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