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United States Department of Agriculture

Agricultural Research Service

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Research Project: Rapid Molecular Pathotyping of Major Salmonella Enterica Serotypes Based on Single-Nucleotide Polymorphisms (Snps) in the Adenylate Cyclas

Location: Egg Safety and Quality

Project Number: 6040-41420-017-01
Project Type: Trust

Start Date: Jun 01, 2012
End Date: Dec 31, 2014

Objective:
1. Identify single nucleotide polymorphisms (SNPs) within the Salmonella enterica cyaA gene unique to the major poultry food safety-related serotypes or S. Enteritidis pathotypes, design primers/probes targeting those SNPs, and develop a hybridization protocol based on the Luminex MagPlex® system. 2. Determine the efficacy and efficiency of this hybridization protocol in detecting the presence, quantity, and relative distribution of the targeted S. enterica serotypes under different cultural and environmental conditions. 3. Expand the utility of the hybridization protocol to include one, or a combination of the following: (1) Additional SNPs within the cyaA gene; (2) SNPs within other target genes such as SEN4316, SEN3898, SEN4256, and SEN1164; (3) Detection of serotypes on environmental samples across the “food to fork” continuum within the poultry industry.

Approach:
Previous work at the USDA-ARS in Athens, GA has shown that small scale genetic changes within the cyaA gene, known as single nucleotide polymorphisms (SNPs), can be used to effectively pathotype different strains of S. Enteritidis (Morales et al., 2007). When combined with just 4 other SNPs, cyaA is central for identifying 7 different types of S. Enteritidis. As a first step to the development of the hybridization protocol, we identified conserved sequence regions upstream (positions 2087-2106) and downstream (positions 2365 – 2387) of this SNP-containing region to create a 300-bp amplicon. Using the cyaA gene sequence alignment single SNPs unique to the 5 strains we are targeting will be identified, and we will design 20-bp probes to target each of these SNPs. Once each individual SNP-probe set is validated, we will sequentially combine multiple SNP probe sets and validate their efficacy for qualitatively identifying different mixtures of S. enterica within a sample until a single, optimized multiplex assay is created to include all 5 SNP probe sets.

Last Modified: 12/20/2014
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