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Rapid and robust detection assays for Salmonella Enteritidis (SE) in shell eggs are essential to enable a quick testing turnaround time (TAT) at the earliest checkpoint and to ensure effective food safety control. Real-time polymerase chain reaction (qPCR) assays provide a workaround for the protracted lead times associated with conventional Salmonella diagnostic testing. However, DNA-based analysis cannot reliably discriminate between signals from viable and dead bacteria. We developed a strategy based on an SE qPCR assay that can be integrated into system testing to accelerate the detection of viable SE in egg-enriched cultures and verify the yielded SE isolates. The specificity of the assay was evaluated against 89 Salmonella strains, and SE was accurately identified in every instance. To define the indicator for a viable bacteria readout, viable or heat-inactivated SE were spiked into shell egg contents to generate post-enriched, artificially contaminated cultures to establish the quantification cycle (Cq) for viable SE. Our study has demonstrated that this technique could potentially be applied to accurately identify viable SE during the screening stage of naturally contaminated shell eggs following enrichment to provide an early alert, and that it consistently identified the serotypes of SE isolates in a shorter time than conventional testing. [ABSTRACT FROM AUTHOR]