An aptamer-exonuclease III (Exo III)–assisted amplification-based lateral flow assay for sensitive detection of Escherichia coli O157:H7 in milk

Abstract

Escherichia coli O157:H7 (E. coli O157:H7), one of the most widespread foodborne pathogens, can cause a series of diseases and even lead to death. In this study, a highly sensitive method was developed by combining aptamer-exonuclease III (Exo III)–assisted amplification with lateral flow assay (LFA) based on gold nanoparticles (AuNP). The compound of single-stranded (ss) DNA-anti-E. coli O157:H7 aptamer (ssDNA-aptamer) was formed by hybridization between designed target ssDNA and aptamer. When E. coli O157:H7 was present, target bacteria were bound with the aptamer, and the free target ssDNA was hybridized with the probes of the designed hairpin (HP) structure. Exo III digests the 3′ double-stranded blunt end of the complex and releases the enzyme product. Because the remaining sequence of the HP of the designed enzyme product was the same as the target ssDNA sequence, the target ssDNA could be amplified. Finally, the enhanced target ssDNA was combined with AuNP-LFA to achieve visual detection of E. coli O157:H7. The quantitative ability of this platform for E. coli O157:H7 was 7.6 × 101 cfu/mL in pure culture, and the detection limit in milk was 8.35 × 102 cfu/mL. This LFA was highly specific to E. coli O157:H7, and the time for detection of E. coli O157:H7 in milk was 4 h. Hence, this system has important application prospects in the detection of pathogenic bacteria in dairy products.