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

Loredana Baffoni1*, Verena Stenico1, Erwin Strahsburger2, Francesca Gaggìa1, Diana Di Gioia1, Monica Modesto1, Paola Mattarelli1 and Bruno Biavati1

Author Affiliations

1 Department of Agricultural Sciences, University of Bologna, viale Fanin 42, 40127, Bologna, Italy

2 Laboratorio de Microbiología Molecular y Biotecnología Ambiental, Departamento de Química and Center of Nanotechnology and Systems Biology, Universidad Técnica Federico Santa María, Avenida España 1680, Valparaíso, Chile

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BMC Microbiology 2013, 13:149  doi:10.1186/1471-2180-13-149

Published: 1 July 2013



Bifidobacterium 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.


This 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.


The 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 spp; hsp60; PCR-RFLP; Taxonomy