Identifying salmonella faster

An Iowa State University researcher and a local company are working together on a faster method to detect and genetically identify salmonella from contaminated foods.

The researcher, Byron Brehm-Stecher, an assistant professor of food science and human nutrition, wants to replace the current system of salmonella detection with a new approach that can provide DNA sequencing-like results in hours rather than days.

Brehm-Stecher is working with Advanced Analytical Technologies Inc., which is providing advanced biomedical instruments and reagents for the research.

The recent results of the research, funded by the Grow Iowa Values Fund, will be presented at the August meeting of the International Association for Food Protection in Anaheim, Calif.

How we identify salmonella now

Currently, definitive genetic identification of food-borne pathogens is done using traditional DNA sequencing methods first developed in the 1980s.

“If you want (DNA) sequence information now, you first need to run a polymerase chain reaction (PCR) on total DNA extracted from a sample of contaminated food,” says Brehm-Stecher.

“This amplifies DNA from the pathogen you’re looking for and will let you know if salmonella is present or not.”

“However, further details about the pathogen are lacking, like what strain is present. To dig deeper, you need to run a cycle sequencing reaction – similar to a long PCR reaction – and send the output from this to a DNA sequencing core facility. Results are available about two days later,” he explains.

This process is not fast enough when you consider the pace of today’s food production and distribution networks.

“We are able to get foods from the farm to the table – really any table around the globe – in a remarkably short period of time,” adds Brehm-Stecher.

Faster detection of specific strains can mean recognizing an outbreak sooner and stopping tainted food from being delivered and consumed.

New approach; bridging the gap

The new method might be helpful for investigative agencies, says Brehm-Stecher.

“Especially for the type of investigation where things are still in motion. The food has been shipped and you may not know where it is. It may be in a truck, on a shelf or in some consumer’s pantry, so time really is of the essence,” he says.

“Next-generation sequencing tools are available, but these are still too complex and expensive for routine use in the food industry,” explains Brehm-Stecher.

“New approaches that are able to bridge the gap between the limitations of traditional PCR and next-generation sequencing could enhance food safety efforts by providing both rapid presence/absence testing and detailed genetic characterization of isolates.”

The method being developed at Iowa State starts with a rapid PCR reaction that amplifies a salmonella-specific gene, generating millions of fluorescently labelled copies of this DNA in about 20 minutes.

Next, instead of cycle sequencing, the PCR product is purified for five minutes, SNAP71 (a reagent developed by Advanced Analytical) is added, and the DNA is heated for 10 minutes at 100ºC.

This reaction chemically cuts the labelled salmonella DNA at all adenine and guanine sites (A’s and G’s) in the DNA chain.

The result is a complex soup of fluorescently labelled DNA fragments of all sizes.
These fragments are then separated in a high-voltage electric field by sieving them through a polymer matrix (a gel) contained in glass capillaries that are 50 microns – not much thicker than a human hair.

This process separates the DNA fragments according to their size, from smallest to largest, and each piece is detected as it passes in front of an intense light source.

For a PCR product that’s 300 bases long, this separation and detection process takes approximately 90 minutes.

Because the SNAP71 reagent cleaves the salmonella DNA only at adenine and guanine, and not at thymine and cytosine sites (T’s and C’s), the method is not a direct replacement for DNA sequencing.

Instead, the process rapidly generates a reproducible pattern of DNA fragments, says Brehm-Stecher.

Salmonella strains having slightly different DNA sequences within a given gene will yield different patterns of fragments, allowing discrimination of different strains of salmonella.

From “food to finish,” the whole process can be accomplished in about two and a half hours.

The ultimate goal of the project is faster detection and characterization of human pathogens from “farm to fork to physician.”