Abstract
Single-stranded DNA aptamers as affinity molecules for the rapid, reliable detection of intestinal bacteria are of particular interest to equip health systems with novel robust and cheap diagnostic tools for monitoring the success of supplementation strategies with selected probiotic gut bacteria in the fight against major widespread threats, such as obesity and neurodegenerative diseases. The human gut bacterium Parabacteroides distasonis (P. distasonis) is positively associated with diseases such as obesity, non-alcoholic fatty liver disease and multiple sclerosis with reduced cell counts in these diseases and is thus a promising potential probiotic bacterium for future microbial supplementation. In this paper we report on the evolution of a specific polyclonal aptamer library by the fluorescence based FluCell-SELEX directed against whole cells of P. distasonis that specifically and efficiently binds and labels P. distasonis. The aptamer library showed high binding affinity and was suited to quantitatively discriminate P. distasonis from other prominent gut bacteria also in mixtures. We believe that this library against a promising probiotic bacterium as a prototype may open new routes towards the development of novel biosensors for the easy and efficient quantitative monitoring of microbial abundance in human microbiomes in general.
Highlights
Parabacteroides distasonis (P. distasonis) has been described as a Gram-negative, obligately anaerobic, non-spore-forming, non-motile, and rod-shaped bacterium [1]
A total of fourteen rounds of the FluCell-SELEX were performed with counterselections against control bacteria according to the conditions given in SELEX with round 14 tentatively being suspected to be sufficiently specific for the intended application to serve as a polyclonal library of affinity molecules for specific labeling of
The specificity of the polyclonal aptamer library for the surface targets of P. distasonis cells was inspected for each round between 12–14 in a discrimination assay, where an ensemble of five other abundant gut bacteria including A. muciniphila, A. stercoricanis, B. producta, R. microfusus, and R. intestinalis were used as negative microfusus, R. intestinalis were used as negative
Summary
Parabacteroides distasonis (P. distasonis) has been described as a Gram-negative, obligately anaerobic, non-spore-forming, non-motile, and rod-shaped bacterium [1]. Inspired by Liu et al the screening process involved rigorous counter selections [31], in this study using mixtures of other high abundant gut bacteria containing Akkermannsia muciniphila, Allobaculum stercoricanis, Blautia by fluorescence analysis to accurately measure the binding of aptamers to the target cells. Bound to the target cell are by PCR and after biotinelution primer and subsequently separated with avidinintroduced coated magnetic to obtain thebiotin-primer focused library This screening separated process iswith repeated obtainmagnetic a polyclonal thatthe binds This and labels subsequently avidintocoated beadslibrary to obtain focused library. The specificity of this aptamer library was subsequently analyzed by ing process is repeated to obtain a polyclonal library that binds and labels the target modern bioanalytical techniques, including fluorometric analysis and fluorescence microscopy. The specificity of this aptamer library was subsequently analyzed by modern bioanalytical techniques, including fluorometric analysis and fluorescence microscopy
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