Salmonella poultry detection methods, advances

DNA-based detection techniques provide a catch-all approach for identifying different types of Salmonella in poultry products.

trans961 | | There are more than 2,500 known different Salmonella commonly referred to as serovars, and some of these Salmonella serovars are highly associated with poultry products.
trans961 | | There are more than 2,500 known different Salmonella commonly referred to as
serovars, and some of these Salmonella serovars are highly associated with poultry products.

Simple identification of the presence of Salmonella species may not be enough for future foodborne pathogen control strategies. Knowing not just whether Salmonella are present but how many are on respective raw chicken carcasses or resulting poultry products may be equally critical for assessing potential risk to the consumer.

Techniques such as multiplex polymerase chain reaction (PCR) assays are useful for identifying more than one type of foodborne pathogen, and they shorten the time-to-results in food sampling and reduce costs when compared to single PCR-based assays.

Another technique is quantitative PCR, which provides additional analyses in that the initial DNA amount can be determined, or quantitated.

Optimizing detection of foodborne pathogens

The Center for Food Safety at the University of Arkansas in Fayetteville recently conducted a series of studies funded by the U.S. Poultry & Egg Association on ways to optimize the detection of foodborne pathogens with an emphasis on Salmonella species. Led by Dr. Steven C. Ricke, the Donald “Buddy” Wray chair in food safety and director for the University of Arkansas's Center for Food Safety in the Institute of Food Science and Engineering, and Si Hong Park, postdoctoral associate, this research group investigated ways to use DNA-based detection techniques as a catch-all approach for identifying different types of Salmonella in poultry products.

Salmonella’s challenge to poultry

Salmonella can wreak havoc on the economic stability of the poultry industry through the production process if contamination and subsequent human illness occurs. These occurrences could result in litigation, and the medical expenses that can result from consumers are a major concern. Also, the inability to properly identify foodborne pathogens such as Salmonella in poultry products often leads to economic loss due to recalls and overall reductions in product yields.

There are more than 2,500 known different Salmonella commonly referred to as serovars. Some of these Salmonella serovars are highly associated with poultry products, and thus catch the eye of researchers who aim to develop applicable detection strategies for the poultry industry. Salmonella serovars such as Enteriditis, Typhimurium, Heidelberg and Kentucky are commonly associated with poultry products; the methods developed by the University of Arkansas focused on identifying Enteriditis, Typhimurium and Heidelberg due to their association with foodborne illnesses.

Progress in Salmonella detection methods

Current detection methods approved by the U.S. Department of Agriculture's Food Safety and Inspection Service (USDA-FSIS) involve an enrichment step to increase the number of bacterial cells in a food sample, which is followed by conventional PCR that enables the identification of Salmonella. Once a Salmonella-positive sample is indicated by PCR, classical serotyping methods are then used to identify the specific type of Salmonella in that particular sample.

Primers, a specific sequence of DNA base pairs, are utilized in PCR reactions to amplify the target DNA to an amount that is detectable by the PCR assay. Improvements on PCR assays have been made in the past, and developing primers that are highly specific for certain targets is a proactive approach for many food detection strategies.

A number of Salmonella isolates have been sequenced, and this data is useful for developing serovar-specific primers that can be used in PCR assays. As whole genome sequencing continues to be implemented for foodborne pathogen lineage and outbreak investigations, databases will be available for scientists to evaluate the regions of DNA that are unique to certain microorganisms and result in more broad identification methods.

Multiplex PCR assays

Salmonella Enteriditis, Typhimurium and Heidelberg have unique regions of DNA that can be targeted with primers, and these primers have been combined in a single PCR assay (known as a multiplex assay) for the simultaneous distinction and identification of these serovars. Multiplex PCR assays are useful for identifying more than one type of foodborne pathogen, or in this instance, more than one serovar with the addition of primers specific for Salmonella Enteriditis, Typhimurium and Heidelberg. Multiplex PCR assays shorten the time-to-results in food sampling and have been estimated to reduce the assay cost by 60 percent when compared to single PCR-based assays.

Previous multiplex assays have been developed, but the multiplex PCR assay optimized by the University of Arkansas' Center for Food Safety integrates two necessary components. A Salmonella genus primer, which identifies Salmonella regardless of serovar, has been integrated into this method to account for more rare, and thus, unexpected Salmonella serovars that may result in product contamination.

The Salmonella genus-specific primer is an important component of these assays to identify other Salmonella serovars that may not typically be associated with poultry. Additionally, a Salmonella subspecies I primer has been added. Salmonella subspecies I isolates can lead to disease in warm blooded animals, and identification of these isolates with this assay will provide heightened awareness for public health and risk management practices. The key to this type of PCR strategy is to avoid future surprises by emerging strains of Salmonella that suddenly appear on poultry products.

Quantitative PCR assays

In addition to multiplex PCR assays, quantitative PCR (qPCR) assays could be utilized with serovar-specific primers. In theory, if a single microorganism is present in a sample, it will be identifiable with conventional single and multiplex PCR assays in a presence/absence (positive/negative) manner.

Quantitative PCR provides additional analyses in that the initial DNA amount can be determined due to the real-time analysis that take places after each PCR cycle and subsequent amplification of DNA. For example, if it only takes 17 cycles for a sample 1 in the qPCR assay to reach threshold compared to 23 cycles for sample 2, it can be concluded that sample 1 had more target DNA initially compared to sample 2 since it took fewer cycles to reach threshold.

Standard curves are generated to help determine how cycle threshold correlates with actual amounts of the foodborne pathogens, and the samples can subsequently be quantitated. Implementing qPCR would be useful for providing a data set of the baseline Salmonella load on poultry products, which could then be connected to risk assessment models.

In many instances, detection strategies will only find what they are looking for, and so this particular method provides means to identify all Salmonella serovars, common or not.

Optimization of Salmonella detection methods                   

Detection methods will likely always progress to benefit the poultry industry, and future approaches can aim at determining methods that account for other costly foodborne pathogens besides Salmonella such as Campylobacter. Additionally, these genetic targets should be evaluated throughout the optimization process to ensure that these foodborne pathogens contain the target DNA – a concern is that foodborne pathogens will have DNA-rearrangements, which could potentially alter the DNA target the primers are geared toward, and result in false negatives.

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