion, the accuracy profile is a statistical tool that is easy to use and totally adapted to validate real-time PCR.

Mots clés: analysis; Escherichia coli; Enterococcus faecium; Bifidobacterium adolescentis; Bacteroides fragilis; microbiology; real time PCR; bacteria; feces

Domaines: Bactériologie

Manuel J Saint-Cyr, Agnès Perrin-Guyomard, Paméla Houée, Maleck V. Vasseur, Michel Laurentie. Use of Accuracy Profile Procedure to Validate a Real-Time PCR Method to Quantify Bacteria in Feces. Journal of AOAC INTERNATIONAL, 2014, 97 (2), pp.1-7. ⟨10.5740/jaoacint.13-310⟩. ⟨hal-00939269⟩