{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,10]],"date-time":"2026-10-10T15:28:15Z","timestamp":1791646095173,"version":"4.3.4"},"reference-count":38,"publisher":"American Society for Microbiology","issue":"23","license":[{"start":{"date-parts":[[2008,12,1]],"date-time":"2008-12-01T00:00:00Z","timestamp":1228089600000},"content-version":"tdm","delay-in-days":0,"URL":"https:\/\/journals.asm.org\/non-commercial-tdm-license"}],"content-domain":{"domain":["journals.asm.org"],"crossmark-restriction":true},"short-container-title":["Appl Environ Microbiol"],"published-print":{"date-parts":[[2008,12]]},"abstract":"<jats:title>ABSTRACT<\/jats:title><jats:p>The virulence factor internalin A (InlA) facilitates the uptake of<jats:italic>Listeria monocytogenes<\/jats:italic>by epithelial cells that express the human isoform of E-cadherin. Previous studies identified naturally occurring premature stop codon (PMSC) mutations in<jats:italic>inlA<\/jats:italic>and demonstrated that these mutations are responsible for virulence attenuation. We assembled &gt;1,700<jats:italic>L. monocytogenes<\/jats:italic>isolates 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 of<jats:italic>inlA<\/jats:italic>genes from isolates with attenuated invasion capacities revealed three novel<jats:italic>inlA<\/jats:italic>PMSCs which had not been identified previously among U.S. isolates. Since ribotypes include isolates with and without<jats:italic>inlA<\/jats:italic>PMSCs, we developed a multiplex single-nucleotide polymorphism (SNP) genotyping assay to detect isolates with virulence-attenuating PMSC mutations in<jats:italic>inlA<\/jats:italic>. The SNP genotyping assay detects all<jats:italic>inlA<\/jats:italic>PMSC 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 novel<jats:italic>inlA<\/jats:italic>PMSC mutations. PMSCs in<jats:italic>inlA<\/jats:italic>were significantly (ribotypes DUP-1039C and DUP-1045B;<jats:italic>P<\/jats:italic>&lt; 0.001) or marginally (ribotype DUP-1062D;<jats:italic>P<\/jats:italic>= 0.11) more common among food isolates than human clinical isolates. SNP genotyping revealed a fourth novel PMSC mutation among U.S.<jats:italic>L. monocytogenes<\/jats:italic>isolates, 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 differentiate<jats:italic>L. monocytogenes<\/jats:italic>isolates carrying virulence-attenuating PMSC mutations in<jats:italic>inlA<\/jats:italic>from strains representing the most significant health risk.<\/jats:p>","DOI":"10.1128\/aem.01138-08","type":"journal-article","created":{"date-parts":[[2008,10,4]],"date-time":"2008-10-04T01:09:42Z","timestamp":1223082582000},"page":"7365-7375","update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":46,"title":["Development and Implementation of a Multiplex Single-Nucleotide Polymorphism Genotyping Assay for Detection of Virulence-Attenuating Mutations in the<i>Listeria monocytogenes<\/i>Virulence-Associated Gene<i>inlA<\/i>"],"prefix":"10.1128","volume":"74","author":[{"given":"A.","family":"Van Stelten","sequence":"first","affiliation":[{"name":"Department of Animal Sciences, Colorado State University, Fort Collins, Colorado 80523"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"K. 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