Abstract
BackgroundBOX-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 (Bacillus cereus; Escherichia coli; isolates of the family Geodermatophilaceae). 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 Modestobacter multiseptatus isolated from three different microsites in an altered carbonatic wall from Cagliari, Italy, and 45 strains of Streptococcus thermophilus isolated from 34 samples of the hand-made, yogurt-like product Matsoni, collected in different locations in Georgia.ResultsFluorophore 6-FAM proved more informative than HEX and BOX-PCR both in agarose gel electrophoresis (p < 0.004 and p < 0.00003) and in capillary electrophoresis (compared only with HEX, p < 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 M. multiseptatus strains according to the microsite of isolation and S. thermophilus strains according to the geographical origin of Matsoni, but resulted intermixed when a BOX-PCR dataset was used.ConclusionF-BOX-PCR represents an improved method for addressing bacterial biogeography studies both in term of sensitivity, reproducibility and data analysis.
Highlights
BOX-A1R-based repetitive extragenic palindromic-PCR (BOX-PCR) is one of the most used techniques in biogeography studies of microbial isolates
In the case of M. multiseptatus, while standard BOXPCR failed to find a clear strain clustering on the basis of sampling site [23], F-BOX-PCR gave a very well defined grouping with a clear relation with the microsite of isolation
SFmiimogpuilharirleuitsy4isUoPlaGteMdAfrtormeetshoeftBypOicXa-lPyCogRhpuart-tleikrnesp(rAo)duanctdMF-aBtsOoXni-PpCroRdupcaettderins3(4Bd) ioffef r4e5nbt afacrtemrsiailnstGraeionrsgbiaelonging to S. therSimilarity UPGMA trees of BOX-PCR patterns (A) and F-BOX-PCR patterns (B) of 45 bacterial strains belonging to S. thermophilus isolated from the typical yoghurt-like product Matsoni produced in 34 different farms in Georgia
Summary
BOX-A1R-based repetitive extragenic palindromic-PCR (BOX-PCR) is one of the most used techniques in biogeography studies of microbial isolates. Different DNA-based typing methodologies are available and BOX-PCR is the most commonly used technique due to its simplicity, efficiency and low cost. This is a particular version of repetitive extragenic palindromicPCR (rep-PCR) [7] that uses the BOX-A1R primer [8]. BOX-PCR patterns are not affected by the culture age of the strain to be analyzed [15] and fingerprinting output can be analyzed by computer assisted methods [16] These features make BOXPCR a frequently used tool in biogeography studies in environmental microbiology [5,17,18,19,20]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
