Rapid and simple quantitative identification of Listeria monocytogenes in cheese by isothermal sequence exchange...

Foodborne pathogens detection is important to ensure food safety and human health. In this study, we designed a comet structure to rapidly and sensitively detect foodborne Listeria monocytogenes. This method combined isothermal sequence exchange amplification (SEA) and surface-enhanced Raman spectroscopy. Listeria monocytogenes DNA could be rapidly amplified at a constant temperature via SEA with a pair of modified primers, which rendered the precise thermal control instrumentation unnecessary. Efficient SEA amplification generated a large number of DNA duplexes that could be easily captured by streptavidin-modified magnetic bead and Au^MB@Ag-isothiocyanate fluorescein antibody (anti-FITC). Au^MB@Ag-anti-FITC was used as a signal probe, which generated a significant excitation signal at 1,616 cm^-1 for quantitative detection and analysis. The results displayed sensitive detection of L. monocytogenes in cheese from 2.0 × 10¹ cfu/mL to 2.0 × 10⁶ cfu/mL within 1.0 h with a detection limit of 7.8 cfu/mL. Furthermore, this comet structure displayed the desirable specificity as its specific primers and amplified DNA ends were attached to streptavidin-modified magnetic beads and Au^MB@Ag-anti-FITC, respectively. We expected that the method devised would provide a promising new approach to screening for L. monocytogenes and guarantee the microbiological safety of dairy products.