Assessment of Bifidobacterium Species Using groEL Gene on the Basis of Illumina MiSeq High-Throughput Sequencing

Abstract

The next-generation high-throughput sequencing techniques have introduced a new way to assess the gut’s microbial diversity on the basis of 16S rRNA gene-based microbiota analysis. However, the precise appraisal of the biodiversity of Bifidobacterium species within the gut remains a challenging task because of the limited resolving power of the 16S rRNA gene in different species. The groEL gene, a protein-coding gene, evolves quickly and thus is useful for differentiating bifidobacteria. Here, we designed a Bifidobacterium-specific primer pair which targets a hypervariable sequence region within the groEL gene that is suitable for precise taxonomic identification and detection of all recognized species of the genus Bifidobacterium so far. The results showed that the novel designed primer set can specifically differentiate Bifidobacterium species from non-bifidobacteria, and as low as 104 cells of Bifidobacterium species can be detected using the novel designed primer set on the basis of Illumina Miseq high-throughput sequencing. We also developed a novel protocol to assess the diversity of Bifidobacterium species in both human and rat feces through high-throughput sequencing technologies using groEL gene as a discriminative marker.

Effects of Different Doses of Fructooligosaccharides (FOS) on the Composition of Mice Fecal Microbiota, Especially the Bifidobacterium Composition

“…This appears to be the first description of the composition of Bifidobacterium in mice. Hu et al [ 15 ] found that the predominant Bifidobacterium species were B. pseudocatenulatum , B. adolescentis , and B. longum in adult human feces, while the dominant species in the gut of rats was B. animalis (>90%), suggesting that the composition of Bifidobacterium varies substantially among different hosts. It is well known that FOS can selectively stimulate the growth of bifidobacteria in hosts.…”

“…The PCR conditions were 95 °C for 5 min; 95 °C for 30 s, 52 °C for 30 s, 72 °C for 30 s, repeat for 30 cycles; and 72 °C for 10 min. To distinguish the species within the genus Bifidobacterium , the groEL gene was amplified with the primers Bif-groEL-F (5′-TCC GAT TAC GAY CGY GAG AAG CT-3′)/Bif-groEL-R (5′-CSG CYT CGG TSG TCA GGAACA G-3′) [ 15 ]. The bacterial genome was set as the template.…”

“…Junick et al developed a method to quantify the human fecal Bifidobacterium species using quantitative real-time PCR analysis targeting the groEL gene [ 14 ], while Hu et al analyzed the bifidobacterial composition of human and rat gut using the groEL gene through high-throughput sequencing on the MiSeq platform [ 15 ]. To the best of our knowledge, there are no reports on the bifidobacterial community in mice or its changes upon oligosaccharide treatment.…”

Human gastrointestinal microbiota modulation with dietary prebiotics : an intimate interaction throughout life

“…Colorectal cancer (CRC) is one of the most common forms of cancer [75]. High-calorie diets, rich in animal fat, red and processed meat or alcohol, and low in dietary fiber, whole grains and vegetables are considered a main risk factor [75,76]. Gut microbiota structure and function is largely dependent on diet, and certain bacterial metabolites are known to be proinflammatory and tumor inducing.…”

“…In addition, identification of species or strains is not reliable [74]. Species-specific primer pairs targeting a hypervariable sequence of a fast evolving gene can be used instead, or in addition to universal primers to provide information about specific species or strains [24,75]. Other approaches have been developed recently that have the potential to provide a better resolution of the sequencing data by generating sequencing reads which span nearly the entire length of the 16S rRNA gene [76], and also do not require PCR amplification, thus avoiding a primer bias [77].…”