Faster salmonella strain detection now possible with new technique
providing both rapid presence/absence testing and detailed genetic characterization of isolates.”
You do not have to go further than the local newspaper to see the depth of the problem. Recent national outbreaks of salmonella in foods include peanut butter (2007 and 2009), alfalfa sprouts (2009), black pepper and hydrolyzed vegetable protein (HVP) (2010). Adding to the problem is the fact that peanut butter, black pepper and HVP are all base ingredients used in many other food products. Salmonella in these ingredients has led to thousands of product recalls, hundreds of illnesses and several deaths, Brehm-Stecher said.
The method being developed at Iowa State University starts with a rapid PCR reaction that amplifies a salmonella-specific gene, generating millions of fluorescently labeled 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 labeled 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 labeled 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, Brehm-Stecher said.
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. “We’re very excited about this approach and about the rapid progress we’ve made since the project began,” said Brehm-Stecher. “The funding for this project has enabled us to work very closely with Advanced Analytical and accelerate application of their instruments to solving important food safety problems.”
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.
The ultimate goal of the project is faster detection and characterization of human pathogens from “farm to fork to physician.”
Advanced Analytical’s instruments are based on technology originally developed at Iowa State University in the lab of Ed Yeung, the Robert Allen Wright Professor and Distinguished Professor in Liberal Arts and Sciences and professor at the U.S. Department of Energy’s Ames Lab.
