AMES, IOWA – An Iowa State University researcher is working on a faster method to detect and genetically identify Salmonella from contaminated foods using technology available through a local company. 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 D.N.A. sequencing-like results in hours rather than days.
Advanced Analytical Technologies Inc., from Ames, Iowa, is providing advanced biomedical instruments and reagents for the research.
Definitive genetic identification of food-borne pathogens is currently being done using traditional D.N.A. sequencing methods first developed in the 1980s.
"If you want [D.N.A.] sequence information now, you first need to run a polymerase chain reaction [P.C.R.] on total D.N.A. extracted from a sample of contaminated food," Mr. Brehm-Stecher said. "This amplifies D.N.A. 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,” he added. “To dig deeper, you need to run a cycle sequencing reaction – similar to a long P.C.R. reaction – and send the output from this to a D.N.A. sequencing core facility. Results are available about two days later.
"This is not fast enough to keep up with the pace of today's food production and distribution networks,” he continued. “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.”
The method being developed at Iowa State University starts with a rapid P.C.R. reaction that amplifies a Salmonella-specific gene, generating millions of fluorescently labeled copies of this D.N.A. in about 20 minutes.
Next, instead of cycle sequencing, the P.C.R. product is purified for five minutes, a reagent developed by Advanced Analytical is added, and the D.N.A. is heated for 10 minutes at 100ºC. This reaction chemically cuts the labeled Salmonella D.N.A. at all adenine and guanine sites (A's and G's) in the D.N.A. chain.
The result is a complex soup of fluorescently labeled D.N.A. 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. This process separates the D.N.A. 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 P.C.R. product that's 300 bases long, this separation and detection process takes approximately 90 minutes.
Because the SNAP71 reagent cleaves the Salmonella D.N.A. only at adenine and guanine, and not at thymine and cytosine sites, the method is not a direct replacement for D.N.A. sequencing. Instead, the process rapidly generates a reproducible pattern of D.N.A. fragments, Mr. Brehm-Stecher said.
Salmonella strains having slightly different D.N.A. 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 team at Iowa State University includes post doctoral researcher Hyun Jung Kim and master's student Brittany Porter. The group is also working with Cleveland Clinic in Ohio.