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Abstract
BackgroundBifidobacterium represents one of the largest genus within the Actinobacteria, and includes at present 32 species. These species share a high sequence homology of 16S rDNA and several molecular techniques already applied to discriminate among them give ambiguous results.The slightly higher variability of the hsp60 gene sequences with respect to the 16S rRNA sequences offers better opportunities to design or develop molecular assays, allowing identification and differentiation of closely related species. hsp60 can be considered an excellent additional marker for inferring the taxonomy of the members of Bifidobacterium genus.ResultsThis work illustrates a simple and cheap molecular tool for the identification of Bifidobacterium species. The hsp60 universal primers were used in a simple PCR procedure for the direct amplification of 590 bp of the hsp60 sequence. The in silico restriction analysis of bifidobacterial hsp60 partial sequences allowed the identification of a single endonuclease (HaeIII) able to provide different PCR-restriction fragment length polymorphism (RFLP) patterns in the Bifidobacterium spp. type strains evaluated. The electrophoretic analyses allowed to confirm the different RFLP patterns.ConclusionsThe developed PCR-RFLP technique resulted in efficient discrimination of the tested species and subspecies and allowed the construction of a dichotomous key in order to differentiate the most widely distributed Bifidobacterium species as well as the subspecies belonging to B. pseudolongum and B. animalis.
Highlights
Bifidobacterium represents one of the largest genus within the Actinobacteria, and includes at present 32 species
Available hsp60 sequences had been retrieved from Chaperonin database (cpnDB) database and GeneBank, thanks to the work of Jian et al [25]
In silico analysis The analysis and comparison of restriction profiles obtained with in silico digestion of bifidobacterial hsp60 sequences allowed the identification of a set of appropriate frequent-cutter endonucleases that recognize non degenerated sequences
Summary
Bifidobacterium represents one of the largest genus within the Actinobacteria, and includes at present 32 species. 16S rDNA of Bifidobacterium spp. has a high similarity, ranging from 87.7 to 99.5% and bifidobacterial closely related species (e.g. B_catenulatum and B. pseudocatenulatum) or subspecies (e.g. B_longum and B. animalis subspecies) even possess identical 16S rRNA gene sequences [17,18]. For this reason different molecular approaches have been tested based on repetitive genome sequences amplification, such as ERICPCR [19,20], BOX-PCR [21,22] or RAPD fingerprinting analysis [23]. These fingerprinting methods have the disadvantage of a low reproducibility, and they need
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