Development and optimisation of a rapid and sensitive detection platform for <i>Escherichia coli</i> O157:H7 using RPA- <i>Pf</i> Ago

Abstract Escherichia coli O157:H7 (E. coli O157:H7) is a significant foodborne pathogen that causes severe illnesses worldwide. In this study, a novel detection system combining recombinase polymerase amplification and Pyrococcus furiosus Argonaute (PfAgo) was developed for the rapid and sensitive identification of the conserved rfbE gene. The system integrates dual-fluorescence probes labelled with 6-carboxyfluorescein (FAM) and X-rhodamine (ROX) for parallel signal verification, which enhances detection reliability and minimises the risk of false positives. The optimised system achieved a limit of detection of 6.14 × 101 copies/μl, 100% specificity and repeatability, with a coefficient of variation below 10%. Validation using artificially contaminated milk and bottled water samples demonstrated complete agreement with the results of polymerase chain reaction, yielding 100% accuracy. With high sensitivity and specificity, this system enables rapid, accurate point-of-care screening for food safety and clinical diagnostics, offering broad potential in public health surveillance and foodborne pathogen control.