Design and evaluation of an oligonucleotide-microarray for the detection of different species of the genus Kitasatospora.
An oligonucleotide-microarray method was developed for the detection of Kitasatospora species in soil samples. The 16S-23S rDNA internal transcribed spacer (ITS) sequence of these antibiotics-producing actinomycetes was applied to design short oligonucleotide probes. Two different 26-mers were synthesized, specific to each species used. Additionally, four oligonucleotide probes were designed to evaluate the system. The oligonucleotides were spotted onto slides of the ArrayTube microarray system and examined with a new silver-labeling detection technique. Prior to hybridization analysis, the 16S-23S rDNA were amplified by polymerase chain reaction both from bacterial cells and environmental samples using two actinomycetes specific primers containing a 5' biotin labeling. The type strains of eight Kitasatospora species included in this study were K. phosalacinea DSM 43860, K. setae DSM 43861, K. cochleata DSM 41652, K. cystarginea DSM 41680, K. azatica DSM 41650, K. mediocidica DSM 43929, K. paracochleata DSM 41656, and K. griseola DSM 43859. The actinomycetes-specific primers were shown to amplify the entire 16S-23S rDNA ITS region from all tested strains. More importantly, the described technique allows the detection of Kitasatospora strains from soil samples by extracting metagenomic DNA followed by a PCR amplification step. This indicates that the oligonucleotide-microarray method developed in this study is a reliable tool for the detection of Kitasatospora species in environmental samples.
Günther
Sebastian
S
Department of Molecular Natural Products Research, Hans-Knöll-Institut für Naturstoff-Forschung e.V. Beutenbergstr. 11a, D-07745 Jena, Germany.
Groth
Ingrid
I
Grabley
Susanne
S
Munder
Thomas
T
D004269:DNA, Bacterial / Q000032:analysis / Q000302:isolation & purification; D021903:DNA, Ribosomal Spacer / Q000032:analysis / Q000235:genetics; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D015345:Oligonucleotide Probes / Q000235:genetics*; D016133:Polymerase Chain Reaction; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D012338:RNA, Ribosomal, 23S / Q000235:genetics; D012988:Soil Microbiology; D013045:Species Specificity; D013306:Streptomycetaceae / Q000145:classification* / Q000254:growth & development / Q000302:isolation & purification
D004269:DNA, Bacterial; D021903:DNA, Ribosomal Spacer; D015345:Oligonucleotide Probes; D012336:RNA, Ribosomal, 16S; D012338:RNA, Ribosomal, 23S