Fluorescent-BOX-PCR for resolving bacterial genetic diversity, endemism and biogeography

• Main Authors: Mora Diego, Chanishvili Nina, Merabishvili Maya, Borin Sara, Tamagnini Isabella, Gtari Maher, Malkhazova Iana, Brusetti Lorenzo, Cappitelli Francesca, Daffonchio Daniele
• Format: Article
• Language: English
• Published: BMC 2008-12-01
• Series: BMC Microbiology
• Online Access: http://www.biomedcentral.com/1471-2180/8/220

<p>Abstract</p> <p>Background</p> <p>BOX-A1R-based repetitive extragenic palindromic-PCR (BOX-PCR) is one of the most used techniques in biogeography studies of microbial isolates. However the traditional separation of BOX-PCR patterns by agarose gel electrophoresis suffers many limitations. The aim of this research was to set up a fluorescent BOX-PCR (F-BOX-PCR) assay in which separation of PCR products is automated in a capillary electrophoresis system. F-BOX-PCR was compared with the traditional BOX-PCR using bacterial strains with different G+C content (<it>Bacillus cereus</it>; <it>Escherichia coli</it>; isolates of the family <it>Geodermatophilaceae</it>). Resolution, discriminatory power and reproducibility were evaluated by assaying different electrophoretic runs, PCR reactions and independent DNA extractions. BOX-PCR and F-BOX-PCR were compared for the analysis of 29 strains of <it>Modestobacter multiseptatus </it>isolated from three different microsites in an altered carbonatic wall from Cagliari, Italy, and 45 strains of <it>Streptococcus thermophilus </it>isolated from 34 samples of the hand-made, yogurt-like product Matsoni, collected in different locations in Georgia.</p> <p>Results</p> <p>Fluorophore 6-FAM proved more informative than HEX and BOX-PCR both in agarose gel electrophoresis (<it>p </it>< 0.004 and <it>p </it>< 0.00003) and in capillary electrophoresis (compared only with HEX, <it>p </it>< 2 × 10^-7). 6-FAM- and HEX-based F-BOX-PCR respectively detected up to 12.0 and 11.3 times more fragments than BOX-PCR. Replicate separations of F-BOX-PCR showed an accuracy of the size calling of ± 0.5 bp until 500 bp, constantly decreasing to ± 10 bp at 2000 bp. Cluster analysis of F-BOX-PCR profiles grouped <it>M. multiseptatus </it>strains according to the microsite of isolation and <it>S. thermophilus </it>strains according to the geographical origin of Matsoni, but resulted intermixed when a BOX-PCR dataset was used.</p> <p>Conclusion</p> <p>F-BOX-PCR represents an improved method for addressing bacterial biogeography studies both in term of sensitivity, reproducibility and data analysis.</p>