ABSTRACTThe virulence factor internalin A (InlA) facilitates the uptake ofListeria monocytogenesby epithelial cells that express the human isoform of E-cadherin. Previous studies identified naturally occurring premature stop codon (PMSC) mutations ininlAand demonstrated that these mutations are responsible for virulence attenuation. We assembled >1,700L. monocytogenesisolates from diverse sources representing 90 EcoRI ribotypes. A subset of this isolate collection was selected based on ribotype frequency and characterized by a Caco-2 cell invasion assay. The sequencing ofinlAgenes from isolates with attenuated invasion capacities revealed three novelinlAPMSCs which had not been identified previously among U.S. isolates. Since ribotypes include isolates with and withoutinlAPMSCs, we developed a multiplex single-nucleotide polymorphism (SNP) genotyping assay to detect isolates with virulence-attenuating PMSC mutations ininlA. The SNP genotyping assay detects allinlAPMSC mutations that have been reported worldwide and verified in this study to date by the extension of unlabeled primers with fluorescently labeled dideoxynucleoside triphosphates. We implemented the SNP genotyping assay to characterize human clinical and food isolates representing common ribotypes associated with novelinlAPMSC mutations. PMSCs ininlAwere significantly (ribotypes DUP-1039C and DUP-1045B;P< 0.001) or marginally (ribotype DUP-1062D;P= 0.11) more common among food isolates than human clinical isolates. SNP genotyping revealed a fourth novel PMSC mutation among U.S.L. monocytogenesisolates, which was observed previously among isolates from France and Portugal. This SNP genotyping assay may be implemented by regulatory agencies and the food industry to differentiateL. monocytogenesisolates carrying virulence-attenuating PMSC mutations ininlAfrom strains representing the most significant health risk.