Enriched Aptamer Libraries in Fluorescence-Based Assays for Rikenella microfusus-Specific Gut Microbiome Analyses

Abstract

Rikenella microfusus is an essential intestinal probiotic with great potential. The latest research shows that imbalance in the intestinal flora are related to the occurrence of various diseases, such as intestinal diseases, immune diseases, and metabolic diseases. Rikenella may be a target or biomarker for some diseases, providing a new possibility for preventing and treating these diseases by monitoring and optimizing the abundance of Rikenella in the intestine. However, the current monitoring methods have disadvantages, such as long detection times, complicated operations, and high costs, which seriously limit the possibility of clinical application of microbiome-based treatment options. Therefore, the intention of this study was to evolve an enriched aptamer library to be used for specific labeling of R. microfusus, allowing rapid and low-cost detection methods and, ultimately the construction of aptamer-based biosensors. In this study, we used Rikenella as the target bacterium for an in vitro whole Cell-SELEX (Systematic Evolution of Ligands by EXponential Enrichment) to evolve and enrich specific DNA oligonucleotide aptamers. Five other prominent anaerobic gut bacteria were included in this process for counterselection and served as control cells. The aptamer library R.m-R13 was evolved with high specificity and strong affinity (Kd = 9.597 nM after 13 rounds of selection). With this enriched aptamer library, R. microfusus could efficiently be discriminated from the control bacteria in complex mixtures using different analysis techniques, including fluorescence microscopy or fluorometric suspension assays, and even in human stool samples. These preliminary results open new avenues toward the development of aptamer-based microbiome bio-sensing applications for fast and reliable monitoring of R. microfusus.

Robust Fluorometric Aptamer Assay for Direct and Rapid Detection of Clinical Isolates of Candida spec.

“…With the finding that polyclonal aptamer libraries originating directly from SELEX processes may outperform the individual aptamers [ 52 ] selected from these libraries, we introduced such enriched or “polyclonal” aptamer libraries as valuable tools for the fluorescent labeling of target structures and thus the specific quantification of a series of microbial symbionts and pathobionts [ 52 , 53 , 54 , 55 , 56 , 57 ]. Moreover, the functionality of this concept was further demonstrated with different tissues of plant roots [ 58 ] and by using specific enriched libraries for the construction of electronic gFET-based biosensors to measure pre-diabetes-related biomarkers [ 59 ].…”

High-Energy Supplemental Feeding Shifts Gut Microbiota Composition and Function in Red Deer (Cervus elaphus)

“…Notably, we found that the relative abundance of Rikenella microfusus, Muribaculum intestinale, and Escherichia coli in the gut microbiota of red deer was significantly higher during SF than during NSF (Table S2). R. microfusus is an essential intestinal probiotic with great potential [72]. In previous studies in the cervids, it was found that Escherichia coli, Yersinia enterolitica, Y. ruckerii, Aeromonas sobria, Enterococcus faecium, Staphylococcus aureus, and Lysteria monocytogenes are potential pathogenic bacteria [23,73].…”

Aptamer decorated PDA@magnetic silica microparticles for bacteria purification

“…Since the complex components on the cell surface may bias the library towards highly expressed epitopes, several combinatorial strategies have been reported such as precision-SELEX [ 30 ] or cross-over SELEX [ 31 ]. Recently, both SELEX and Cell-SELEX have been implemented to generate the possible aptamers for different pathogens and their subspecies, such as Escherichia coli [ 32 ], Staphylococcus aureus [ 33 ], Bacillus cereus [ 34 ], Streptococcus agalactiae [ 35 ], Rikinella microfusus [ 36 ], and many other medical and food-born pathogenic bacteria [ 37 , 38 ], to utilize them in biosensing or purification applications.…”