(Opt:1.00%) (Tol 0.55%-0.55%) (H>0.0% S>0.0%) [0.0%-100.0%]. Discussion The Vibrio genus is a complex group of marine-associated bacteria currently comprised of 74 species. The genus appears to be poised for continued growth as novel species are added regularly http://www.vibriobiology.net/. Consequently, this study was undertaken to develop a means by which these species
could be efficiently, reliably, and accurately identified and differentiated. To date, analyses of IGS located between the 16S-23S rRNA gene loci have drawn considerable attention as one such means to accomplish this particular goal. Unfortunately, these analyses Linsitinib chemical structure tend to be more laborious (i.e., restriction endonuclease analysis followed by probe-based detection) requiring a considerable time commitment. Moreover, many of these protocols generate extraneous artifacts
that make interpretation of results often times difficult selleck screening library and unreliable. To date, the most commonly used primers for the amplification of the IGS have been those described by Jensen et al. . The 16S rRNA gene primer (G1) was generated for a highly conserved region of the 16S rRNA gene locus approximately 30-40 bp upstream of the IGS using the 16S rRNA gene sequence data generated by Dams et al  from a broad range of bacterial and eukaryotic genera (107 species). In contrast, as the 23S rRNA gene sequence is much less conserved than that of the 16S rRNA gene, the 23S primer (L1) nearly was designed from the 23S rRNA gene sequences of only five bacterial and four plant species previously determined by Gutell et al . As these primers were not based solely on Vibrio 16S and 23S rRNA gene sequences, a new set of Vibrio-specific primers was designed from an alignment
of 16S and 23S Vibrio rRNA gene sequences. PCR reactions were optimized using these primers such that the amplification products from four reference strains (V. Selleck MK-8776 parahaemolyticus BAA239 (O3:K6), V. cholerae ATCC 25874, V. vulnificus ATCC 43382 and V. fischeri ATCC 700601) were consistent with the number and sizes of those that could be theoretically derived from genomic sequences available at the NCBI database (V. parahaemolyticus RIMD 2210633 (Chromosome I: NC_004603; chromosome II: NC_004605), V. cholerae O395 (chromosome 1: NC_009456; chromosome 2: NC_009457), V. vulnificus CMCP6 (chromosome 1: NC_004459; chromosome 2: NC_004460) and V. fischeri ES 114 (chromosome 1: NC_006840; chromosome 2: NC_006841)). As an example, the chromosome coordinates, relative size, and number of IGS regions targeted by this assay for V. parahaemolyticus, V. vulnificus, and V. cholerae are depicted in Figure 7. In every case, IGS banding patterns correlated perfectly with expected fragment size (compare Figure 7 to Figures 1 and 3). Afterwards, the testing of each remaining reference species demonstrated unique banding patterns for all strains included.