elegans, L. coleohominis (Facklamia hominis, F. languida, F. miroungae) ≤ 35 this study LCC1030 CCTGTATCCCGTGTCCCG Cy3, FAM 1030-47 Lactococcus lactis, L. garvieae 40-55 this study EUB338 GCTGCCTCCCGTAGGAGT Cy3, FAM 338-55 Most Eubacteria ≤ 50  a Bold printed bases indicate the position of locked-nucleic-acids. b 16S rRNA target position (Escherichia coli numbering). c Taxa in parentheses are detected by the probe but
have not been described to colonize the human oral LY3009104 in vitro cavity . d Optimum formamide www.selleckchem.com/products/rg-7112.html concentration in hybridization buffer. Figure 1 outlines the concept for the design of the probes targeting oral lactobacilli. Two broad Lactobacillus probes (LGC358a and LAB759) were generated with the idea
that they should complement each other and thus limit the potential of misidentifications . Elongated by one and shifted by four bases LGC358a is a derivative of probe LGC354a . Probes LGC358b (staphylococci and related bacteria) and LGC358c (streptococci) are analogously related to LGC354b and LGC354c described by Meier et al. . As observed often with probes to larger phylogenetic groups, initial experiments with both probes detected besides the targeted lactobacilli significant numbers of false SCH727965 cell line positives (predominantly cocci) when applied to oral plaque samples (see below). In silico analyses suggested that these false hybridizations were due to single sequence mismatches and could possibly be avoided by the application of unlabeled competitor probes that are fully complimentary to the targeted 16S rRNA
segment of the false positive organisms. Applied in excess together with the labeled FISH probe such competitor probes can increase the differentiation between true- and potential false positives . Thus, LGC358a used in conjunction with LGC358b-comp should recognize selectively most Lactobacillaceae organisms and in addition detect parts of the non-oral families Leuconostocaceae and Carnobacteriaceae, whereas LAB759, when applied together with LAB759-comp (which should suppress recognition of Streptococcus mutans as well as Eikenella, Kingella, Sitaxentan and Neisseria sp.) is supposed to identify all oral lactobacilli except Lactobacillus salivarius and the majority of L. fermentum strains. Application of these competitor probes to various types of plaques samples proved to be successful in providing specificity for lactobacilli (see below). The other probes for lactobacilli were designed to identify bacteria from all major deep branching clusters of the phylogenic tree (Figure 1). Three probes recognize deeply branched, individual species (L. fermentum, L. salivarius and Lactobacillus vaginalis), which, however, belong to the most frequently detected oral lactobacilli.