Identification of species belonging to the Bifidobacterium genus by PCR-RFLP analysis of a hsp60 gene fragment

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.

Bifidobacterium myosotis sp. nov., Bifidobacterium tissieri sp. nov. and Bifidobacterium hapali sp. nov., isolated from faeces of baby common marmosets (Callithrix jacchus L.)

“…Conditions for amplification and sequencing of the partial hsp60 gene were described by Baffoni et al (2013); primers H60F…”

Isolation and identification of cultivable Bifidobacterium spp. from the faeces of 5 baby common marmosets (Callithrix jacchus L.)

“…Representative isolates were identified according to the hsp60 RFLP-PCR method developed by Baffoni et al [10] and Stenico et al [11] allowing a rapid identification at the species level of 36 Bifidobacterium spp. A 16S rRNA gene sequence analysis was carried out for isolates with unknown hsp60 RFLP-PCR profiles.…”

Bifidobacterium eulemuris sp. nov., isolated from faeces of black lemurs (Eulemur macaco)

“…hsp60 RFLP-PCR analysis, developed by Baffoni et al (2013), was described as a rapid tool for the efficient identification of bifidobacteria at the species and subspecies levels. The method was used to discriminate LMM_E3 T from B. lemurum DSM 28807 T after virtual digestion of the obtained hsp60 gene sequences with Hae III according to Baffoni et al (2013).…”

“…The method was used to discriminate LMM_E3 T from B. lemurum DSM 28807 T after virtual digestion of the obtained hsp60 gene sequences with Hae III according to Baffoni et al (2013). The theoretical fragment patterns were different, 32-42-60-104-139-158 bp for LMM_E3 T and 10-42-81-104-139-158 bp for B. lemurum DSM 28807 T , suggesting the placement of the two strains in separate taxa (Fig.…”