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Development of a Multiplex Real-Time PCR Assay with Internal Amplification Control for the Detection of Shigella Species and Enteroinvasive Escherichia coli

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Abstract:

Shigella species, particularly S. sonnei and S. flexneri, remain some of the leading bacterial etiological agents of gastrointestinal diseases in the United States and globally. The isolation and detection of these foodborne pathogens are critical for preventing the spread of disease and facilitating epidemiological investigations aimed at determining the source of a Shigella infection outbreak. A multiplex real-time PCR-based assay was developed that targets all four species of Shigella plus enteroinvasive Escherichia coli. The assay incorporates primers directed to the ipaH genes located on both the virulence plasmid and chromosome, the plasmid-encoded virulence gene mxiC, a mutated mxiC gene (mxiC::kan) that differentiates wild-type strains from a laboratory control strain, and an internal amplification control. More than 50 isolates of all four Shigella species were tested for inclusivity and specificity of the multiplex PCR assay, and more than 30 non-Shigella isolates were tested for exclusivity of the assay. The sensitivity of the assay was 1 to 3 CFU and 5 to 50 fg of target (total) DNA for the ipaH, mxiC, and mxiC::kan gene targets. The assay performed equally well and with no measurable inhibition in the Shigella target reactions when rinsates of several high-risk produce commodities (parsley, cilantro, alfalfa sprouts, and lettuce) were added to the reactions. This multiplex PCR assay is sensitive and specific and has the added dimension of discriminating all Shigella species from the positive control strain so that in any sample analysis other strains can be excluded as a source of contamination.

Document Type: Research Article

Affiliations: Division of Microbiology, Center for Food Safety and Applied Nutrition, U.S. Food and Drug Administration, College Park, Maryland 20740, USA

Publication date: September 1, 2010

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    First published in 1937, the Journal of Food Protection®, is a refereed monthly publication. Each issue contains scientific research and authoritative review articles reporting on a variety of topics in food science pertaining to food safety and quality. The Journal is internationally recognized as the leading publication in the field of food microbiology with a readership exceeding 11,000 scientists from 70 countries. The Journal of Food Protection® is indexed in Index Medicus, Current Contents, BIOSIS, PubMed, Medline, and many others.

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