PCR primers targeting the 16S rRNA gene for the specific detection of streptomycetes.
Streptomycetes are filamentous actinobacteria commonly found in soil and biotechnically important, but they also have adverse effects on human health. In this work, two primer pairs, StrepB/StrepE and StrepB/StrepF combined with Bst YI restriction endonuclease digestion, targeting the 16S rRNA gene of streptomycetes were designed. The specificity of the primers was determined by polymerase chain reaction (PCR) amplification from Streptomyces strains and near relatives. All streptomycetes tested positive and non-streptomycetes were not amplified except three strains that, however, gave Bst YI restriction endonuclease digestion results distinct from streptomycetes. Moreover, both primer pairs gave an amplification product of the expected size only when Streptomyces VTT E-99-1334 DNA was present in the template DNA mixture isolated from six bacterial and three fungal strains. The primers were further successfully used to amplify from DNA isolated from two soil and two building material samples. The 40 sequenced amplification products obtained with the primer pair StrepB/StrepE showed greater than 96.1% similarity to streptomycete 16S rRNA sequences. Seventy PCR amplification products obtained with the primers StrepB/StrepF were analysed by sequencing and restriction analysis. All 54 PCR products having >95.7% similarity to streptomycete sequences were cleaved with Bst YI. No false-positive results were achieved. Both primer sets proved to be specific for streptomycetes, and applicable for the detection of streptomycetes in environmental samples.
Rintala
H
H
National Public Health Institute, Department of Environmental Health, 70701 Kuopio, Finland. helena.rintala@ktl.fi
Nevalainen
A
A
Rönkä
E
E
Suutari
M
M
D017931:DNA Primers / Q000138:chemical synthesis* / Q000235:genetics; D005798:Genes, Bacterial / Q000235:genetics; D020459:Genes, rRNA; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*; D012329:RNA, Bacterial / Q000235:genetics; D012336:RNA, Ribosomal, 16S / Q000235:genetics*; D012988:Soil Microbiology; D013302:Streptomyces / Q000235:genetics* / Q000302:isolation & purification*
D017931:DNA Primers; D012329:RNA, Bacterial; D012336:RNA, Ribosomal, 16S