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 × 10 1 cfu/mL in pure culture, and the detection limit in milk was 8.35 × 10 2 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.

Aptamer-Based Lateral Flow Assays: Current Trends in Clinical Diagnostic Rapid Tests

“…Most of these LFA methods have been developed to identify pathogenic bacteria and viruses that have caused many health and economic problems worldwide. For example, Ren et al ( 2021) developed an aptamer-based LFA for sensitive detection of Escherichia coli O157:H7 (E. coli O157:H7), which is one of the most widespread foodborne pathogens [49]. In order to increase the assay sensitivity, they combined aptamer-exonuclease III (Exo III)-assisted amplification with AuNPs-based LFA.…”

Advances, applications, and limitations of portable and rapid detection technologies for routinely encountered foodborne pathogens

“…The technology was only tested in pure culture setups, and it may need longer assay time with potential pre-treatment steps during real applications. In milk samples, STEC O157:H7 was detected using an aptamer-exonuclease III (Exo III)–assisted amplification-based lateral flow assay ( Ren et al, 2021 ). Its hairpin sequence post enzymolysis was identical to the target ssDNA sequence, thus allowing signal amplification and achieving a LOD of 7.6 × 10 1 CFU/ml in pure culture and 8.35 × 10 2 CFU/ml in milk samples.…”

Bridge-DNA synthesis triggered by an allosteric aptamer for the colorimetric detection of pathogenic bacteria

“…However, in addition to the intensive labor and prolonged testing time, the plate cultivation method fails to detect bacteria in a viable but nonculturable (VBNC) state, yet these may revive upon changes in the environment. 6 To address this question, many culture-independent methods have been or are being developed, such as enzyme-linked immunosorbent assay (ELISA), 7,8 ow cytometry (FCM), 9,10 surface-enhanced Raman scattering (SERS), 11,12 and gold nanoparticle (AuNP) colorimetric detection. 13,14 However, the amplication and detection of bacterial pathogens with a concentration lower than 10 2 cfu mL −1 remains a great challenge.…”