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Quantitative real-time PCR assays targeting thegroEL gene for the specific enumeration of 12 human fecalBifidobacterium species were developed. The housekeeping genegroEL (HSP60 in eukaryotes) was used as a discriminative marker for the differentiation ofBifidobacterium adolescentis ,B. angulatum ,B. animalis ,B. bifidum ,B. breve ,B. catenulatum ,B. dentium ,B. gallicum ,B. longum ,B. pseudocatenulatum ,B. pseudolongum , andB. thermophilum . The bifidobacterial chromosome contains a single copy of thegroEL gene, allowing the determination of the cell number by quantification of thegroEL copy number. Real-time PCR assays were validated by comparing fecal samples spiked with known numbers of a givenBifidobacterium species. Independent of theBifidobacterium species tested, the proportion ofgroEL copies recovered from fecal samples spiked with 5 to 9 log10 cells/g feces was approximately 50%. The quantification limit was 5 to 6 log10 groEL copies/g feces. The interassay variability was less than 10%, and variability between different DNA extractions was less than 23%. The method developed was applied to fecal samples from healthy adults and full-term breast-fed infants. Bifidobacterial diversity in both adults and infants was low, with mostly ≤3Bifidobacterium species andB. longum frequently detected. The predominant species in infant and adult fecal samples wereB. breve andB. adolescentis , respectively. It was possible to distinguishB. catenulatum andB. pseudocatenulatum . We conclude that thegroEL gene is a suitable molecular marker for the specific and accurate quantification of human fecalBifidobacterium species by real-time PCR.

Quantification of Human Fecal Bifidobacterium Species by Use of Quantitative Real-Time PCR Analysis Targeting the groEL Gene