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Molecular characterisation of bacterial community composition of five cheeses by DGEE and cloning

DNA was extracted from 3mm surface areas of each of the cheeses. A nested PCR approach was used with actinobacterial specific primers (Stach et al, 2003) in order to amplify a 650 base pair fragment of the 16S rRNA gene. Total community DNA amplification was carried out with a reaction mixture consisting of 1µl of 20pmol/µl of S-C-Act-235-a-S-20 (5�-CGC GGC CTA TCA GCT TGT TG-3�), 1µl of 20 pmol/µl S-C-Act-878-a-A-19 (5�-CCG TAC TCC CCA GGC GGG G-3�) primers, 1µl of 1.25 pmol/µl of dNTP�s (Sigma), 5µl of 10X NH4 + buffer (Sigma), 1.5µl of 50mM of MgCl2 0.25µl of Taq polymerase (Sigma) and 1µl of genomic DNA. The final volume was made up to 50µl with sterile distilled water. DNA amplification were performed using a Perkin Elmer thermal cycler (Applied Biosystems, USA_ Amplification was done using touchdown PCR. PRC products were quantified by electrophoresis on a 1.5% (wt/vol) agarose gel stained with ethidium bromide.

Parallel DGGE was performed essentially as described previously by Muyzer et al, 1993 using a Biorad Dcode universal mutation detection system TM. Band patterns were compared and specific bands cut out, amplified and sequenced. Sequences were assigned to genera and species on the basis of similarity to 16S sequences in the RDP database. Community composition was compared with the identification n of cheese isolates. Both uncultured and members belonging to the class Actinobacteria in any stages of ripening. In general, more groups were identified by DGGE when compared to the identificatio of cultivable strains. Some of the groups which were not identified could be attributed to the bias in DGGE or cultivation techniques. A number of different groups not previously isolated from cheese were detected by DGGE. All of the new taxa highlighted in studies of the culturablestrains were not detected by DGGE. It is clear from the data that both culture independent (DGGE) and culture independent (isolation) methods are needed to reveal the full extent of bacterial diversity on the surface of smear ripened cheeses.

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