Aptamer-based technology for detecting Bacillus subtilis in soil.

The uncertainty associated with the impact of a bioinoculant on soil microbial community and, as a consequence, on soil quality, as well as the need to define its persistence, has prompted the demand for an accurate detection and tracking of the presence and the quantification of a target microbial inoculant in soil. Although DNA or RNA-based molecular detection are well established and commonly applied in this regard, alternative ligands such as DNA-aptamers have several advantages over them, such as low cost, ease of modification, ease of immobilisation on lab-on-chip or nanosensors, high stability and not thermolability. In this study, we used a toggle-cell SELEX method to isolate, select and characterise ssDNA (single-strand DNA) aptamers to detect a Bacillus subtilis strain which is being tested as a plant growth promoting rhizobacteria (PGPR) formulation. Two ssDNA aptamers (patenting application n.102022000022590) showed strong affinity and specificity for B. subtilis strains, with values of the kinetic parameters Kd (dissociation constant) in the nanomolar range and Bmax (maximum intensity of binding) around 1. Validation of the suitability of the aptamers was validated on three inoculated soils characterised by different chemical-physical features and in soil from a field trial with the formulated B. subtilis PCM/B 00105 strain. These are considered significant features to monitor B. subtilis strains in soil, practical to optimise bioinoculant application methods, support regulatory processes and foster the shift of agricultural production toward more sustainable cropping systems. KEY POINTS: • First DNA aptamers binding a B. subtilis strain included in a bioinoculum formulation. • First DNA aptamer binding B. subtilis in soil. • Aptamer may be a method for microbial inoculant detection in soil.