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Development of genus specific primers for staphylococci, microbacteria and arthrobacter

Members of the genera Arthrobacter, Staphylococcus and Microbacterium are some of the dominant genus that were identified on the surface of Tilsit, Livarot, Limburger, Reblochon and Gubbeen cheeses. Full length 16S rDNA sequences of Arthrobacter, Microbacterium and Staphylococci. were downloaded from the RDP (, EMBL and NCBI ( databases for alignment using the Clustal W and Clustal X programs (Thompson et al 1994, 1997). Strength of the alignment was also checked by other manual alignment programs, notably PHYDIT: Phylogenetic Molecular Sequence Editor (Chun, J. 1995. Eighty two representative Staphylococcus sequences, 66 Microbacterium sequences and 54 Arthrobacter sequences were independently aligned. Using these alignments 16S rDNA-targeted PCR primers were designed from the conserved regions. Primers were compared to the aligned SSU_rRNA database of the RDP using the CHECK_PROBE utility (Maidak et al, 1997). Previously published Arthrobacter and Microbacterium (Kolloffel et al, 1997) genus specific primers were tested for comparison.

Primers were designed for members of all three genera. Phylogenetic trees were constructed to identify adjacent genera to test for non-specificity. Results from in silico analysis showed that the primers were specific for the relevant genus. These primers proved to be more specific than the previously published primers, which targeted genera like Agrococcus. Previously published Arthrobacter.Micrococcus specific primers also showed non-specificity with many other genera.


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