Real-time PCR assays for strain-specific quantification of probiotic strains in human faecal samples

Strain-specific real-time PCR assays were developed for the probiotic strains Bifidobacterium breve 99, Lactobacillus rhamnosus LC705 and Propionibacterium freudenreichii ssp. shermanii JS, used in combination, for quantification of the strains in human faecal samples. Unique randomly amplified polymorphic DNA (RAPD) sequences or a phage-related sequence of the strains were used as a basis for designing strain-specific primers and probes. The real-time PCR conditions were optimised and the specificities of the assays were confirmed by analysing DNA from related bacterial strains and from faecal samples originating from an intervention study. The assays allowed quantification with a linear range of six orders of magnitude, from 101 to 107 target genomes per assay. The detection limit was 4 × 105 genome copies per g of faeces. The strain-specific quantitative real-time PCR assays developed were suitable for identification and quantification of probiotic strains in human faecal samples in a controlled intervention trial.