Sequence-specific capture of oligonucleotide probes (SCOPE): A simple and rapid microbial rRNA quantification method using molecular weight cut-off membrane

Appl Environ Microbiol. 2021 Jul 28:AEM0116721. doi: 10.1128/AEM.01167-21. Online ahead of print.ABSTRACTA method named sequence-specific capture of oligonucleotide probes (SCOPE) was developed for quantification of microbial rRNA molecules in a multiplex manner. In this method, molecular weight cut-off membrane (MWCOM) was used for the separation of fluorescent-labeled oligonucleotide probes hybridized with rRNA from free unhybridized probes. To demonstrate proof-of-concept, probes targeting bacteria or archaea at different taxonomic levels were prepared and were hybridized with rRNAs. The hybridization stringency was controlled by adjusting reaction temperature and urea concentration in the mixture. Then, the mixture was filtered through the MWCOM. The rRNA and hybridized probes collected on the MWCOM were recovered and quantified using spectrophotometer and fluorospectrometer, respectively. The method showed high accuracy in detecting specific microbial rRNA in a defined nucleic acid mixture. Furthermore, the method was capable of simultaneous detection and quantification of multiple target rRNAs in a sample with sensitivity up to a single-base mismatch. The SCOPE method was tested and benchmarked against the reverse transcription-quantitative PCR (RT-qPCR) for the quantification of Bacteria, Archaea and some key methanogens in anaerobic sludge samples. It was observed that the SCOPE method produced comparatively more reliable and coherent results. In this way, the SCOPE m...
Source: Applied and Environmental Microbiology - Category: Microbiology Authors: Source Type: research