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U.S. Department of Health and Human Services

About FDA

New Field Ready Methods for Enteric Pathogen Detection and Identification

Description: 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.

Accomplishment: All the PCR assays developed under this key project can be performed in any microbiology laboratory with PCR capability, i.e., most laboratories within ORA and FERN. These more rapid detection and identification methods will result in quicker trackback and recall, potentially leading to reduction in exposure to contaminated food and therefore number of illnesses.

  • Singleplex RT-PCR assays were developed and published for highly sensitive and specific detection of E. coli O157:H7 and Salmonella. In addition, combining these assays with propidium monoazide (PMA) treatment permitted the selective detection of viable pathogens from foods (Appl Environ Microbiol. 2012. 78:5297-304; BMC Microbiol. 2013. 13:273).

  • Further RT-PCR-based methods developments using high resolution melting curve analysis were developed in standard 96-well plate format for Shigella spp. and approximately seven major serovars of Salmonella enterica using signature SNPs.

  • Conventional PCR and novel microarray designs were developed for molecular serotyping and comprehensive subtyping of E. coli pathogens (including non-O157 STEC). The microarray designs were validated and transferred to FDA field laboratory in Irvine, CA.

  • The PCR assay that was developed and published (Food Microbiology. 2012. 31:199-209) can determine Salmonella enterica serotypes from both pure isolates and enrichment cultures. The method can determine 33 of the most common serotypes associated with foodborne illness, and work is continuing to add additional serotypes to the assay. This serotyping assay is currently undergoing a multi-laboratory validation coordinated through the Office of Food and Veterinary Medicine (OFVM) Method s Validation Coordination Committee (MVCC), and will be transferred to Office of Regulatory Affairs (ORA) and Food Emergency Response Network (FERN) laboratories following validation.

  • In collaboration with laboratories within the FERN, a database has been built for molecular serotyping of Salmonella enterica using the Diversilab system. The Diversilab was evaluated as part of a CFSAN exercise examining technologies capable of cluster analysis and molecular serotyping, a manuscript is in preparation.

  • Milestone DescriptionMilestone DateMilestone StatusMilestone Completion Date

    A. Enteric Pathogen Detection:


    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)


    Completed 3/30/2012

    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


    Completed 6/30/2012

    iii. Adapt RT-PCR assays for the selective identification of viable pathogens


    Completed 9/30/2012

    iv. Develop multiplex RT-PCR assays for the combined detection of E. coli O157:H7, Shigella, and Salmonella pathogens


    Completed 9/30/2013

    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


    Completed 4/30/2014

    B. Microarray technology transition:


    i. Co-develop high-density resequencing by hybridization microarray platform with TessArae, LLC for commercialization


    Completed 8/30/2012

    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


    Completed 9/30/2012

    iii. Test and inhouse validate streamlined microarray


    Completed 1/18/2013

    iv. Transition and deploy array platforms using Affymetrix GeneAtlas Instrumentation System for parallel utility to pulsed-field gel electrophoresis (PFGE) workflow and database development


    Completed 4/30/2014

    C. Molecular Serotyping of Salmonella enterica:


    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.


    Completed 6/30/2012

    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.


    Completed 9/28/2012

    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.


    Completed 6/30/2013

    Key Projects Legend

    Milestone StatusDefinition
    Not Yet StartedWork for specific milestone has not yet been started.
    CompletedMilestone and/or overall project is completed.
    On TrackMilestone - On track for completion by milestone deadline. Quarter status - Project is on track for completion based on overall milestone status.
    On HoldMilestone - On hold, but deadline for completion has not passed. Quarter status - Project is on hold, based on overall milestone status.
    DelayedMilestone - Delayed as it has not been completed and deadline has passed. Quarter status - Project is delayed based on overall milestone status.



    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.


    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.


    Of, relating to, or being within the intestine.

    Note: The data provided on this website is produced on an ongoing basis for performance management purposes and is subject to change due to updates of preliminary estimates, corrections, or other reasons. In addition, FDA may change the type or amount of data provided on this website at any time. Information marked as "Completed" may include measures and/or key projects for which activities are ongoing but no longer tracked as part of FDA-TRACK.