Mechanisms and applications of convection PCR utilizing trimmed β-lactoglobulin aptamers for ultra-fast detection of milk allergens

The delayed reporting of milk allergen residue detection results is a significant factor contributing to substantial losses for food production enterprises. Cumbersome operational procedures and high testing costs often lead to the abandonment of tests, resulting in frequent allergic reactions among milk-sensitive populations. Therefore, there is an urgent need for a novel, rapid, and low-cost detection method to mitigate enterprise losses and protect allergic individuals. This study investigates the formation mechanism of convection PCR, driven by Fast Taq polymerase in standard PCR tubes, and develops a fluorescence detection device for convection PCR. Through molecular docking and experimental validation of the trimmed β-LG aptamers, we elucidate their potential multi-site binding mechanism. Utilizing this mechanism, we designed a dual-strand hybridization magnetic bead detection system (dsA-SA-B), employing easily synthesized and amplified aptamer complementary strands as an indicator signal; Signal amplification was achieved within 15 min, while the entire detection process was completed in less than 50 min. This detection method allows for the rational selection of aptamer sequences based on actual testing needs, enabling the detection of hydrolyzed milk powder. The method demonstrated a linear detection range for β-LG from 5 to 100 μg/mL, with a detection limit as low as 0.25 μg/mL, effectively identifying hydrolyzed milk powder. This approach transforms protein detection into nucleic acid detection, and lays a solid foundation for multiplex PCR detection of eight major food allergens, facilitating the widespread detection of milk allergens and ultimately contributing to improved food safety and public health.