A 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 method allows a simple and rapid detection and quantification of target microbial rRNAs for environmental microbial population analysis without any need for enzymatic reactions.
Importance
Microorganisms play integral roles in the earth's ecosystem. Microbial population and their activities significantly affect the global nutrient cycles. Quantification of key microorganisms provides important information that is required to understand their roles in the environment. Sequence-based analysis of microbial population is a powerful tool, but it only provides information on relative abundance of microorganisms. Hence, the development of a simpler and quick method for the quantification of microorganisms is necessary. To address the shortcomings of a variety of molecular methods reported so far, we developed a simple, rapid, accurate and multiplexed microbial rRNA quantification method to evaluate the abundance of specific microbial population in complex ecosystems. The developed method demonstrated high specificity, reproducibility, and applicability to such samples. The method is useful for quantitative detection of particular microbial members in the environment.