Selective and sensitive detection of Alicyclobacillus spores by aptamer-based approaches

Continuous monitoring of product quality in the beverage industry is of great importance to meet consumer expectations. Although Alicyclobacillus spp. are Gram-positive, thermo-acidophilic bacteria that are non-pathogenic, they pose a significant quality problem due to their heat-resistant endospores, which can survive typical fruit juice processing conditions. These bacteria can withstand high temperatures and acidic environments, thereby causing serious quality issues in juice production. Conventional detection methods, which rely on culturing, isolation, and species identification, are time-consuming and may take up to a week to yield results. This delay can lead to disruptions in the supply chain and economic losses. This study aimed to develop and characterize DNA aptamers capable of recognizing Alicyclobacillus spores. Aptamer selection was performed using a magnetic bead-based spore-SELEX (Systematic Evolution of Ligands by EXponential enrichment) method. Throughout the selection process, flow cytometric binding assays and agarose gel electrophoresis were employed to validate selection efficiency. Post-selection analyses revealed that the selected aptamers exhibited high affinity and specificity toward the target spores, confirming the successful development of sequence-specific aptamers. The binding performance of FAM-labeled Apt-1, Apt-2, and Apt-3 aptamers to Alicyclobacillus spores was evaluated under varying concentrations and environmental conditions. In SELEX buffer, standard calibration curves with high correlation coefficients demonstrated that the aptamers possessed stable signal properties suitable for quantitative analysis. Time-dependent binding assays indicated that equilibrium was reached at 45 minutes. While Apt-2 showed the lowest Kd value in SELEX buffer, Apt-3 displayed the highest binding affinity in complex food matrices such as orange and apple juice. These findings contribute to both the aptamer development process and the understanding of their application potential, highlighting the feasibility of implementing a rapid and reliable quality control method in the fruit juice industry.