TUCKER, Ga. — The USDA’s Food Safety Inspection Services (FSIS) has declared Salmonella as an adulterant in raw breaded stuffed chicken products that exceed 1 CFU/gram, necessitating the development of methods for quantifying Salmonella in poultry samples.
To date, limited research has been conducted on the development of these methods. Currently, only three relative quantification-based PCR assays are available for quantification of Salmonella levels, which provides a very low number of options. Recent independent validation studies on one of the available methods have shown a lack of reproducibility, further limiting available options for the poultry industry. Meanwhile, another current option for detecting and quantifying Salmonella uses a complex DNA extraction protocol.
Diagnostic laboratories rely heavily on PCR or real-time PCR-based methods for the detection of target foodborne pathogens. One of the major and overlooked issues associated with a high number of foodborne infections is the current food testing approach, which relies on “presence / absence” testing of the Salmonella in food or food enrichments and ignores the Salmonella concentration in the contaminated samples.
A food sample that is positive for Salmonella or any other foodborne pathogen by PCR may contain anywhere between one and a million or more cells of the pathogen. Food contaminated with a pathogen concentration above the infective dose is a much greater public health hazard compared to food with a pathogen concentration below its infective dose. Hence, in addition to detecting pathogens, quantification of the pathogen concentration in a food sample is critically important for understanding the hazards associated with food and the intervention measures needed to mitigate the hazard.
The poultry industry needs methods that are free of intellectual property and can be used for reliable Salmonella quantification across various poultry samples without any standard curves.
During the USPOULTRY Spring Research Competition, a project titled, “Digital PCR Method for the Detection and Estimation of Salmonella Load in Poultry Samples,” was funded. Dr. Prashant Singh, associate professor in the Department of Health, Nutrition, and Food Sciences, at Florida State University, is the lead investigator for this project.
This funded grant aims to standardize and validate a partition-based digital PCR assay for the absolute quantification of Salmonella in chicken breast, ground chicken, and boot swab samples. The approach will include optimization of the digital PCR workflow for three matrices. In phase one, DNA isolation protocol, lysis buffer volume, enrichment media volume, enrichment time, and DNA volume for digital PCR reaction will be optimized. In the second phase, the optimized protocol will be validated with lab-inoculated poultry samples using cold-stressed strains of Enteritidis, Typhimurium, and I 4,[5],12:i:-, serotypes and samples from processing facilities and farms.
The cost of a digital PCR instrument is lower than some qPCR instruments and has comparable reagent costs. Digital PCR is highly resistant to the presence of PCR inhibitors (i.e., protein, fat), making the digital PCR assay more reproducible.
Additionally, the digital PCR assays can detect the target in a sample with high background non-target DNA, does not require any sample dilution as the reaction can handle micrograms of crude DNA whereas real-time PCR stop working at 100-200 nanograms of DNA, and has a very similar workflow commonly used by food testing lab professionals. Further, it has been shown that the digital PCR assay typically generates more precise and reproducible results.
The digital PCR workflow standardized in Dr. Prashant Singh’s laboratory for the quantification of Salmonella addresses the critical needs of the federal and independent third-party food testing laboratories.
This USPOULTRY-funded grant may result in the development of an assay for the detection and quantification of Salmonella load in poultry samples, allowing the industry and regulatory agencies to improve compliance testing, enhance food safety and decrease the number of Salmonella-associated outbreaks and infections.
Compared to real-time PCR, digital PCR technology is more advanced, unaffected by PCR inhibitors and crude DNA, and generates more reliable results without any standard curves, which can be a huge benefit for the industry. The workflow developed will enable the poultry industry to have a protocol that can be used across various sample types and generate reproducible results across all their processing facilities at a price and completion time comparable to real-time PCR assay.
Dr. Denise Heard is vice president of research with the U.S. Poultry & Egg Association based in Tucker, Ga. She can be reached by e-mail at dheard@uspoultry.org.
