3% of the actinobacterial sequences would be matched with primer Ac1186r, allowing zero mismatches. In silico reverified 16S rRNA gene sequences of 164 different type strains from the class Actinobacteria were correctly identified. Despite the short fragment length (270 bp), all of the theoretically amplified 16S rRNA gene fragments could be affiliated correctly at genus level. Species identification with the new primer system was not possible. But the 16S rRNA gene similarity classification system is per se limited by the resolution at genus level (Fox et al., 1992; Stackebrandt et al., 1997). The in situ specificity of the new primer system was clearly displayed by cloning and sequencing
selleck products analyses of PCR products
obtained from environmental samples as well as by screening 16S rRNA gene clone libraries obtained from 18 different building material samples. Investigations of environmental clone libraries showed that 87% of all obtained sequences were affiliated to known Actinobacteria; the remaining clone sequences were affiliated to as yet uncultured Actinobacteria (Table 4). In clone libraries from 18 building material samples, about 90% of the investigated sequences were assigned to Actinobacteria; only 2.7% nontargets were amplified. The high primer specificity was supported by detailed sequence analyses. Sequence information from compost and compost bioaerosol clone libraries revealed members of the genera Actinomadura, Saccharomonospora, Saccharopolyspora Streptomyces, selleck chemicals llc Thermobifida, Thermocrispum, Thermomonospora, Rhodococcus, Corynebacterium and Pseudonocardia, which have already been reported in this environment (Albrecht & Kämpfer, 2000; Dees & Ghiorse, 2001; Song et al., 2001). In addition, 21 further genera were detected using the new primer system Com2xf/Ac1186r: sequences of the genera Polymorphospora (18.7%) Dactylosporangium (13.5%) and Acidothermus (12.5%) were predominant in the clone library of mature compost material. Analyses of sequences
gained from a duck house revealed the presence of the genera Arthrobacter, Brevibacterium, Corynebacterium, Curtobacterium, Dietzia, Frigoribacterium and Microbacterium, which Ketotifen have been reported earlier in this environment (Andersson, 1999; Martin et al., 2010). Species of the genera Arthrobacter, Microbacterium, Mycobacterium, Nocardia, Nocardiopsis, Promicromonospora, Pseudonocardia, Rhodococcus, Streptomyces and Cellulomonas, shown in previous studies to be colonizers of the indoor environment, were all detected in this study, both in the clone library generated from plaster material and in screened clone libraries from the different building material samples (Anderson et al., 1997; Anderson, 1999; Peltola, 2001; Hyvärinen et al., 2002; Lorenz et al., 2003a; Suihko et al., 2009). In addition, 47 further genera were detected in water-damaged building material in the present study.