Use of accuracy profile procedure to validate a real-time PCR method to quantify bacteria in feces.
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.
Manuel J
MJ
Saint-Cyr
Agnès
A
Perrin-Guyomard
Paméla
P
Houée
Malek V
MV
Vasseur
Michel
M
Laurentie
D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001419:Bacteria / Q000145:classification / Q000302:isolation & purification*; D002511:Cephalosporins / Q000494:pharmacology; D004269:DNA, Bacterial / Q000235:genetics / Q000302:isolation & purification*; D005243:Feces / Q000382:microbiology*; D051381:Rats; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013045:Species Specificity
D000900:Anti-Bacterial Agents; D002511:Cephalosporins; D004269:DNA, Bacterial; C068212:cefquinome