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 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.
Hahn
D
D
Agricultural University Wageningen, Department of Microbiology, The Netherlands.
Kester
R
R
Starrenburg
M J
MJ
Akkermans
A D
AD
D000192:Actinomycetales / Q000235:genetics / Q000302:isolation & purification*; D001483:Base Sequence; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D015345:Oligonucleotide Probes; D012329:RNA, Bacterial / Q000032:analysis; D012336:RNA, Ribosomal, 16S / Q000032:analysis*; D012680:Sensitivity and Specificity; D012988:Soil Microbiology
D009838:Oligodeoxyribonucleotides; D015345:Oligonucleotide Probes; D012329:RNA, Bacterial; D012336:RNA, Ribosomal, 16S