Sequence-specific cleavage of 16S rRNA for rapid and quantitative detection of particular groups of anaerobes in bioreactors.
We developed a rapid and simple method for rRNA-based quantitative detection of a specific group of microorganisms in complex ecosystems. The method relies on the sequence-specific scission of 16S rRNA with ribonuclease H (RNase H) and oligonucleotides that specifically hybridize with targeted rRNA molecules. RNAs from a complex community were first mixed with an oligonucleotide and were subsequently digested with RNase H to achieve sequence-dependent rRNA cleavage at the hybridization site. For the quantitative detection of targeted rRNAs, the resulting RNA fragment patterns were analyzed by gel-electrophoresis, which separated and quantified cleaved and intact rRNA fragments. This method enabled the quantitative detection of microbes in a complex microbial community by a relatively simple and fast experimental procedure. We then applied the cleavage method to actual anaerobic microbial communities such as digested sewage sludge and UASB sludges. The results demonstrated that the present method was fully applicable to anaerobic digestor ecosystems containing complex anaerobic microorganisms.
Sekiguchi
Y
Y
Institute of Biological Resources and Functions, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Ibaraki 305-8566, Japan.
Uyeno
Y
Y
Sunaga
A
A
Yoshida
H
H
Kamagata
Y
Y
D019149:Bioreactors; D004926:Escherichia coli / Q000235:genetics / Q000302:isolation & purification; D017021:Methanosarcina barkeri / Q000235:genetics / Q000302:isolation & purification; D019640:Methanosarcinales / Q000235:genetics / Q000302:isolation & purification; D009693:Nucleic Acid Hybridization; D015345:Oligonucleotide Probes; D009841:Oligonucleotides / Q000235:genetics / Q000378:metabolism; D019642:RNA, Archaeal / Q000032:analysis*; D012329:RNA, Bacterial / Q000032:analysis*; D012336:RNA, Ribosomal, 16S / Q000032:analysis; D016914:Ribonuclease H / Q000378:metabolism*; D012722:Sewage / Q000382:microbiology*
D015345:Oligonucleotide Probes; D009841:Oligonucleotides; D019642:RNA, Archaeal; D012329:RNA, Bacterial; D012336:RNA, Ribosomal, 16S; D012722:Sewage; D016914:Ribonuclease H