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Applied and Environmental Microbiology, December 2008, p. 7365-7375, Vol. 74, No. 23
0099-2240/08/$08.00+0     doi:10.1128/AEM.01138-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Development and Implementation of a Multiplex Single-Nucleotide Polymorphism Genotyping Assay for Detection of Virulence-Attenuating Mutations in the Listeria monocytogenes Virulence-Associated Gene inlA{triangledown} ,{dagger}

A. Van Stelten and K. K. Nightingale*

Department of Animal Sciences, Colorado State University, Fort Collins, Colorado 80523

Received 20 May 2008/ Accepted 24 September 2008

The virulence factor internalin A (InlA) facilitates the uptake of Listeria monocytogenes by epithelial cells that express the human isoform of E-cadherin. Previous studies identified naturally occurring premature stop codon (PMSC) mutations in inlA and demonstrated that these mutations are responsible for virulence attenuation. We assembled >1,700 L. monocytogenes 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 inlA genes from isolates with attenuated invasion capacities revealed three novel inlA PMSCs which had not been identified previously among U.S. isolates. Since ribotypes include isolates with and without inlA PMSCs, we developed a multiplex single-nucleotide polymorphism (SNP) genotyping assay to detect isolates with virulence-attenuating PMSC mutations in inlA. The SNP genotyping assay detects all inlA 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 inlA PMSC mutations. PMSCs in inlA were 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. monocytogenes 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 L. monocytogenes isolates carrying virulence-attenuating PMSC mutations in inlA from strains representing the most significant health risk.

* Corresponding author. Mailing address: Department of Animal Sciences, Colorado State University, 108B Animal Sciences Building, Fort Collins, CO 80523-1171. Phone: (970) 491-1556. E-mail: kendra.nightingale{at}colostate.edu

{triangledown} Published ahead of print on 3 October 2008.

{dagger} Supplemental material for this article may be found at http://aem.asm.org/.

Applied and Environmental Microbiology, December 2008, p. 7365-7375, Vol. 74, No. 23
0099-2240/08/$08.00+0     doi:10.1128/AEM.01138-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.





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