Extraction of ribosomal RNA from soil for detection of Frankia with oligonucleotide probes

Sequences of 16S rRNA of the nitrogen-fixing Frankia strain Ag45/Mut15 and the ineffective Frankia strain AgB1.9 were used to design a genus-specific oligonucleotide probe. Hybridization experiments of this Frankia probe and a second probe, specific for Nif+-Frankia strains only, were used to detect...

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Bibliographic Details
Published in:Archives of microbiology 1990, Vol.154 (4), p.329-335
Main Authors: Hahn, D, Kester, R, Starrenburg, M.J.C, Akkermans, A.D.L
Format: Article
Language:English
Subjects:
Actinomycetales - genetics
Actinomycetales - isolation & purification
Bacteriological methods and techniques used in bacteriology
Bacteriology
Base Sequence
Biological and medical sciences
detection
extraction
Frankia
Fundamental and applied biological sciences. Psychology
Laboratorium voor Microbiologie
Microbiological Laboratory
Microbiologie
Microbiology
Molecular Sequence Data
Nucleic Acid Hybridization
nucleotide sequences
Oligodeoxyribonucleotides - chemistry
Oligonucleotide Probes
ribosomal RNA
RNA, Bacterial - analysis
RNA, Ribosomal, 16S - analysis
Sensitivity and Specificity
Soil Microbiology
strains
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Summary:Sequences of 16S rRNA of the nitrogen-fixing Frankia strain Ag45/Mut15 and the ineffective Frankia strain AgB1.9 were used to design a genus-specific oligonucleotide probe. Hybridization experiments of this Frankia probe and a second probe, specific for Nif+-Frankia strains only, were used to detect Frankia specific target sequences in RNA isolations from soil. A method is described for direct isolation of RNA from a loamy soil and a peat. Yields of about 10 ng RNA/g wet soil are obtained without detectable contamination with humic acids. Isolation of RNA after initial extraction of bacteria from soil resulted in significantly lower RNA yields, compared to the direct isolation procedure. Hybridization with both probes against rRNA isolations from Frankia-containing soil could detect target sequences within RNA isolations from 1 g wet soil with an estimated detection limit of 10(4) cells.
ISSN:0302-8933
1432-072X
DOI:10.1007/BF00276527