Salmonella enterica is a common foodborne pathogen that can cause food poisoning in humans. The organism also infects and causes disease in animals. Rapid and sensitive detection of S. enterica is essential to prevent the spread of this pathogen. Traditional technologies for extraction and detection of this pathogen from complex food matrices are cumbersome and time-consuming. In this study, we introduced a novel strategy of biphasic assay integrated with accelerated strand exchange amplification (ASEA) method for efficient detection of S. enterica without culture or other extraction procedures. Food sample is rapidly dried, resulting in a physical fluidic network inside the dried food matrix, which allowing polymerase and primers to access the target DNA and initiate ASEA. Dried food matrix is defined as the solid phase, while amplification products are enriched in the supernatant (liquid phase) and generate fluorescence signals. The analytical performances demonstrated that this strategy was able to specifically identify S. enterica and did not show any cross-reaction with other common foodborne pathogens. For artificially spiked food samples, the strategy can detect 5.0 × 101 CFU/mL S. enterica in milk, 1.0 × 102 CFU/g in duck, scallop or lettuce, and 1.0 × 103 CFU/g in either oyster or cucumber samples without pre-enrichment of target pathogen. We further validated the strategy using 82 real food samples, and this strategy showed 92% sensitivity. The entire detection process can be finished sample-to-answer within 50 min, dramatically decreasing the detection time. Therefore, we believe that the proposed method enables rapid and sensitive detection of S. enterica and offers a great deal of promise for the food safety industry.

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