title: Detection of Saccharopolyspora rectivirgula by quantitative real-time PCR.
abstract: The thermophilic actinomycete species Saccharopolyspora rectivirgula has been associated with the exogen allergic alveolitis (EAA). EAA is caused by the inhalation of high amounts of airborne spores that can be found for example in environments of agricultural production, compost facilities, mushroom cultivation rooms, or rooms with technical air moistening. Because of the medical relevance of S. rectivirgula, a reliable detection system is needed. Therefore, a quantitative real-time polymerase chain reaction (qPCR) primer system was designed, targeting the 16S rRNA gene of the type strain S. rectivirgula DSM 43747(T) and six other S. rectivirgula reference strains. Our investigation showed that S. rectivirgula presumably own four operons of the 16S rRNA gene, which has to be considered for estimation of cell equivalents. Furthermore, the DNA recovery efficiency from these strains was tested in combination with bioaerosol or material sample as well as the influence of non-target DNA to the recovery rate. Results showed a recovery DNA efficiency of 7-55%. The recovery rate of DNA in a mixture with non-target DNA resulted in ∼87%. In summary, a high amplification efficiency using real-time PCR was found, for which estimated concentrations revealed cell numbers of 2.7 × 10(5) cells m(-3) in bioaerosol and 2.8 × 10(6) cells g(-1) fw(-1) in material samples from a duck house. The specificity of the new developed quantification system was shown by generation of two clone libraries from bioarosol samples, from a duck house, and from a composting plant. Totally, the results clearly show the specificity and practicability of the established qPCR assay for detection of S. rectivirgula.
authors.affiliation: Bundesanstalt für Arbeitsschutz und Arbeitsmedizin, Nöldnerstrasse 40-42, Berlin, Germany. schaefer.jenny@baua.bund.de
authors.forename: Jenny
authors.initials: J
authors.lastname: Schäfer
authors.forename: Peter
authors.initials: P
authors.lastname: Kämpfer
authors.forename: Udo
authors.initials: U
authors.lastname: Jäckel
mesh_terms: D000391:Air Microbiology; D000395:Air Pollutants, Occupational / Q000032:analysis*; D000822:Animal Husbandry; D000818:Animals; D015139:Blotting, Southern; D003001:Cloning, Molecular; D017931:DNA Primers; D004269:DNA, Bacterial / Q000032:analysis / Q000235:genetics; D004372:Ducks; D004784:Environmental Monitoring / Q000379:methods; D005203:Farmer's Lung / Q000382:microbiology; D006799:Housing, Animal; D006801:Humans; D008969:Molecular Sequence Data; D010802:Phylogeny; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D016930:Saccharopolyspora / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D012680:Sensitivity and Specificity; D017421:Sequence Analysis / Q000379:methods; D012987:Soil; D012407:rRNA Operon
chemical_list: D000395:Air Pollutants, Occupational; D017931:DNA Primers; D004269:DNA, Bacterial; D012336:RNA, Ribosomal, 16S; D012987:Soil