About FDA

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.