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

摘要

Quantitative real-time PCR assays targeting the groEL gene for the specific enumeration of 12 human fecal Bifidobacterium species were developed. The housekeeping gene groEL ( HSP60 in eukaryotes) was used as a discriminative marker for the differentiation of Bifidobacterium adolescentis , B. angulatum , B. animalis , B. bifidum , B. breve , B. catenulatum , B. dentium , B. gallicum , B. longum , B. pseudocatenulatum , B. pseudolongum , and B. thermophilum . The bifidobacterial chromosome contains a single copy of the groEL gene, allowing the determination of the cell number by quantification of the groEL copy number. Real-time PCR assays were validated by comparing fecal samples spiked with known numbers of a given Bifidobacterium species. Independent of the Bifidobacterium species tested, the proportion of groEL copies recovered from fecal samples spiked with 5 to 9 log_(10) cells/g feces was approximately 50%. The quantification limit was 5 to 6 log_(10) 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 ≤3 Bifidobacterium species and B. longum frequently detected. The predominant species in infant and adult fecal samples were B. breve and B. adolescentis , respectively. It was possible to distinguish B. catenulatum and B. pseudocatenulatum . We conclude that the groEL gene is a suitable molecular marker for the specific and accurate quantification of human fecal Bifidobacterium species by real-time PCR.