Development of a new PCR primer system for selective amplification of Actinobacteria

Abstract

The occurrence of Actinobacteria in water-damaged building materials as well as the clinical relevance of some Actinobacteria (e.g. Saccharopolyspora spp., Mycobacterium spp., Nocardia spp., etc.), led us to develop a detection system to examine the actinobacterial community. A new primer system, Com2xf/Ac1186r (16S rRNA gene based) specific for Actinobacteria was designed. The adequacy for the intended use of the primer system was first investigated in silico using sequences of 164 different species belonging to 75 different genera of the class Actinobacteria. To test the primer specificity in complex environmental samples, four 16S rRNA gene clone libraries were generated (plaster material, compost material, compost plant- and duck house bioaerosols). Overall, 87% of obtained sequences were assigned to actinobacterial genera. To verify the applicability of the new designed primer system in water-damaged building material, 16S rRNA gene clone libraries of 18 different water-damaged materials were screened for their affiliation to Actinobacteria. A total of 88% of all 'Actinobacteria-positive' detected plasmid inserts were affiliated correctly. Results of SSCP-fingerprinting clearly showed differences of the species detected by the Actinobacteria-specific primer system within the different samples. Overall results obtained in this study indicate the applicability of the developed primer system for its intended use. © 2010 Federation of European Microbiological Societies.