Develop methodologies for rapid detection and identification of foodborne pathogens using PCR (polymerase chain reaction) and quantitative real time (RT)-PCR technologies available in field laboratories; transition microarray technology into field utility for enhanced rapid primary identification and subtyping. Further, develop new methods for Salmonella enterica serotyping using conventional PCR equipment available in ORA and FERN laboratories, as well as evaluate several new technologies for the identification and serotyping of Salmonella enterica. This project is being done in collaboration with ORA and FERN in anticipation of technology transfer for use by ORA and FERN laboratories. This project will provide methods that utilize current equipment and provide data on the feasibility of using newer technology in ORA and FERN laboratories. The methods developed will enhance or replace front-end analyses for pathogen identification while introducing emerging new technologies to the field component. ON TRACK ON TRACK A. Enteric Pathogen Detection: sub i. Investigate new RT-PCR methodologies by transitioning comprehensive Salmonella serovar-specific single nucleotide polymorphism (SNP) panel to RT-PCR platform using high resolution melting curve analysis (HRMA) 3/30/2012 Completed 3/30/2012 sub ii. Develop RT-PCR suite of singleplex molecular assays for the detection and identification of E. coli O157:H7, Shigella, and Salmonella pathogens using new gene- SNP-based targets 6/30/2012 Completed 6/30/2012 sub iii. Adapt RT-PCR assays for the selective identification of viable pathogens 12/31/2012 Completed 9/30/2012 sub iv. Develop multiplex RT-PCR assays for the combined detection of E. coli O157:H7, Shigella, and Salmonella pathogens 3/30/2013 On Track sub v. Further investigate HRMA capacity to distinguish E. coli serotypes and Shigella subgroups; otherwise develop standard PCR-based molecular serotyping for "big six" Shiga toxin-producing E.coli (STEC) and Salmonella 12/31/2013 Not Yet Started B. Microarray technology transition: sub i. Co-develop high-density resequencing by hybridization microarray platform with TessArae, LLC for commercialization 6/30/2012 Completed 8/30/2012 sub ii. Submit final design specifications and procure streamlined next-generation custom Affymetrix microarray design of genomic signatures (genes, SNPs, molecular serotyping) for rapid identification of enteric foodborne pathogens 8/30/2012 Completed 9/30/2012 sub iii. Test and inhouse validate streamlined microarray 12/31/2012 Completed 1/28/2013 sub iv. Transition and deploy array platforms using Affymetrix GeneAtlas Instrumentation System for parallel utility to pulsed-field gel electrophoresis (PFGE) workflow and database development 12/31/2013 Not Yet Started C. Molecular Serotyping of Salmonella enterica: sub i. Evaluate molecular methods including an in-house conventional PCR method, the Diversilab repetitive element PCR (repPCR), and commercially available microarrays for identification and serotyping of Salmonella enterica using multiple isolates of the most clinically relevant serotypes. 6/30/2012 Completed 6/30/2012 sub ii. Evaluate the molecular serotyping methods using Salmonella enterica from produce and materials collected from tomato farms including soil, water, and phyllospheres and rhizopheres collected from tomato plants. The methods will be tested using a variety of sample matrices including pure cultures, pre-enrichment broths, and selective media cultures. 12/31/2012 Completed 9/28/2012 sub iii. Compare the molecular serotyping methods using clinical and environmental samples to determine if the source of the isolate affects the results. Any differences will be further examined using sequence analysis. 6/30/2013 On Track PCR Polymerase chain reaction – the amplification of a specific DNA sequence, termed target or template sequence, that is present in a complex mixture, by adding two or more short oligonucleotides, also called primers, that are specific for the terminal or outer limits of the template sequence. The template-primers mixture is subjected to repeated cycles of heating to separate (melt) the double-stranded DNA and cooling in the presence of nucleotides and DNA polymerase such that the template sequence is copied at each cycle. microarray A microarray is a multiplex lab-on-a-chip. It is a 2D array on a solid substrate (usually a glass slide or silicon thin-film cell) that assays large amounts of biological material using high-throughput screening methods. enteric Of, relating to, or being within the intestine. Single Nucleotide Polymorphism (SNP) A DNA sequence variation occurring when a single nucleotide - A, T, C, or G - in the genome (or other shared sequence) differs between members of a species (or between paired chromosomes in an individual). For a variation to be considered a SNP, it must occur in at least 1% of the population. http://www.fda.gov/AboutFDA/Transparency/track/ucm206221.htmFDA-TRACK CFSAN Dashboard ਍††਍਍਍਍਍਍਍